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+Sesame allergy is a life-threatening disease that has been growing globally with poorly understood mechanisms. To protect sensitive consumers, sesame is regulated in many countries. There were four research goals for this work on sesame allergy: (i) to map the timeline, and the extent of its global rise; (ii) to dissect the complexity of the disease, and its mechanisms; (iii) to analyze the global regulation of sesame; and (iv) to map the directions for future research and regulation. We performed a literature search on PubMed and Google Scholar, using combinations of key words and analyzed the output. Regulatory information was obtained from the government agencies. Information relevant to the above goals was used to make interpretations. We found that: (i) the reports appeared first in 1950s, and then rapidly rose globally from 1990s; (ii) sesame contains protein and lipid allergens, a unique feature not found in other allergenic foods; (iii) it is linked to five types of diseases with understudied mechanisms; and (iv) it is a regulated allergen in 32 advanced countries excluding the USA. We also provide directions for filling gaps in the research and identify implications of possible regulation of sesame in the USA.There is sound evidence that the Western world is in the midst of an ongoing widespread food allergy, and that some of the Eastern world is noticing its rise as well [1,2,3,4]. For example, recent reports show that the prevalence of food allergies in the United States of America (USA) have increased to 8% in children and 10.8% among adults from their respective prevalence rates of 6% and 3.7% just 15 years ago [5,6]. This data translate to an increase of 33% among children, and a staggering 191.8% rise among adults during this short historical period. A similar rise in food allergy prevalence has been noted in Canada, Australia, European countries, New Zealand, and Israel [4]. Furthermore, in the Eastern world, once thought to be free from food allergies, increasingly evidence shows their rise in Japan, Singapore, South Korea, and India [1,7,8]. A few African countries have recently reported cases of food allergies as well [9,10]. Thus, food allergies are on the rise in multiple continents, and therefore warrant serious considerations for further research and food safety regulation.Since avoidance of food allergens is critical in protecting allergic consumers, the USA, Canada, European Union (EU) countries, the United Kingdom (UK), Australia, New Zealand, and Japan have developed their own lists of priority/major allergenic foods for regulatory purposes [11,12,13,14,15,16] (Table 1). There are many allergic foods that are commonly regulated across all these countries. However, some foods are not. For example, in the USA, sesame, mustard, lupine, celery, mollusks, and sulfites are not presently regulated as food allergens. During the past two years, sesame allergy has been increasingly discussed in the USA, and the United States Food and Drug Agency (US FDA) has been considering whether or not to include sesame into the list of major allergens for regulation [2,11].Sesame is a major global agricultural crop whose production has increased from 2.3 million tons in 1994 to more than 5.5 million tons in 2017 [17]. Interestingly, sesame allergies are not reported from the top ten sesame-producing countries in the world, including India and China, which are the top two largest sesame producers [17]. Sesame seeds are available in three different colors—black, white, and brown (Figure 1). They contain 50% to 60% oil. Both seeds and oils are commonly used in food preparations. For example, sesame seeds are added to foods (such as bread, bagels, buns, pizza, etc.) or used directly in making food items (such as tahini, hummus, dry food powder, and confectionaries) [18]. Sesame oil is used in salad dressings and for the deep-frying of foods. Sesame oil has long been regarded as safe and inert [18,19]. Therefore, it is used very commonly in pharmaceutical and cosmetic industries. For example, oil is used in ointments, in intramuscular injections to deliver hormones and drugs, in lipsticks, body oils, and in moisturizing creams [18]. Furthermore, sesame oil is widely used in ancient medical practices of Indian (Ayurveda) and Chinese systems for pain disorders [18,20,21]. Furthermore, exposure to sesame also occurs in occupational settings (e.g., bakeries and oil industry) where it is dealt with as an occupational hazard for workers [22,23,24]. Thus, environmental exposure of humans to sesame and sesame containing products has been increasing globally.There were four research goals for this article: (i) to map the timeline, and the extent of the global rise of sesame allergy; (ii) to dissect the complexity of sesame allergy, and identify the gaps in the mechanisms of the disease; (iii) to compare and contrast the global regulation of sesame and to identify the challenges; and (iv) to map the directions for future research and regulation.We performed a literature search on PubMed and Google Scholar using the key words. We retried 265 documents from the PubMed using the key words ‘Sesame AND allergy’. Other combination of key words retrieved 212 to 215 documents from the PubMed. We retrieved 4490 articles from Google Scholar search using the combination of key words ‘Sesame AND allergy AND hypersensitivity AND human’. We analyzed the output, removed the duplications and identified the relevant articles in English language for further analysis. All used articles have been cited in the references. The relevant information pertaining to the study goals was then used to produce collective data and make interpretations to accomplish the goals. Published information by food safety regulatory agencies on their respective webpages was used to collect, analyze, and interpret the information on the regulation of sesame.Results from this work has provided the concise and an up to date information on the published evidence related to the timeline of the global rise of sesame allergy, the natural history of sesame allergy, the diverse nature of the sesame allergens, the complexity of diseases with largely unknown underlying mechanisms, the gaps in the scientific knowledge, and the challenges and implications for potentially regulating sesame as a major allergenic food in the USA. We think that this timely article will stimulate advances in sesame allergy research, as well as provide the scientific background and a context to the ongoing discussions on whether or not to regulate sesame as a major food allergen in the USA, and perhaps in other countries as well.Sesame has been used by humans since the ancient times as a food, as well as a medicine. Earliest records of use dates back to 2450 BC [25]. We have tabulated the origin and the major milestones of the global rise of sesame allergies [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48] (Table 2). Reports of adverse reactions to sesame started appearing from the 1950s and then rose dramatically during the past thirty years. Ironically, although sesame is not currently regulated in the USA, the very first report on sesame seed and oil allergy and anaphylaxis came from this country [26]. The first report describes a sudden, multi-organ onset of symptoms in a subject by 30 min of consumption of food containing sesame seed and sesame oil. Reactions included gut and skin symptoms, as well as anaphylaxis. Skin testing was positive with sesame seed extract. The author raised safety concerns for sesame for the first time in the modern medical history. A year later, a second case with symptoms of asthma, as well as food allergic reaction caused by sesame seed was also reported from the USA [27].The first report of an allergic reaction to sesame oil was also from the USA [28]. These authors surprisingly found that sesame oil, which had been used as a control oil for gold therapy in rheumatoid arthritis patients, had triggered generalized urticaria and pruritus in 25% of control subjects (5/25) when intramuscularly injected. They warned that, in contrast to the popular medical notion that sesame oil was inert and safe, serious adverse reactions to sesame oil must be considered.Since then, the number of reports on sesame allergy from multiple countries have steadily grown in the literature. However, estimation of the prevalence of sesame allergy did not begin until the later part of the 1990s. Although the first estimation of population prevalence of sesame allergy in the UK showed it a low level, later studies reported significantly increased prevalence. For example, based on perception, a prevalence rate of 0.6% among children >15 years old was reported subsequently [49]. Later studies, based on skin prick testing, estimated a prevalence of 0.2–0.9% among the children of the UK [50,51,52]. However, based on oral challenge confirmations, the prevalence was at the rate of 0.1% [52]. Similarly, in Australia also, increased prevalence among infants (0.8%) has been reported since the first prevalence study [53,54].In light of the extensive use of sesame in the Middle Eastern diet, there is a growing interest in studying sesame allergy in those countries with the report from Israel. The sesame allergy was the third most common food allergy amongst Israeli children after egg and milk; and sesame was the second most common trigger of anaphylaxis after milk [33]. A recent study showed that the prevalence of sesame allergy in Israel among young adults (17–18 years) was at 0.09% [55]. In Lebanon, sensitization (i.e., IgE, and skin prick testing positivity) to sesame among infants, children, and adults was at 3.9%, 2.65% and 1.9%, respectively; and anaphylaxis was the only clinical symptom [37]. Later studies have identified that the sesame allergy and anaphylaxis are also a growing problem in Saudi Arabia, Iran, Kuwait, and Turkey [39,41,42,48]. Thus, the extensive use of sesame in the food and the increased reporting of sesame allergy among the Middle Eastern population suggests a strong correlation of disease with widespread exposure to sesame via the food.Although the first US prevalence study on sesame allergy, based on self-reporting, found a rate of 0.1% among the general population, a very recent report and the largest study to date (78,851 subjects), showed that 0.49% of the US population reported sesame allergy [2]. Based on at least one stringent symptom, they found that 0.23% of the population had confirmed sesame allergy. These two studies, conducted about a decade apart, suggest substantial increase (230–490%) in the prevalence of sesame allergy during the past decade in the USA [2]. Sesame allergies have also become a significant public health problem in Canada and Mexico [36,40].In Asian countries, sesame allergies appear to be not very common, with the exception of Singapore, where nearly 3.7% of food allergic children had sensitization to sesame as measured by skin prick testing [56]. Interestingly, there are no reports on sesame allergy from India, China, or other countries where it is commonly grown and used. It is unclear whether or not this is due to absence of actual disease, or simply a lack of reporting due to inadequate diagnosis and testing. Nevertheless, as discussed above, there is now growing evidence that the burden of sesame allergy appears to be increasing in European countries, the Americas, Australia, and Middle Eastern countries with a high risk of life-threatening anaphylaxis (Table 2).Most food allergies start early in infancy or childhood (during the first three years of life after birth), and so do most sesame allergies [3,5,57,58]. However, adult-onset food allergies are also fairly common [3]. A recent study shows that 1 in 4 sesame allergies are indeed adult-onset types [2]. A similar frequency of adult-onset food allergy has been previously reported for shellfish, milk, wheat, tree nuts, and soy [58].Some food allergies are outgrown more commonly than others. For example, even though children develop allergies to egg, milk, soy, and wheat early on in life, most of them outgrow their food allergies before becoming adults [3,4,58]. In contrast, most (70–80%) children with allergies to peanut, tree nut, fish, shellfish, and sesame do not outgrow their food allergies [2,3,49,58,59,60]. In general, there is no evidence for a major gender-bias for either sesame allergies or for other major food allergies [2,3,18].Thus, similar to other major food allergies, sesame allergies also tend to be life-long health problems for both children and adults. All must avoid the offending allergenic foods for the rest of their lives until they outgrow their food allergies as verified by their doctors. Therefore, sesame allergy subjects face the same set of challenges of avoiding the allergen as do subjects with other major types of food allergies.Sesame allergy does not appear as just one type of disease. Rather, current evidence from the literature shows that it includes at least five types of distinct clinical entities based on the nature of clinical presentations as summarized below and illustrated in Figure 1.In the first type, clinical symptoms appear immediately within minutes to 3 h after exposure to sesame seeds. Symptoms include angioedema, vomiting, diarrhea, urticaria (hives), conjunctivitis, systemic anaphylaxis, and airways allergic reactions including asthma [1,2,3,4,5,18,58]. This reaction is potentially deadly.The second type of reaction is of immediate nature, however, it is triggered by sesame oil, where systemic anaphylaxis, including generalized pruritus, erythema, vomiting, and dizziness occurs within minutes to hours after eating the sesame oil containing foods [18,61,62]. This reaction is also potentially deadly.The third type of reaction includes mainly skin symptoms (i.e., allergic contact dermatitis) a day or two after exposure to the sesame oil [29,63,64,65]. This is not a life-threatening reaction. However, without the prompt diagnosis and avoidance of exposure to the sesame oil, it can become a chronic inflammatory skin condition.The fourth type involves acute reactions with colic and diarrhea within 1–4 h upon eating sesame. This condition is known as acute food protein-induced enterocolitis syndrome (AFPIES) with explosive (or projectile) vomiting as the most common symptom [66,67]. This reaction can be potentially fatal in infants and children.Finally, the fifth type includes chronic inflammation of the esophagus leading to dysphagia and is associated with excessive number of eosinophils in the esophagus—a condition known as eosinophilic esophagitis (EOE) [2,5,68,69]. Without diagnosis and elimination of sesame from the diet, this can become a chronic, debilitating condition. However, it is not a life-threatening reaction.Current evidence shows that sesame produces at least two distinct groups of allergens—protein allergens present in the sesame seeds and lipid allergens present in the sesame oil. There are eight protein allergens identified in sesame to date—Ses i 1 to Ses i 8, with molecular weights ranging from 7 to 57 kDa (Table 3). There is the possibility that additional protein allergens may be present in the sesame seed that remain to be characterized [70,71,72,73,74].Six of the sesame protein allergens (Ses i 1, Ses i 2, Ses i 3, Ses i 6, Ses i 7, and Ses i 8) are hydrophilic in nature and therefore are soluble in aqueous solutions. They belong to 4 different protein families that function as seed storage proteins—2S albumin, 11S globulin, 7S vicilin, and profilin [70,71,72,73,74]. Seed storage proteins are an important source of nutrients during seed germination. Allergy to seed storage protein is easily detected by conventional skin testing with aqueous extracts.Two sesame protein allergens, Ses i 4 and Ses i 5, are hydrophobic in nature, and are associated with oil. These are known as oleosins [75,76]. They function as structural proteins present in the oil bodies and stabilize the oil droplets. They are linked to severe anaphylaxis. However, allergy to oleosins is not detectable by conventional skin testing done with aqueous extracts because of their insolubility. They are major allergens in France and minor allergens in the Netherlands [75,77].Sesame oil contains at least three lipid allergens. Previous studies have identified these lipid allergens as unsaponifiable lignan molecules with a small ring structure—Sesamin, Sesamol, and Sesamolin [29,65]. These investigators used purified lipid allergens and confirmed their ability to cause allergic contact dermatitis by conducting skin patch testing in sensitized subjects. There is no current evidence that other major allergenic foods, including peanut oil, contain any lipid allergens [3,4,78]. There are reports on local and systemic contact dermatitis in cashew nut industry workers. There is also report of such a reaction upon eating pesto sauce contaminated with cashew nutshell oil, which contains lipid allergens similar to urushiol present in poison ivy [79,80,81].As noted above, sesame has been associated recently with AFPIES and EOE [66,67,68,69]. However, the biochemical nature of allergens responsible for triggering these immune reactions is unknown.The mechanisms of the pathogenesis of sesame allergy are largely understudied. It is simply assumed to be similar to other food allergies. Most food allergies manifest in two general forms based on the time of appearance of clinical symptoms after exposure and the underlying immune mechanisms involved in eliciting them [3,4,82]. They are: (i) immediate (or Type I or IgE antibody-mediated) hypersensitivity reactions, where symptoms appear within minutes to 3 h; and (ii) delayed (or Type IV or cell mediated) hypersensitivity reactions, where symptoms of reaction appear after 48–72 h of exposure to the food allergens [83,84]. Most food allergies are of the Type I nature. However, some food allergies such as milk allergies can involve both Type I and Type IV reactions [84].Mechanisms underlying these two distinct types of reactions are completely different. However, both involve two essential steps: (i) sensitization phase; and (ii) disease elicitation phase [3,4,82]. Although specific mechanisms of these phases for sesame allergy have not been widely studied, they are thought to be similar to other allergies. Therefore, using general mechanisms of food allergy development, these phases are described below for sesame allergy.Mechanisms through which sesame seed proteins specifically trigger sensitization in humans are largely unknown. However, based on mechanisms in a mouse model of sesame allergy, and based on the general mechanisms of other food allergies, the following model is proposed (Figure 2) [3,4,82,85,86].Exposure of a genetically susceptible subject to sesame protein allergens via the oral and/or skin routes results in capture of the allergenic proteins by the professional antigen presenting cells (APCs) (e.g., Langerhans cells in the skin, dendritic cells, macrophages/monocytes in the gut) [3,4,82] (Figure 2). These APCs process the allergen and present its fragments as peptides in association with the Major Histocompatibility Complex (MHC) class-II molecule to allergen specific naïve T helper (Th) lymphocytes. In association with costimulatory cell surface molecules and secreted cytokines, these naïve Th lymphocytes differentiate to become sesame-specific Th-2 lymphocytes. These Th2 lymphocytes in turn help sesame-specific B lymphocytes to produce sesame-specific IgE antibodies. These sesame specific IgE bodies are secreted and released into the blood stream and distributed throughout the body. These IgE antibodies are captured by the high affinity IgE receptors expressed on the mast cells present in the connective tissues and the basophils in the blood. Once an individual has reached this state, this person is regarded as ‘sensitized to sesame protein’ and the process of sensitization to sesame protein is complete. It is noteworthy that at this time, this person has not expressed any clinical symptoms of the disease, but the entire immune process has occurred in the body of the susceptible subject because of exposure to sesame protein allergens [3,4].Re-exposure of a previously sensitized subject to the sesame allergens via the skin, oral, nasal, or other routes of exposure, results in the entry of the allergenic protein into the local tissues where mast cells loaded with sesame-specific IgE antibodies are located, and into the blood where basophils loaded with sesame-specific IgE antibodies are located [3,4,82] (Figure 2). Direct cross-linking of IgE-IgE receptor molecules by the allergenic proteins results in cellular activation and the release of histamine and other powerful mediators of allergic inflammation. It only takes a few minutes to 3 h for these processes to be triggered. Consequently, clinical symptoms of Type I reactions appear immediately after exposure to the sesame allergen. Entry of significant amounts of the allergen into the blood triggers systemic anaphylaxis that can be life-threatening [3,4,82].Sesame oil causes a delayed hypersensitivity reaction known as allergic contact dermatitis [29,63,64,65]. No other major allergenic food-derived oils, with the exception of cashew nutshell oil, appear to have this capacity [3,4,78,79,80,81]. Sesame oil allergens that trigger these reactions have been identified as Sesamin, Sesamol, and Sesamolin [29]. These are small ring-structured lipid allergens known as lignans [18,29]. The mechanisms through which they cause sensitization and elicitation of reactions are unknown.Lipid allergens in general are small hydrophobic chemicals present in plant oils (sesame oil, cashew nutshell oil, poison ivy, poison oak, poison sumac) and cosmetics such as fragrance, toothpaste, and hair dyes (e.g., balsam of Peru, urushiol of poison ivy, farnesol, benzyl benzoate, benzyl cinnamate) [86,87,88,89,90,91]. The mechanisms through which they trigger allergic reactions have been deciphered for some of the lipid allergens, but not for sesame lipid allergens [86,87,88,89,90,91]. Furthermore, some of these lipid allergens show structures similar to that of sesame lipid allergens. Therefore, here we have discussed mechanisms about how lipid allergens are known to trigger allergic contact dermatitis (or contact hypersensitivity) reactions in humans or in animal models, and have illustrated this in Figure 3.Entry of lipid allergens into the body of susceptible subjects leads to their uptake by the APCs, such as Langerhans cells and presentation to specific naïve T cells to initiate their proliferation and differentiation into effector and memory T cells [86,87,88,89,90,91] (Figure 3). There are two models to explain specifics on how APCs are able to do this. The first model suggests that lipid allergens, which are considered as haptens because of their very small sizes of <500 Daltons, form covalent bonds to host proteins, resulting in an immunogenic hapten-carrier complex molecule that is then presented by the APCs in the context of MHC class II molecules to T cells that get activated [86]. The second, and most recent, model suggests that lipids allergens can also directly bind to CD1a molecule, a non-classical MHC molecule expressed by the Langerhans cells (a type of APC) in the skin, by displaying the self-lipids normally bound to the CD1a inside its molecular cleft or hole [87,88,89,90,91]. This results in the presentation of lipid allergens to which T cell reactions are initiated, resulting in the formation of specific effector and memory T cells. Once an individual has reached this state, this person is regarded as ‘sensitized to the lipid allergen’ and the process of sensitization to the lipid allergen is complete. At this time, however, no disease symptoms are expressed; only the entire immune process has occurred in the body of the susceptible subject because of the exposure to the lipid allergen.Re-exposure of a previously sensitized subject to the same lipid allergens results in their entry and presentation of by the APCs to the pre-formed allergen specific effector and memory T cells [86,87,88,89,90,91] (Figure 3). This results in their activation, proliferation, and release of cytokines and chemokines into the local tissues and eventually into the blood. These inflammatory molecules recruit more immune cells (including T helper subsets, eosinophils, monocytes, macrophages) to the tissue that further cause damage. Consequently, this tissue damage is expressed clinically as swelling, erythema, itching, and urticaria as in allergic contact dermatitis. It takes 48–72 h for these cellular and molecular responses to reach the peak. Consequently, this type of allergic disease is known as delayed hypersensitivity.Interestingly, there are two reports of sesame oil causing anaphylactic reaction without the need of IgE antibodies [61,62] (Figure 1). In the first study, authors investigated a case of recurrent anaphylactic attacks from sesame oil consumption [61]. They found 2-fold stronger skin test positivity to commercial sesame oil (4+) vs. commercial sesame seed protein (2+) and strong histamine release from sesame oil (40%) vs. the sesame seed protein (6%). Specific IgE (Radio Allergo Sorbent Test) results were negative. Therefore, they concluded that the mechanism of anaphylaxis to sesame oil was unknown. In the second study, authors investigated a case of anaphylaxis upon eating foods containing sesame oil but not the sesame seed [62]. They found the blood test negative for IgE to sesame protein (ImmunoCap-FEIA). They optimized a basophil activation test and found that sesame oil activated basophils to a higher level (51%) than the sesame seed (44%). They concluded that anaphylaxis to sesame oil can occur in the absence of IgE antibodies. The mechanism was not explained.Sesame is also increasingly linked to two other less understood types of allergic reactions: acute food-protein induced enterocolitis syndrome (AFPIES) and eosinophilic esophagitis (EOE) [66,67,68,69]. Both of these are non-IgE allergic reactions mediated by unknown mechanisms.The AFPIES is characterized by vomiting, lethargy, and diarrhea in infants and children within 1–4 h after feeding. This requires immediate medical help because it can be life-threatening due to dehydration and shock [66]. Many major allergenic foods (milk, egg etc.), can trigger this, and so also can sesame. However, authors note that there is lack of awareness on this issue as this is a relatively newly identified immune disorder [67]. Furthermore, another recent study found that compared to subjects with other food allergies, subjects with sesame allergy had 2.5-fold higher rates of AFPIES [2]. Although these allergic reactions are linked to sesame, the biochemical nature of the allergens mediating them are unknown (Figure 1).The food induced EOE is a non-IgE mediated reaction. It is caused by the reaction of eosinophils to the food components. Several major allergenic foods (e.g., wheat, milk, egg, soy) can trigger this reaction. Recent studies have identified sesame also as a suspected food trigger of EOE [2,68,69]. One study showed that compared to subjects with other major food allergies, sesame allergy subjects have seven times higher rates of EOE [2]. However, the components of sesame (i.e., the allergen) responsible for causing EOE are unknown. The molecular mechanism of EOE pathogenesis is a subject of intense research at present and readers are referred to an excellent recent review [92] (Figure 1).Thus, there is growing evidence that allergic reactions caused by sesame are diverse and complex. Cellular and molecular mechanisms underlying these reactions are largely understudied. Therefore, future research is warranted in this area.As opposed to several other major allergenic foods (e.g., peanut, milk, egg etc.), research on sesame allergy remains very basic. Published papers typically report the sesame allergy prevalence, characterization of the allergens, and description of the cases illustrating clinical presentation, diagnosis, and treatment. We have illustrated eight specific directions where advancement of research is needed, rather urgently, in view of the ongoing global rise of sesame allergies and their ability to trigger life-threatening anaphylaxis (Figure 4).Food allergies are complex genetic disorders where unknown environmental factors trigger the disease onset only in genetically susceptible subjects [93,94,95,96]. The role of genetics in resistance vs. susceptibility to develop sesame allergies needs to be determined. Knowledge about what kind of environmental factors favor development of sesame allergy are largely unknown [97,98,99,100,101,102,103,104,105].Several exposure conditions (e.g., environmental toxicants, triclosan, parabens, food preservatives, misuse of antibiotics, Vitamin D deficiency, sunlight exposure, dog ownership, excess use of antacids etc.,) that are currently being explored for other food allergies should provide the starting points of research [97,98,99,100,101,102,103,104,105].Host microbiome plays a critical role in the development of multiple diseases including food allergies [106,107]. Their role in prevention as well as causing sesame allergies remains unknown in humans or in animal models. Therefore, this represents a fascinating area of research to undertake in the future.Effective clinical interventions (e.g., vaccines, immunotherapy) are underway for peanut allergies and other food allergies with variable success [3,4]. Therefore, development of vaccines and effective immunotherapy protocols (e.g., oral, sublingual, and epicutaneous immunotherapy) for sesame allergies are urgently needed.Food processing has been shown to influence food protein allergenicity [108,109,110,111,112,113,114]. For example, boiling vs. roasting of peanuts reduces vs. increases allergenicity, respectively [107,110,111,114]; and extrusion processing appears to reduce hazelnut allergenicity [112]. Therefore, effect of thermal and nonthermal food processing methods on sesame allergenicity must be evaluated in future research.The last, but not the least, important area is the development of hypoallergenic sesame products. There are hypoallergenic food products available for milk and egg allergic subjects [115,116]. Hypoallergenic wheat lines and peanuts are being developed using genetic engineering methods [117,118]. Similar approaches could be used to develop hypoallergenic and potentially nonallergenic sesame products. Previous studies show that different wheat genotypes may naturally differ in allergenicity [119]. Whether or not sesame varieties naturally differ in allergenicity in vivo is largely unexplored at present. Therefore, evaluating differences in allergencity among existing sesame varieties and efforts to produce novel potentially hypo/nonallergenic varieties using conventional cross-hybridization and plant breeding methods may be undertaken in the future.Animal models of sesame allergy would be of immense value in all these areas of research [82,120]. However, only one mouse model of sesame allergy and anaphylaxis to proteins has been reported so far [34]. Therefore, more emphasis needs to be placed on developing both primate and nonprimate animal models and their use to conduct the basic, preclinical translational research on sesame allergy.In essence, there is conspicuous absence of significant basic, preclinical, and clinical research on sesame allergies at present. The current rapid rise in sesame allergies warrants that the food industry, the government agencies, and the private research enterprise needs to take note of this gap in science and devote funds to address these research challenges so that the future generations are empowered to prevent and manage sesame allergies.Since exposure to sesame allergens is required to trigger allergic reactions including life-threatening anaphylaxis, preventing exposure is an effective method to protect sensitive consumers. It is not easy to avoid sesame, as opposed to other major allergens, because it is often added in small amounts as a garnishing item to many foods, dressings, condiments, and as a ‘natural flavor’ [121]. Despite labeling, accidental exposures are common [122,123,124]. It is noted that both initial and accidental reactions to sesame are inadequately managed for children at present [125]. Thus, the challenges faced by sesame allergic subjects and their families are very similar to that faced by other food allergic subjects. Consequently, food safety regulatory authorizes in Canada, the EU countries, the UK, Australia, New Zealand, and Japan currently regulate sesame as a major or priority food allergen [18,126]. Appropriate labeling on the food products is an effective method of prevention by informing sensitive consumers to avoid the food product.The Food Allergen Labeling and Consumer Protection Act (FALCPA) of the USA came into effect from January 2006 to improve the regulation of major food allergens [11]. However, it did not include sesame into the list of major allergens. The FALCPA regulates specifics on the labeling of allergenic foods on food packages. Absence of such labeling is a leading cause of class I food recalls—the highest state of alert issued in the USA [11]. However, sesame is not federally regulated by FALCPA. A new federal bill, the Food Allergy Safety, Treatment, Education, and Research (FASTER) act, has been introduced recently in the US Congress that proposes to add sesame to the list of major allergenic foods, although the final decision on regulation has not been made as yet [44].The lack of the federal regulation of sesame, but the growing prevalence of the disease at an alarming rate in the USA as discussed earlier, resulted in the creation of a new state law in Illinois, USA [45]. On 26 July 2019, the Illinois Governor signed HB2123, which requires any packaged food containing sesame to identify it on the label. This action amended the Illinois Food, Drug, and Cosmetic Act by changing the Section 11 of the law. Per this change, “a food is considered misbranded: _ (s) if it contains sesame, is offered for sale in package form but not for immediate consumption, and the label does not include sesame” (lines 19–21) [45].A zero-tolerance policy is in place for the regulation of major allergenic foods in all countries where they are regulated [126]. This applies to sesame also in those countries where it is regulated (Table 1). The zero-tolerance policy is based on the fact that threshold oral elicitation doses for food allergens have not been universally standardized and are not well accepted at present. Oral provocation tests show that there is a large variability in the oral threshold elicitation doses amongst food allergic subjects, with some reacting to lower milligram quantities of allergenic food extracts [126]. A similar variability has been noted for sesame allergy patients in oral challenge studies [127,128,129]. Latest studies show that the minimal reaction elicitation dose (ED) in 5% of the sensitized population (ED05) for sesame vs. peanut is as follows: 4.2 mg (95% CI, 0.6–57.7 mg) for sesame vs. 3.9 mg (95% CI, 2.8–7.1 mg) for peanut [130]. Thus, oral disease elicitation potency of sesame appears to be similar to that of the other currently regulated major allergenic foods. These facts suggest that a zero-tolerance policy might be necessary for the regulation of sesame, similar to other major allergenic foods, if it were to be regulated in the USA.Currently, food safety regulations apply only to protein allergens. Regulation of oils from major allergenic foods is complicated. In the USA, highly refined oils derived from the major allergenic foods are exempt from FALCPA labeling requirements as long as there is evidence that the method used to produce that oil establishes absence of protein contaminants [11]. In Canada, all unrefined oils are subjected to enhanced food allergen labeling requirements [14]. However, highly refined oils, except peanut oil, are not subjected to enhanced food allergen labeling requirements, but declaration as ingredient is required [14]. In the EU countries, Australia, and New Zealand, fully refined soy oil is exempt from labeling and no information is available for sesame oil or oils derived from other major allergenic foods in those countries [12,13,15].Sometimes, oil-induced reactions could result from trace amounts of proteins present in them due to inadequate processing, and that are falsely attributed to oils. Furthermore, some hydrophobic protein allergens, known as oleosins, can also be present in oils. Nevertheless, as discussed earlier, sesame oil has been implicated in causing true allergic reactions—both immediate as well as delayed types of reactions (Figure 1). Furthermore, an oral challenge test using oil in sesame allergy subjects also shows that immediate allergic reactions do occur upon oil feeding in controlled studies: some react to 3 mL while others at 15 mL of sesame oil in oral provocation testing [127,128,129]. In addition, a variety of adverse reactions to sesame oil have been reported in the literature: lipoid pneumonia from sesame oil pulling, pneumonitis, eosinophilia, and eosinophilic pneumonia after in vitro fertilization treatments that had exposed subjects to the sesame oil, and subcutaneous granuloma formation due to intramuscular injection of sesame oil [131,132,133,134,135,136]. Thus, there is considerable evidence that the sesame oil is immunologically active in vivo and that it can elicit immune mediated adverse reactions in a variety of exposure situations.Thus, together these lines of evidence show that the mechanisms of action of sesame oil on the immune system needs to be further elucidated. Furthermore, they also raise the question: should sesame oil be regulated for food safety in addition to the sesame protein?The current food allergen regulation model is based on the ability of foods to trigger IgE-mediated reactions that tend to be life-threatening. There is growing evidence that non-IgE mechanisms to sesame and to other foods/drugs also can mediate life-threatening reactions in humans [137,138]. Therefore, should non-IgE mediated mechanisms also dictate food safety regulation?Mandatory food allergen labelling has significantly advanced food safety for food allergy consumers. A related issue is the precautionary labelling of allergens, which are increasingly noticed on packaged foods. Occasionally, sesame is identified on the food labels in the USA, under the precautionary allergen labelling concept, which is not legally required in the USA. It is a voluntary decision made by the food industry. However, a recent outstanding study showed that consumers misunderstand such precautionary allergen labelling and that up to 40% of consumers purchased the products with precautionary labels [139]. Therefore, to enable enhanced food safety for sesame allergic consumers in the USA, mandatory labelling may be of more benefit than the voluntary precautionary allergen labels.There is substantial evidence that the prevalence and the severity of sesame allergy has been rising at the global level, including in the USA. There is scientific evidence that sesame contains both protein as well as lipid allergens that can trigger distinct types of allergic reactions. However, the specific mechanisms underlying the pathogenesis of sesame allergy remains to be determined. There is a major gap in animal models development and their application in basic and preclinical research studies. The human clinical research on sesame allergy is urgently needed to develop effective therapeutic and preventive methods for both IgE as well as non-IgE mediated reactions. Currently, 32 countries regulate sesame as a major or priority allergen. However, sesame is not federally regulated as yet in the USA. Nevertheless, the state of Illinois recently enacted a state law to regulate sesame in that one state in the USA. This situation, along with the recent research showing an alarming rise in the prevalence during the past decade, has placed more pressure on regulating sesame as a major allergen in the USA. This review is expected to provide the background and the context to stimulate further discussions on whether or not to regulate sesame in the USA and in other countries where it is not currently regulated.Conceptualization, V.G. and H.G.A.; methodology, V.G. and H.G.A.; validation, V.G. and H.G.A.; formal analysis, V.G. and H.G.A.; investigation, V.G. and H.G.A.; resources, V.G.; data curation, V.G. and H.G.A.; writing—original draft preparation, V.G.; writing—review and editing, V.G. and H.G.A.; visualization, V.G. and H.G.A.; supervision, V.G.; project administration, V.G.; funding acquisition, Not Applicable. All authors have read and agreed to the published version of the manuscript.This project was conducted without any funding.The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Sesame allergy: diversity of the allergens and the complex disease spectrum. The brown, white, and black sesame seeds contain protein allergens, lipid allergens, and allergens of unknown nature. Current evidence shows that sesame is linked to at least five types of clinical presentation of diseases (from left to right): non-IgE immediate hypersensitivity to oil, delayed hypersensitivity to lipid allergens, IgE mediated immediate hypersensitivity to protein allergens, and acute food protein-induced enterocolitis syndrome, and eosinophilic esophagitis caused by unknown allergens.Mechanism of IgE mediated immediate hypersensitivity reaction elicited by sesame seed protein allergens: Type I Hypersensitivity. This happens in two phases. (1) the sensitization phase: exposure to sesame protein allergens results in their uptake by the antigen presenting cells (APCs). The APCs process and present the allergen peptide fragments along with the Major Histocompatibility Complex (MHC) class II molecules to the allergen specific naïve T cells. With the help of the costimulatory molecules and the local cytokines (e.g., IL-4), they differentiate to become T helper (Th)-2 cells. These cells help the allergen specific B cells to produce IgE antibodies. The B cells become plasma cells and secrete large amounts of IgE antibodies that are distributed systemically and picked up by the mast cells and the basophils, which express high affinity receptor for IgE. At this stage, the subject is considered ‘sensitized to sesame’ but has no apparent clinical symptoms of the disease; and (2) the disease elicitation phase: re-exposure of the sensitized subject to the sesame allergen results in direct binding of the allergen fragments to the IgE present on mast cells and basophils. Cross-linking of the surface IgE receptor results in cellular activation, degranulation, and release of mediators resulting in the clinical expression of the disease within minutes to 3 h after exposure to the protein allergens. Therefore, this disease is known as immediate hypersensitivity reaction.Mechanism of delayed hypersensitivity reactions elicited by sesame lipid allergens: a plausible model. This happens in two phases. (1) the sensitization phase: exposure to sesame lipid allergens results in their uptake by the antigen presenting cells (APCs). They present the lipid allergen along with the non-classical MHC molecule, CD1a, to the allergen specific naïve T cells that differentiate to become the effector T (ET) cells and memory T (MT) cells. There are no disease symptoms at this phase; and (2) the disease elicitation phase: re-exposure of the sensitized subjects to the sesame lipid allergen results in their uptake and presentation by the APCs (along with the CD1a?) to the ET and MT cells resulting in their activation. The MT cells differentiate to become ET cells. Activated ET cells release cytokines and chemokines that cause recruitment of additional immune cells to the local tissue. The consequent tissue damage from all these processes occurs in about 48–72 h after exposure to the lipid allergen and, therefore, it is known as the delayed hypersensitivity reaction.Directions for the future basic and translational research on sesame allergy. Advances in basic and translational (preclinical and clinical) research on sesame allergies are urgently needed because the prevalence and the severity of sesame allergies are growing at an alarming rate globally. This figure shows major directions for conducting future studies using both human and nonhuman (animal, cell line, plant) systems as illustrated.Food safety regulation of sesame and other allergenic foods.# Examples of crustaceans: shrimp, crayfish, crab, and lobster. * Examples of mollusks: white clams, scallops, oysters, and mussels. ** European Union includes these 27 countries: Austria, Belgium, Bulgaria, Croatia, Republic of Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, and Sweden.Global rise of sesame allergies: the origin and the milestones.FASTER = Food Allergy Safety, Treatment, Education and Research; CFIA = Canadian Food Inspection Agency; NIH = National Institutes of Health; FALCPA = food allergen labeling and consumer protection act.Protein and lipid allergens present in sesame seed: biochemical nature, diversity, and functions.* Not an official allergen of the International Union of Immunological Societies (IUIS) as yet. ** Sesamol is a thermally degraded product of Sesamolin.

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+Several animal food allergens assemble into amyloids under gastric-like environments. These aggregated structures provide Gad m 1 with an enhanced immunoglobulin E (IgE) interaction due to the fibrillation of the epitope regions. However, whether these properties are unique to Gad m 1 or shared by other food allergens has not yet been addressed. Using Bos d 5, Bos d 12 and Gal d 2 as allergen models and Gad m 1 as the control, aggregation reactions and the sera of milk, egg and fish allergic patients have been analyzed, assessing the IgE interactions of their amyloids. We found that amyloids formed by Bos d 12 and Gal d 2 full-length and truncated chains are recognized by the IgEs of milk and egg allergic patient sera. As with Gad m 1, in most cases amyloid recognition is higher than that of the native structure. Bos d 5 was not recognized under any fold by the IgE of the sera studied. These results suggest that the formation of IgE-binding amyloids could be a common feature to animal food allergens.More than 5% of the population in industrialized countries suffer from a type I food allergy, an immunoglobulin E (IgE)-mediated hypersensitivity disease resulting from the loss of oral tolerance to food proteins [1,2,3,4]. A limited number of foods are responsible for the majority of reactions, with 95% of food allergies being caused by only eight foods including eggs, milk, fish, crustacean shellfish, peanuts, tree nuts, wheat and soybeans [3,5,6]. All these foods contain many proteins, but only a few of them are allergens [3,5,6]. Known food type I allergens have, in general, molecular weights below 70 kDa, are stable molecules that resist cooking and digestion, and stimulate the immune response inducing the production of allergen-specific IgE [3,5]. Regardless of the limited number of represented native structural scaffolds, sequence difference thresholds, abundance and stability properties, the molecular signature of protein allergenicity remains yet unresolved [3,5,6,7,8,9].All food proteins are exposed to pH 1.3–2 at the gastric compartment during digestion, a condition that is usually used in vitro to trigger the refolding of proteins and of their fragments into amyloid aggregates [10,11]. Amyloids are insoluble fibrillary protein structural states displaying protease resistance and, with one exception, a cross-β sheet backbone [11,12,13]. An amyloid formation is a generic feature of many, if not all, natural polypeptide chains if placed under the appropriate environment and concentration [10,11,12]. In fact, food allergens of animal sources such as the β-parvalbumin Gad m 1 from fish (Gadus morhua); α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), αS2-casein (Bos d 10), β-casein (Bos d 11) and κ-casein (Bos d 12) from bovine milk, and the ovalbumin (Gal d 2) and lysozyme (Gal d 4) from chicken egg-whites rapidly form amyloid aggregates under a variety of conditions, some of which are those of the gastric compartment [14,15,16,17,18,19,20,21,22]. Bos d 5 and Bos d 12 also form heterogeneous or mixed amyloid fibrils under ultraheat, mimicking treatments used in the dairy industry [23]. Gad m1 amyloids displayed an enhanced proteinase resistance and a 1000-fold increased IgE-specific binding compared to that of the monomer precursor [14,15,16]. Importantly, unrelated amyloid fibrils such as those formed by Aβ42 and PrP are not recognized by the IgE present in the sera of fish allergic patients, underlining the sequence specificity of the recognition process [14]. A battery of approaches including peptide arrays, proteomics and the use of mutant chains concluded that regions forming the amyloid cores overlapped with the IgE epitopes [15,16]. Notwithstanding, whether this property is specific to Gad m 1 or is shared by other food allergens has not yet been addressed. Given their well-characterized aggregation processes, commercial availability and the knowledge of the major IgE epitopes, we have chosen Bos d 5, Bos d 12, and Gal d 2 as animal food allergen models to analyze the IgE-binding properties of their amyloid state using the sera of allergic patients.All experimental protocols and methods were performed following the guidelines of the University Hospital La Paz and the Spanish National Research Council and applied with approval from the Ethics Committee of the University Hospital La Paz (protocol number PI-3065). Anonymity was preserved as established in the ethical permission.Sera were obtained from patients recruited from Hospital La Paz in Madrid (Spain). Fish, milk, and egg allergic patients were selected based on case history, a positive skin prick test with commercial extracts and determination of specific IgE (sIgE) by ImmunoCAP 100 (Thermo Fisher, Uppsala, Sweden) according to the manufacturer instructions (Table 1).Bos d 5 (P02754), Bos d 12 (P02668), Gal d 2 (P01012) and Gad m 1 (A5I874) sequences were retrieved from UniProtKB database [24]. IgE binding epitopes were taken from previous SPOT-membrane and array assays [15,16,25,26,27,28]. Amyloid cores were predicted using the ZipperDB algorithm [29,30]. IgE-binding regions and amyloid forming segments were used to generate binary functions (0,1) of the polypeptide chain (residue number including the signal peptide) using Origin 2019 software. The relative organization (overlap/flank relation) between regions was visualized using a 3D stacking plot.Gal d 2 (A-2512), Bos d 5 (L3908) and Bos d 12 (C0406) were purchased from Sigma-Aldrich. Gad m 1 (A5I874) was prepared as described [31]. Before their use all proteins were extensively dialyzed at 4 °C against either 25 mM Tris, 0.1 M NaCl pH 7.5 or 0.1 M Gly pH 1.5 using dialysis membranes with an 8 kDa pore diameter (Spectra Por). Dialyzed solutions were centrifuged at 13,500 rpm at 4 °C for 20 min to clear possible existing aggregates and the protein concentrations of the supernatants were determined with the Bradford assay. Proteins at pH 7.5 were referred as the native states (N), whereas the solutions at pH 1.5 were used for amyloid formation.Protein solutions at 5–8 mg/mL solution in 0.1 M Gly pH 1.5 were incubated at 90 °C for 5 h. After the heating step, all protein samples were stored at room temperature for 36 h to allow fibril maturation. Fibrils were harvested in the pellet fraction of an ultracentrifugation at 100,000× g for 1 h at 4 °C and resuspended in 50 mM Gly pH 1.5. When required, mature fibrils were placed in 1.5 mL eppendorf tubes and sonicated for 15 min in a sonicating water bath.Circular dichroism (CD) measurements were performed using a Jasco J-820 spectropolarimeter equipped with a Peltier-controlled thermostatted cell holder. Far-UV CD spectra were recorded using a 0.3 mg/mL protein concentration solution in 25 mM Gly pH 1.5 at 25 °C. Spectra were corrected for the base line contribution and analyzed as described taking 110 Da as the residue average molecular weight [14].For AFM visualization, 30 μL of the aggregate solutions prepared at 0.05 mg/mL in 2.5 mM Gly pH 1.5 were absorbed onto freshly cleaved mica via a 5–10 min incubation at room temperature. The surfaces were then rinsed with double-distilled water and dried. Images were obtained in the tapping mode using a JPK Nanowizard 2 microscope and HQXSC11 B (Mikromash) cantilevers (2.7 N/m force constant and 70 kHz resonance frequency). An AFM analysis was performed using the free program WSxM 4.0 (Nanotec)Typically, 3 μg of native and aggregated proteins were separated by SDS-PAGE under denaturing conditions using Mini-Protean® TGX Stain-FreeTM (#456-8126) gels. Bands were visualized using Coomassie Brilliant Blue R-250 (CBB) staining solution (BioRad, Hercules, CA, USA), and the images were recorded with the Molecular Imager ChemiDoc XRS-Plus (BioRad, Hercules, CA, USA).Samples of 0.1–0.2 µg protein/dot were loaded in duplicate onto nitrocellulose membranes. After blocking for 1 h in TBS-T (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Tween 20) containing 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature, the membranes were incubated for 2 h with either the distinct serum (diluted 1:10 in blocking buffer) or with the anti-amyloid fibril antibody (OC antibody) (AB2286 Merck Millipore, 1:2000 dilution, Darmstadt, Germany). After extensive washing with TBS-T, membranes were incubated for 1 h with horseradish peroxidase (HRP)-labeled goat antimouse IgG (Sigma-Aldrich, 1:5000 dilution, St. Louis, MO, USA) or mouse monoclonal B3102E8 antihuman IgE (Abcam, 1:2000 dilution, Cambridge, UK) and developed using Clarity Western-ECL (BioRad, Hercules, CA, USA). The signals were recorded with the ChemiDoc XRS-Plus imager and analyzed using the volume tools provided by ImageLab software.Previous work with Gad m 1 showed a peculiar overlapping between the major IgE binding epitopes and the segments forming amyloid fibrils [15]. To test whether other food allergens share this feature the sequences of Bos d 5, Bos d 12, and Gal d 2 taken as allergen models were transformed into the binary (0/1) functions of IgE-binding epitopes and amyloid cores using previous epitope reports [15,16,25,26,27,28] and the predictions of the ZipperDB algorithm [29,30], respectively. To visualize their relative organization (flanking and/or overlapping), the obtained functions of the residue number were plotted using a 3D stacked bar representation (Figure 1). As previously shown, the 109 amino acid chain of Gad m 1 contains two major IgE-binding epitopes which are overlapped and flanked at their C-terminus by regions predicted as amyloid cores [14,15,16]. These sequences are displayed in Table 2. Bos d 5, made by a 178 amino acid residues chain that undergoes the removal of the 1–16 N-terminal signal sequence, contains two major IgE-binding epitopes and six segments predicted to form amyloids. It must be noted that ZipperDB yields hexapeptides as minimum cores [29,30], but the aggregation property can extend at their both N- and C-terminal side as experimentally shown [32]. The N-terminal IgE-binding epitope is overlapped by an amyloid core, whereas the C-terminal IgE-binding epitope is mainly flanked by aggregating segments with a partial C-terminal overlapped. Importantly, both regions contain polymorphic sites (Q75H, G80D and A134V) characteristic of distinct isoforms [24] and Cys residues which are engaged in disulfide bonds and may modify both IgE binding and amyloid formation features (Table 2). Bos d 12 is produced as a precursor chain (190 amino acids) with an N-terminal signal sequence (1–21 residues), the same as Bos d 5. The Bos d 12 mature chain harbors three major IgE binding epitopes, being the two N-terminal epitopes overlapped by amyloid forming regions (Figure 1, Table 2). The C-terminal epitope which contains a large number of glycosylation sites, is mainly flanked by minimal adhesive regions [33]. The 385 amino acid mature chain of Gal d 2 contains four IgE binding epitopes, three of which are overlapped by the predicted amyloid cores as depicted in Table 2. Then, this in silico analysis shows that the milk and egg allergen models share with the fish allergen Gad m 1 the sequence overlap of IgE binding epitopes and amyloid forming cores and suggests that their aggregates may harbor IgE-binding properties [15,16].To analyze the interaction of the distinct food allergens with the IgE contained in the sera of food allergic patients, both their native and amyloid structures were prepared. The native fold (N) required as the control was prepared by an extensive dialysis in 25 mM Tris, 0.1 M NaCl pH 7.5, followed by centrifugation to clear the solutions from unspecific aggregates. Regarding the amyloid fold (A), a variety of conditions in acid media differing in the temperature and length of incubation, ionic strength, presence or absence of alcohols, protease treatments and protein concentration have been specifically used for each of the food allergens [14,17,18,19,20,21,22]. Aiming to find a balance between yield and kinetics, reduce the working protein concentration below 10 mg/mL and a find a general application to all proteins, we tailored a procedure consisting of the use of dialyzed protein solutions prepared at 5–8 mg/mL in 0.1 M Gly pH 1.5 (removal of ligands), heating at 90 °C for 5 h (denaturation), followed by 36 h of growth at room temperature and isolation of the aggregates by ultracentrifugation.The use of this procedure allowed the isolation of insoluble aggregates amounting to about 10% of the initial precursors and consisting in a mixture of full length and truncated chains (Figure 2a). The isolated aggregates displayed far-UV CD spectral features of secondary structures governed by a β-sheet (Figure 2b) and specifically reacted with an anti-amyloid fibril OC antibody in dot-blot assays (Figure 2c). In addition to the general amyloid features provided by the anti-OC reactivity, AFM imaging showed a variety of fibrillary shapes (Figure 3). Bos d 5 aggregates consisted of thick and linear fibrils filaments with variable lengths (70 nm to 1 µm length). Bos d 12 and Gal d 2 formed fibrils of 200 nm length and 5 nm diameters with a high number of lateral associations. On the contrary, Gad m 1 insoluble aggregates appeared as long flexible thin curved fibrils different from the fibrillary polymers generated under an acid pH in the absence of heating treatment [14].Then, the use of simple and systematic procedures permits the preparation of the native and amyloid folds of all the food protein allergens for their further analysis.To analyze the recognition of the formed amyloids with the IgE of the sera of the food allergic patients described in Table 1 and compare it with the corresponding native folds, we used dot blot assays (Figure 3). In these assays, 0.1 µg of both the native and amyloid folds of the distinct allergens were dotted in duplicate and the interactions with the sera probed using blocking buffers containing bovine serum albumin (BSA).It must be underlined that the serum of allergic patients contains a collection of IgEs (total), some of which specifically but variably recognize the offending food allergen (sIgE) [3]. In this sense, the intensity of the recognition might be modulated by the existence of neutralizing IgG4 [3]. For these reasons and despite the fact that a higher number of sera were used in a preliminary analysis targeting individual allergens, the collective study was performed with the reagents displayed in Table 1 with a single use of the 1/10 dilutions.Since Gad m 1 is a major allergen in fish allergy, membranes were first probed with the sera of fish allergic patients (Figure 4). As previously shown, for a similar load of Gad m 1 structures amyloids are specifically recognized by the IgEs contained in the sera of fish allergic patients [14,15]. As expected, no signal was detected when the membranes were probed with the IgEs of the sera from milk and egg allergic patients.The serum IgEs of milk allergic patients recognized the amyloid fold of Bos d 12. For patients 2 and 3, the IgE binding to the Bos d 12 amyloid is 5-fold higher in the native state as judged from signal quantitation. On the contrary, the IgEs of the sera of patient 1 interact largely with the native fold of Bos d 12 and also slightly recognize the amyloid fold Gal d 2. These recognition differences between the sera of patient 1 and the sera of patients 2 and 3 might result from the large differences in the anti-Bos d 12 IgE levels (Table 2). Importantly, Bos d 5 under native and amyloid folds was not recognized by the sera IgE of any of the patients despite the ImmunoCap revealed presence of anti-Bos d 5 IgEs (Table 2). Increasing the relative loads of both Bos d 5 folds using both the similar membrane format and membranes devoid of Bos d 12 forms did not altered the result. The absence of Bos d 5 recognition by the sera IgE suggests differences in the isoform composition of the product provided by Sigma-Aldrich and the reagent used in the ImmunoCap approach [24].Similar to Bos d 12 and Gad m 1, Gal d 2 amyloids are the general binding target of the IgEs contained in the sera of egg allergic patients. For sera 5 and 6, the IgE interaction with Gal d 2 amyloids was 10-fold higher than with the native fold, whereas in serum 4 the preference amounted to a 3-fold increase. Interestingly, the serum of patient 5 which contained a higher level of total IgEs also recognized Bos d 12 amyloids.Taken together, these results show that, as described for Gad m 1 in fish allergy, Bos d 12 and Gal d 2 amyloids are the structural states highly recognized by the IgEs present in the sera of milk and egg allergic patients, respectively. Notwithstanding, the relative specific recognition of the amyloid state compared to the native fold by the sera IgEs varies among the distinct food allergic patients.In this pilot study, we sought to investigate whether the formation of IgE-binding amyloids as found for Gad m 1 is a feature shared by other animal food allergens. Our results show that Bos d 12 and Gal d 2 yield amyloids which are recognized by specific IgEs contained in the sera of milk and egg allergic patients, respectively. For most patient sera, the amyloid fold represents the major IgE-reactive state. Bos d 5 behaved distinctly since both native and amyloid folds did not react with the IgE of the milk allergic patient sera used, despite the sIgE levels provided by the ImmunoCAP characterization, suggesting differences in the isoform composition of both reagents [24].The search for the molecular signatures that predispose food proteins to become allergens has yielded several characteristics including abundance in food, structure, resistance to processing and digestion and the presence of multiple linear IgE binding epitopes [5,6,7,8,9]. These features have been established and omit the effects that transit through the gastric compartment can cause. One of the effects is the pH-induced refolding of the allergens and of their fragments into their amyloid states [10,11]. Indeed, of almost all the proteins contained in their sequence, at least one segment capable of forming amyloid fibrils [34]. However, only 5.3% of these segments are found in the surface of the native 3D structures and less than 0.1% of them displayed the proper sticky geometry [34]. Consequently, the formation of amyloid fibrils under physiological conditions is common for intrinsically disordered proteins such as Bos d 12 [19,35]. However, globular proteins such as most food allergens require a partial unfolding or a nicking process to expose the amyloid forming segments otherwise buried in the structure interior or stabilized by bound ligands. The efficiency of this refolding mainly depends on the sequence, the abundance of the protein and the interference of off-pathways such as those driving the less favorable amorphous aggregates and nonproductive truncations [11,12,17,36]. For complex mixtures such as food, factors such as processing treatments and the presence of native state ligands and of anti-amyloid compounds might also modulate the efficiency [12,17,19]. Tailoring a protocol using acid pH, low protein concentrations, short incubations and random truncations, for general use, facilitates the amyloid fibril purification required for their systematic characterization.The isolated Bos d 5, Bos d 12, Gal d 2 and Gad m 1 amyloid states are not unique and differ in their shape, polymorphism degree and surface reactivity as shown by AFM imaging and IgE binding. Indeed, the multiplicity of potential fibrillation-prone segments predicted by the ZipperDB algorithm and the fragmentations showed by SDS-PAGE entails complex aggregation events far beyond those uniquely containing IgE-binding sites. On the other hand, the formed aggregates may display different elongation and dissociation rates which will dictate the landscape of molecular species existing under gastrointestinal-like conditions [12,14]. In this sense, easy dissociating amyloid fibrils will function as IgE-binding epitope depots, whereas those displaying a slow dissociation will function as epitope backbones. These nonexclusive possibilities might coexist in any of the allergens if fibrillation is seeded through different sticky segments.An in silico analysis using previously described IgE-binding epitopes and the amyloid cores predicted by the ZipperDB algorithm detected multiple regions with a functional overlap in each allergen chain. The overlap degree should be taken in qualitative terms, since each of the variables used employ different length windows to define function. In this sense, the considered IgE epitopes were determined using 20 amino acid residues sequential peptides with a three-amino acid shift and sera from paediatric patient cohorts [25,26,27,28]. On the contrary, the prediction of amyloid cores uses a six-amino acid peptide window. Studies with Gad m 1 using 12-length peptides and a two-amino acid sequential shift between two consecutive peptides, an immobilization density of ≈10 nmol per spot (≈ 400 nmol/cm2), sera from fish allergic patients for IgE-epitope mapping and the antifibril OC-antibody for amyloid detection identified the segments 25FDHKAFFTKVGLAAKSSA42 and 67FLQNFSAGARAL78 as the segments with dual functions [15,16]. Both sequences are N-terminal shifted to the predicted sticky segments, indicating the expansion of the aggregation properties beyond the detected cores. Using a similar approach for milk and egg allergens and the sera from a varied allergic patient cohort will allow a closer view of the functional overlap.The potential conformational multiplicity of the regions forming the IgE-binding epitopes questions the validity of mapping the epitopes using the native 3D structures of food allergens as unique template. In fact, the IgE binding sequences DHKAFFTKV and FLQNFS form parts of helical structures in the native state of Gad m 1 whereas the amyloid fold adopts a cross β-sheet structure [16,37]. In fact, consideration of the IgE-epitope amyloid folds will simultaneously explain their protease resistance and higher avidity and affinity for any ligand binding process [12,14,15,32]. On the other hand, the sequence differences of the segments with functional overlaps identified in the distinct allergens sustain the binding specificity of the IgE repertoire present in the sera of the allergic patients sensitized to distinct foods. Additionally, for the given food allergen differences in the covalent structure (isoforms, covalent modifications, etc.), the concentration in the food source and local industrial processing may impact the functionality of the aggregating regions and therefore their stability. Dynamic changes in the quality of the offending foods may explain variations in the IgE-epitope repertoires of different patient cohorts.In summary, this works provides the pilot proof of concept by which the amyloid fold of segments of animal food allergens can define the novel structural properties of IgE-binding epitopes. Notwithstanding, their solid assignment will require a deeper study with a higher number of sera samples, as will their simultaneous characterization using SPOT-membranes/arrays using a similar design (peptide length and offsets, and surface density, among other critical parameters) and the synthetic reconstruction of the segments of interest as performed with Gad m 1 [14,15,16]. Indeed, IgE-epitope repertoires depend on the regional origin of the patient cohort and change with the patient’s age.Conceptualization, M.G.; methodology, M.G., M.C.; formal analysis, R.P.-T., M.C.; investigation, R.P.-T., M.C.; resources, J.L.H., D.L.-A., M.P., R.R.P.; writing—original draft preparation, M.C., M.G.; writing—review and editing, R.P.-T., M.C., M.P., P.R.-P., M.G.; funding acquisition, J.L.H., R.R.-P. and M.G. All authors have read and agreed to the published version of the manuscript.This research was funded by the Spanish AEI/EU-FEDER BFU2015-72271-EXP, AEI/EU-FEDER PID2019-103845RB-C21, and CDTI-TOLERA grants.We would like to acknowledge Natalia Canales and Patricia Pedraz (Laboratorio de Microscopía de Fuerza Atómica, IMDEA Nanociencia) for their technical support.J.L.H. comes from the Research Center of Angulas Aguinaga company, the company plays no role in any result and conclusion. The rest of the authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Relative organization of the IgE-binding epitopes and the predicted amyloid cores in the sequences of Bos d 5, Bos d 12, Gad m 1 and Gal d 2 allergens. IgE-binding epitopes are shown as red bars. The regions predicted as amyloid cores are depicted as blue bars. Residue numbers correspond to those of the precursor chains including the signal peptides.Amyloid aggregation of the milk, egg and fish allergens. (a) Typical analysis by SDS-PAGE and Coomassie Brilliant Blue R-250 (CBB) staining of the distinct food allergens before (N) and after aggregation (A). Lanes of interest are labelled with the corresponding food allergen. MW correspond to the prestained protein ladder with the molecular weights (bottom to top): 10, 15, 20, 25, 37, 50, 100, 150 and 250 kDa. The unlabeled lanes were left to display the original gel. (b) Far-UV Circular dichroism (CD) spectra of Bos d 5 (black), Bos d 12 (red), Gal d 2 (orange) and Gad m 1 (blue) aggregates in 25 mM Gly pH 1.5. (c) Typical dot blot membranes probed the anti-amyloid fibril OC antibody. Dots were performed in duplicates using 0.1 µg of protein of both their native (N) and aggregated (A) states.Atomic Force Microscopy (AFM) imaging of the insoluble aggregates formed by the milk, egg and fish allergens. X and Y dimensions are indicated in the white bar, whereas the Z-axis color scale is shown at the right hand side of each panel.Dot-blot analysis of the IgE interaction of native and amyloid folds of milk, egg and fish allergens using the sera of food allergic patients. Sample dotting was performed in duplicates using 0.1 µg of the native (N) and amyloid (A) structures of the food allergens. Sera are indicated in numbers (from 1 to 8) and grouped according to the offending food (milk, egg and fish).Clinical features and immunoglobulin E (IgE) profiles of the sera of food allergic patients recruited for the study.a A, anaphylaxis; AE, angioedema; E, eczema; OAS, oral allergy syndrome; RD, respiratory symptoms. b ImmunoCap determinations. nd, not determined.Sequence regions with IgE binding and amyloid forming properties. IgE-binding epitopes are depicted with grey background; amyloid forming regions are shown in blue.

Med-MDPI/allergies/allergies-01-01-00003.txt

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+Allergen immunotherapy may modify the natural course of allergic diseases and induce remission. It includes subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). For asthma, allergen immunotherapy using house dust mite (HDM) improves clinical symptoms and airway hyperresponsiveness and decreases drug requirements. Furthermore, it has been suggested that allergen immunotherapy also has the following effects: (1) the effect can be maintained for more than a year even if the treatment is terminated, (2) the remission rate of childhood asthma can be increased, (3) new allergen sensitization can be suppressed, and (4) asthma development can be prevented if allergen immunotherapy was performed in the case of pollinosis. Allergen immunotherapy differs from conventional drug therapy, in particular the effect of modifying the natural course of allergic diseases and the effect of controlling complicated allergic diseases such as rhinoconjunctivitis. The general indication for HDM-SCIT in asthma is HDM-sensitized atopic asthma with mild-to-moderate disease and normal respiratory function. HDM allergens should be involved in the pathogenesis of asthma, and a duration of illness of less than 10 years is desirable. HDM-SLIT is available for allergic rhinitis but not for asthma in Japan. However, as the efficacy of SLIT for asthma has been fully proven internationally, SLIT is also applied in asthmatics with complicated allergic rhinitis in Japan.Bronchial asthma has become a well-controlled disease in general because of advances in drug therapy centered on inhaled corticosteroid (ICS). However, ICS does not modify the natural course of asthma and is being positioned as a so-called symptomatic treatment [1,2]. Furthermore, ICS does not provide therapeutic benefits for allergic rhinoconjunctivitis, which is often complicated in asthmatic patients. Allergen immunotherapy is the only existing treatment that can be expected to induce immunological remission, that is, a possible cure of allergic diseases [3]. Moreover, allergen immunotherapy has therapeutic potency for a variety of allergic diseases simultaneously observed in an allergic individual. This treatment includes subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Immunotherapy differs from drug therapy in that it may modify the natural course of allergic diseases by targeting allergen-specific Th2-type immune responses. In this article, we review the effect of allergen immunotherapy and its role in treating bronchial asthma. We also describe the current status of allergen immunotherapy for asthma in Japan.In asthma, meta-analyses have demonstrated that SCIT improves clinical symptoms and airway hyperresponsiveness and decreases drug requirements [4,5]. For example, Abramson et al. reported that the odds ratio for symptom improvement by SCIT with any allergen was 3.2 (95% CI 2.2–4.9), the odds ratio for drug reduction in SCIT using house dust mite (HDM) was 4.2 (95% CI 2.2–7.9), and the odds ratio for improvement of airway hypersensitivity was 6.8 (95% CI 3.8–12.0) [4].The effect of the addition of SCIT with HDM (HDM-SCIT) to the guideline treatment was reported in patients with mild or moderate HDM-sensitized asthma [6]. In the immunotherapy group, a decrease in the frequency of inhalational β2-agonists and a significant improvement in peak flow were observed. Furthermore, in pediatric asthma, adding HDM-SCIT to the guideline treatment reduces the requirement for ICS and improves the morning peak flow [7]. Therefore, HDM-SCIT has an additional effect even after the standard treatment is already performed. Furthermore, as described below, allergen immunotherapy has a controlling effect on other allergic diseases such as rhinoconjunctivitis, which is often complicated in asthma, a maintenance effect for more than a year even after discontinuation of treatment, and may have an inhibitory effect on sensitization to new allergens. Therefore, allergen immunotherapy has shown significantly different clinical meaning from drug therapy represented by ICS.However, the efficacy and effectiveness of SCIT in asthma remain controversial. Current evidence is derived from several small randomized controlled trials (RCTs), not only registration trials. Additionally, even in registration RCT, the effect of SCIT seems to be small to moderate. Furthermore, several biases including potential publication bias are also suggested in the meta-analyses [5].Nonetheless, the United States adult asthma management guideline (EPR3) states that SCIT should be considered for allergic asthma in steps two to four (mild persistent-moderate persistent equivalent) of the six treatment steps [8]. The European Academy of Allergy and Clinical Immunology (EAACI) guideline states that HDM-SCIT is recommended as an add-on to regular asthma therapy for adults with controlled or partially controlled HDM-driven allergic asthma [9].Unlike other drug therapies, allergen immunotherapy may have the action of modifying the natural course of allergic diseases. Allergen immunotherapy remains effective for more than a year even after the end of treatment. For example, 3-year allergen immunotherapy for rhinitis/conjunctivitis improves symptoms and suppresses conjunctiva-induced allergic responses for 7 years after treatment discontinuation [10]. Furthermore, Durham et al. conducted RCTs and reported that 3-year allergen immunotherapy by SLIT results in a symptom-relieving effect for 1 or 2 years after treatment discontinuation [11,12]. The effect of inducing asthma remission in childhood asthma has also been reported. Allergen immunotherapy in pediatric patients with allergic rhinitis/asthma increases the rate of asthma remission after 5 years of treatment, and the remission can be maintained for 5 years after discontinuation [13].Generally, allergen sensitization annually increases in patients with allergic asthma. However, allergen immunotherapy may have the long-term clinical effect of suppressing the spread of new allergen sensitizations. In a 15-year observational study, Marogna et al. reported that all allergic patients enrolled in the study and treated only by drug therapies were further sensitized with one or more new allergens in 15 years (100%). However, 3-, 4-, and 5-year allergen immunotherapy reduced the frequency of new allergen sanitization to 21%, 13%, and 12%, respectively [14].Furthermore, allergen immunotherapy is effective in preventing asthma development in children with hay fever. In a 3-year observation study in children aged 6–14 years with rhinitis due to hay fever, 32 of 72 children in the control group developed asthma, whereas 19 of 79 children in the immunotherapy group developed asthma. There was a significantly lower rate of asthma development in the immunotherapy group (odds ratio 2.52; p < 0.05) [15]. Furthermore, this preventive effect was maintained 7 years after the termination of immunotherapy. This study shows that allergen immunotherapy in patients with rhinitis may be effective in reducing the risk of developing asthma. The modifying effect of allergen immunotherapy on the natural course of allergic diseases remains controversial. Long-term effects were not observed in all patients. The strongest evidence for long-term effects was obtained from a follow-up study after RCT by Durham et al., as described above. However, the efficacy of 3-year allergen immunotherapy using SLIT was assessed for only 2 years after discontinuation [12]. Two-year allergen immunotherapy using SLIT did not improve nasal response to allergen challenge at 1 year after discontinuation [16]. Concerning the prevention of new allergen sensitization, Di Bona et al. systemically reviewed the effect on developing new allergen sensitization and reported that the evidence was of a low-grade level and the risk of bias was high [17]. Small studies and studies with a shorter follow-up showed the highest benefit of allergen immunotherapy [17]. Moreover, a meta-analysis by Di Lorenzo et al. did not find evidence to support the preventing effect of new allergen sensitization in children [18]. Valovirta et al. reported that allergen immunotherapy using grass SLIT in patients with rhinoconjunctivitis without asthma suppressed the risk of experiencing asthma symptoms and using asthma medication, even up to 2 years after discontinuation. However, no effect was seen on the time to asthma onset [19]. Therefore, the actual effect of allergen immunotherapy on the modification of the natural course of allergic diseases must be further elucidated.In allergic rhinitis, allergen immunotherapy is already the standard treatment. Asthma has a very high rate of complication with rhinitis [20]. In patients with allergic rhinitis, nasal mucosal allergen exposure induces smooth muscle contraction and eosinophil infiltration in the lower respiratory tract and bronchial hypersensitivity [21]. In addition, in asthmatics without rhinitis, nasal mucosa has eosinophil infiltration, and direct administration of sensitized allergen into the trachea induces nasal inflammation and eosinophil infiltration into nasal mucosal tissues [22]. Therefore, in this manner, airway inflammation is worsened by nasal allergen exposure or nasal inflammation and vice versa, and the concept of “one airway, one disease” is well recognized [21].In patients with asthma with allergic rhinitis, the treatment of rhinitis improves asthma symptoms and airway hyperresponsiveness and reduces asthma exacerbation [23]. According to the results of our questionnaire survey, patients with poor control of asthma symptoms are aware that asthma symptoms worsen as their nasal symptoms worsen, and asthma symptoms tend to improve with nasal treatment [24]. Therefore, the management of rhinitis is important for the treatment of asthma with rhinitis, and allergen immunotherapy is a reasonable strategy to control rhinitis and asthma. In clinical practice, comprehensive treatment should be considered for not only bronchial asthma but also complicated allergic diseases in individual asthmatics.The indication for HDM-SCIT in atopic asthma is mild-to-moderate persistent type with percent predicted forced expiratory volume in one second (%FEV1) of ≥70%. Treatment should be started in the stable period. Generally, a strong effect can be expected in a patient who is not sensitized to other allergens and is sensitized to HDM alone. We observed that the clinical effect, based on the rate of obtaining step-down of asthma severity, is significantly lower in patients with >10 years of disease or forced expiratory volume in one second (FEV1) of <70% [25]. Therefore, this therapy is more effective when applied as an intervention in the early phase of atopic asthma, in which airway remodeling has not developed. Moreover, as described above, in asthmatic patients with allergic rhinitis, an effect on rhinitis can be simultaneously expected.Patients with heart, liver, kidney, thyroid, or collagen disease should be excluded, and therapy initiation during pregnancy should be avoided. However, treatment can be continued if the patient has already reached maintenance therapy before pregnancy. Furthermore, as the therapeutic effect of ICS on asthma is diminished in smoking patients, it is assumed that the effect of immunotherapy in smoking patients is not sufficiently exerted.Allergen immunotherapy increases the concentrations of serum allergen-specific IgG or IgG4 and IgA antibodies (Abs) (Figure 1) [26,27,28,29] and transiently increases concentrations of allergen-specific IgE Abs. Several studies have demonstrated the inhibitory capacity of IgG or IgG4 for IgE-dependent immune responses. IgG or IgG4 can compete with IgE for allergen, inhibiting allergen-IgE complex formation [30]. Thus, it prevents cross-linking of high-affinity IgE receptors (FcεRI) on basophils and mast cells, which suppress histamine release, and blocks binding of allergen-IgE complexes to low-affinity receptors (FcγRIIb) on B cells [31], which suppress IgE-facilitated antigen presentation to T cells.Local production of Th2 cytokines, such as IL-4 and IL-5, or the numbers of Th2 cells are decreased by allergen immunotherapy (Figure 1) [32,33,34]. We found that immunotherapy attenuates HDM-specific production of thymus and activation-regulated chemokine, a potent chemokine activator of Th2 cells, from peripheral blood mononuclear cells (PBMCs) obtained from patients with HDM-sensitized allergic asthma, suggesting that immunotherapy can reduce accumulation of Th2 cells during allergen exposure [3]. Furthermore, immunotherapy suppresses allergen-induced Th2 cytokines such as IL-5 from PBMCs of allergic patients [35]. Therefore, immunotherapy can induce systemic immunological changes in response to allergens and provides some clinical benefits in allergic asthma. In addition to the effects on Th2-mediated immune responses, allergen immunotherapy induces regulatory T cells (Tregs) (Figure 1) [27,29,36,37,38]. Tregs are divided into two subsets: natural regulatory T cells (nTregs), which express the transcription factor forkhead box P3 (FOXP3), and inducible regulatory T cells (iTregs), which produce IL-10, IL-35, and TGF-β. For example, allergen immunotherapy increases local FOXP3+ T cells [36,37]. Allergen immunotherapy also increases local IL-10-expressing T cells [27,38] and TGF-β+ T cells [29]. However, the involvement of Tregs in allergen immunotherapy for Th2 suppression is probably regulated by multiple factors, including allergen and time of assessment. Furthermore, the role of regulatory B cells (Bregs), which also produce IL-10 and can suppress immune responses, has also been highlighted [39,40]. In bee venom-tolerant patients, IL-10-producing B cells, which express CD25 and CD71 but not CD73, are increased and associated with IgG4 production [39,40]. In addition to IL-10, Bregs reveal their suppressive property through TGF-β and IL-35 [39]. Moreover, allergen immunotherapy increases allergen challenge-induced expression of IL-12 mRNA in the skin [41]. These findings suggest that allergen immunotherapy suppresses T cell-mediated airway inflammation via downmodulation of Th2 cells and induction of Tregs or Th1 cells. As for type-2 innate lymphoid cells (ILC2), which are also important sources of Th2 cytokines such as IL-5 and IL-13, allergen immunotherapy decreases the number of ILC2 in peripheral blood [42], although this finding is controversial [43].Eosinophilic airway inflammation is an important feature of bronchial asthma. Infiltration of activated eosinophils in the airways is associated with asthma severity. Allergen immunotherapy suppresses airway inflammation, including the numbers of infiltrated eosinophils and concentrations of eosinophil-specific granule proteins (Figure 1) [3]. For circulating eosinophils to accumulate in asthmatic airways, they must adhere to and then migrate across vascular endothelial cells. These processes are largely regulated by cytokines/chemokines produced by various cells, including Th2 cells [44,45,46]. Increased adhesion of peripheral blood eosinophils and increased chemotactic activity of eosinophils into the airways are observed during the allergen exposure period in birch pollen asthma, and allergen immunotherapy suppresses increased eosinophil adhesion and chemotactic activity [3,32]. We reported that stimulation of PBMCs from HDM-sensitized allergic asthmatics with HDM increases eosinophil adhesion-inducing activity, eosinophil chemotactic activity, and eosinophil transendothelial migration-inducing activity, and the increase in these eosinophil activities was attenuated by allergen immunotherapy [3,47]. These findings suggest that the modification of Th2-mediated immune responses to specific allergens by allergen immunotherapy can suppress eosinophil accumulation in the airways. Biomarkers to predict effects of allergen immunotherapy are clinically important. The EAACI Task Force reported a consensus statement on biomarkers of allergen immunotherapy [48,49]. Biomarkers were grouped into seven domains: (1) IgE (total IgE, specific IgE, and specific IgE/total IgE ratio), (2) IgG subclasses (allergen-specific IgG1 and specific IgG4, including the specific IgE/IgG4 ratio), (3) serum inhibitory activity for IgE (assessed by IgE-facilitated antigen-binding (IgE-FAB) assay), (4) basophil activation, (5) cytokines and chemokines, (6) cellular markers (Tregs, Bregs, and dendritic cells), and (7) in vivo biomarkers, which include provocation tests. Although the optimal biomarker for the prediction of the effects has not been identified, specific IgE/total IgE ratio, allergen-specific IgG4 including the specific IgE/IgG4 ratio, IgE-FAB, and basophil activation are thought to be potentially useful [48,49].An early increase in specific IgE levels is observed during allergen immunotherapy, and the seasonal increase in IgE subsequently slows thereafter [28,50]. Then, specific IgE gradually decreases over several years [51], although there is no clear association between changes in specific IgE levels and the clinical response [29,38]. In contrast, several studies suggested that the ratio of specific IgE/total serum IgE at baseline correlates with clinical response to immunotherapy [52,53], although these findings were not reproduced in other studies [54,55]. Allergen-specific IgG subtypes including IgG4 are increased during allergen immunotherapy as compared with baseline values (Figure 1) [56,57,58]. We reported that there was a high correlation between the increase in log provocative doses causing a 20% decline in FEV1 and increase in the ratio of HDM-specific IgG4 to IgG1, suggesting that the increase in IgG4 is associated with the improvement of airway hyperresponsiveness in asthma [3]. However, a correlation between allergen IgG4 concentrations and clinical outcomes has not been reported in all studies [56,57,59]. For example, in a long-term follow-up study that evaluated patients up to 6 years after discontinuation of allergen immunotherapy, there was no correlation between allergen IgG4 concentrations and clinical outcomes [59]. As for specific IgE/IgG4 ratio, a decreased ratio was reported after allergen immunotherapy and associated with a reduction in late cutaneous skin reactions [48].IgG-associated IgE-inhibitory activity can be assessed by IgE-FAB assay (Figure 1) [49,60]. The IgE-FAB assay measures the ability of IgG-containing serum obtained after allergen immunotherapy to inhibit FcεRII-dependent binding of allergen-IgE complexes to B cells, although this assay is currently confined to specialized laboratories [28,49]. Another approach is the enzyme-linked immunosorbent-facilitated antigen-binding (ELIFAB) assay [49,61]. Several studies suggest a good correlation between IgE-FAB and ELIFAB results and the clinical response to immunotherapy as compared with serum IgG or IgG4 levels [28,49,61]. This is probably because IgE-FAB or ELIFAB measures the function of affinity and/or avidity of Ab binding. Basophil activation can be assessed by measuring the expression of surface markers, such as CD63 and CD203c, using whole blood. Although CD63 expression indicates basophil degranulation (Figure 1) [62], CD203c is a specific basophil marker that also indicates IL-3-dependent activation. Intracellular staining of phycoerythrin-conjugated diamine oxidase (DAO) has recently been highlighted (Figure 1) [63,64]. DAO binds to its substrate histamine, such that allergen stimulation reduces intracellular DAO levels in basophils proportional to the amount of intracellular histamine released. This reduction has been detected during both SCIT and SLIT [63,64].As SCIT has a risk of systemic reactions, although infrequent, SLIT has been developed mainly in Europe as a safer alternative method to SCIT.It was reported from the 1990s that SLIT with HDM (HDM-SLIT) improves symptom scores and airway hyperresponsiveness in patients with HDM-sensitized allergic asthma [65]. Since then, in asthmatic patients with rhinitis due to hay fever, SLIT has been shown to improve asthma symptoms, decrease the use of bronchodilators, and improve respiratory function compared with symptomatic treatment. Furthermore, Marogna et al. compared the effects of SLIT and ICS in patients with mild asthma and concomitant rhinitis due to grass pollen [66]. After a run-in season, patients were randomized to either 800 μg/day budesonide, an ICS, during the pollen season or continuous grass SLIT for 5 years. Asthma symptoms significantly decreased in both groups; however, improvements were greater in the SLIT group at 3 and 5 years. Furthermore, a decrease in both nasal symptoms and nasal eosinophils was observed only in the SLIT group.SLIT may be inferior to conventional SCIT in terms of efficacy [67,68,69]. For example, Di Bona et al. reported that SCIT is more effective than SLIT as assessed by symptom scores or medication scores [68]. However, because SLIT is less painful, convenient, and highly safe, it can be used in many countries in general clinical practice.Recently, the HDM-SLIT tablet developed by the Danish ALK was reported to be effective in bronchial asthma in a large-scale clinical study [70,71]. This HDM-SLIT tablet (6 standardized quality (SQ)) significantly reduces ICS use in asthma (SLIT 42%, placebo 15%, p = 0.0011) [70]. Furthermore, this HDM-SLIT tablet [6SQ] suppresses moderate-to-severe asthma exacerbation associated with ICS reduction (hazard ratio 0.72, p = 0.045) [71]. Based on this evidence, the Global Initiative for Asthma includes the description that HDM-SLIT should be considered in adult HDM-sensitized patients with allergic rhinitis, provided that %FEV1 is >70% [72].The standardized HDM allergen for SCIT was not available in Japan until 2015. Before 2015, SCIT using house dust (HD), collected from the general house, was utilized as an alternative therapeutic agent. The main component of HD is mites, but there were problems with product quality, and it was necessary to improve the effect and safety by standardizing allergens. The standardized purified HDM allergen for SCIT was prepared in 2015 and is currently used for the treatment of asthma. For SLIT, two HDM-SLIT tablets were approved for allergic rhinitis, but not for asthma, in 2015. The tablet developed by Torii Pharmaceutical Co., Ltd., Tokyo, Japan (MITICURE®) is the same as the tablet manufactured by ALK, in which the effect on asthma has been fully proven in Europe as described (10,000 Japanese allergy unit (JAU); the maintenance dose in Japan is equivalent to 6SQ in Europe). Recently, the effect of the HDM-SLIT tablet on asthma has also been determined in Japan. In HDM-sensitized atopic asthma with rhinitis, the addition of MITICURE® to the standard treatment improved the symptom scores of asthma, fractional exhaled nitric oxide, FEV1, and airway wall thickening in chest CT [73], suggesting that HDM-SLIT can suppress not only airway inflammation but also airway remodeling of asthma. Furthermore, a study of the effect of MITICURE® on asthma exacerbation associated with ICS reduction demonstrated that this treatment suppresses asthma exacerbation in patients who used short-acting β2-agonists during the observation period [74], which is consistent with the previous study in Europe [71]. Japanese cedar pollen (JCP) is widely scattered in the spring in Japan. Pollinosis by JCP is a representative seasonal rhinitis in Japan. People living in urban areas also suffer from pollinosis because JCP is scattered over tens of thousands of kilometers. One epidemiological study reported that the prevalence of Japanese cedar pollinosis was 26.5% [75]. Furthermore, its prevalence increased by about 10% in 10 years. This situation in Japan is unique in that Japanese cedars are planted forests and not natural ones. Similar to other pollens, JCP has been reported to exacerbate asthma. For example, Hojo et al. reported that the asthma control level, measured by the visual analog scale of Self-Assessment of Allergic Rhinitis and Asthma Questionnaire and Asthma Control Test score, worsened during the JCP-scattering season in asthmatic patients with allergic rhinitis by JCP, although 84% received treatment for rhinitis [76]. Asthma control during the pollen season was impaired in 18–38% of asthmatics with seasonal rhinitis with JCP [76]. Although the mechanisms for asthma exacerbation by JCP have not been fully clarified, several possible mechanisms are proposed. For example, fine orbicules (about 1 μm) adhering to the surface of JCP can reach the lower respiratory tract and directly induce asthma exacerbation. In addition, the effects of nasal obstruction, mediator released locally in the nose, and increased systemic cytokine production may be involved in JCP-related asthma exacerbation.Regarding JCP-related asthma, we have confirmed that treatment with JCP-SLIT almost completely abrogates the appearance of asthma exacerbation during the JCP-scattering season [77], supporting the certain prevention effect of SLIT on asthma exacerbation. Collectively, these findings indicate that HDM- or JCP-SLIT should be considered for asthmatic patients with rhinitis.One important problem which should be addressed is adherence to allergen immunotherapy [78,79]. As described above, most studies reported that more than 3 years of treatment is required to exert a modifying effect on the natural course of allergic diseases. For example, Kiel et al. reported that only 23% and 7% of patients receiving SCIT and SLIT, respectively, continued treatment for 3 years [78]. Sena et al. reported poor adherence using manufacturer sales data: SLIT prescription sales decreased from 100% to 44%, 28%, and 13% in the first, second, and third years, respectively, suggesting that <20% of patients had good adherence after 3 years [79]. Therefore, barriers to allergen immunotherapy adherence and strategies to improve compliance must be further investigated.In Japan, the treatment continuation rates of JCP-SLIT may be high. Yuta et al. reported that good adherence by direct calculation from prescription for 2 years was observed in 83% of patients [80]. We confirmed the similar treatment continuation rates of JCP-SLIT [81]. Although the reason of the difference is difficult to ascertain, the symptoms of patients with rhinitis by JCP were not sufficiently alleviated by drug treatment alone: consequently patients may become enthusiastic to receive JCP-SLIT. In our case, careful explanation on the role of SLIT and the importance of continuation for at least 3 years are usually performed. We assessed the predictors of adherence to JCP-SLIT prospectively and found that age younger than 40.5 years was the cutoff value for predicting poor adherence to JCP-SLIT [81]. Therefore, to reduce the discontinuation rate, the necessity of long-term treatment continuity should be clearly communicated prior to commencing treatment, especially for patients younger than 40 years.In HDM-sensitized asthma, HDM-SCIT improves clinical symptoms and airway hyperresponsiveness and decreases drug requirements. Furthermore, HDM- or JCP-SLIT can decrease asthma exacerbation and drug requirements. Current pharmacotherapy, such as ICS, provides powerful anti-symptomatic benefits in asthma; however, it does not modify the natural course of allergic diseases. In contrast, allergen immunotherapy targets the immunological background including the pathological activation of Th2 cells. Thus, it is expected to lead to long-term amelioration of asthma and allergic diseases. It is hoped that allergen immunotherapy is more widely applied in the treatment of asthma as a strategy for comprehensive management of allergy symptoms and modification of disease course.K.N. wrote the manuscript. M.N. edited the manuscript. All authors have read and agreed to the published version of the manuscript.This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology (15K09228).M.N. received honoraria from Torii Pharmaceutical Co., Ltd. K.N. has no conflict of interest.Mechanisms of allergen immunotherapy. Allergen immunotherapy induces T regs or Th1 cells and suppresses Th2 cells, eosinophils, mast cells, and basophils. It also induces allergen-specific IgG4 and suppresses allergen-specific IgE. Yellow text indicates potential biomarkers for allergen immunotherapy. DAO, diamine oxidase; ELIFAB, enzyme-linked immunosorbent facilitated antigen-binding; FAB, facilitated allergen binding.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-01-01-00004.txt

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+The new journal’s title Allergies (ISSN 2313-5786) answers the titular question because among all the existing journals devoted to allergies, few pretend to be exhaustive and cover all possible aspects of allergology. Allergies is intended to fill this gap by providing the most comprehensive possible coverage of a science in constant evolution.Over the past decade, allergology has grown steadily with the development of diagnosis based on molecular allergens and the new concepts introduced in the regulation of allergic response at the cellular and molecular levels. Awareness of the social and financial impact of allergies in all their forms, which currently comprise the fourth leading cause of chronic human diseases in the world, has played an obvious role in these developments.Different areas of allergies such as food allergies have intensely developed with the discovery of new food allergens, linked to the introduction of novel foods or novel food preparation processes. Similarly, the complexity of non-IgE mediated allergic responses associated with celiac disease and severe gluten intolerance has quickly become, beyond a trending effect, an important area of research. Other areas, such as the potential allergenicity associated with the introduction of new proteins expressed in genetically modified plants (GMPs) into the human diet, deserve special attention.Asthma and its associated pollinosis treatment have also undergone sustained research efforts, with the increasing development of sublingual immunotherapy in the treatment of grass pollen-induced allergic rhinoconjunctivitis.The allergological aspects of viral, bacterial and fungal infections comprise another area of research that is closely related to the COVID-19 epidemic and could be further developed.In addition, attention should be paid to the clinical aspect of allergology regarding reports of well-documented clinical cases of anaphylaxis associated with the identification and characterization of its responsible allergen(s). These clinical reports, which were previously widely available in allergological journals, are now more rarely published, and this is regrettable because of their obvious clinical interest.Accordingly, the list of topics addressed by Allergies was chosen to encompass all of these aspects of allergies, and members forming the topics and editorial boards were carefully selected because of their recognized skills and expertise in these different areas. Particular attention was paid to the selection of topics editors, who are particularly promising young allergologists. Allergies is a young international journal that aims to reach the allergologist community for the benefit of us all. We are confident that this journal will grow rapidly and contribute to the dissemination of peer-reviewed allergology data.The author declares no conflict of interest.
+        
+      Pierre Rougé is Professor emeritus at the UMR PhamaDev of the University Paul Sabatier (UPS) and the Institute of Research and Development (IRD) in Toulouse. His research interests comprise: (i) the structure–function relationships of allergens (e.g., IgE-binding epitopes and IgE-binding cross-reactivity), (ii) celiac disease, (iii) molecular modelling and docking, (iv) food allergies, and (v) predictive structural approaches (h-index: 52).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-01-01-00005.txt

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+Nasal obstruction is a frequent disorder that interferes with the daily patient’s quality of life. The key element in the pathophysiology of the disorder is the inferior turbinate hypertrophy related to multiple conditions such as allergic rhinitis (AR). Many patients are managed using conventional drug therapies such as antihistamines, decongestants, and intranasal steroid sprays, anticholinergic agents, mast cell stabilizers, and desensitizing vaccines. When traditional therapy failed to relieve AR symptoms, surgical inferior turbinate reduction (ITR) is indicated. A vast variety of surgical techniques have been reported in the literature for AR such as resectioning, coagulating, and laser procedures. We aimed to revise all surgical options in AR management. We confirm that no ideal standard technique for turbinate reduction has been developed so far regarding the multitude of different surgical procedures. Furthermore, no prospective and comparable long-term studies are present in the literature; it is challenging to recommend evidence-based surgical techniques.Nasal obstruction is a common disease that interferes with the daily patient’s quality of life [1].The key element in the pathophysiology of the disorder is the inferior turbinate hypertrophy related to multiple conditions such as allergic rhinitis (AR), pseudo-allergy, and non-allergic rhinitis eosinophilic, and iatrogenic syndrome [2,3,4].The inferior turbinates (IT) execute a central function in modulating airflow and nasal resistance through vasodilation and consequent nasal obstruction, congestion, and discomfort [5].Signs of AR result consequent to an inhaled allergen are characterized by a T helper 2 response and higher serum IgE levels and eosinophilia [6,7].The interaction between allergens and nasal mucosa is mediated by antigen-presenting cells (dendritic cells, APCs) and naïve T cells [8].Consequently, the T lymphocytes change into T helper type 2 (Th2) cells and produce interleukin 4 (IL-4), IL-5, and IL-13, promoting the B cell phenotype producing related IgE. Subsequently, IgE bound to the distinct receptors (FcεRI) of mast cells, and basophils cause cell activation; we illustrated Il5-13 pathways between eosinophils and mast cells in Figure 1 [9].Re-exposure to sensitized allergens leads to cascade disorders and allergic rhinitis (RA) manifestations.Many patients are treated with the use of conventional drug therapies such as antihistamines, decongestants and intranasal steroid sprays, anticholinergic agents, mast cell stabilizers, and desensitizing vaccines [10].Although medical treatment is often useful in restoring good nasal breathing, sometimes nasal obstruction becomes disabling, leading inexorably to use and abuse local nasal spray with a consequent high risk of iatrogenic outcomes.When traditional therapy fails to reduce AR symptoms, inferior turbinate reduction (ITR) is one of the simplest procedures made for the surgical treatment of nasal blocking. A series of techniques concerning reducing turbinates are currently feasible as cryosurgery, electrocautery, laser turbinectomy, partial or total turbinectomy, and Vidian’s neurectomy give different results. Due to direct manipulation of the mucous membrane during surgery, adverse accidents such as bleeding, pain, crusting, smell change, dry nose, and synechia can happen [11,12,13,14].Eosinophils, mast cells, and other immune cells play a critical role in allergic rhinitis: surgical treatment help prevent eosinophil and immune cell-mediated allergic diseases. Mladina et al. in 78 patients treated with turbinate surgery found improvements in 90% of cases of post-operative cytologic findings both in allergic and non-allergic subjects [15]. Moreover, Cassano et al. stated symptoms’ reduction in 51.4% of allergic rhinitis [16]. In contrast, the authors found improvements in 42.8% with non-allergic rhinitis with eosinophils (NARES) and 64.3% of mast cells with eosinophils (NARESMA).The primary goals of turbinal surgery, according to the clinical practice guidelines (2015), are based on observational studies, with a preponderance of benefit over damage, evaluating: -Quality of life (QoL) and nasal breathing improvement [17];-Symptomatic gain achieved through various mechanisms: exuberant mucous tissue reduction determines a reduction of the contact points for the allergens present in the inhaled air [9];-Reduction in the use of drugs with better patient compliance;-Preservation of the nasal mucosa using techniques that maintain mucociliary activity unchanged;Quality of life (QoL) and nasal breathing improvement [17];Symptomatic gain achieved through various mechanisms: exuberant mucous tissue reduction determines a reduction of the contact points for the allergens present in the inhaled air [9];Reduction in the use of drugs with better patient compliance;Preservation of the nasal mucosa using techniques that maintain mucociliary activity unchanged;The scar tissue that develops inside the submucosal layer leads to damage of both to the vascularization and the glandular structures, preventing their regrowth through fibrosis with the reduction of the amount of specific IgE present on the nasal mucosa and of eosinophils [18].Mucosa’s preservation that remains perfectly functioning allows, after the ITH procedure, both the maintenance of enough air space to support air’s humidification and purification and the maintenance of airways physiological resistance.The purpose of the narrative review performed was to list and better clarify the specific characteristics of each surgical technique described in the literature to date in the treatment of AR refractory to medical therapy (Figure 2).Techniques for turbinate reduction show that there is no absolute technique that supports long-term success but that the outcome is mainly linked to complications that occur in the short–long term (Table 1) [19].In a comprehensive review, Mol and Huizing described 13 different surgical techniques for ITR [17].Based on these criteria, turbinate reduction studies only meet the terms of evidence levels 3 and 4: only two studies have yet reached evidence level 2 where long-term follow-ups of 6 and 5 years were conducted, respectively [10,20].Each technique is judged on two basic criteria:how effective the method was in decreasing hypersecretion from respiratory obstruction, headaches, and sneezing episodes;side outcomes occurring in the short and long term.how effective the method was in decreasing hypersecretion from respiratory obstruction, headaches, and sneezing episodes;side outcomes occurring in the short and long term.It would be a mistake to concentrate entirely on expanding the nasal passages in endoscopic findings, rhinomanometry, and acoustic rhinometry.A wider nasal cavity does not necessarily mean the nose works better: It is now known that total turbinectomy is nearly out-of-date and radical resection of the inferior turbinate can cause atrophic rhinitis, chronic osteitis, or paradoxical nasal obstruction. The following complication is the hallmark of empty nose syndrome (ENS).In order to systematize the different surgical techniques, it is possible to outline a subdivision of techniques into three surgical groups: lateralization or lateropexy (it is named only for the sake of completeness), resection, and coagulation procedures.Resection and surgical reduction of the inferior turbinates are techniques that date back to the early nineteenth century. Total turbinectomy (full turbinate separated directly at the base of the lateral nasal wall) and partial turbinectomy (mucosal lamina and bones resected in the front third of the turbinate) are not, to date, widely used due to the obvious disadvantages that can be applied meeting.In literature, there is a highly inappropriate success rate that can vary between 0% and 89% [21]. Turbinectomy is not compatible with the primary goal of preserving nasal function; some patients suffered from atrophic rhinitis and secondary ozaena with symptoms of nasal dryness, crusting, bleeding, pain, and headache [22]. Talmon et al., performed, over 6 years, 357 total inferior bilateral turbinectomies in patients with chronic nasal obstruction: 351 patients (98.3%) underwent the procedure without any significant complication, six patients (1.7%) experienced postoperative bleeding that required emergency surgery [22]. Huizing and de Groot referred to a total turbinectomy as a “nasal crime”: they, therefore, suggest never to remove more than half of the inferior turbinate [23].Many surgeons prefer submucous inferior turbinectomy (SIT) as a resective method performed on the anterior third of turbinate through a vertical incision of the mucous membrane of the turbinate head. In this way, IT nasal mucosa and submucosa remain uninjured, and the turbinal volume is reduced without alternating functionality.Passali et al. performed a randomized comparative study on 382 patients with 4-year follow-up where it was shown that submucosal resection linked with lateralization of IT conferred long-term results regarding better nasal breathing, faster healing of mucociliary clearance, and the local secretion of IgA [10].Mori et al. explained submucosal inferior turbinectomy (SIT) inhibited allergic reactions in the IT of patients with AR improving the QOL in patients with severe allergic rhinitis: [24] this study group reported long-term results over a 5-year follow-up period, evaluating a significant improvement in both symptomatology and subjective mucosal response after exposure to inhalant allergens, after submucosal resection in a classic way. The mean total nasal symptom score (maximum 9) was significantly lower at one year after surgery (7.5 +/− 1.6 vs. 1.8 +/− 1.8, p < 0001) compared with before surgery and a significant improvement in nasal symptoms was noted at the 3-year (2.8 +/− 2.3, p < 0.0001) and 5-year (3.3 +/−1.6, p < 0.0001) time points [24].Submucosal reduction of the inferior turbinate is generally well tolerated with few long-term side effects and provides beneficial effects for RA patients.Microdebrider is an essential tool in the field of nasosinus surgery; it was introduced in daily use in turbinal surgery as MAIT (microdebrider-assisted inferior turbinoplasty).The MAIT technique can be performed in a dual-modality both intraturbinally and on the surface of the external turbinate with an endoscope in order to remove the IT abundant tissue. In the intraturbinal MAIT, after a small submucosal incision, microdebrider performs a millimeter resection, and extraturbinal MAIT can accurately extract the hypertrophic piece. Benefits include real-time aspiration with precise tissue removal [25,26].Lee et al. showed that extraturbinal MAIT appeared more powerful for long-term relief of allergic symptoms than intraturbinal techniques [28]. Sixty patients diagnosed with perennial AR were chosen, 30 patients were treated with intraturbinal MAIT (group 1) and the other 30 with extraturbinal MAIT (group 2). All symptoms decreased significantly in both groups at 3, 6, and 12 months; above all in group 2 at 12 months (p < 0.001) [27].Although the microdebrider is a very useful technique for ITR in AR, the bony portion of the turbinate is spared. In this case, the addition of out fracture or lateropexy can be considered [28].Chen et al. (2008) evaluated the long-term effectiveness of microdebrider-assisted inferior turbinoplasty with lateralization (MAITL) compared to submucosal resection for HIT. They showed MAILT appears to be as efficient as submucosal resection at decreasing nasal symptoms and limiting total nasal resistance and saccharin transit times for more than three years in patients with perennial AR. Compared to classic submucosal resection, the data in the literature suggest improved mucociliary time thanks to less surgical trauma and less tissue removed [28].Coagulation procedures are a set of methods based on deep high-frequency heat (electromagnetic waves in the frequency range of 106–1010 Hz) that determine the formation of wounds: during their healing, the venous sinusoids, present in the submucosal, sclerotize slowly, causing in the long term, the volumetric reduction of the turbinate.Submucosal diathermy (SMD) is a coagulating procedure based on monopolar or bipolar caustics use: the needle electrode inserted into submucosal space, starting at the level of the turbinate body up to its extremity, with an application during 5 s [29].Fradis et al. evaluated the short-term outcomes of SMD underlying good results in 78% of cases 2 weeks after surgery, but the effectiveness of the procedure was decreased to 76% two months after surgery. [31] According to their nasal airflow patency, one hundred patients with nasal blocking were subdivided into two groups: 49 patients underwent inferior bilateral turbinectomy (BIT) and 51 patients underwent SMD. All 100 patients were followed for two months after surgery. Two weeks after surgery, the operation’s success for nasal obstruction was 96% for the BIT patients and 78% for the SMD patients (p < 0.005). Both procedures are comparably safe and efficient, helping the chronic nasal obstruction, although SMD is more effective in the short term and could be disappointing on a long-term basis [30].Another study confirmed long-term results. It was non-encouraging: 533 patients with HIT treated with carbon dioxide (CO2) and (Nd: YAG) laser techniques and compared their long-term results with those of submucosal diathermy. Among the different techniques, compared to submucosal diathermy, both laser methods produced better long-term results: two years postoperatively, the overall benefit rate, as defined by patient satisfaction, was 79.6% for the CO2 Laser, 68.3% for the Nd: YAG laser, and 36% for submucosal diathermy [31].In the IT electrocoagulation, the inferior turbinate’s medial wall is cauterized in an anteroposterior direction using bipolar forceps. The heat generated by coagulation leads to fibrosis and necrosis first, scarring of the turbinate mucosa a second time [32].This electrocoagulation type is a rather risky procedure as it consciously compromises mucosal integrity. After the procedure, both atrophic and/or metaplastic changes of the mucosa and submucosa could be found up to a loss of efficacy of mucociliary transport.A prospective study evaluated the effectiveness of electrocautery, cryotherapy, and radiofrequency techniques, comparing patients’ reply to these three surgical modalities of ITR [34]: 90 patients manifesting nasal blocking with or without allergic symptoms, at the end of 12 months, 56.6% of patients treated with electrocautery and 56.6% of patients treated with cryotherapy registered 75% improvement in nasal obstruction, whereas 59.99% of patients treated with radiofrequency showed 100% gain. So, radiofrequency showed a better personal increase in nasal obstruction when compared to cryotherapy and electrocautery [33].Golding-Wood introduced Vidian neurectomy in 1961 to relieve severe nasal hypersecretion in vasomotor rhinitis [34]. He proposed the vidian nerve through Caldwell-Luc’s procedure, removing the posterior and some of the antrum’s medial wall to uncover the pterygopalatine fossa, opening of the vidian canal, aiming for parasympathetic resection of the vidian nerve [34].The posterior nasal nerve is a peripheral branch from the sphenopalatine ganglion, which starts the nasal cavity through a separate hole 4 to 5 mm below the sphenopalatine foramen/ethmoid crista after the nerve bifurcation into the lacrimal nerve. The posterosuperior nasal nerves innervate the upper and middle turbinates and foramina; other parasympathetic nerve fibers of the nose branch off from the greater palatine nerve and enter the nasal cavity through the canaliculi of the perpendicular plate of the palatine bone such as the posterior–inferior nasal nerves, these nerves innervate the meatus and the inferior turbinate [35]. Endoscopic endonasal surgery’s development replaced the classic Vidian neurectomy with posterior nasal nerve neurectomy (PNN) [36]. This surgery overcame the complications of Vidian neurectomy and is minimally invasive.Kawamura et al. reported in 2000 on the effectiveness of posterior nasal neurectomy linked with submucosal inferior turbinectomy for AR patients who had severe hyperreactive nasal symptoms refractory to medical treatment, in which neural bundles of the posterior nasal nerve were selectively cut or cauterized at the sphenopalatine foramen using an endoscopic nasal approach, leaving the secretory nerve fibers to the lacrimal glands intact [36]. In 2007, Kikawada reported an endoscopic PNN technique that resets the posterior nasal nerve near the sphenopalatine arteries and can control intraoperative bleeding under direct vision. A questionnaire survey was conducted in 94 patients with AR 2 years after surgery evaluating the efficacy of the procedure in about 80% of cases [37]. A retrospective study conducted in 212 patients with AR symptoms evaluated the endoscopic resection of PNN and mini-inferior turbinoplasty technique [38]. Patients were considered two weeks before and after surgery, followed up from 1st, 2nd, 6th, and 12th month postoperatively through a subjective evaluation with sinonasal outcome questionnaire SNOT-22: the mean SNOT-22 score after the procedure also significantly reduced from 50 to 8 at 12 months. In addition, 39.6% (84/212) of the patients had outlived almost free from all symptoms without medication at 12 months (p-values < 0.01) [38]. Argon plasma coagulation (APC) uses a high-frequency monopolar caustic which, through the ionized argon gas (the so-called plasma) conducts the current flow without direct contact with the mucosa, with a distance of 2–10 mm between the applicator tip and the fabric, reaching temperatures up to 3000 °C. This thermocoagulation involves forming a zone of local necrosis that during the wound’s healing results in a cicatricial reduction of the surrounding mucosa.In 2003, Bergler suggested much evidence on APC use in head and neck surgery. In the literature, reduction of turbinates, removal of leukoplakia or laryngeal papilloma, and treatment of epistaxis in patients with genetic telangiectasia are described [39]. In a prospective study of 121 patients (AR in 24 patients, vasomotor rhinitis in 75 cases, and chronic decongestant nose spray in 22 patients) with a follow-up period of 16 months, Bergler et al. concluded 76% of patients felt better nasal breathing after one week. After six weeks, the turbinates were re-epithelialized by healthy mucosa in 63% of the patients. After 12 months, 83% of the patients stated that they had better nasal airflow than preoperatively [40]. The most crucial advantage of APC turbinoplastly is the chance of performing a procedure without direct connection, while cryosurgery or electrocautery requires direct contact of the applicator with the IT [41]. Gierek et al. evaluated 100 patients (70 patients with bilateral hypertrophy of turbinates and 30 patients control group without breathing problems). Rhinomanometric parameters, clearance of saccharine test results, and cytological examinations were analyzed, obtaining results confirming the high effectiveness of APC for inferior turbinates reduction [41]. The use of inert gas argon during APCt results in a lack of smoke formation, which in the case of the use of electrocautery or Laser may hinder the precise orientation in the operative field.Edyta Jura-Szołtys et al. evaluated bronchial asthma symptoms control in patients with chronic rhinitis after argon plasma coagulation turbinectomy (APCt) [42]. The effect of APCt was assessed in 47 adults with drug-resistant chronic rhinitis and bronchial asthma 3-month post-procedure. Subjective improvement of nasal blockage three months after APCt was recognized in 87% and of rhinorrhoea in 75% of patients. Rhinomanometry showed a 219 ± 19 cm3/s increase of flow and 0.75 ± 0.06 Pa/cm3/s reduction of resistance. The predominance of patients with insufficient bronchial asthma control decreased from 79% to 4%. The decrease was associated with diminished frequency of eosinophils (20% in nasal cytology from 83% pre-procedure to 28% in the follow-up). Reduction in drug-resistant rhinitis symptoms after APCt is followed by significant improvement of asthma control [42]. Radiofrequency ablation of inferior turbinates (RFAIT) is a minimally invasive surgical technique that reduces turbinate size and decreases nasal obstruction with the direct application of high frequency (temperature about 75 °C), inducing mucosa’s thermonecrosis [43].RFAIT is a widely used thermal ablation technique to reduce nasal obstruction symptoms, acting on reducing the volume of the lower turbinates [43]. This technique has the advantage of being an outpatient procedure that can be performed quickly and can be performed under local anesthesia, increasing the patient’s quality of life [44].Overall, encouraging long-term results are reported. A prospective study (Lin et al. 2010) was performed on 146 patients with allergic rhinitis refractory to medical therapy who underwent RFAIT. Visual analog scale (VAS) was used to assess the allergic symptoms, including nasal obstruction, rhinorrhea, sneezing, after RFAIT at 6 months and 5 years postoperatively: the mean VAS score changed from 5.90 (2.79) to 3.79 (2.97) for rhinorrhea; from 5.15 (2.77) to 3.50 (2.77) for sneezing; from 3.67 (3.03) to 2.41 (2.30) for itchy nose; and from 2.94 (3.02) to 2.02 (2.42) for itchy eyes (all p < 0.001) [14].The long-term evaluation showed that RFAIT for AR or NAR appeared to improve the smell, reduce nasal resistance, and had subjective benefits in 82% of patients in a long time (60 months) [45].Turk et al. showed radiofrequency seems to be an adequate and safe treatment option for ITH of 59 patients with AR or non-allergic rhinitis (23 with AR, 36 with NAR), providing a better perception of all nasal symptoms (nasal obstruction, rhinorrhea, nasal itching, sneezing) measured by VAS score: 43. All parameters were analyzed as obstruction degree, acoustic rhinometry; VAS score reduced after a third and sixth month in a statically significant way (p < 0.001) [46]. De Corso et al., in their study, confirmed the insignificant distress and low risk of side effects of RFA, showing good efficiency of the method in the preponderance of patients for at least 36 months after surgery. A total of 305 patients (114 allergic and 191 non-allergic) who underwent RFAIT completed the NOSE-scale questionnaire pre-and postoperatively after 1 month and yearly for 5 years. Postoperatively, there was a notable improvement in nasal stuffiness, nasal obstruction, and mouth breathing (p < 0.05). In the following 2 years, it was perceived a worse temporal trend in terms of recurrence rate, and in particular in AR patients with a significant difference vs. NAR patient (p < 0.05) [47]. Coblation technology is based on the concept of molecular activation electrodissection acting on the submucosal layer. This technique has achieved rising popularity in recent years and is mainly adopted in the pediatric population thanks to greater patient compliance and less pain [48,49]. Sim’eon et al. studied the effectiveness of Coblator (Arthro-Care Corporation, Austin, TX) on the radiofrequency device in 9 AR patients with a mean age of 12.7 years. Long-term results (6 months) were observed, with a net reduction in nasal resistance and subjective symptoms such as itching, sneezing, hyposmia, and rhinorrhea [49]. RFAIT and coblation procedures are well tolerated with minimal adverse effects and can be performed safely in the operating room or even outpatient.The use of lasers in ITH reduction deserves a separate mention. The Laser produces a beam of light that is absorbed from the tissue depending on the wavelength of the laser light. The energy released in this process leads to thermal damage to the fabric. It is also possible to apply laser light pulsed or continuous [50,51]. The goal of laser therapy is to prevent excessive damage to the turbinal mucosa or bone exposure during the turbinates’ volumetric reduction. The laser emission can be applied according to linear, spot, anteroposteriorly, or the anterior third of the IT [52]. Actually, there are six several laser methods available: the carbon dioxide laser (CO2), the argon laser, the neodymium: yttrium aluminium garnet laser (Nd: YAG), potassium titanyl phosphate laser (KTP), the diode laser, and the holmium: yttrium aluminium garnet laser (Ho: YAG) [53]. The CO2 laser, a gas laser, is the most frequently used laser mode used in head and neck surgery. The laser beam (9.60–10.60 μm), given through a surgical microscope, avoids laser power being partially received by the fiberoptic cable: a custom self-retaining nasal speculum permits the surgeon to have both hands free, making laser surgery easier [54]. The published long-term results expose a substantial variety and range between 50% and 100%. Almost all studies are retrospective, non-comparative and describe different techniques of laser surgery [52]. Testa et al. showed evidence that CO2 laser turbinectomy can positively influence QoL patients with AR and chronic obstructive rhinitis. A total of 308 patients (168 with AR, 140 with chronic rhinitis) were recorded. Laser turbinectomy reconstructed nasal flow and induced a change in total score, which was statistically significant, for specific and general symptoms at the first, second, and third follow-up (p < 0.01) [55]. In a prospective study, Takeno et al. described CO2 laser partial turbinectomy in perennial and seasonal RA patients and noted less pronounced improvement in the seasonal RA group. It used acoustic rhinometry to estimate postoperative differences in nasal passage. Four months after treatment, the minimum cross-sectional area and nasal cavity volume had increased, respectively, by 61.7% and 30.7% in the perennial AR, and by 30.7% and 16.2% in the seasonal group: CO2 laser therapy may be useful for acute seasonal exacerbations but the effects may not be therapeutic at most until later in the allergy season [56]. The argon laser is an ion laser with a wavelength of 0.48–0.52 μm. The argon laser is absorbed by hemoglobin, and this makes it an ideal tool for vascular alterations. It was already used in 1981 for Rendu Osler disease as described by Parkin-Dixon [57]. It has not been widely accepted in turbinal surgery in AR, but we mention it anyway for the sake of completeness of this paper.The neodymium: yttrium aluminium garnet laser (Nd: YAG-laser) is a solid-state laser with a wavelength in the infrared field of 1.06 μm that can penetrate to a depth of 1 cm. Since the thermal reaction is more significant than other laser systems, it is recommended to limit the energy to 1000 watts/cm2 and reduce the application time by 0.5 s [58]. Laser light is carried out by employing a flexible fiber under endoscopic vision with or without contact on the mucosal surface. The energy released is absorbed by the submucosal venous plexus causing vasculitis sclerotizes in the exposed area and reduces the volume of the turbinate [59]. Olthoff et al. (1999) used the Nd: YAG Laser by contacting the inferior turbinate in a total of 83 patients with AR and vasomotor rhinitis. After a one-month check-up, an 80% success rate was found both in the AR group and in the group with vasomotor rhinopathy [60]. Vagnetti et al. carried out a study on 121 patients (30 patients with AR, 91 with NAR) treated with Nd:YAG laser and controlled up to one year. At 1-year follow-up, the complication rate in all our patients treated with this laser technique was very low, and we achieved a steady improvement in nasal patency in 104 patients (85.9%). The relapse rate was approximately 14%, but it was observed that 65% of the patients who experienced long-term failure were affected by allergic rhinitis [61]. The Holmium: YAG (Ho: YAG Laser) solid-state Laser features a wavelength of 2.123 nm and a reduced infiltration depth of 0.4 mm. Its minimum thermal energy decreases the turbinate volume, causing the formation of fibrin and scabs that heal in a period ranging from 3 to 6 weeks [62]. Sroka et al. compared Ho: YAG Laser with diode laser in two groups of patients after six months and 3-year long-term follow-up. In the first therapy group, a total of 80 patients (allergic rhinitis 46% vs. vasomotor rhinitis 54%) were treated by a pulsed Ho: YAG laser, and the second group of 113 patients (allergic rhinitis 52% vs. vasomotor rhinitis 48%), instead, were treated by diode laser. Three years after laser surgery, a subjective improvement of nasal airflow had been described by the patients in 67.5% after Ho: YAG- and in 74.4% after diode laser treatment [63]. In a retrospective review between Ho: YAG and submucosal diathermy, Rejali evaluated 19 patients with AR (8 patients treated with Laser vs. 11 treated with diathermy). Effectiveness in the laser (n = 8) and diathermy group (n = 11) was comparable. The complication/morbidity score was lower in the laser group (1.92 versus 3.48 (P ¼ 0.04, CI: 0.01, 2)). The long-term benefit was 50% and 36% in the laser and diathermy group, respectively. Ho: Yag laser treatment is equally efficacious but causes fewer complexities and morbidity than surface diathermy [64]. Sapci et al. addressed this problem by comparing three techniques: CO2 laser ablation, partial turbinectomy, and RFAIT. The study was conducted on three groups of 45 adult patients with nasal obstruction and stuffiness related to enlarged turbinates. Group A was treated with laser ablation, group B was treated with radiofrequency tissue ablation, and group C was the control group non-treated with surgical therapy. At 12 weeks after surgery, the nasal mucociliary transport time results were compared in the same patients. In patients on whom RFAIT and partial turbinectomy were applied, the average nasal mucociliary transport time was 10.33 min, whereas it was 11.33 min on the partial turbinectomy side. Rhinomanometric measurements demonstrated a significant decrease in nasal resistances at 12 weeks on both sides in groups A and B. All three techniques were valid for the turbinate volumetric reduction, the Laser presented, compared to the other greater damage to the mucosa [54]. The diode laser produces infrared light with a wavelength of 810/940 nm responsible for thermal reactions up to a depth of 5 mm [57]. Caffier’s research group studied the use of diode lasers extensively: 42 patients were evaluated who obtained, after the procedure, a success rate of 88% at 6 months and 74% after 12 months from treatment [65]. In another work conducted by the same study group, the effects of turbinoplasty using diode lasers were analyzed in 40 AR patients (20 patients with perennial pAR vs. 20 patients with seasonal sAR). Examinations were performed preoperatively and at follow-ups 1, 12, and 24 months after surgery, including physical parameters (rhinomanometry, video endoscopy, allergy tests) and subjective VAS scale. Throughout follow-up, objective rhinomanometry and subjective scores for nasal obstruction, rhinorrhea, sneezing, itching, and overall satisfaction gained significantly with time (p < 0.0005). The improvement was most remarkable for nasal obstruction, initially higher in pAR but more sustained in sAR. After 2 years, 30% sAR and 40% pAR subjects had been receiving pharmacotherapy due to recurrent symptoms: the improvement was perpetuated two years after the surgical procedure [66]. Another recent paper confirmed the use of diode laser in 60 patients with AR resistant to medical therapy. In this descriptive cross-sectional study, there was a significant improvement in symptoms like nasal obstruction, nasal discharge, sneezing, and decreased sense of smell measured with the Visual Analogue scale [67]. Potassium titanyl phosphate (KTP) Laser has a wavelength of 532 nm. [68] The laser beam is effectively absorbed by pigments such as hemoglobin and melanin-producing localized coagulation, making it very effective, especially in highly vascularized tissues such as the turbinate [69,70,71]. Vijayakumar et al. evaluated the performance of laser KTP for the volumetric reduction of the turbinates in subjects with AR. Thirty patients were selected, SNOT22 and saccharine transit time (STT) were evaluated before surgery and at a follow-up at 1 week, 1 month, and 3 months. The patients showed meaningful differences in the symptoms (p = 0.0001) at each follow-up. Saccharin transit test at one month prolonged significantly (p = 0.0001), but at the third month, saccharin transit time returned to normal limits (17.96 min) [71]. Another recent study compared the KTP laser with the diode laser [72]. This randomized controlled trial included 209 patients with ITH. Nasal Obstruction Symptom Evaluation (NOSE) score and STT were observed. Postoperatively, the median NOSE score was 50 in the diode group and 40 in the KTP group, and at three months, 15 in the diode group and five in the KTP group. KTP Laser registered a 93% increase in the NOSE score as compared to a 77% improvement in the diode laser group. On the other hand, both lasers altered the nose’s mucociliary clearance for up to three months of postoperative follow-up. KTP laser was more productive than diode laser in improving the NOSE scores but with a slightly increased rate of complications in the early postoperative period [72].Turbinates inferior hypertrophy is a clinical condition usually observed in allergic rhinitis (AR), in non-allergic rhinitis (NAR), and in anatomic-structural dysfunction of nasal cavities (septal deviation). Medical treatment with antihistamines, systemic decongestants, topical decongestants, or local corticosteroids often gives only mild relief for patients who use them. The surgical reduction of the lower turbinates is often required benefit in the medium to long term. A wide variety of surgical techniques have been described in the literature. These include total and partial turbinectomy, submucosal turbinectomy, submucosal resection with microdebrider, cryotherapy, and submucosal electrosurgery and turbinectomies using different types of lasers.The perfect surgery for the lower turbinates should produce the least number of side effects and discomfort and protect the nose’s physiological functions such as its humidification, the heating of the inspired air, and the respect of mucociliary clearance. The principal goal in turbinate surgery is to reduce the concal submucosal volume while protecting the integrity of the nasal mucosa.When analyzing the advantages, disadvantages, and complications in the short- long term of the various surgical techniques used to treat turbinal hypertrophy, especially in allergic subjects, it is complicated to draw definitive conclusions.An ideal study should meet the following criteria: the study should be prospective or comparative; patients should be randomly assigned to the study groups, and all should suffer from the same disease, avoiding comparing results in subjects with RA and NAR; preoperative and postoperative parameters should include the patient’s symptoms (VAS score), functional tests (respiration, mucociliary transport, nasal defense, etc.), complications, and up to 3–5 follow-up years. We realized it is difficult to meet all these requirements.However, we all agree that only the studies of this type would really help us find answers to many more remaining questions.Conceptualization, A.M., M.D.L., and S.C.; methodology, C.G. and C.M.G.; software, E.P. and I.L.M.; validation, F.M., V.B., C.V., G.I., and C.R.; writing—original draft preparation, I.L.M., C.M.G., G.C., A.M., and D.L.M.; writing—review and editing, S.C. and A.M. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The authors declare no conflict of interest.Immunophlogistic cascade of the allergic pathway. Abbreviations: CCL11, C-C motif chemokine 11; CCL17, C-C motif chemokine 17; CCL22, C-C motif chemokine 22; IL-4, Interleukin 4; IL-5, Interleukin 5; IL-9, Interleukin 9; IL-13, Interleukin 13; SCF, stem cell factor; TSLP, Thymic stromal lymphopoietin.Main surgical techniques currently described in the literature in the surgical treatment of allergic rhinitis. Abbreviations: PNN, posterior nasal nerve neurectomy; APC, Argon plasma coagulation; RFAIT, radiofrequency ablation of the inferior turbinate; CT, coblation technique; Ho:YAG-laser, Holmium:yttrium-aluminium-granat-laser; Nd:YAG-laser, Neodymium: yttrium aluminium garnet laser; KTP laser, Potassium titanyl phosphate laser.Main studies identified in the literature and principal features described.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Sequential IgE-binding epitopes were identified on the molecular surface of the Pis v 1 (2S albumin), Pis v 2 (11S globulin/legumin) and Pis v 3 (7S globulin/vicilin)—major allergens from pistachio (Pistacia vera) seeds—using the Spot technique. They essentially consist of hydrophilic and electropositively charged residues well exposed on the surface of the allergens. Most of the epitopic regions identified on Pis v 1 and Pis v 3 do not coincide with the putative N-glycosylation sites and thus are not considered as glycotopes. Surface analysis of these epitopic regions indicates a high degree of conformational similarity with the previously identified epitopic regions of the corresponding allergens Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) from the cashew (Anacardium occidentale) nut. These results offer a molecular basis for the IgE-binding cross-reactivity often observed between pistachio and cashew nut. They support the recommendation for prescribing pistachio avoidance in cashew allergic patients. Other conformational similarities were identified with the corresponding allergens Ses i 1 (2S albumin), Ses i 3 (vicilin) and Ses i 6 (legumin) from sesame (Sesamum indicum), and Jug r 1 (2S albumin), Jug r 2 (vicilin) and Jug r 4 (legumin) from walnut (Juglans regia). Conversely, conformation of most of the epitopic regions of the pistachio allergens often differs from that of epitopes occurring on the molecular surface of the corresponding Ara h 1 (vicilin), Ara h 2 (2S albumin) and Ara h 3 (legumin) allergens from peanut (Arachis hypogaea).The IgE-mediated anaphylaxis to tree nuts, such as almond (Prunus dulcis), Brazil nut (Bertholletia excelsa), cashew nut (Anacardium occidentale), hazelnut (Corylus avellana), pecan nut (Carya illinoinensis), pistachio nut (Pistacia vera), walnut (Juglans regia), has now become a public health concern, responsible for a major proportion of often severe anaphylactic shocks in both children and adults [1,2,3]. Although belonging to different botanical families, tree nuts share with peanut (Arachis hypogaea) and other edible seeds from lentil (Lens culinaris), pea (Pisum sativum), kidney bean (Phaseolus vulgaris), soybean (Glycine max), sesame (Sesamum indicum) and buckwheat (Fagopyrum esculentum), three groups of 2S albumins, 7S globulins or vicilins, and 11S globulins or legumins that have been recognized for a long time as being the most frequent allergen sources in seeds [4]. However, except for peanut and hazelnut, seed allergens from other tree nuts are less well known. This is the case with pistachio allergens, which are responsible for sometimes severe allergic manifestations. Ingestion of roasted pistachio nuts (Pistacia vera) has been reported to cause potentially harmful anaphylactic reactions [5,6], but pistachio is also currently incorporated in ice creams, bakery products and various exotic dishes. Food-dependent exercise-induced anaphylaxis to pistachio has also been reported [7]. However, anaphylaxis to pistachio is most often associated to cashew anaphylaxis [8,9].To date, three major pistachio seed storage protein allergens have been identified in pistachio, corresponding to a 2S albumin (Pis v 1), a 11S globulin (Pis v 2) and a 7S globulin (Pis v 3) [10,11]. Another 11S globulin closely related to Pis v 2, Pis v 5, was further reported to occur in pistachio seed [acces. Q8GZP6]. In addition, another major allergen, Pis v 4, which consists of a [Mn]SOD (superoxyde dismutase) unrelated to seed storage proteins, was identified in pistachio [12]. These allergens closely resemble the corresponding allergens from peanut (the 7S globulin Ara h 1, the v2S albumin Ara h 2, and the 11S globulin Ara h 3) [13,14,15] and other tree nuts, and cross-react, especially, with the 7S globulin Ana o 1, the 11S globulin Ana o 2 and the 2S albumin Ana o 3 of cashew (Anacardium occidentale) [16,17], which belongs to the same botanical family of Anacardiaceae. Additional cross-reactivity with the homologous allergens from more distantly-related seeds, has been also reported [18,19,20,21,22].The present work was aimed at identifying the IgE-binding epitopes of the major allergens of pistachio, Pis v 1 (2S albumin), Pis v 2 (11S globulin/legumin), and Pis v 3 (7S globulin/vicilin), as the molecular basis for their cross-reactivity with other corresponding tree nut allergens.Raw seeds from pistachio (Pistacia vera), cashew nut (Anacardium occidentale) and sesame (Sesamum indicum) were freely obtained from SOFICOR-Menguy’s company (Toulouse, France).Immune sera were drawn after informed consent of patients experiencing anaphylaxis to cashew nut (Table 1). Their reactivity towards pistachio was checked in Western blot experiments using a freshly prepared pistachio protein extract in Tris buffered saline (pH 7.5) as an allergen source. The specificity of the used sera had been assessed in previous publications [8,23].Sera from patients allergic to cashew were used as probes to identify the IgE-binding cross-reacting epitopic stretches from Pis v 1, Pis v 2, Pis v 3 and Ana o 3, immobilized on cellulose sheets. Sera from patients allergic to sesame (Table 2), were used as probes to identify the IgE-binding epitopic stretches from Ses i 3 and Ses i 6, immobilized on cellulose sheets.Protein extract was prepared from frozen seeds by two grinding steps of 40 s each in a Fast Prep-24 homogenizer (MP Biomedicals, Illkirch, France), in 20 mM Tris-HCl buffered saline (pH 7.4). The resulting slurry was centrifuged at 15,000× g for 10 min at 4 °C. The supernatant fraction was carefully collected while avoiding the floating lipid layer, filtered through a 0.2 μm membrane, and stored at −20 °C until used.The protein content of the pistachio extract was estimated using the bicinchonic acid kit reagent (Pierce) [24] with bovine serum albumin as a standard. The protein extract was checked by SDS-PAGE in 15% polyacrylamide gels using Tris-glycine as trailing ion [25] and staining with Coomassie blue. Coomassie blue stained bands were digested with trypsin in the gel and mass mapped by MALDI-TOF analysis as previously described [26]. The software Protein Prospector was used for the identification of the protein using the NCBI non-redundant database.IgE-containing sera from allergic patients were used in Western blotting as probes for the pistachio allergens. Following 1D SDS-PAGE, proteins were transferred onto a Protran nitrocellulose 0.2 μm membrane (Sigma-Aldrich, L’Isle d’Abeau Chesnes, France) at 20 V during 45 mn using a 48 mM Tris/39 mM glycine/20% (v/v) methanol mixture. After an overnight incubation in 10 mM PBS (pH 7.4) containing 0.2% (v/v) tween and 5% (v/v) skimmed milk, the membrane was soaked in the patient IgE-containing sera diluted 1:10 in the same buffer and incubated for 2 h in a moist chamber. After three washings of 10 min each with the same buffer, the membrane was soaked in rabbit HRP-labelled anti-human IgE diluted 1/5000 in the buffer and incubated for 1 h under gentle stirring. Following three washings of 10 min each with buffer, the immunolabelled spots were detected using the ECL Plus detection (ThermoScientific, Illkirch, France) after 3 min exposure in cassette. All the handling was carried out at room temperature.Overlapping 15-mer peptides, frameshifted by three residues, covering the entire amino acid sequences of Pis v 1, Pis v 2 and Pis v 3 were prepared by using the SPOT technique [27]. The protocol previously described in detail [28] was followed with the exception of the utilization of the Multipep automatic Spot synthesizer (Intavis). Briefly, peptides were assembled using the Fmoc chemistry on a cellulose membrane harboring an amino polyethylene glycol moiety. The C-terminal residue of each peptide was coupled to the activated membrane. After Fmoc deprotection, the following amino acids were sequentially added. At the end of the synthesis, side chain protecting groups were removed by a trifluoracetic acid treatment while the linkage of the peptides to the membrane was maintained.The membrane was soaked overnight into 20 mL of Tris-buffered saline (TBS) containing 2 mL blocking buffer (ThermoFisher, Montigny-le-Bretonneux, France) and 1 g sucrose (pH 7.0), and then washed three times with TBS containing 0.1% (v/v) tween (TBSTw). A 1:10 (v/v) diluted pool of patient sera was added in the presence of an anti-protease cocktail Roche (Sigma-Aldrich, L’Isle d’Abeau Chesnes, France) and the membrane was incubated in a moist chamber for 2 h. After three washes with TBSTw (pH 7.0) the membrane was stirred in a 1:4000 dilution of mouse monoclonal anti-human IgE labelled with alkaline phosphatase (Sigma-Aldrich, L’Isle d’Abeau Chesnes, France) for 1 h. After three washes with TBSTw (pH 7.0), the interacting peptide spots were colored for 30 min by adding the 5-bromo-4-chloro-3-indolylphosphate (BCIP) substrate for alkaline phosphate (Promega). The membrane was washed three times with deionized water and dried for scanning. The membrane can be used repeatedly after a regeneration step consisting in one wash in dimethylformamide for 1 min, three washes in deionized water, three washes in 8M urea containing 1% (w/v) SDS and 1% (w/v) b-mercaptoethanol and three subsequent washes in a mixture of ethanol-acetic acid-water (50:10:40, v/v/v). A similar protocol was used to map IgE-binding epitopes along the amino acid sequence of Ana o 3 (2S albumin) from Anacardium occidentale, and Ses i 3 (7S globulin/vicilin) and Ses i 6 (11S globulin/legumin) from sesame (Sesamum indicum) seed.The amino acid sequences of Pis v 1 (acces. ABG73108.1), Pis v 2 (acces. ABU42022.1) and Pis v 3 (ABO36677.1) were taken from the NCBI protein database (http://www.ncbi.nlm.nih.gov/pubmed, accessed on 7 January 2021). The multiple amino acid sequence alignment was performed with MacVector, using the T-coffee program [29]. Unrooted phylogenetic trees were built up from the multiple alignment of 2S albumins, vicilins (7S globulins) and legumins (11S globulins), using the uncorrected neighbor joining method integrated in MacVector.Homology modelling of Pis v 1, Pis v 2, Pis v 3, Ana o 3 and Ses i 3 was performed with YASARA [30], using appropriate templates from the Protein Data Bank (PDB) [31]. Five different models were built for Pis v 1, using 2S albumin from Moringa oleifera (PDB code 5DOM) [32], Ara h 2 from Arachis hypogaea (PDB code 3OB4) [33], Ber e 1 from the Brazil nut Bertholletia excels (PDB code 2LVF) [34], the seed storage albumin from Helianthus annuus (PDB code 5U87) (to be published), and the sweet protein mabinlin II from Capparis masaikai (PDB code 2DS2) [35], as appropriate templates. Nine different models were built for Pis v 3 (vicilin, 7S globulin) using vicilin from eggplant Solanum melongena (PDB code 5CAD, 5VF5) [36,37], vicilin-like protein from Capsicum annuum (PDB code 5YJS) [38], Car i 2 (vicilin) from the pecan nut Carya illinoinensis (PDB code 5E1R) [39], and vicilin from Pinus koraiensis (PDB code 4LEJ) [40], as appropriate templates. Nineteen different models were built for Pis v 2 (legumin, 11S globulin) using 11S globulin from Wrightia tinctoria (PDB code 5WXU) [41], cocosin (legumin) from the coconut Cocos nucifera (PDB code 5WPW) [42], subunit-β of 11S globulin from Cucurbita maxima (PDB code 2E9Q) [43], 11S globulin from Amaranthus hypochondriacus (PDB code 3QAC) (to be published), and prunin-1 (11S globulin) from almond Prunus dulcis (PDB code 3FZ3) [44], as appropriate templates. Five models were built for Ana o 3 (acces. AAL91665.1), using Ara h 2 from Arachis hypogaea (PDB code 3OB4) [32], mabinlin II from Capparis masaikai (PDB code 2DS2) [35], 0.19 α-amylase inhibitor from wheat Triticum aestivum (PDB code 1HSS) [45], and Ara h 6 (conglutin) from peanut Arachis hypogaea (PDB code 1W2Q) [46], as appropriate templates. Ten different models were built for Ses i 3 (acces. AAK15089.1), using the same templates as those selected for Pis v 3 [36,37,38,39,40]. Twenty-five different models were built for Ses i 6 (AAD42944.1), using five different templates 5WXU [40], 5WPW [41], 2E9Q [42], 3FZ3 [43], and Ara h 3 from Arachis hypogaea (PDB code 3C3V) [47]. Finally, a single hybrid model was built up from the different previous models, for each of the modelled proteins. PROCHECK [48], ANOLEA [49], and the calculated QMEAN scores [50,51], were used to assess the geometric and thermodynamic qualities of the three-dimensional models (Table 3). Molecular surfaces and Coulombic charges (negative, positive and neutral surfaces colored red, blue and white, respectively) were calculated and rendered with Chimera [52].To date, five allergens from pistachio seeds have been identified and characterized, two major allergens Pis v 1 (2S albumin) [10] and Pis v 2 (11S globulin/legumin), and three additional allergens Pis v 5 (11S globulins, legumin) [acces. B7SLJ1], Pis v 3 (7S globulin, vicilin) [11] and Pis v 4 (SOD) [12], as mentioned in the WHO/IUIS Allergen Nomenclature database (http://www.allergen.org, accessed on 9 January 2021) [53]. In addition, another vicilin-like, two additional 11S globulins and oleosins, have been identified from the genome assembly available for pistachio (GCF_008641045.1) but we do not know whether the identified potential allergens are actually expressed in pistachio seeds.The pistachio allergens Pis v 1, Pis v 2 and Pis v 5, and Pis v 3, expressed in mature pistachio seeds, accumulate gradually in protein bodies during the seed ripening process, as seed storage proteins (Figure 1A). Major allergens of protein bodies are easily detected in immunofluorescence experiments, using sera from patients allergic to cashew as primary antibody and an anti-human secondary antibody coupled to Alexa 633, respectively (Figure 1B).Upon SDS-PAGE, the major allergens from pistachio are well separated and readily interact with the IgE-containing patient sera in Western blot experiments (Figure 2). However, depending on the patient sera, different allergens are predominantly recognized by the IgE antibodies. Accordingly, several different patient sera should be used to accurately identified the IgE-binding epitopic stretches of Pis v 1, Pis v 2 and Pis v 3 mapped on the corresponding activated membranes.Homology modelling of the Pis v 1 allergen from appropriate templates yielded a typical 2S albumin structure built up of two α-helix-containing polypeptide chains linked together by two conserved disulfide bridges (Figure 3C,F). As predicted from the GlyProt server (http://www.glycosciences.de/modeling/glyprot/php/main.php, accessed on 9 January 2021) [54], the N-terminal putative N-glycan site at 13NLS should be actually glycosylated.Four main IgE-binding epitopic stretches were identified along the amino acid sequence of Pis v 1 in Spot experiments using sera that predominantly recognized Pis v 1 in Western blots as a probe (Figure 3A,B). These 3 stretches correspond to four distinct IgE-binding regions exposed on the molecular surface (Figure 3D,G). Most of these epitopes contain charged residues and coincide with the electropositively and electronegatively charged regions occurring at the molecular surface of the allergen (Figure 3E,H).The modelled 2S albumin allergen from cashew nut, Ana o 3, exhibit a very similar fold (Figure 4C,F), and four main IgE-binding epitopic stretches were similarly identified along the amino acid sequence of Ana o 3 (Figure 4D,G), which also correspond to electropositively and electronegatively charged regions occurring on the molecular surface of the allergen (Figure 4E,H). Very similar pictures were previously obtained with other 2S albumin allergen Jug r 1 from walnut (Juglans regia) [55] and Ses i 1 from sesame (Sesamum indicum) [56].Obviously, Pis v 1 and other 2S albumin allergens exhibit a quite superposable three-dimensional fold core, even though their amino acid sequences share rather low percentages of both identity and similarity (Figure 5). However, Pis v 1, Ana o 3 and Ses i 2, are readily clustered in the unrooted phylogenetic tree built for 2S albumin allergens (Figure 6), that suggest strong phylogenetic affinities between these proteins. In this respect, it is noteworthy that Pis v 1 and Ana o 3 belong to the same Anacardiaceae family.The phylogenetic relationships observed between Pis v 1 and Ana o 3 are reflected in the high similarity of their IgE-binding epitopic stretches along their amino acid sequences (Figure 7), which most probably account for the IgE-binding cross-reactivity occurring beetwen Pis v 1 and Ana o 3.Other less phylogenetically closely related 2S albumin allergens, such as Ses i 1 and Ara h 2, share much less similarity with Pis v 1 and Ana o 3.The modelled vicilin allergen Pis v 3 corresponds to a homotrimer built from the tail to tail non covalent association of three identical single-chain protomers made of a core of two cupin motifs, extended at both ends by two side arms made up of α-helices (Figure 8C,F). Each protomer contains a putative N-glycosylation site at 243NIT, which is predicted to be actually glycosylated according the GlyProt server (http://www.glycosciences.de/modeling/glyprot/php/main.php, accessed on 9 January 2021) [54]. This type of structural organization currently occurs in many other 7S globulin/vicilin allergens from other tree nuts and legume allergens [57].Up to nine main IgE-binding stretches identified along the amino acid sequence of the Pis v 3 protomer using the Spot method (Figure 8A,B), correspond to more or less exposed IgE-binding epitopic areas arrayed on both faces of the Pis v 3 homotrimer (Figure 4C,F). In fact, no information is available on the exposition of epitopes #1 and #2 at the surface of Pis v 3 since both epitopes occur in the N-terminal region of the polypeptide chain which is lacking in the three-dimensional model built for Pis v 3 by homology modelling. Two other epitopic region corresponding to epitopes #4 (colored magenta) and #8 (colored sienna), are almost completely buried and very little exposed on the surface of the allergen (Figure 4D,G). Like for other pistachio allergens, the well exposed epitopic regions corresponding to epitopes #3, #5, #6, #7 and #9, respectively, mostly coincide with both electronegatively (colored red) and electropositively (colored blue) charged regions, and their coalescence creates more extended epitopic regions on the molecular surface of the allergen. Other single surface-restricted epitopes (colored medium blue) are usually well exposed and mostly coalescent with the more extended IgE-binding patches. In addition, the exposed region of epitope #7 (colored sienna) could correspond to a CCD since it contains the putative 367NIT N-glycosylation site occurring on the amino acid sequence of Pis v 2.Up to twelve IgE-binding stretches were similarly identified along the amino acid sequence of the corresponding protomer Ses i 3 from sesame seeds, using the Spot method (Figure 9A,B) and, similarly, some epitopic regions corresponding to epitopes #4 (colored magenta) and #6 (colored purple), are poorly exposed at the surface of Ses i 3 whereas other exposed epitopes #2, #3, #5, #7, #8, #9, #10, #11 and #12 (Figure 9C,D,F,G), readily coincide with electronegatively (colored red) and electropositively (colored blue) charged regions arrayed on the molecular surface of Ses i 3 (Figure 9E,H).According to the common and superposable fold of the 7S globulin/vicilin allergens together with their epitopic similarities, the multiple alignment of these seed allergens exhibits a high degree of both identity and similarities, especially for Pis v 3 and Ana o 1, which belong to the same family of Anacardiaceae, and to a lesser extent, for Cor a 11 and Jug r 6 (Juglandaceae) and Ses i 3 (Pedaliaceae) (Figure 10). Accordingly, Pis v 3 and Ana o 1, and Cor a 11, Jug r 6 and Ses i 3, are distributed in two phylogenetically closely related clusters in the unrooted phylogenetic tree built for the 7S globulin/vicilin allergens (Figure 11).The phylogenetic relationships observed between Pis v 3 and Ana o 1, are reflected in the similarity of their IgE-binding epitopic stretches along their amino acid sequences (Figure 12), that most probably accounts for the IgE-binding cross-reactivity reported between Pis v 3 and Ana o 1. Much less similarity occurs between Pis v 3 and other less phylogenetically closely related vicilin allergens like Jug r 2 (Juglandaceae), Ses i 3 (Pedaliaceae) and Ara h 1 (Fabaceae) (Figure 12).Interestingly, the first epitopic stretch identified at the N-terminal end of Pis v 3 amino acid sequence (epitope #1), exhibits some amino acid identities with linear IgE-binding epitopes previously identified at the N-terminal end of Ana o 1, Ara h 1 and Len c 1 (Figure 12). This is also the case of an epitopic stretch recently characterized at the N-terminus (26–83) of Ara h 1, recognized as a major epitope responsible for the IgE-binding activity of the 7S basic peanut protein fraction [58]. In fact, this IgE-binding epitope coincides with two other linear IgE-binding epitopes that had been previously identified along the Ara h 1 amino acid sequence [59,60].The cupin allergen Pis v 2 consists of a legumin homotrimer resulting from the non covalent association of three protomers built up from two cupin motifs (Figure 13C,F). In fact, each protomer consists of a large acidic N-terminal and a shorter basic C-terminal subunit covalently associated by a single disulfide bridge. Finally, two homotrimers associated face to face to build an hexameric structure corresponding to a dimer of homotrimers. Like in other legumin hexamers [57], the face to face association of both homotrimers should predominantly result from electrostatic interactions occurring between the oppositely charged faces of the homotrimers, one face being predominantly electronegative (colored blue) whereas the other is essentially electropositive (colored red) (Figure 13E,H).Up to ten main IgE-binding epitopic stretches were identified along the amino acid sequence of the Pis v 2 protomer together with single epitopic spots (colored grey in Figure 13B), using the Spot method (Figure 13A,B). They correspond to IgE-binding patches well exposed at the surface of Pis v 2, except for epitopes #1 (colored red) and #3 (colored green), which are predominantly buried and much less exposed on the surface (Figure 13C,D,F,G). As with other allergens with cupin motifs, the more exposed IgE-binding patches coincide with the localization of electropositively (colored blue) and electronegatively (colored red) regions on the the molecular surface (Figure 13E,H).Ten distinct IgE-binding epitopic stretches were revealed along the amino acid sequence of Ses i 6, the legumin allergen from sesame, associated to some IgE-binding single-spots, using the Spot method (Figure 14A,B). Except for epitope #2 (colored blue), other epitopes #1, #3, #4, #5, #6, #7, #8, #9 and #10, are nicely exposed on the surface of the Ses i 6 homotrimer (Figure 14C,D,F,G) and most of them coincide with electropositively (colored blue) and electronegatively (colored red) charged regions on the surface of Ses i 6 (Figure 14E,H).Multiple amino acid sequence alignment of 11S globulin/legumin allergens shows a high degree of both identity and homology/similarity between the members of this protein family, especially between the members of the Anacardiaceae (Ana o 2, Pis v 2, Pis v 5), Juglandaceae (Jug n 4, Jug r 4) and Betulaceae (Cor a 9) families (Figure 15). Accordingly, both Ana o 2, Pis v 5, Jug n 4, Jug r 4 and Cor a 9, are closely clustered in the dendrogram built for the 11S globulin/legumin allergens (Figure 16). However, Pis v 2 appears as being more distantly related to Ana o 2, compared to Pis v 5.In agreement with the phylogenetic relationships observed between Pis v 2, Pis v 5, Ana o 2, Jug n 4, Jug r 4, and Cor a 9, the sequential IgE-binding epitopic stretches identified along their amino acid sequences exhibit pronounced similarities (Figure 17), that most probably account for the IgE-binding cross-reactivity reported between some of these legumin allergens [61]. However, less similarity occurs between Pis v 2 and other phylogenetically related legumin allergens whereas Pis v 5, the other legumin allergen from pistachio, appears as more closely related to other legumin allergens than Pis v 2. Compared to Pis v 2, Pis v 5 exhibits a very similar amino acid sequence with, however, a few amino acid changes. In spite of these changes, regions in Pis v 5 corresponding to the IgE-binding epitopes identified in Pis v 2, look like very similar (Figure 17). Conversely, Ara h 3, which belongs to the rather distantly related Fabaceae family, exhibits a higher degree of epitopic similarity with the other legumin allergens (Figure 17).Due to the high degree of conservation and phylogenetic relatedness of their amino acid sequences, 2S albumin, vicilin and legumin allergens, are readily superposable with Rmsd values below 1.0 Å, e.g., for the pruned atom pairs corresponding to the conserved cupin motifs of the Pis v 3/Ana o 1 (0.906), Pis v 3/Jug r 2 (0.979), Pis v 3/Ses i 3 (0.955) and Pis v 3/Ara h 2 (0.929) vicilin pairs, respectively (Figure 18).A detailed surface analysis of epitope #1 of Pis v 1 reveals that it shares a similar topographical distribution of the amino acid residues along the α-helix, associated to partial conformational similarities, with the corresponding epitope #1 from Ana o 3 (Figure 19A,B). Similarly, epitope #8 from Pis v 2, exhibits a similar topographical distribution of amino acids and partial conformational similarities with the corresponding epitope from Ana o 2 (Figure 19C). Obviously, these epitopic similarities should account for the IgE-binding cross-reactivity often reported among the different allergens from pistachio and cashew nut [61]. However, these conformational similarities could only be observed between closely related epitopes sharing a high degree of identity. In other cases of couple of epitopes sharing a moderate level of amino acid sequence identity, no relevant conformational similarities could be identified.In addition to the IgE-binding epitopic similarities observed among the different members of the same allergen family, other cross-reactivity has been previously reported between the CD4(+) T cell epitopes from cashew nut, hazelnut and pistachio [62].These IgE-binding epitopic amino acid identities, sometimes associated with conformational similarities, come in support to the well recognized cross-reactivities among the allergens from different species belonging to the same family of Anacardiaceae (pistachio, cashew), Juglandaceae (walnut), Fabaceae (peanut), Pedaliaceae (sesamum) and Betulaceae (hazelnut), previously reported from immunodiffusion and crossed immuno-electrophoresis experiments, Western blotting experiments, recognition of recombinant allergens and clinical assessments [63,64,65] (Figure 20).The allergenicity of the vicilin allergen Ara h 1 from peanut (Arachis hypogaea), has been attributed in part to the particular arrangement of the monomers in the typical homotrimeric structure of vicilin/7S globulin proteins [66]. The quaternary association of monomers in the homotrimer has been proved to mainly depends on hydrophobic interactions responsible for the swapping of monomers by their distal ends, allowing the major IgE-binding epitopes located along these buried extremities to escape the proteolytic degradation by digestive enzymes. However, in the case of Pis v 3 (vicilin) and Pis v 2 (legumin), an additional mechanism could explain the resistance to digestive proteolysis. Looking at the distribution of the putative cleavage sites for pepsin predicted to occur at the molecular surface of Pis v 3 and Pis v 2, using the web server PeptideCutter of Expasy (https://web.expasy.org/peptide_cutter/, accessed on 15 January 2021), suggests that some of the IgE-binding epitopes previously identified does not contain cleavage sites and could remain in attacked by the protease, e.g., epitopes #3, #4, #6, #9 from Pis v 3, and epitopes #1, #2, #3, #5, #6, #7 from Pis v 2 (Figure 21).However, due to the occurrence of numerous putative cleavage sites for trypsin predicted in the IgE-binding epitopes of both Pis v 3 and Pis v 2, most of these epitopes should be degraded later, during the intestinal digestion in the presence of trypsin, except for epitopes #1, #2 and #8 from Pis v 2, which do not contain any K or R residues (Figure 13).Using the Spot technique with a panel of IgE-containing sera from patients allergic to pistachio and cashew nut, sequential IgE-binding epitopes were identified on the molecular surface of the modelled Pis v 1 (2S albumin), Pis v 2 (legumin) and Pis v 3 (vicilin) allergens, respectively. These epitopic amino acid stretches essentially consist of hydrophilic (N,Q,S,T) and charged residues (D,E,K,R). In this respect, these residues account for ~60% of the epitopic stretches identified on Pis v 3. Accordingly, most of these epitopic regions coincide with the electropositively and electronegatively charged areas occurring on the surface of the allergens. Moreover, and whatever the size of the allergens, a few IgE-binding epitopes locate in the same area and their coalescence should create more extended epitopic surfaces susceptible to correspond to discontinuous epitopes. However, this clustering tendency is more or less pronounced depending on the allergens (see Figure 3D,G for Pis v 1, Figure 8D,G for Pis v 3, and Figure 13D,G for Pis v 2). A similar epitopic coalescence was observed for the counterpart allergens of Pis v 1 (Figure 4D,G for Ana o 3), Pis v 3 (Figure 9D,G for Ses I 3) and Pis v 2 (Figure 14D,G for Ses I 6). Finally, some IgE-binding epitopes well exposed on the surface of the cupin allergen protomers, e.g., epitope #8 of Pis v 3 (which contains the putative N-glycosylation site at 243NIT), become partly buried upon the oligomeric association of the protomers. Accordingly, the allergenic potency of the Pis v 3 protomer should slightly differ from that of the Pis v 3 homotrimer. Obviously, these discrepancies depend on the mode of association of the protomers in the vicilin and legumin homotrimers [62]. In addition, the occurrence of a N-glycan chain on each Pis v 3 protomer at the N-glycosylation site 243NIT located on epitope #8, which is predicted to be actually glycosylated by GlyProt (http://www.glycosciences.de/modeling/glyprot/php/main.php, accessed on 15 January 2021) server, should participate in the allergenic potency of the allergen. N-glycans have been demonstrated to participate in the IgE-binding activity of N-glycosylated epitopes of e.g., the structurally related Ara h 1 vicilin allergen from peanut [63]. The N-glycan chain of the 13NLS glycosylation site of Pis v 1, which is adjacent to the IgE-binding epitope #1, could similarly participate in the allergenicity as a carbohydrate determinant (CDD), even though there are no evidence for such a role of the N-glycan chains of pistachio allergens.The approach which has been used to identify the linear or continuous epitopes, which combines the Spot technique with molecular modelling, provides results which need to be interpreted with caution since it does not allow to identify discontinuous or conformational epitopes, depending on the length of the synthetic peptides used as IgE-binding probes. In addition, while the molecular modelling techniques have improved significantly during the past decade, they still remain less reliable than the conventional approaches by X-ray crystallography or NMR to identify and map the IgE-binding epitopes on the molecular surface of the allergen-IgE complexes. However, the coupling of Spot and molecular modelling techniques is a reasonable and expedient compromise to get an insight into the IgE-binding epitopes from allergenic proteins.Depending on the localization of the exposed pepsin and trypsin cleavage sites on the three-dimensional model built for the Pis v 3 and Pis v 2 protomers (Figure 21), a central core structure remaining unaltered upon the digestive proteolytic attack by pepsin is predicted to occur in both allergens. Interestingly, this core structure still retains some epitopes that should remain protected from the proteolytic attack till Pis v 3 is recognized by the immuno-competent cells of the gastro-intestinal tract. However, except for a few IgE-binding epitopic stretches from Pis v 2, apparently devoid of lysine and arginine residues, most of the epitopes remaining unaltered upon the pepsin attack, should be further degraded due to their richness in electropositively charged residues.A strong overall IgE-binding cross-reactivity between pistachio and cashew allergens has been often reported from immunoelectrophoretic analyses, Western blotting experiments, and specific IgE measurements [63,64,65]. Both vicilins (Pis v 3, Ana o 1) [11] and 2S albumins (Pis v 1, Ana o 3) [67] have been incriminated as the main IgE-binding cross-reacting allergens. A detailed surface analysis of the epitopes occurring on the molecular surface of these allergens, revealed a rather high degree of amino acid sequence identities, associated or not with conformational similarities depending on the degree of sequence identity occurring between their epitopes. Both sequence identities and conformational similarities establish the molecular basis of the IgE-binding cross-reactivity between the allergens from pistachio and cashew, which belong to the same botanical family of Anacardiaceae. However, the surface analysis performed on the legumin allergens revealed no conformational similarities between most of the sequential IgE-binding epitopes of Pis v 2 and Ana o 2. A conformational IgE-binding epitope (called 2B5) was identified on the molecular surface of Ana o 2 that mainly consists of 24 amino acid residues (20EPDNRVEYEAGTVEAWDPNHEQFR43) located at the N-terminus of the large (acidic) subunit, which is expressed only when associated to the small (basic) subunit [68,69]. The IgE-binding epitopes #1 and #2 of Pis v 2 were identified on the corresponding amino acid sequence stretch (30EPKRRIESEAGVTEFWDQNEEQLQ53) which displays 55% identity (underlined bold letters) with the 2B5 epitope.Obviously, the amino acid sequence identities between the IgE-binding epitopes from allergens of different origin depend on the degree of conservation the allergens have retained during evolution. Accordingly, the occurrence of IgE-binding epitopes sharing similar physicochemical properties has been pointed out as a key factor contributing to the cross-reactivity among peanut and tree nut allergens [61]. In this respect, 2S albumin allergens (Pis v 1, Ana o 3) and vicilin allergens (Pis v 3, Ana o 1), are predicted to display a degree of conservation higher than that predicted for the legumin allergens (Pis v 2, Ana o 2). Most conserved residues essentially occur along the structurally conserved secondary features, e.g., the α-helices of 2S albumins and the β-strand cupin motifs of the vicilins. These conserved regions are predominantly built up from hydrophilic residues. According to the sequence and conformational similarities observed among 2S albumin and vicilin allergens, Pis v 1 and Pis v 3 of pistachio closely cluster with the corresponding Ana o 3 and Ana o 1 allergens of cashew in the dendrograms built up from the amino acid sequence alignments with the neighbor joining method (Figure 6 and Figure 11). Although Pis v 2 and Ana o 2 allergens appear as being more distantly related in relation with the lower degree of sequence similarities that relate both allergens, another legumin allergen related to Pis v 2, Pis v 5, is closely clustered to Ana o 2 and other tree nut allergens in the legumin dendrogram (Figure 16).Due to the high degree of conservation of the 2S albumin and cupin allergen structures, the occurrence of amino acid sequence similarities observed between the pistachio allergens Pis v 1, Pis v 2 and Pis v 3 and other homologous allergens from either closely or distantly related families, has an influence on their allergenicity and cross-reactivity. Especially, the location of the amino acid sequence similarities in regions corresponding to IgE-binding epitopes, greatly determine their capacity to cross-react with the corresponding regions from other homologous allergens. Depending on the degree of sequence identity between epitopes belonging to homologous allergens, the resulting cross-reactivity will be strong, moderate or low. In this respect, the sequence similarities observed between the homologous allergens of Anacardiaceae, e.g., between Pis v 1 and Ana o 3 or between Pis v 2 and Ana o 2, account for the high level of cross-reactivity and cross-allergenicity observed between pistachio and cashew. The IgE-binding cross-reactivity the major allergens from pistachio, Pis v 1, Pis v 2 and Pis v 3, share with homologous proteins from peanut, tree nuts and proteins from, sesame, buckwheat, peppercorn or mango, has a clinical incidence and in particular, helps to discriminate between allergies and co-sensitizations [19,22,70,71,72,73,74,75,76,77,78]. Whereas co-sensitization frequently occurs between pistachio and cashew, it is clinically relevant in only one-third of cases [72]. However, skin prick tests (SPT) performed on French children indicated that a low reaction dose to cashew in cashew-allergic children would be a predictive factor of allergy to pistachio [78]. Accordingly, oral food challenge to pistachio should be avoided in cashew-allergic children exhibiting a low reaction dose to cashew nut.Methodology and validation, A.B., H.B.; investigation, C.N. and C.G.; software, P.R.; original-draft preparation, C.G. and P.R.; writing—review and editing, P.R. All authors have read and agreed to the published version of the manuscript. This research received no external funding.Informed consent was obtained from parents of subjects involved in the study.This article is dedicated to the memory of Fabienne Rancé, who actively contributed to the methodology and validation of the results and provided us with the patient sera used in this work. Many thanks to Alain Jauneau (CNRS, Toulouse, France), who provided us with the micrography and histo-immunochemical pistachio slices.The authors declare no conflict of interest.(A) Toluidine blue-staining of seed storage proteins located into the protein bodies (PB) of cotyledon cells of pistachio. CW: cell wall. Tiny white globules arrayed in the cytoplasm correspond to oleosin-containing oil bodies. (B) Binding of IgE from patient sera allergic to cashew nut and pistachio, to the major allergen-containg protein bodies of cotyledon cells (red spots). Alexa-labelled anti-IgE was used as probe to reveal the binding of IgE-containing sera from cashew allergic patient to protein bodies.SDS-PAGE of a protein extract from pistachio nut (line 1) and Western blots of allergic patient sera that preferentially react with Pis v 1 (line 3) and Pis v 2 (lane 4). Molecular weight markers are indicted on lane 2. Protein fractions corresponding to the Pis v 1, Pis v 2 and Pis v 3 allergens were identified by MALDI-TOF after trypsic digestion in gel of the protein bands (line 1). * and ** indicate the most IgE-reactive protein fractions.(A) IgE-binding peptides (boxed violet spots) revealed on the Pis v 1 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Pis v 1. Epitopes are colored red (epitope 1), blue (epitope 2), green (epitope 3) and magenta (epitope 4), respectively. (C,F) Ribbon diagram of the front (C) and back (F) face of the modelled Pis v 1 showing the localization of the IgE-binding epitopic regions numbered and colored as in (B). (D,G) Surfaces occupied by the colored IgE-binding epitopes on the front (D) and back (G) face of Pis v 1 numbered and colored as in (B). (E,H) Mapping of Coulombic charges on the front (E) and back (H) face of Pis v 1. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.(A) IgE-binding peptides (boxed violet spots) revealed on the Ana o 3 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Ana o 3. Epitopes are colored red (epitope 1), blue (epitope 2), green (epitope 3) and magenta (epitope 4), respectively. (C,F) Ribbon diagram of the front (C) and back (F) face of the modelled Ana o 3 showing the localization of the IgE-binding epitopic regions numbered and colored as in (B). (D,G) Surfaces occupied by the IgE-binding epitopes on the front (D) and back (G) face of Ana o 3 numbered and colored as in (B). (E,H). Mapping of Coulombic charges on the front (E) and back (H) of Ana o 3. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.Multiple amino acid sequence alignment of 2S albumin allergens Ana o 3 from Anacardium occidentale; Ara d 2 and Ara d 6 from Arachis duranensis; Ara h 2, Ara h 6 and Ara h 7 from Arachis hypogaea; Ara i 2 and Ara i 6 from Arachis ipaensis; Ber e 1 from Bertholletia excelsa; Bra j 1 from Brassica juncea; Bra n 1 from Brassica napus; Bra r 1 from Brassica rapa; Car i 1 from Carya illinoinensis; Cor a 14 from Corylus avellana; Cuc ma 5 from Cucurbita maxima; Fag e 2 from Fagopyrum esculentum; Fag t 2 from Fagopyrum tataricum; Gly m 8 from Glycine max; Hel a 2S from Helianthus annuus; Jug n 1 from Juglans nigra; Jug r 1 from Juglans regia; Lup a δC from Lupinus albus; Lup an δ1 from Lupinus angustifolius; Ric c 1 from Ricinus communis; Ses i 1 and Ses i 2 from Sesamum indicum; Sin a 1 from Sinapis alba. Identical amino acids are grey shadded. Aminoacid sequences of allergens belonging to the same botanical family, are similarly colored in red (Anacardiaceae), green (Betulaceae), magenta (Pedaliaceae), orange (Juglandaceae) and blue (Fabaceae).Dendrogram of 2S albumins from Brazil nut (Ber e 1 from Bertholletia excelsia), buckwheat (Fag e 2 from Fagopyrum esculentum, Fag t 2 from F. tartaricum), cashew nut (Ana o 3 from Anacardium occidentale), Castor bean (Ric c 1 from Ricinus communis), hazelnut (Cor a 14 from Corylus avellana), lupine (convicilin Lup a δC from Lupinus albus, convicilin Lup an δ1 from Lupinus annuus), mustard (Sin a 1 from Sinapis alba), peanut (Ara d 2 and Ara d 6 fom Arachis duranensis; Ara h 2, Ara h 6 and Ara h 7 from A. hypogaea; Ara i 2 and Ara i 6 from A. ipaensis), pecan nut (Car i 1 from Carya illinoinensis), pistachio (Pis v 1 from Pistacia vera), pumpkin (Cuc ma 5 from Cucurbita maxima), sesamum (Ses i 1 and Ses i 2 from Sesamum indicum), soybean (Gly m 8 from Glycine max), sunflower (Hel an 2S from Helianthus annuus), turnip and rapeseed (Bra j 1 from Brassica juncea and Bra r 1 from B. rapa; Bra n 1 from B. napus), walnut (Jug n 1 from Juglans nigra and Jug r 1 from J. regia). 2S-albumins clustered to Pis v 1 are red circled.Multiple alignment of 2S albumin allergens Pis v 1, Ana o 3, Ara h 2, Cor a 14, Jug r 1 and Ses i 1. The Ig-E-binding epitopes delineated along the amino acid sequence of Pis v 1 (red boxes) and Ana o 3, Ara h 2, Cor a 14, Jug r 1 and Ses i 1 (black boxes) are indicated. Amino acid residues occurring in regions of 2S albumins homologous to the IgE-binding epitopes of Pis v 3, are shaded pale green.(A) IgE-binding peptides (boxed violet spots) revealed on the Pis v 3 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Ana o 3. Epitopes are colored red (epitope #1), blue (epitope #2), green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), deep green (epitope #7), sienna (epitope #8) and dark blue (epitope #9), respectively, and numbered as in B. Other single-spot IgE-binding peptides are colored gray. (C,F) Ribbon diagram of the front (C) and back (F) face of the modelled Ana o 3 showing the localization of the colored IgE-binding epitopic regions numbered and colored as in B. IgE-binding epitopic patches corresponding to single-spot IgE-binding peptides, are colored grey. (D,G) Surfaces occupied by the colored IgE-binding epitopes on the front (D) and back (G) face of Pis v 3. Epitopes are numbered as in (B). (E,H) Mapping of Coulombic charges on the front (E) and back (H) of Pis v 3. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.(A) IgE-binding peptides (boxed violet spots) revealed on the Ses i 3 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Ana o 3. Epitopes are colored red (epitope #1), blue (epitope #2), green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), deep green (epitope #7), sienna (epitope #8), deep blue (epitope #9), cyan (epitope #10), black (epitope #11) and grey (epitope #12), respectively. (C,F) Ribbon diagram of the front (C) and back (F) face of the modelled Ses i 3 homotrimer, showing the localization of the colored IgE-binding epitopic regions. Epitopes are numbered and colored as in (B). (D,G) Surfaces occupied by the colored IgE-binding epitopes on the front (D) and back (G) face of Ses i 3. Epitopes are numbered and colored as in (B). (E,H) Mapping of Coulombic charges on the front (E) and back (H) face of Ses i 3. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.Multiple amino acid sequence alignment of 7S globulin/vicilin allergens Ana o 1 from Anacardium occidentale, Ara d 1 from Arachis duranensis, Car i 2 from Carya illinoinensis, Cic a 1 from Cicer arietinum, Coc n 1 from Cocos nucifera, Cor a 11 from Corylus avellana, Gly m 5 and Gly m Bd 28K from Glycine max, Gos h Vic from Gossypium hirsutum, Jug n 2 from Juglans nigra, Jug r 2 and Jug r 6 from Juglans regia, Len c 1 from Lens culinaris, Lup a 1 from Lupinus albus, Lup an 1 from Lupinus angustifolius, phaseolin from Phaseolus vulgaris, Pin k 2 from Pinus koraiensis, Pis s 1 and Pis s 2 from Pisum sativum, Pis v 3 from Pistacia vera, Ses i 3 from Sesamum indicum, Zea m g1 from Zea mays. Identical amino acids are grey shadded. Aminoacid sequences of allergens belonging to the same botanical family, are similarly colored in red (Anacardiaceae), green (Betulaceae), magenta (Pedaliaceae), orange (Juglandaceae) and blue (Fabaceae).Dendrogram of 7S globulin/vicilin allergens from cashew nut (Ana o 1 from Anacardium occidentale), chickpea (Cic a 1 from Cicer arietinum), coconut (Coc n 1 from Cocos nucifera), cotton (Gos h Vic. from Gossypium hirsutum), garden pea (Pis s 1 and Pis s 2 from Pisum sativum), hazelnut (Cor a 11 from Corylus avellana), kidney bean (phaseolin from Phaseolus vulgaris), Korean pine (Pin k 2 from Pinus koraiensis), lentil (Len c 1 from Lens culinaris), lupine (Lup a 1 from Lupinus albus and Lup an 1 from L. annuus), maize (vicilin Zea m γ1 from Zea mays), peanut (Ara h 1 from Arachis hypogaea), pecan nut (Car i 2 from Carya illinoinensis), pistachio (Pis v 3 from Pistacia vera), pumpkin (Cuc ma 5 from Cucurbita maxima), sesamum (Ses i 3 from Sesamum indicum), soybean (Gly m 5 and Gly m Bd 28K from Glycine max), sunflower (Hel an 2S from Helianthus annuus), turnip and rapeseed (Bra j 1 from Brassica juncea and Bra r 1 from B. rapa; Bra n 1 from B. napus), walnut (Jug n 2 from Juglans nigra, Jug r 2 and Jug r 6 from J. regia). Vicilins clustered to Pis v 1 are red circled.Multiple alignment of 7S globulin/vicilin allergens Pis v 3, Ana o 1, Ara h 1, Ses i 3, Cor a 11, Jug n 2 and Len c 1. Ara h 1 e, corresponding to the IgE-binding epitope recently identified at the N-terminal end of Ara h 1 sequence, contains 3 extra aminoacids compared to the N-terminal sequence of Ara h 1. The Ig-E-binding epitopes delineated along the amino acid sequence of Pis v 3 (red boxes), Ara h 1 e (blue box), and Ana o 1, Ara h 1, Ses i 3, Cor a 11, Jug n 2 and Len c 1 (black boxes), are indicated. Amino acid residues occurring in regions of vicilins homologous to the IgE-binding epitopes of Pis v 3, are shaded pale green.(A) IgE-binding peptides (boxed violet spots) revealed on the Pis v 2 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Pis v 2. Epitopes are colored red (epitope #1), blue (epitope #2), green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), olive green (epitope #7), sienna (epitope #8), dark blue (epitope #9) and orange (epitope #10), respectively. (C,F) Ribbon diagram of the front (C) and back (F) face of the modelled Pis v 2 showing the localization of the colored IgE-binding epitopic regions. Epitopes are numbered and colored as in (B). (D,G) Surfaces occupied by the colored IgE-binding epitopes on the front (D) and back (G) face of Pis v 2. Epitopes are numbered and colored as in (B). (E,H) Mapping of Coulombic charges on the front (E) and back (H) faces of Pis v 2. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.(A) IgE-binding peptides (boxed violet spots) revealed on the Ses i 6 Spot membrane. (B) Mapping of the corresponding continuous IgE-binding epitopic regions (colored boxed white letters) along the amino acid sequence of Ses i 6. Epitopes are colored red (epitope #1), blue (epitope #2), green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), deep green (epitope #7), sienna (epitope #8), dark blue (epitope #9) and grey (epitope #10), respectively. (C,F). Ribbon diagram of the front (C) and back (F) face of the modelled Ses i 6 showing the localization of the colored IgE-binding epitopic regions. Epitopes are numbered and colored as in (B). (D,G). Surfaces occupied by the colored IgE-binding epitopes on the front (D) and back (G) faces of Pis v 1. Epitopes are numbered and colored as in (B). (E,H) Mapping of Coulombic charges on the front (E) and back (H) faces of Ses i 6. Electropositive and electronegative regions are colored blue and red, respectively. Neutral regions are white.Multiple amino acid sequence alignment of 11S globulin/legumin allergens Act c 12 from Actinidia chinensis, Ana o 2 from Anacardium occidentale, Ara h 3 from Arachis hypogaea, Ave s 11S from Avena sativa, Ber e 2 from Bertholletia excelsa, Car i 4 from Carya illinoinensis, Cic a 6 from Cicer arietinum, Coc n 4 from Cocos nucifera, Cor a 9 from Corylus avellana, Cuc ma 4 from Cucurbita maxima, Fag t 1 from Fagopyrum tataricum, Gly m 6 from Glycine max, Jug n 4 from Juglans nigra, Jug r 4 from Juglans regia, Lup a ��C from Lupinus albus, Lup an αC from Lupinus angustifolius, Pis v 2 and Pis v 5 from Pistacia vera, Pru du 6, Ses i 6 and Sesi i 7 from Sesamum indicum, Sin a 2 from Sinapis alba. Identical amino acids are grey shaded. Amino acid sequences of allergens belonging to the same botanical family, are similarly colored in red (Anacardiaceae), green (Betulaceae), magenta (Pedaliaceae), orange (Juglandaceae) and blue (Fabaceae).Dendrogram of 11S globulin/legumin allergens from almond (Pru du 6 from Prunus dulcis), Brazil nut (Ber e 2 from Bertholletia excelsia), buckwheat (Fag t 1 from Fagopyrum tartaricum), cashew nut (Ana o 2 from Anacardium occidentale), chickpea (Cic a 6 from Cicer arietinum), coconut (Coc n 4 from Cocos nucifera), hazelnut (Cor a 9 from Corylus avellana), kiwi (Act c 12 from Actinidia chinensis), lupine (convicilin Lup a αC from Lupinus albus, convicilin Lup an αC from Lupinus angustifolius, mustard (Sin a 2 from Sinapis alba), oat (Ave s 11S from Avena sativa), peanut (Ara h 3 from Arachis hypogaea), pecan nut (Car i 4 from Carya illinoinensis), pistachio (Pis v 2 and Pis v 5 from Pistacia vera), pumpkin (Cuc ma 4 from Cucurbita maxima), sesamum (Ses i 6 and Ses i 7 from Sesamum indicum), soybean (Gly m 6 from Glycine max), walnut (Jug n 4 from Juglans nigra and Jug r 4 from J. regia). Legumins clustered to Pis v 1 are red circled.Multiple alignment of 11S globulin/legumin allergens of Pis v 2, Pis v 5, Ana o 2, Ara h 3, Cor a 9, Jug r 4 and Ses i 6. The Ig-E-binding epitopes delineated along the amino acid sequence of Pis v 2 (red boxes) and Ana o 2, Ara h 3, Cor a 9, Jug r 4, Ses i 6 (black boxes) are indicated. Amino acid residues occurring in regions of legumins homologous to the IgE-binding epitopes of Pis v 2, are shaded green. Amino acid residues common to all the legumin sequences (blue boxes) are indicated.(A) Superposition of the ribbon diagrams of the 2S albumin allergens Pis v 1 (colored red), Anao 3 (colored blue), Ara h 2 (colored yellow), Jug r 1 (colored green) and Ses i 1 (colored purple). (B) Superposition of the ribbon diagrams of the 7S globulin/vicilin allergens Pis v 3 (colored red), Ana o 1 (colored blue), Ara h 1 (colored yellow), Jug r 2 (colored green) and Ses i 3 (colored purple). (C) Superposition of the ribbon diagrams of the 11S globulin/legumin allergens Pis v 2 (colored red), Ana o 2 (colored blue), Ara h 3 (colored yellow), Jug r 4 (colored green) and Ses i 6 (colored purple).(A,B) Analysis of the topographical distribution and conformational similarities of amino acids of epitope #1 from Pis v 1 (A), compared to epitope #1 from Ana o 3 (B). The amino acid sequences of epitopes #1 from Pis v 1 and Ana o 3, are indicated. (C,D) Analysis of the topographical distribution and conformational similarities of amino acids of epitope #8 from Pis v 2 (C), compared to epitope #8 from Ana o 2 (D). The amino acid sequences of epitopes #8 from Pis v 2 and Ana o 2, are indicated.Cartoon summarizing the different types, limited, mild or strong, of cross-reactivities susceptible to occur between the members of the different families of Anacardiaceae (green), Juglandaceae (red), Fabaceae (blue) and Pedaliaceae (orange). Hazelnut belongs to the Betulaceae.(A–D). Localization of predicted cleavage sites by pepsin (red patches) on the three-dimensional surface of front (A) and back face (B) of Pis v 3, and front (C) and back face (D) of Pis v 2 monomers. The IgE-binding epitopic regions identified on the surface of Pis v 3 are numbered and colored pale green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), dark green (epitope #7), brown (epitope #8) and dark blue (epitope #9), respectively. The IgE-binding epitopic regions identified on the surface of Pis v 2 are numbered and colored red (epitope #1), pale blue (epitope #2), pale green (epitope #3), magenta (epitope #4), yellow (epitope #5), purple (epitope #6), dark green (epitope #7), brown (epitope #8), dark blue (epitope #9) and orange (epitope #10), respectively.List of sera from subjects allergic to cashew nut and pistachio.* M: male, F: female, ** ImmunoCAP Phadia F203, *** ImmunoCAP Phadia F202.List of sera from subjects allergic to sesame.* ImmunoCAP Phadia F10.Geometric and thermodynamic qualities of the three-dimensional models built by homology modeling with YASARA.The number of residues (*) refer to those occurring in the different model monomers.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+The global COVID-19 pandemic has brought respiratory disease to the forefront of public health, but asthma prevalence has been rising globally for decades. Asthma is mediated by errant immune activation and airway remodeling, but the influences of environment, nutrition, and comorbidities (e.g., asthma-chronic obstructive pulmonary disorder-overlap [ACO]) are still poorly understood. Even as a new generation of biologic-based treatments offer better airway control and reductions in mortality, a lack of prophylactic treatments and mechanistic understanding complicates efforts to prevent pathogenesis. This review will explicate and synthesize current knowledge on the effect of ACO and biologics (omalizumab, mepolizumab, reslizumab, benralizumab, and dupilumab) on pathogenesis, treatment, and prognosis.Asthma is an endemic disease, affecting over 339 million children and adults worldwide according to the World Health Organization, and industrialized countries are not exempt, as evidenced by Japan where, in 2017, over 1 million people in Japan were newly diagnosed with asthma [1]. Of these, 427,000 were under the age of 15 and diagnosis rates year-on-year have been climbing since 1984 [2]. Treatment is symptomatic and effective cures and prevention measures have not been reported in spite of advancements in immunology. Over the last 20 years, multiple advancements in immunosuppressive treatment have appeared, yet the prevalence of this incurable disease has climbed steadily, particularly in children. Numerous causes, from inheritance to allergies to industrial pollution, have been considered as key triggers for disease pathogenesis but the incredible diversity of responses inherent to the immune system confounds efforts to pin the blame on a single source. For this reason, we here will briefly review the current body of knowledge on asthmatic mechanisms, known/putative causes, environmental influence, standard treatments, newly developed biologic treatments, and overlap with chronic obstructive pulmonary disease (COPD). It is our opinion that targeted biologics will have the greatest impact on asthma treatment in the next 10 years and, if it cannot be prevented, then targeted therapies will provide the highest levels of control with the least side effects.Allergic asthma is a chronic inflammatory disease of the lungs and airway that causes symptoms such as wheezing, breathlessness, coughing and chest tightness [3]. It is classified phenotypically and these categories have been extensively reviewed [4]. Allergic asthma is further subclassified according to the component of inflammatory cells in the sputum such as eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic phenotypes [5]. These errant Type 2 immune responses manifest into three phenotypes: eosinophilic inflammation, IgE antigen-specific, and airway hyperresponsiveness [5,6]. (Figure 1) The role of histamine in the pathology and exacerbation of these types of asthma has been well studied and reviewed [7,8]. Although asthma can also be grouped according to genotype, the most common types are thought to be molecularly mediated through environmental or immune microenvironmental cues [9,10]. Regardless of type, the common manifestation of an asthma attack is characterized by chronic inflammation of the airway, leading to hyper-responsiveness and remodeling of the respiratory tract [3]. However, phenotypic manifestations may differ as adult asthma is evidenced by mucosal and sputum neutrophil infiltration whereas juvenile asthma is associated with atopy [3].Biochemically, allergic asthma is primarily mediated by mast cells, CD4 + T helper-2 (Th2) cells, and the secretion of interleukin 4 (IL-4), IL-5, and IL-13 [13]. First, T-lymphocytes that are recruited by IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) secreted from challenged epithelium cause the release of Type 2 (T2) cytokines, attracting eosinophils and mast cells within the airway tract [13]. Moreover, these T2-cytokines mediate IL-5 release, which stimulates release of IL-4 and IL-13 by innate lymphoid, CD4+ T, and mast cells (among diverse other types such as basophils and NK T cells), as well as eosinophilic inflammation [11,13,14]. IL-4 and IL-13 then cause B-lymphocytes to produce IgE when allergens are detected in the body and, after binding to the IgE receptor (FcεR1), mucosal mast cells migrate to the bronchus and mediate the inflammatory cytokine cascade comprised of IL-4, IL-5, IL-13, growth factors, neutrophils, histamine, cysteinyl-leukotrienes (Cys-LT), and prostaglandin D2 (PG-D2) [13]. Second, mast cells release proteases such as tryptase and chymase [13]. Tryptase is a mast cell-specific protease that mediates airway hyperresponsiveness whereas chymase activates transforming growth factor-β (TGF-β) that initiates airway remodeling [13]. TGF-β then triggers the hyperplasia and hypertrophy of smooth muscle cells in the airway while vascular-endothelial growth factor (VEGF) increases the number of blood vessels, narrowing the airway [13]. As a result, plasma leakage from the postcapillary venules causes the airway lumen to swell [13]. Third, large numbers of group 2 innate lymphoid cells (ILC2) are also found in asthma patients and are developmentally controlled by epithelial cytokines such as IL-25, IL-33, and thymic stromal lymphopoietin in addition to TGF-β [13] (Figure 1). Lastly, chronic lung inflammation thickens the epithelial layer with stiff collagen III and V through irreversible fibrosis [13]. This collagen deposition, as well as the addition of fibronectin and tenascin C, causes permanent airway fibrosis and, as a consequence, irreversible airway constriction [13,15]. The cells, secreted cytokines, and targets involved in this pathway are summarized in Table 1.The mechanism behind asthma pathogenesis varies by subtype, onset age, and mediating illness or allergy. Viral infections at a young age are thought to play an important role in the development of childhood asthma since rhinoviral infection engenders atopic childhood asthma via reductions in interferon-β and interferon-γ in bronchial epithelial cells [16,17]. Respiratory syncytial viruses, human rhinoviruses, human metapneumoviruses, parainfluenza viruses, and coronaviruses are the main respiratory viruses associated with wheezing and asthma exacerbation [18]. Thus, the mechanism behind virally induced asthma is the putative destruction of bronchial epithelial cells via release of pro-inflammatory/pro-necrotic cytokines, leading to epithelial cell and tight junction damage [19]. Biologically, relationships between genetic regulatory events, such as DNA methylation of FOX and RUN family genes associated with T cell development, and childhood asthma pathogenesis have been reported [20]. Since the use of eosinophil counts in bronchoalveolar lavage fluid (BALF) from juvenile patients under six years of age with wheezing symptoms were not found to be predictive of asthma, these new genetic approaches might be more predictive of asthma pathogenesis in very young children [21].Early exposure to bacterial antigens or strains of symbiotic flora in “dirty” environments may also mediate asthmatic development. Two studies, PARSIFAL and GABRJELA, have reported that children living on farms have lower asthma prevalence than children living in the same area but not on farms (adjusted odds ratios of 0.49 and 0.76, respectively) [22]. Furthermore, the mattress dust surveyed in this study contained more bacteria and a higher variety of fungi for children living on farms than the children from the reference group [22]. Another meta-analysis showed similar results where children raised on farms had a 25% lower asthma rate than children raised on non-farming land [23]. Likewise, the Amish, a population with a traditional farming lifestyle that eschews technology, showed the strongest protection against allergic asthma [23]. Interestingly, whey proteins and microRNA that are included in unprocessed cow milk are also thought to protect children against juvenile asthma development and the ALEX study highlighted the importance of exposure to these allergens during the first years of life [23]. Similarly, the PARSIFAL study expressed the exposure to these allergens during pregnancy is effective to reduce asthma prevalence [23]. Mechanistically, the PARSIFAL study demonstrated how children on farms had high expression of Toll-like receptors, such as IRAK1, IRAK2, and RIPK1 from activated CD4 T helper cells, that are responsible for activating innate immune responses against allergic asthma [23]. Moreover, farm dust increases respiratory epithelial layer barrier capacity via A20 regulation, resulting in a stronger defense against viral infections that drive asthma pathogenesis [23].Interestingly, not all species of microbes are protective against autoimmune diseases like asthma. Wilson and colleagues reviewed several studies showing that S. aureus colonization may enhance the risk of atopic dermatitis (an asthma risk factor) by actively promoting mast cell degranulation, spurring the inflammation that may precipitate other autoimmune reactions [24]. Location may be crucial in this case as S. pneumoniae, H. influenzae, and M. catarrhalis colonization of upper airways carries an increased risk of asthma pathogenesis while gut colonization by Faecalibacterium, Lachnospira, Veillonella, and Rothia genera (termed FLVR) within the first 100 days after birth were shown to be protective against asthma [24]. In addition, fungal microorganisms, especially Aspergillus and Pneumocystis spp., have been well reviewed and are known to increase sensitization, exacerbation, and resistance to control among asthmatics [25]. As the mystery behind gut biome–immune interactions are unraveled, more data on the specific genera or species that provide protection against autoimmune diseases could lead to novel targeted therapies with probiotic or microbial supplementation as asthma prophylaxis and treatment.Despite multiple studies and reviews proving or disproving the protective effect of early pet antigen exposure on asthmatic pathogenesis, there are microbe/immune interactions, environmental mechanisms, and possible individualized interactions that have not been fully delineated. Details of factors that may influence this effect have been extensively reviewed elsewhere [22,23,26,27,28].In contrast, the origins for adult-onset asthma are atopy, obesity, aspirin exacerbated respiratory disease (AERD), gastroesophageal reflux disease (GERD), occupation, and smoking [29]. Compared to childhood asthma, adult-onset asthma is more common in females than males, has a smaller remission rate, and less relation between allergy and atopy [29]. Moreover, adult asthma tends to cause heavier lung damage than childhood asthma while progressing much faster [30]. Additionally, adult lifestyle risk factors, such as smoking, stress, pollution, female sex hormones, and occupational irritant exposure, are thought to be contributive [30]. Studies on microbial influence and asthma pathogenesis in adults are limited but promising; a study by Pekkanen and colleagues found that Clostridium cluster XI microbes within the gut were protective against asthma [31]. Again, since the gut flora are an integral part of the immune system, supplementation trials with known immune-modulating and commensal bacteria, such as B. fragilis, S. epidermidis (that counters S. aureus biofilms), and Bifidobacter genera, may provide some protection against pathogenesis or synergistic relief when paired with standard therapies [32].COPD can be thought of as a progressive decompensation of the pulmonary system due to damage mediated primarily by accumulated exposure to smoke (tobacco or biomass burning), pollution, or some supranormal lung function phenotypes [33]. In particular, cigarette smoking, a key driver of COPD, creates chronic, long-term assault by thousands of toxic compounds deep within the pulmonary bronchiole network. This recruits sentinel macrophages and neutrophils to the epithelium of the airway, where tertiary lymphoid organs form and T cell-mediated, inflammation-driven airway remodeling (through perforin-mediated alveolar apoptosis and subsequent fibrosis) precipitates emphysema (restricted airflow) as the primary symptom [33,34]. This “insidious” sub-clinical inflammation ensures that decades of permanent damage may be inflicted before symptoms are noticed. Catabasis, the resolution and resetting of immune-mediated inflammation, includes lipoxins, resolvins, TGF-β, and endothelial growth factors, but chronic exposure to smoke or pollution may overwhelm these mechanisms, especially since nicotine paralyzes the lung cilia that would otherwise clear tar and other damaging compounds from the airway [33]. Similarities between asthma and COPD pathogenesis come from the common inflammation and damage of the airway as the respiratory epithelial layer in both COPD and asthma share a common feature of goblet cell metaplasia and squamous cell metaplasia [35,36]. However, the chemical mediators stimulated in COPD and asthma differ in that COPD triggers neutrophils, CD8+ T-lymphocytes, macrophages, IL-8, and TNF-α but asthma mediates the release of eosinophils, mast cells, CD4+ T-lymphocytes, macrophages, histamine, IL-4, IL-5, and IL-13 [35].Some viral pneumoniae, bacterial, or fungal exposures may also lead to COPD pathogenesis as higher T and B lymphocyte counts and B cell infiltration into lung tissue could result in generation of autoimmune antibodies against structural components such as elastin or even epithelial cells [33]. In such patients, the secretion of autoantibodies may even target neutrophil granule proteins (e.g., MMP9, CTSH) with secreted IgG, as well as shift targeting by these antibodies to extracellular compartments [37].Since COPD is irreversible, it may also cause synergistic exacerbation of other lung-associated diseases other than asthma, such as obstructive sleep apnea, allergies, or heart disease [33,38].Asthma and COPD share several symptoms, namely coughing, shortness of breath, tightness or a feeling of pressure in the chest, and wheezing [38]. However, asthma is distinct in that it is exacerbated by triggers while COPD symptoms are chronic (but may be worse in the morning or at other times) and also include excessive sputum, dyspnea, mouth breathing/gasping, and associated anxiety and depression [39]. Both diseases limit physical activity, daily activities, and have the potential to reduce quality of life unless controlled.Although symptomatically similar, it is important to remember that asthma arises primarily from mast cell and Th2-mediated inflammation while COPD is induced by lymphocytic infiltration (Th1, Th17, CD4/CD8 T cells) and damage due to environmental exposure [40]. However, the common site of both diseases (lungs) introduces the concept of, statistical overlap with, and treatment strategies of asthma-COPD comorbidity.COPD and asthma comorbidity is known as asthma-COPD-overlap (ACO). ACO patients experience worse exacerbations, lower quality of life, and greater lung damage than with either disease alone [41]. However, while both asthma and COPD are categorized as obstructive pulmonary diseases, asthma airway flow issues are reversible whereas COPD is not [19]. Within Japan, a recent analysis found that, of 30,405 hospitalized patients over the age of 40, 2.3% of ACO, 1.2% of asthma-only, and 9.7% of COPD-only patients died from all causes, reflecting a higher severity of COPD in the pulmonary hierarchy of disease [42]. According to The Global Initiative for Asthma and the Global Initiative for Chronic Obstructive Lung Disease, ACO is diagnosed by history of chronic airway disease and the coexistence of both asthma and COPD symptoms [43]. The ACO prevalence worldwide varies from 3.1% to 55.5% and, in Japan, it is around 15.4% to 20.7% [44]. Moreover, a study found that the ACO prevalence in patients that were already diagnosed with asthma was 27.1% in Japan [44]. This disease status exists in either a COPD-dominant or asthma-dominant form and each may have unique medical, genetic, and environmental causes. A large US study by Diaz-Guzman et al. that compared death rates among ACO, asthma, and COPD sufferers revealed that ACO carried the highest death risk (HR 1.83), followed by COPD alone (HR 1.44) and asthma alone (HR 1.16) [45]. ACO patients are found to have a 13% higher chance of asthma exacerbation and hospitalization than patients with only asthma or COPD [35]. ACO prevalence in Japan was reported to be higher in men whereas, in some regions, the prevalence was higher in women [44]. This variation may be ascribed to higher use of tobacco products among men, which is the main cause of COPD in Japan [44]. However, biomass smoke, often from agricultural fires, is another causative agent of COPD and may promote ACO pathogenesis in other areas such as North America and Europe [44,46].Similar treatments are used for ACO and asthma itself as the main treatments for ACO are also the simultaneous use of inhaled corticosteroids (ICS) and long-acting β2 agonists (LABA) or long-acting muscarinic antagonists (LAMA) [47]. Additionally, there is a link between juvenile-onset asthma and ACO in that childhood asthma increases the risk of future COPD diagnosis [48]. Of 10,199 smokers, 7% claimed to have asthma since childhood and, through gene analysis, different gene loci were found that were associated with COPD (IL1RL1, IL13, LINC01149, near GSDMB, and in the C11orf30-LRRC32 region) [48]. This common genetic heritage may be responsible for the beneficial effect of typical asthma treatments on ACO exacerbations. While the divergent pathogenesis mechanisms of asthma and COPD mean that drugs specific for mast cells will not treat COPD, global immunosuppression may relieve the autoantibody-mediated inflammation seen in COPD while preventing asthmatic exacerbations.The differences between adult-onset asthma and ACO, especially the COPD-dominant form, are found in the age of onset, symptoms, reversibility of airway obstruction, and the presence of atopy [49]. Adult-onset asthma has less comorbidity than COPD since COPD is more often comorbid with vascular and lifestyle diseases such as cardiac failure, osteoporosis, obesity, depression, lung cancer, and pneumonia [49]. To distinguish between adult-onset asthma and ACO, medical history, reversibility of airflow, smoking history, response to treatments, and the presence of atopy must be carefully weighed [49]. Additionally, there are differences between COPD-dominant ACO and asthma-dominant ACO. Research conducted by Kim et al. proved that asthma-predominant ACO patients often belong to a lower socioeconomic status and have lower FEV1 and FVC% (predicted), as well as quality of life, than COPD-predominant ACO patients [50]. Asthma-predominant ACO patients, on the other hand, have a lower hospitalization rate, lower medical costs, and barriers to effective health care than COPD-predominant ACO patients [50].Airflow through the lungs and bronchus is the main criterion for diagnosis and respiratory efficiency thresholds, coupled with signature symptoms, are the gold standards for worldwide diagnosis. In Japan, for example, adult asthma is diagnosed through criteria such as repetitive symptoms of paroxysmal dyspnea, wheezing, chest tightness and cough, reversible airflow limitation, airway hyperresponsiveness, atopy, airway inflammation, and exclusion of other diseases [9]. Reversible airflow limitation is measured by peak expiratory flow (PEF) and FEV1: if FEV1 increases by 12% or more after administration of a β-agonist inhaler, the patient is diagnosed as having asthma [9]. Airway hyper-responsiveness is tested using nitric oxide (FeNO), acetylcholine, and histamine thresholds while airway inflammation is tested by eosinophil and mast cell counts in sputum [51]. For juvenile-onset asthma, symptoms are similar to adults but there is more emphasis on family history and total serum IgE levels to aid diagnosis [51]. As current diagnostic technology is thought to be sufficient to catch new cases, translational medicine is currently focused on genomic screening to predict prevalence and treatment responses [52,53].The current standard treatment worldwide for asthma, as noted in the National Asthma education Prevention Program and in the Global Initiative for Asthma, is a stepwise and measured approach [54]. This protocol starts with corticosteroid inhalers followed by long-acting β agonists, leukotriene modifiers, long-acting anticholinergics, and finally oral corticosteroids for the most severe cases [54]. Some research into antihistamines has also been conducted with hopes to inhibit exacerbations but these compounds only seem to be effective in allergic asthma cases [8]. However, recent studies have developed personalized biological and cytokine-specific therapies, derived from antibodies (as denoted by -mab in their generic name), that selectively bind to the IgE receptor to inhibit its activation and prevent Type 2 asthma attacks [54]. As research progresses, standard management modalities have improved over time and mortality from asthma-related causes is dropping even as morbidity increases. For example, The Vital Statistics of the Japanese Ministry reported a decrease in asthma patient mortality in Japan from 4.5–5.0 per 100,000 patients in 1994 to 1.2 per 100,000 patients in 2016 while, in the US, a decrease in asthma-related mortality from 1999–2015 was seen in all ethnicities and genders for a total rate of 1.5 per 100,000 persons [55,56].The following section is a survey of the newly developed, targeted therapies being tested for effectiveness in controlling exacerbations. Drugs are listed along with their trade names in the order they were approved by the FDA for asthma [57]. An overview of the direct targets of each compound is available in Figure 2. A recent review by Tu and colleagues suggests that a treatable traits concept, where phenotypic or endotypic features are used to create multi-dimensional biomarker profiles for treatment that can be tested in animal models and this may drive development of newer biologics [58]. A gold-standard analysis of promising ACO treatments (such as macrolides or muscarinic antagonists) has been reviewed by Leung and Sin [59].Originally developed from a murine source, this IgE-specific human, monoclonal antibody prevents activation of the inflammatory pathway by both neutralizing IgE and downregulating the IgE receptor on basophils [60]. As free IgE has been known to result from IgG class switching mediated by IL-4 and IL-13, as well as release from allergen-stimulated IgE-positive B cells, driving down this response short circuits the activation of mast cells and basophils that activate inflammation and lower T-cell activation thresholds which drive Th2 responses [61]. Omalizumab thus downregulates the sensitization of the immune system through preventing chronic activation of Th2 responses that drive both exacerbation and the histological changes seen in asthmatic airways.A meta-analysis conducted by the Global Evaluation of Treatment Effectiveness reported that 77% of severe asthma patients treated with omalizumab had improvement in symptoms after four to six months [62]. In a phase IV clinical trial (Clinicatrials.gov ID: NCT00264849), the use of long-term corticosteroid was reduced 32.3% more by the addition of omalizumab to optimized asthma treatment (OAT) compared to OAT alone in severe atopic asthma [63]. Omalizumab was also proven effective in non-atopic asthma in a phase IV clinical trial (Clinicatrials.gov ID: NCT01007149) [64]. Patients treated with omalizumab had significant decreases in high affinity IgE receptor (FcεRI) and plasmacytoid dendritic cells (pDC2) levels (p < 0.001) compared to the placebo group coupled with FEV1 improvements from the baseline by 9.9% [64].Although omalizumab may hold promise for ACO, an Australian study of the Xolair Registry found that patients with overlapping asthma and COPD had improved asthma control but did not experience improvements in FEV1 or other related parameters [65]. No clinical trials using omalizumab specifically for ACO have been reported but several case reports and retrospective studies have shown benefits in these patients [66,67,68] This monoclonal antibody is generated from human N-glycosylated IgG1 kappa chains and was designed to be highly specific to IL-5, neutralizing its binding capacity to IL-5 receptor alpha [69]. As a result, eosinophil maturation is disabled in the bone marrow and eosinophil levels are decreased in blood and bronchial mucus [70]. However, as mepolizumab is highly effective at neutralizing IL-5, immune pathways crucial to anti-parasitic response may be compromised, a side effect that must be taken into account within developing countries or pediatric patients [71].The MENSA study (Clinicaltrials.gov ID: NCT01691521) in 576 patients found a 53% reduction in exacerbations compared to placebo when administered subcutaneously versus a 47% reduction via intravenous administration, along with a 100 mL increase in FEV1 [72]. In mild asthma patients, mepolizumab reduced eosinophils in both the blood and sputum [73]. Likewise, in mild atopic asthma patients, mepolizumab reduced eosinophils in the blood, the bronchoalveolar fluid, and the bone marrow [73]. Mepolizumab is currently registered as a key add-on drug to treat severe eosinophilic asthma for juveniles older than 12 years of age in the United States and for adults in Europe [73]. A retrospective analysis of a French early access program for mepolizumab add-on therapy in cases of severe eosinophilic asthma found that exacerbations of eosinophilic asthma patients, which averaged 5.8 per year, dropped to 0.6 per year after 24 months of follow up along with a significant reduction in primary corticosteroid usage (92.8% at baseline vs. 34.7% after 24 months of follow up) [74]. This effect was also seen in the COSMEX study (Clinicaltrials.gov ID: NCT01691508), where 339 patients received mepolizumab for 2.2 years and found either maintenance of or reductions in oral corticosteroids needed, along with significant reductions in exacerbations [75]. As the primary mode of action is eosinophil depletion, determining the thresholds of eosinophilia that would indicate mepolizumab as suitable for concomitant use is of supreme importance but determining these levels is still controversial [76]. Mepolizumab is also useful regardless of systemic IgE concentration as its local mode of eosinophil control in the airways would lower levels of IgE within the lungs. However, IL-13-mediated pathways would not be affected, as seen in a clinical study that observed no changes in fractional exhaled nitric oxide (FeNO) levels after mepolizumab treatment in 102 patients [77].The METREX (Clinicaltrials.gov ID: NCT02105948) trial of 836 patients and METREO (Clinicaltrials.gov ID: NCT02105961) trial of 674 patients sought to establish the effect of mepolizumab in preventing COPD exacerbations driven by eosinophilia. METREX found that a 100 mg dose was effective at lowering moderate-to-severe exacerbations of COPD (1.40 per year vs. 1.71 per year in the placebo group) whole METREO also found that 100 mg was most effective in preventing exacerbations (1.19 per year vs. 1.49 per year in the placebo group) [78]. Both trials did not observe severe adverse effects that were significantly different from placebo. A smaller trial of 18 COPD patients with eosinophilic bronchitis, however, found no significant improvements in lung function or exacerbation rates even with significant depletion of sputum eosinophils [79]. However, as the conflicting results indicate, the role of mepolizumab in ACO is suspect as asthma benefits are not seen in the COPD realm, an effect possibly due to the partial role of eosinophils in COPD progression. The FDA, in spite of the METREX and METREO studies, voted against approval of mepolizumab for COPD treatment in 2018 due to questions over the data collection and inability to duplicate the observed efficiencies seen in both studies, noting that observed improvements may have been due to asthmatic improvements instead of direct action on COPD [80].Reslizumab is a humanized monoclonal antibody IL-5 (mAb) of IgG4 kappa with a high affinity for IL-5α and a terminal half-life of 24.5 to 30.1 days [73]. Reslizumab functions in severe asthma patients by selectively inhibiting IL-5α receptors to suppress eosinophil production in the bone marrow [81]. For those COPD patients with eosinophilic-mediated inflammation (around 40%), reslizumab may act to reduce risks at eosinophil levels of 300 cells/microliter as reported in the COPDGene and ECLIPSE studies [82].In a phase III study, reslizumab significantly reduced asthma exacerbations by 54%, improved lung function by 0.11 L, and improved quality of life scores [73]. Pelaia et al. reported that reslizumab significantly increased forced expiratory flow at 25% to 75% of forced vital capacity (FEF25–75), proving increased airflow in the peripheral airways [81]. A recently published clinical trial (Clinicaltrials.gov ID: NCT01237039) found that, in 477 asthmatics aged 12–75 years, reslizumab reduced exacerbation risk significantly compared to placebo (77.5% reduction in risk vs. 15.2% in the placebo group) while also reducing primary corticosteroid therapy burden (254 mg per-patient in the reslizumab group vs. 611 mg per-patient in the placebo group) [83]. Side effects for these clinical trials were all mild to moderate.No clinical trials have yet been reported for reslizumab efficacy in ACO, but, as ACO places a dual inflammatory burden on the pulmonary system, relieving asthma and reducing medication burden on individual patients may have a beneficial effect in controlling overall ACO impact on quality of life. Additionally, as 20% of COPD exacerbations are thought to be eosinophil driven, some relief from ACO may be found with reslizumab [82].IL-5 stimulates eosinophil production and the recombinant monoclonal antibody benralizumab was created to block the IL-5 receptor-α to arrest eosinophil differentiation in bone marrow [84]. Unlike other IL-5-neutralizing biologics (reslizumab and mepolizumab), benralizumab directly depletes eosinophils by antibody-dependent cell-mediated cytotoxicity [69] and also binds to NK cells, macrophages, and neutrophils with its heavy chain Fcγ-receptor [69,84]. As a result, eosinophil populations are rapidly reduced (more than 95%) in both systemic circulation and tissue [84]. However, excessive levels of Type 2 inflammation from inflammatory factors (such as smoking exposure, genotype, gender, age, weight, and comorbidities) may result in poor control of eosinophilic asthma even with benralizumab and cluster analyses to fully define and predict therapy response have been reported [85,86,87]. Additionally, benralizumab carries no real benefits for non-Type 2-related asthma (non-eosinophil-mediated) and clinicians cannot rely on FeNO and IgE levels to monitor treatment as there have been studies that show no changes in these biomarkers during treatment [85,86,88].A phase III SIROCCO and CALIMA trial examined 2295 uncontrolled asthma patients treated with corticosteroids and long acting β2 agonists (LABA) where one group received 30 mg benralizumab every eight weeks, one received it every four weeks and one received a placebo [89]. Patients that received benralizumab every eight weeks had a 42% average reduction in exacerbation rates and experienced a 0.14 L increase in FEV1 compared to placebo [89]. Benralizumab was most effective in patients that were on high doses of oral corticosteroids, had polyposis, had a predicted FVC of less than 65%, had more than two exacerbations in the previous year, and were diagnosed after 18 years of age [89]. A different phase III SIROCCO trial that gave benralizumab every four or eight weeks showed how patients with eosinophil counts of more than 300 cells/μL treated every four weeks experienced an average of 45% less exacerbations compared to placebo and this reduction was 51% for the eight-week cohort [90]. Patients who participated in the SIROCCO and CALIMA trials were further studied in the BORA trial over two years to examine the efficacy and safety of benralizumab [91,92]. Patients were either given 30 mg of benralizumab subcutaneously every four or eight weeks and blood eosinophils were counted [91,92]. As a result, 50% of the patients did not have any exacerbations and the mean prebronchodilator FEV1 increased by 0.310 L and 0.364 L in the four-week and eight-week groups [91,92].Unfortunately, trials with benralizumab in severe ACO have not returned definitive results. A multi-country, phase IIA trial by Brightling et al. in 2014 examined 101 adults, aged 40–85 with severe ACO and found that benralizumab did not significantly reduce COPD exacerbations (0.95 [95%CI 0.68–1.29, n = 40] with benralizumab vs. 0.92 [95%CI 0.67–1.25, n = 42] with placebo) but gave some small increases in FEV1 [93]. The recently completed GALATHEA (Clinicaltrials.gov ID: NCT02138916, n = 1656) and TERRANOVA (Clinicaltrials.gov ID: NCT02155660, n = 2255) phase III clinical trials reported in a joint publication that 100 mg benralizumab did not significantly improve exacerbations unless ACO patients had significant elevations in eosinophils [94]. Taken together, benralizumab seems to work only in ACO only when the disease is eosinophil driven. However, it may hold value for asthmatic risk factors, such as atopic dermatitis, and this potential is being tested in several ongoing clinical trials (Clinicaltrials.gov: NCT03563066, NCT04605094).Dupilumab, a fully human monoclonal IgG4 antibody that selectively blocks the IL-4/IL-13 co-receptor to prevent signal transduction via IL-4R and IL-13, through JAK/STAT6, is used to treat Th2-mediated asthma [95,96]. IL-4R triggers IgM to IgE class switching and precipitates a type I allergic reaction and eosinophil infiltration within the airway [95]. IL-13 increases mucus production and inducible nitric oxide synthase (iNOS) in the epithelial cells, mediating airway constriction [95]. Moreover, it promotes goblet cell hyperplasia, which leads to the transformation of bronchial fibroblasts into myofibroblasts [95,96]. Remodeling of the airway with these contractile cells results in the airway hyper responsiveness that is the hallmark of asthma symptoms [95]. Dupilumab thus selectively targets and inhibits a key inflammatory pathway.A study of 104 asthmatic patients, 52 of whom were treated with dupilumab, saw a 87% reduction in exacerbation rate compared to placebo [97]. Likewise, dupilumab was found to decrease inflammatory biomarkers such as FeNO concentration, IgE levels (serum, thymus), and activation-regulating chemokines with no serious, drug-related adverse events [97]. In a trial conducted by Rabe et al., 210 asthma patients treated with oral glucocorticoids were randomly chosen to additionally receive either dupilumab or a placebo every two weeks for 24 weeks [98]. The dupilumab patients experienced a 70.1% decrease in the use of glucocorticoid versus a 41.9% reduction in the placebo group [98]. Furthermore, there were 30% more people in the dupilumab group than the placebo group that were able to reduce the amount of glucocorticoid by more than 50% and their exacerbation rate was 59% lower [98]. Similar to the studies by Wenzel et al., the dupilumab group also saw an increase in FEV1 of around 200 mL [97,98]. A similar study was outlined in a report by Bassani et al., where 69% of patients that used dupilumab were able to reduce the amount of glucocorticoid to less than 5 mg a day and 48% were able to stop using glucocorticoids completely [99]. Those patients that were given dupilumab twice a week had a two-fold reduction in severe asthmatic episodes versus placebo [99]. Several human studies were conducted to verify the safety of dupilumab. Rathinam et al. showed that it takes 1 week for 600 mg doses of dupilumab to reach maximum plasma concentration and the bioavailability from subcutaneous injection is around 62.35% [100]. After reaching therapeutic levels, it takes approximately 10–13 weeks to be eliminated from the body to levels of less than 78 ng/mL [100]. A phase IIB, multi-dosage trial conducted in 15 countries with 769 patients reported that groups receiving 200 mg and 300 mg of dupilumab every two weeks saw significant increases in FEV1 compared to placebo [100]. In that study, patients that were given dupilumab every two weeks, regardless of dosage, had fewer exacerbations than other groups [100]. As activation mechanisms are shared between atopic dermatitis and asthma, an extensive review by Matsunaga et al. details successful treatment of both diseases with dupilumab, which was recently (2018) approved in Japan for recalcitrant and poorly responding atopic dermatitis and asthma [96].Dupilumab effectiveness in COPD is currently being studied in the ongoing BOREAS trial (Clinicaltrials.gov ID: NCT03930732) but extensive trials for ACO have not been reported. Dupilumab was found in a clinical trial (Clinicaltrials.gov ID: NCT01312961) to reduce Th2-driven inflammation but has also been reported to increase eosinophils through migration of progenitor cells via IL-4 and IL-13, which may exacerbate COPD symptoms or cause pneumonia [97,101]. As such, no current recommendations for dupilumab in ACO have yet been determined.Asthma prevalence is increasing worldwide even while serious complications and deaths are falling due to innovative new biologic treatments and therapies. With a multifactorial pathogenesis, heterogenous presentation, and long development time, asthma appears to be a disease that must be ameliorated to the fullest extent possible while mechanistic studies are completed that will generate curative therapies. Biologic therapies targeting IL-5 have demonstrated the greatest promise for exacerbation control as evidenced through lower exacerbations (of all severities) and reduced inflammatory biomarkers (eosinophil levels) but adding the complex and not-yet-fully-understood comorbidities of COPD and a lack of solid clinical results has left ACO treatment a complex set of decisions. Future studies will need to address multiple molecular mechanisms simultaneously to determine the best biologic configuration for maximum therapeutic effect. Perhaps high-throughput technologies, such as deep sequencing, proteomics, and metabolomics within the immune and endocrine systems, will provide snapshots of the biochemical and genetic milieu that drive asthma pathogenesis while larger mechanistic studies will detail the progressive deterioration of COPD and its molecular links to asthma exacerbation.Conceptualization, M.K. and B.J.M.; data curation, M.K. and B.J.M.; writing—original draft preparation, M.K. and B.J.M.; writing—review and editing, M.K. and B.J.M.; supervision, B.J.M.; funding acquisition, B.J.M. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable due to the review-only nature of the manuscript.This review reported only previously published results and, as such, informed consent is not applicable. All reported studies involving humans that were used in this review were confirmed to have informed consent statements. Data on reported clinical trials are available from clinicaltrials.gov or their respective publications. No new, unreported datasets were used. The authors declare no conflict of interest.The eosinophil-mediated pathway of asthmatic inflammation [11,12].The specific targets of the current generation of biologics within the inflammatory cycle [11,12].A summary of secretory cells, cytokines, and targets/function. Adapted from Barnes 2017 [13].Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+The house dust mite (HDM) is globally ubiquitous in human habitats. Thirty-two allergens for Dermatophagoides farinae and 21 for Dermatophagoides pteronyssinus have been detected so far. The present minireview summarizes information about the role of Der p 1 as a key coordinator of the HDM-induced allergic response and reports on a series of Italian patients who are allergic to HDMs. We studied the specific IgE profiles in a population of patients with allergic asthma and rhinitis screened for specific immunotherapy (SIT) for HDM allergies, with the aim of obtaining insights into the pathogenic role of Der p1. Patients co-sensitized to other airborne allergens showed a higher prevalence of asthma (9/12 (75%) vs. 2/7 (29%); p < 0.05) than did HDM mono-sensitized patients. The latter group showed higher Der p1 concentrations than that of the co-sensitized group (p = 0.0360), and a direct correlation between Der p1 and Der p2 (r = 0.93; p = 0.0003) was observed. In conclusion, our study offers insights into the role of Der p1 in a population of patients with allergic rhinitis and asthma who were candidates for SIT. Interestingly, Der p1 positivity was associated with bronchial asthma and co-sensitization.The house dust mite (HDM) is globally ubiquitous in human habitats. It is considered the main indoor allergen cause of allergic rhinitis and asthma [1]. At total of 600 million people suffer from allergic rhinitis and 200 million cases are concomitant with asthma, as reported by The World Health Organization (WHO) [2,3]. Allergies are common in 50% of all adult asthmatic patients, and up to 85% of them are allergic to HDMs, independent of differences in geography, temperature, and humidity [3].According to the WHO position paper [4,5], the best approach to allergic rhinitis consists in allergen avoidance, pharmacotherapy (including antihistamines, leukotriene receptor antagonists, and inhaled/intranasal corticosteroids), and allergen immunotherapy. Allergen immunotherapy has been used in selected patients for 50 years to achieve clinical tolerance to causative allergens through administration of allergen extracts. It is effective in alleviating the symptoms of allergic rhinitis and/or asthma and in improving quality of life. Allergen immunotherapy has been used in selected patients to achieve clinical tolerance to allergens through exposure of patients to different amounts of allergen extracts in order to modify the immune system’s response [6,7].The main HDM species include Dermatophagoides pteronyssius (Der p), Dermatophagoides farina (Der f), and Blomia tropicalis, which coexist in most geographical regions [8]. Group 1 (Der p 1, Der f1), group 2 (Der p 2, Der f 2), and group 23 (Der p 23) are considered the dominant allergens [9,10,11].Twenty-one group 1 allergens have been found to have protease activity that can destroy epithelial tight junctions. Thirty-two group 2 allergens may mimic the effect of Toll-like receptor 4 coreceptor MD-2, and new ones are still being reported [12,13]. In group 23, a new allergen has been identified as a main allergen, a gut-derived peritrophin in the outer membrane of mite feces. This allergen reacts with IgE antibodies in 74% of patients allergic to Der p [13,14].The first aim of the present study was to summarize the information about the role of Der p1 as a key coordinator of the HDM-induced allergic response. The second aim was to investigate a series of Italian patients allergic to HDM who were screened for anti-HDM treatment in order to establish the role of specific IgE in Der p groups measured by ImmunoCAP 250.In 1980, Chapman and Platts-Mills were the first to isolate the HDM allergen Der p 1 [8]. Der p 1 has been associated with different paradigms over the past 30 years [15,16,17,18,19,20,21].Der p 1 is a papain-like cysteine protease [10,12]. It is considered to be not only the most abundant HDM allergen in house dust and mite cultures but also a powerful allergenic protein [15,22,23]. High levels of IgE specific for this protease have been developed by more than 80% of patients allergic to HDMs [15,24,25,26].In the last 10 years, several authors identified in vitro the protease precursors of the mite cysteine (Der p1) and serine (Der p 3, Der p 6, and Der p 9) protease precursors [27,28,29,30]. The crystal structure of Der p 1 demonstrates that this allergen is a papain-like cysteine protease, while sequence homologies and protease inhibition assays have shown that Der p 3, Der p 6, and Der p 9 belong to the trypsin-like, chymotrypsin-like, and collagenolytic-like serine protease families, respectively [31]. Der p1, 3, 6, and 9 are synthesized as pre-zymogens, consisting of a signal peptide needed for secretion and an N-terminal propeptide, followed by the mature protease domain. The expression of prosequences inhibit the respective proteases to prevent cell toxicity. Literature data reported the role of Der p 1 as the “maestro” in the maturation processes of the different HDM protease allergens [15,16,17]. The behavior of Der p1 appears to be rather uncommon in the protease world and depends on specific sequences at the C-terminus of the different propeptides [32]. Although the biological roles of these proteases in mites have not been completely unraveled, the allergens probably have a digestive function for the mite, as they are detected in the gut as well as in mite feces.The proteolytically active HDM allergens have a critical role in the initiation of an allergic response. The elucidation of the maturation pathways of these allergens is required and could provide insights into their proteolytic specificities. Moreover, the corresponding protein substrates on the innate target and adaptive immune cells will need to be identified. The initial simplistic representation of Der p 1 as an HDM-digestive cysteine protease with IgE-binding properties has been replaced by a much more complex scenario [26,32]. Recent evidence shows a pivotal role of Der p 1 in the maturation of HDM serine protease allergens, suggesting that Der p 1 protein substrates could shape the allergenic potential of HDM proteins. Huge progress has highlighted the key role of environmental and microbial adjuvant factors in activation of innate immune pathways that are essential for the development of allergic responses [33,34,35,36]. These factors combine with the intrinsic biological activity of the allergens, which stimulates innate immune pathways, leading to allergy.Different mechanisms influence the proteolytic activity of HDM allergens in the development of the allergic response, including cleavage of lung epithelium surfactant proteins (SP-A, SP-D) [37], tight junction protein occludins, and immune receptors expressed by dendritic [38], B [39], and T [40] cells; activation of damage-associated molecular patterns (DAMPS) [41] and protease-activated receptors (PARs) expressed on airway epithelial cells [42]; and inactivation of protease inhibitors such as α1-antitrypsin [43]. Altogether, cleavage of these cell receptors and secreted proteins by HDM proteases influences the development of allergic sensitization and may exacerbate allergic inflammation by promoting a pro-Th2 environment and/or by downregulating Th1/Treg differentiation [44].However, the different cell pathways triggered by active Der p 1 are still unclear due to the difficulty of isolating natural Der p 1, free of serine protease contamination, from HDM allergen extracts Accordingly, these limitations stimulated research into the contribution of Der p 1 protease activity in developing a HDM-induced allergic response by means of papain, a structurally similar cysteine protease that is considered to be a surrogate for Der p 1 and HDM allergen extracts, in the presence or absence of cysteine protease inhibitors. Kubo M. reported that proteolytically active papain stimulates an innate cell network (IL-33–ILC-2–IL-13 axis) that can trigger eosinophilia in patients with early airway inflammation in the absence of specific IgE production [45]. It is intriguing to speculate that a similar mechanism could be activated by Der p 1. Cayrol et al. showed than Der p 1 can regulate the cytokine activity of alarmin IL-33 through cleavage of its sensor domain [46].Controversial data exist on the capacity of Der p 1 to activate PAR-2 (not commonly activated by cysteine proteases), leading to proinflammatory cytokine release. The finding may have been due to residual HDM serin protease contamination of Der p 1. Recently, Der p 1 was shown to indirectly stimulate PAR-1/PAR-4 signaling pathways through thrombin, their canonical activator [47].Bioinformatic tools and peptide substrate libraries are useful for predicting potential new targets of Der p 1 in the human cell-surface proteome, targets that could play a role in allergic airway inflammation [17,43]. Besides the capacity of Der p 1 to cleave IL-10 and IL-12 receptors, offering a new pathway for promotion of Th2-polarization, Der p 1 can cleave cell-surface inflammatory receptors (including IL-2 type II, IL-17, IL-18, and IL-23), suggesting that it is involved in a complex balance of pro- and anti-inflammatory effects [48].Many questions still remain about the possibility of Der p 1 being a digestive enzyme and of its role as a potent activator of other mite allergens and innate immune pathways. However, the drastic effect on allergen content and release from mite fecal pellets through the protease activity of Der p1 confirms the role of Der p 1 as a key coordinator of the HDM-induced allergic response.Sensitization to mite allergens in the first years of life has a significant impact on lung function in pediatric populations who suffer from wheezing and has been associated with poorer long-term clinical outcomes. This might explain why the approach advocated by current guidelines for allergic rhinitis (AR) (ARIA, Allergic Rhinitis and its Impact on Asthma) [49] and allergic asthma (GINA, Global Initiative for Asthma) [50] classifies disease based on the severity of symptoms, often leaving the underlying allergic cause unaddressed.The present study analyzed the specific IgE profiles in a population of patients with allergic asthma who were undergoing rhinitis anti-HDM treatment, with the aim of obtaining insights into the pathogenic role of Der p 1 (Figure 1).All patients gave written informed consent to participate in the study, which was approved by the local ethics committee C.E.A.V.S.E. (code number 180712; Markerlung 17431).A total of 38 patients (M/F: 20/18; median (interquartile range, IQR) age 35 (26–40) years) were screened: eight were mono-sensitized to HDM, 13 were co-sensitized to other airborne allergens, and the others were co-sensitized to other allergens. Fifteen (39.4%) had asthma and the other 23 (60%) had allergic rhinitis. Specific IgE for Der p 1, Der p 2, Der p 10, and Der p 23 were measured by ImmunoCAP 250 (Thermo-Fisher Scientific, Uppsala, Sweden) as previously reported [51]. Levels >0.35 kU/L were considered positive. From August 2019, nineteen patients (M/F: 13/6; median (IQR) age 32 (27–40) years) were enrolled for anti-HDM treatment. The inclusion criteria were persistent allergic rhinitis (moderate-to-severe) to dust mites, despite the use of symptomatic medications, and dust mite allergic asthma not well controlled by inhaled corticosteroids, associated with mild-to-severe dust mite allergic rhinitis. Seven of nineteen patients were mono-sensitized to HDM and 12 were co-sensitized to other airborne allergens. Twelve (63.1%) had asthma. Nasal symptoms (runny nose/nasal drip, nasal congestion/stuffiness, sneezing, and itchy nose) were tallied as the total nasal symptom score (TNSS; max. 12) expressed as median ± standard deviation.The prevalence of rhinitis and/or asthma did not show gender or age differences. TNSS was 7.82 ± 0.51 before starting the anti-HDM treatment. Patients co-sensitized to other airborne allergens showed a higher prevalence of asthma (9/12 (75%) vs. 2/7 (29%); p < 0.05) than did HDM mono-sensitized patients. The latter group showed higher Der p1 concentrations than that of the co-sensitized group (p = 0.0360), and a direct correlation between Der p1 and Der p2 (r = 0.93; p = 0.0003) was observed.An AR exacerbation responder was defined as a patient with no AR exacerbations during the 8-week efficacy evaluation period. All of our patients were classified as responders to anti-HDM treatment (based on the above definition of responders) and seven of them (17%) reached a one-year follow-up. The other ones continued to be followed beyond one year.Our study reports on a cohort of patients screened for anti-HDM treatment. This treatment has been available in Italy since June 2019 and was recently decreed to be free of charge for Tuscan residents. Our data showed a predominance of Der p1 in co-sensitized patients before therapy. Pioneering studies showed that Der p 1 is not only the most abundant allergen from mite fecal pellets but also a major inducer of the IgE response in patients with HDM allergies [17,52]. Although Der p 1 is mainly present in the mite digestive tract, its actual biological function (in relation to the development of asthma and rhinitis) is still unclear. Literature data highlighted the pivotal role of Der p 1 in the maturation of HDM serine protease allergens, suggesting the allergenic potential of HDM proteins could be shaped by the processing of protein substrates [32]. As the first allergen characterized, Der p 1 was naturally chosen to develop a general paradigm for predicting protein allergenicity based on protease activity. Proteolytic activities have subsequently been demonstrated by biochemical and structural characterization of Der p 1 [12,17]. Great progress in immunology concomitantly evidenced the role of environmental and microbial factors in activation of innate immune pathways, which are essential for the development of allergic responses [21,23,46]. Taken together, the Der p 1 paradigm inevitably shifted to a more broadly applicable model of protein allergenicity: the allergy is caused by the association of intrinsic biological activity of an allergen along with its ability to stimulate innate immune pathways. The cysteine protease activity of Der p1 promotes a Th2-allergic response, even if the elucidation of different cell pathways triggered by active Der p 1 is still challenging. Future studies will involve solving the technical problem of isolating natural Der p 1 from HDM allergen extracts (isolation from serine protease contamination or preparation in reproducible batches).Chevigné A et al. suggested potential new targets for Der p 1 in the human cell-surface proteome. These could play a role in allergic airway inflammation [17]. The increased concentrations of Der p1 and the high prevalence of asthma in our co-sensitized HDM patients before treatment with anti-HDM treatment suggest that underlying severe inflammation supports allergic airway diseases.In conclusion, our study offers insights into the role of Der p groups in a population of patients with allergic rhinitis and asthma who were candidate for specific immunotherapy. Interestingly, Der p1 positivity was associated with bronchial asthma and co-sensitization.This research received no external funding.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Ethical Committee of Comitato Etico Regione Toscana Area Vasta Sud Est (C.E.A.V.S.E.) (protocol code 17431, 15-06-2020).Written informed consent has been obtained from the patient(s) to publish this paper.The data presented in this study are available on request from the corresponding author.The authors declare no conflict of interest.A summary of the aim and main results of the present study, including the biological role of Der p1.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Even though allergic disease is identified in the first year of life, it is often in a less forward fashion, with elements of a wait and see approach. If the infant does not have an anaphylactic food reaction, other less dramatic allergic phenomenon is often under-emphasized, waiting for additional concerns. We approached this with a conception to first conduct birthday surveys, attempting to link intrauterine and peri-birth circumstances to affect better allergy recognition in young infants.The conceived fetus has many promising outcomes, but becoming allergically sensitized, and to have an allergic disease develop within the first year of birth, is an unwelcome outcome. A recent manuscript thoroughly discusses food allergic anaphylactic reactions in infants, and the clinical management during and after [1]. We review here the information on how that infant was set up to have a food allergic reaction, including intrauterine immunology, intrauterine IgE sensitization, the presence of IgE sensitization at birth, breast feeding sensitization, transcutaneous sensitization, and the allergic diseases that typically result from food allergic sensitization, including anaphylaxis, within the first year. In the broad sense, the immediate family presence of an allergic disease provides familial, presumably genetic [2], pressure to have the same, or related, allergic disease eventually develop in the infant’s antenatal life. In the case of asthma, recent studies are showing strong perinatal influences [3,4,5,6,7].Atopic dermatitis in a parent seems to being stronger atopic dermatitis potential to their child [5,8]. Allergic rhinitis and food allergy in a parent provide allergic influence on the fetus, but less direct influence for those specific diseases. The genetics of an allergic disease has had remarkable breakthroughs, and many chromosome and gene areas have been identified using population and sib-sib analyses but without a single specific gene identified for a specific disease [9]. Very likely, multiple genes contribute to each disease, along with intrauterine epigenetic influences [5,10,11,12], and intrauterine and infant post-birth environmental pressures [5,11,13,14].Substantial information has accumulated on fetal immunity, largely building off mouse studies [5,10,14,15,16]. A recent review of human immunology was published in Science in 2020 [17]. We recapitulated it here briefly, focusing on the components of the immune system essential for development of allergy. Macrophages and tissue mast cells appear very early in the first trimester. Early mast cells do not have the gene for the alpha unit of IgE receptors, but IgE can be detected after 11 weeks as mature B cells will appear by 9 weeks. Naïve T cells appear about 8 weeks, as does the thymic tissue. Neutrophils and eosinophils are in the bone marrow by 20 weeks. Therefore, by 20 weeks the fetus is relatively armed, and needs antigen to proceed, but in their sterile world only a few bacteria or infectious by-products, and very certainly almost no allergens can invade. The multiple cells involved in the post-uterine life allergic process are present; based on the subsequent discussion, their ability to execute an allergic response scenario appears to be well dampened until after birth.The TH2 (T2) immune system is the critical component of the newborn through adult life that drives the atopic response and contributes to the development of an allergic disease [18]. TH2 lymphocytes are characterized by the prostaglandin D2 receptor CRTH2 [11]. In the fetus, there is a skewing of the lymphocyte (CD4+) population to the TH2 phenotype [11,12]. The neonate, then, in the right (wrong) exposure environment has the functional elements to produce an intrauterine IgE response. Maternal IgG crosses the placenta, while IgE is (theoretically) an excluded [11,15,19]. The fetus receives food, and potentially other foreign antigens, delivered via the placenta interface. The maternal IgG may balance the fetal response to foreign antigen, but IgE can be produced by the fetus [14]. The subsequent discussion on post-uterine allergic disease will focus on the extreme uncommonness of allergic clinical responsivity in the first months of life. This would indirectly suggest that fetal production of sufficient IgE to an allergen, food for example, is primed and ready for post-uterine production and the production and release requires the non-maternal environment and possibly the additional (first) exposure(s) to induce the first post-uterine allergic reaction.Fetal tissue can produce IgE in the late first–early second trimester [14]. It is largely bound, or is under-represented, as a B-cell class switching potential [14,15,17,20], and not overtly released into the direct fetal circulation for mast cell/basophil/eosinophil binding. It’s likely there are brakes to control intrauterine IgE levels to not endanger the developing fetus. The mere fact that fetuses of atopic/allergic mothers or fathers don’t have increased intrauterine demise is likely in-direct proof that the level of specific IgE in infants is low enough, and the volume of allergen food is low enough, to not induce an allergic intrauterine response, and likely other protective factors, such as diminished mast cell IgE binding, are in play [17].Cord blood total IgE is low, as is specific IgE; studies have supported some specific IgE production, although this area has remained controversial [16]. The use of umbilical cord IgE was attempted as a predictor for future atopic disease or in epidemiological studies [16,21] but has not received any recent research direction. A recent search at Clinicaltrial.gov (accessed on 3 January 2021) showed two studies of cord blood IgE in Taiwan [22]. Older studies of cord blood IgE yielded data that was associative for future allergic disease [23] or presented maternal or environmental factors that influenced cord blood IgE [24].A pertinent discussion to the topic of intrauterine IgE sensitization, and subsequent early life allergic reaction, was data from the PASTURE study published in 2008 [11]. The most frequent IgE in cord blood for foods was milk, eggs, hazelnut, wheat, and soy [11]. A study of ex-vivo transplacental transfer from 2000 reports studies document allergen specific T-cell proliferation (implying exposure and, at least, T-cell sensitization) for alpha-casein, beta-casein, kappa-casein, bovine serum albumin, and ovalbumin [25]. A recent study in mice suggested maternal IgE may cross the placenta [26]. If true in humans, the amount and degree of specific binding to mast cells is negligible. In most fetuses and early infants, the non-bound half-life of IgE allows for quick reduction (unless the infant themselves maintain extrauterine production) and no clinical consequences.If a fetus can develop their own food specific IgE, then the food of interest must reach the developing fetus. A review of protein sources to the infants from 1990 largely references work from the 1970s–1980s [27]. The emphasis, at that point, was amino acids. T-cells, as mentioned, recognize larger amino-acid constructs [25], but the translation to B-cell message, class-switching, and IgE production in the fetus is largely unknown.The exact format of maternally ingested protein, that is eaten, digested, absorbed, and passed to the fetus, is largely speculative [28]. A recent interesting case report of abdominal hives after wheat ingestion specifically suggests more rapid absorption of ingested protein can be locally absorbed, and due to enhanced maternal circulation, it could be more easily passed to the fetus [29]. The exact nature of fetal specific IgE promotion lineage, whether to linear or conformational epitopes, then becomes more relevant, based on the size of the protein transferred, intact conformational or shorter amino acid linear sequences. (linear). The available literature strongly supports amino acid transfer, but virtually no processes is suggested for larger components of food or allergen transfer, although it obviously could be based, at least, on cord blood specific IgE recovery.The method of birth has influence on the future development of allergies and allergic disease in children [30]. Otherwise, any swallowed maternal blood containing maternal IgE only adds to the intrauterine IgE levels the infant is born with. The infant is now either specifically IgE sensitized (in theory) or not, and life happens (Figure 1).Several reports have provided immediate allergy epidemiological data in infants. In a United States population study of food-induced anaphylaxis in an urban hospital, birth-age 18 years, 13% of the total (n = 357) were less than the age 12 months [31]. Eggs and cow’s milk were the two most common foods. In a European study, comparing infants (<12 months) to older 1–6 years, 59 of 375 children were <12 months, with a mean age of 6 months [32]. Cow’s milk and hen’s eggs were the two most common.Cow’s milk IgE specific anaphylaxis, generated by infant milk-based formula, is an exceptionally rare occurrence in the first several months of life. It is conceivable that a rapid shift to a non-milk formula may (disguise) lessen the chance of a severe allergic reaction, although the first replacement is often a lactose-free cow’s milk product. In breast-fed infants, with maternal ingestion of milk protein, anaphylaxis is an equally non-existent occurrence [33]. Case reports of a fish allergic reaction to maternally transferred protein have been reported, but not until 4 months or later [34,35], suggesting an immunological brake to similar reactions to other breast-transferred food proteins early in life. The presence of food protein in breast milk has been recently reviewed, with much person-to-person variability, but evidence for, at least, milk, wheat, and egg protein components are detectable [33]. The ability to actually measure the allergenic protein may not be sensitive enough.If the fetus initiated the development of IgE antibodies, or was already sensitized in-utero (in theory, based on recoverable specific IgE in cord blood), then advancement of further sensitization, especially to food, will presumably depend on continued exposure, awaiting the proper moment for clinical expression (Figure 1). The other possibility is that in-utero sensitization occurred, but post-uterine internal immunological control, or a major change in post-uterine exposure un-enhances further IgE production. The third possibility, currently undertaken as a major national and international approach, is to reduce or prevent post-uterine food IgE sensitization and subsequent allergic reactions, using early introduction to all foods [36]. A comprehensive report of further recommendations of this model of primary prevention is available [36].IgE food sensitization is enhanced or initiated by exposure post-birth predominately via two methods. One, skin contact with food allergens still not eaten; two, exposure of food antigens/allergens via breast milk. Infant skin exposure to foods is nearly universal if the food is eaten by others in the home [33,37]. Genetic predispositions to being an atopic infant was of course, pre-programmed, and exposure becomes step 2. If the allergen already had intrauterine IgE priming, the re-exposure post-birth via skin contact is likely.Intact skin is more resistant to food absorption, and the recommendation to apply a moisturizer from birth has received some success [38]. The presence of other skin abnormalities, especially common ones, could enhance absorption. In this regard, infantile seborrheic dermatitis [39] and/or genetic decreases in (pro)filaggrin production are culprits [40].Either with food transcutaneous absorption [41], or air-borne exposure to food substance in the airstream of the house or care facility [42] or transfer of food protein via the breast milk [33], the infant either starts the class switch process that was under-developed in-utero or rapidly begins their de-novo extra-uterine IgE production.The relative lack of anaphylaxis in the first 2–3 months suggests a more delayed production of IgE to the point of sufficiency of inducing anaphylaxis or contributing to atopic dermatitis [43,44].As previously mentioned, IgE-mediated food anaphylaxis does occur prior to 12 months [31,32], and eggs and milk are the most common foods, followed by peanuts [31]. The concept of age-specific IgE-mediated disease in the first year of life has been recently discussed, including anaphylaxis [1,18] (Figure 1).A second presentation of food allergies is within the context of atopic dermatitis. A perplexing clinical presentation in early childhood is an admixture of seborrhea and atopic dermatitis. If the child also has xerosis cutis with lowered filaggrin levels [45], a trifecta of skin permeability exists, which often get lumped into the term eczema and has been discussed as over-lap [46], and although the author was admixing atopic dermatitis and psoriasis [46], the concept works for other combinations. The clinician needs to separate the different disease conditions and provide counseling and therapeutic intervention for each [46].IgE-mediated food allergens can play a role in the atopic dermatitis process, as the permeable skin (with xerosis and/or seborrhea) may have allowed for further transcutaneous sensitization. If a true IgE-mediated food allergy (anaphylaxis) has not occurred, the presence of positive IgE testing results, either specific IGE levels on venous samples or percutaneous allergy tests. the clinician must face the dilemma of avoidance or continuation of feeding the specific food implicated in testing. The recent review by Rajanai et al. suggests a clinical approach for suspected food allergen in breastfed or formula fed infants with atopic dermatitis [33]. A reasonable time of avoidance may assist with improving the AD; re-introduction of a specific avoided food can resume skin itching and AD exacerbation. If a specific food is avoided, or previously eaten, an oral challenge needs to be performed if avoided for 6–12 months due to the potential development of an anaphylactic reaction [47]. It’s also common the positive food has not been eaten by the infant and the only solution is to stop it in the mother’s diet and avoid stored breast milk.The IgE-mediated food reaction in infants focused solely on the gastrointestinal tract is less common and requires a large differential diagnosis. Cow’s milk-induced acute proctocolitis can also include vomiting, but the IgE testing for milk is almost universally negative [48,49,50]. Isolated vomiting without hypotension may occur with milk, and the differential includes food protein enterocolitis (FPIES) [49] or isolated cow’s milk allergy [48,50]. The IgE test will be almost always be negative in FPIES [49], but positive if cow’s milk is the allergen in the later scenario and is a subset of anaphylaxis. The first ingestion of egg, wheat, or peanut may be the concern, and the presence of IgE antibodies helps direct the diagnosis, although all three are less common for FPIES [51] and extremely rare for proctocolitis [52,53]. FPIES has other foods of concern, including oats and rice [51]. A negative skin test is expected.When chronic upper gastrointestinal tract symptoms several considerations for food-induced disease exists. Chronic FPIES, with milk or soy, but with negative allergy tests may be responsible, while eosinophilic esophagitis, or EGID can occur, more often in boys, and likely, but necessarily, accompanied by IgE-mediated positive tests [52]. An esophagogastroduodenoscopy may be necessary with required biopsies.Isolated chronic diarrhea is less likely IgE-mediated, and allergy testing is a potential part of an evaluation, but diarrhea alone is unlikely a single or multiple IgE-mediated food allergen concern. An EGD with or without a colonoscopy may direct an eosinophilia-mediated concern.Neither asthma or allergic rhinitis has specific IgE-mediated food concerns under most circumstances but has been reported and evaluated long-term [53].We illustrate with a case seen by RJH as the manuscript was being written.VFB presented to clinic at 7 months. She was a Caucasian female and a product of a full-term uneventful pregnancy. Mom drank cow’s milk and ate peanuts during pregnancy and continued after pregnancy. She was exclusively breast feeding and had xerosis and truncal eczema and had rashes on face and body when eating “new” foods. She breast-fed while eating or bottle fed with breast milk. She had aggressively refused cow’s milk formula. Both parents had xerosis and hyper-creased palms. Older brother had “eczema”. The baby was well developed and developmentally appropriate. She had patches of eczema, hyper-creased palms, and pictures on the mother’s phone confirmed hives, facially, and on trunk. Skin testing revealed 4+ reactions to peanuts and milk, 3+ to dog and HDM.Infants can present with allergic diseases well before their first birthday, which must argue for earlier IgE sensitization than presentation of the disease (Figure 1). We attempted here to take a conception to early life approach to emphasize the pathways to IgE sensitization and the eventual associated allergic disease onset. Since young infants can present with allergic diseases, an understanding of the complicated course of events that must happen is critical and were examined here, using a broad base of clinical experience, punctuated by an exact illustrative case.Conceptualization, writing and editing: R.J.H. and M.A.P. Resources: P.S. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Not applicable.The authors declare no conflict of interest.A schematic representation of the intrauterine chances to produce specific IgE and the subsequent events until the first birthday for clinical expression of an IgE mediated disease.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Purpose of the article: Acrylate and methacrylate (MA) use in the dental industry is widespread, being utilized in dental prostheses and composite resins, dentin bonding materials, and glass ionomers. However, occupational methacrylate allergy in dental personnel is a growing phenomenon. The aims of this retrospective observational study are to evaluate the risk of occupational contact dermatitis following exposure to methacrylates in dental personnel and to identify possible preventive measures. Materials and Methods: A total of 126 subjects exposed to acrylic and methacrylic resins in their professional context and who reported clinical manifestations were included from our outpatient department database. These were subdivided into two groups: 81 dental technicians and 45 dental hygienists. All the subjects had undergone patch testing with a “methacrylate series” (FIRMA) and readings were taken after 2 days (D2), 4 days (D4), and 7 days (D7). Results: A significantly higher incidence of methacrylate allergy was found in the dental technician group compared to the dental hygienists. Among the dental technicians, 40.7% of the subjects presented skin manifestations. The hands were the most frequently affected sites. Conclusion: Our results confirm the high sensitizing potential of MA in the workplace for dental personnel and in particular an increased professional risk in work where the hands are directly involved (dental technicians). Patch testing as an integrated part of a screening tray is needed for a complete evaluation of occupational skin allergy due to MA in dental personnel. The adoption of proper primary preventive measures, including gloves, protective eyewear, face shields, and disposable gowns, can be useful in preventing new cases of contact dermatitis, which may lead to a change of occupation in dental personnel.Acrylates and methacrylates (MA) are different types of chemical products deriving from the esterification of acrylic and methacrylic acids. Their reactivity varies according to the type of acid used for the esterification process. Acrylic monomer use is widespread, being used in dental prostheses and composite resins, dentin bonding materials, and glass ionomers [1,2,3,4]. Dental personnel are at risk of developing acrylate and methacrylate allergy [5]. Classically, acrylate allergy is characterized by a facial and/or eyelid rash, eczematous finger pulp fissuring (pulpitis), nail dystrophy, and/or periungual dermatitis [6,7]. Sensitized patients may have complications with dental work [8]. Acrylates and MA can also be responsible for occupational asthma [9,10]. The aims of this study are to evaluate the risk of skin allergy following exposure to MA in dental technicians and dental hygienists and to identify the allergens responsible for allergic contact dermatitis and possible preventive measures.In this retrospective observational study, data were obtained from our outpatient department database and included patients that had been evaluated using patch testing for allergic contact dermatitis from January 2017 to December 2018. In particular, we considered dental technicians and dental hygienists exposed to acrylic and methacrylic resins in their professional context referring clinical manifestations. The most commonly reported dermatological manifestations were facial and/or eyelid dermatitis, eczematous finger pulp fissuring (pulpitis) and nail dystrophy.The study population was divided into two groups: 81 dental technicians and 45 dental hygienists for whom it was possible to assume an allergic reaction to MA. In all the subjects, patch tests with a “methacrylate series” (FIRMA) (Table 1) with Finn Chambers® (SmartPractice) on Scanpor® tape (Norgesplaster, Vennesla, Norway) were performed and readings were taken after 2 days (D2), 4 days (D4), and 7 days (D7). The readings were always carried out by the same dermatologist trained in the field. Descriptive statistics were used to describe the patients’ characteristics. The non-parametric Mann–Whitney U test was used to compare the groups. The association between categorical variables was estimated with a chi square test or Fisher exact test. However, when needed, the odds ratios (OR) and their relative 95% confidence intervals (CI 95%) were calculated using a univariate logistic regression model. SPSS statistic software (version 21) was used to perform the statistical analysis.Of the 81 dental technicians (67 males, 14 females; median age 35.7), 33 (40.7%) presented localized dermatitis, respectively, in: the hands (57.6%), forearms (24.2%), neck, and face (12.1%) (Table 1). Of the 45 dental hygienists (12 males, 33 females; median age 23.5), 13 (28.8%) showed clinical features of dermatitis; the most frequently affected sites were the hands (53.8%), forearms (23.1%), head, and neck (15.4%) (Table 1). Patch tests were positive for at least one allergen in 37 individuals: 30 dental technicians (37%) and 7 dental hygienists (15.6%) (Table 2).In 21 of these, the positive patch tests were multiple. The most frequently positive allergens were: 2-hydroxyethyl methacrylate (2-HEMA), ethylene glycol dimethecrylate (EGDMA), methyl methacrylate (MMA), and 2-hydroxypropyl methacrylate (2-HPMA) (Table 3). As previously described by Koppula et al. [11], the cross-reactivity of methacrylates is based on their chemical structure. In particular, they hypothesized that the acrylates with the carboxy ethyl side group react with receptors on antigen-presenting cells to generate antigenically identifiable residues. The odds ratio for the professional risk of exposure was OR = 3.19 (CI 95%, 1.27–8.04; p = 0.02) in dental technicians vs. dental hygienists, showing an increased professional risk in the first group. The results of our study confirm the high incidence of occupational allergy to MA and the growing phenomenon of allergy to acrylates and MA in dental technicians and dentists; these data are in accordance with the scientific literature [5,6,10,12,13]. In our study, the higher incidence was found in the dental technician group, due to the predominant use of the hands, which caused an increased exposure to the substances (21% positive patch tests in the dental technician group vs. 13.3% in the dental hygienist group). This group of workers was exposed to the monomeric form of MA during the preparation of the dental resins. Acrylates and MA are important compounds in dental prosthetic work, so it was not surprising that a greater reactivity in dental technicians was found compared to dental hygienists. The most commonly positive acrylic monomers were EGDMA, 2-HEMA, and 2-HPMA. Animal studies have shown strong cross-reactivity between these three methacrylates [14]. Stevenson, in 1941, reported the first case of acrylate allergy in a patient with an allergy to methyl MA [15]. Therefore, sensitized individuals are often multiallergic and, accordingly, it is difficult to reach definitive conclusions regarding cross-allergy, because concomitant exposure to several acrylic monomers and multiple sensitization could be an alternative explanation [10]. Additionally, the presence of impurities in denture acrylate components, which may contain a variable proportion of other (meth)acrylates than those officially declared, should be considered [16]. Skin manifestations usually involve directly exposed areas that are not covered by clothes or individual protection devices. The typical clinical signs involve the hands; however, facial involvement has also been described. In our patients, neck and face manifestations were also observed. The causes include contamination by hands to the face and airborne allergenic material [17]. In fact, during their professional activity, personnel use cutters which produce dust, thus favoring the possibility of an airborne dermatitis that involves the skin of the neck and face. In fact, following exposure to MA suspended in air, professionals show symptoms even if such areas have not been in direct contact with acrylates. Our results confirm the high sensitizing potential of MA in the workplace for dental personnel, and in particular, an increased professional risk due to the predominant use of the hands for dental technicians. Allergic contact dermatitis caused by MA involves directly exposed areas that are not covered by clothes or individual protection devices and can be correlated with a lack of or inadequate use of personal protective equipment. In order to avoid contact dermatitis, health practitioners should recognize possible occupational hazards in dentistry and should adopt preventive measures.The preventive measures should include the proper use of personal protective equipment, including gloves, protective eyewear, face shields, and disposable gowns.No-touch techniques are important in order to avoid skin exposure to these chemicals [18]. The nitrile rubber gloves provide better protection against permeation by MA with higher resistance to permeation and a lower permeation rate of the monomers [19]. Moreover, gloves should be changed frequently. The adoption of such primary preventive measures can be useful in preventing new cases of contact dermatitis, which may lead to a change of occupation. Patch testing is needed for a complete evaluation of occupational skin allergy due to MA in dental personnel.The principles of the 1975 Declaration of Helsinki revised in 2013 were followed in this study.N.C.: supervision; study design; conceptualization; methodology; writing—original draft; database strategy research; and acquisition of data. M.M.: conceptualization; writing—original draft; methodology; and data curation. F.P.: review and editing; visualization; critical revision of the manuscript; and writing. M.S.: review and editing; visualization; and critical revision of the manuscript; A.C.: resources; study design; conceptualization; methodology; supervision; and data curation. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Ethical review and approval were waived for this study because it was a retrospective study performed on a database.Patient consent was waived because it was a retrospective study performed on a database. Data is contained within the article. The authors declare that they have no competing financial interests or other potential conflict of interest.Localization of allergic contact dermatitis skin manifestations in dental technician and dental hygienist groups.Methacrylate series (FIRMA) patch test positivity for at least one allergen in dental technician and dental hygienist groups.Methacrylate series (FIRMA) patch test-positive allergens.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-01-02-00011.txt

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+Unverified beta-lactam allergies are a substantial public health problem, as the majority of patients labeled as beta-lactam allergic do not have clinically significant allergies that may hinder the use beta-lactam therapy when indicated. Outdated or inaccurate beta-lactam or penicillin allergies can result in serious consequences, including suboptimal antibiotic therapy, increased risk of adverse effects, and use of broader spectrum antibiotics than indicated, which may contribute to antimicrobial resistance. The purpose of this review is to provide an overview of beta-lactam allergy and highlight the role of pharmacists in managing beta-lactam allergies. Studies have shown that pharmacists can play a vital role in allergy assessment, penicillin skin testing, beta-lactam desensitization, evaluation of beta-lactam cross-reactivity and recommending appropriate antibiotic therapy in patients with beta-lactam allergies.Beta-lactam antibiotics are considered a first-line therapy in many bacterial infections and include agents such as penicillins, cephalosporins and carbapenems. However, beta-lactam antibiotics are a common cause of adverse drug reactions and are associated with high rates of antibiotic allergy [1].Unverified beta-lactam allergies are a substantial public health problem, as the majority of patients labeled as penicillin allergic do not have clinically significant IgE-mediated allergies that may hinder the use of beta-lactam therapy when indicated [2]. The vast majority of patients have a subjective and inaccurate understanding of beta-lactam allergy. Approximately 10% of patients report an allergy to penicillin; however, up to 90% of these patients do not have a true allergy [3]. A true allergy, better known as an IgE-mediated or type I reaction, typically occurs immediately or within 1 h of medication administration, and can consist of hives, angioedema, wheezing, shortness of breath, and anaphylaxis [4]. Even in patients with true beta-lactam allergies, the IgE antibodies decrease over time. In patients who have previously tested positive for beta-lactam allergy, there is an expected 10% decrease per year of a subsequent positive reaction. Therefore, with the avoidance of beta-lactams, 80% to 100% of patients will ultimately test negative for beta-lactam allergy 10 years after the initial positive test [3].Outdated or inaccurate beta-lactam allergies can result in serious consequences, such as suboptimal antibiotic therapy, increased risk for adverse effects, and use of broader spectrum antibiotics than needed, which may contribute to antimicrobial resistance [5]. Pharmacists can play a vital role in evaluating the patient and identifying a true allergy, assisting in de-labeling inaccurate allergies, performing a standard of care skin test, deciding when desensitization is an appropriate option, and considering cross-reactivity for optimal antibiotic treatment.The purpose of this review is to provide an overview of the beta-lactam allergy and highlight the role of the pharmacist in managing beta-lactam allergies. A literature search was conducted via PubMed using the following search terms “pharmacist, beta-lactam, penicillin, allergy, impact, management” from February to March 2021. Relevant English language studies were included in this review.Beta-lactam allergies are hypersensitivity reactions that are the consequence of an adaptive immune response. Of the four reaction types described in Table 1, penicillin allergies most often present as type I or type IV hypersensitivity reactions [3]. Type I hypersensitivity reactions are triggered by the interaction of an allergen with allergen-specific IgE bound to mast cells, basophils, and eosinophils. This interaction causes the cells to degranulate and release a potent mixture of histamine and inflammatory mediators [6]. In response to histamine and other inflammatory mediators, there is an increase in vascular permeability and a widespread constriction of smooth muscle, which can ultimately lead to anaphylactic shock [6]. Type I reactions are immediate in nature and may result in symptoms such as urticaria, flushing, dyspnea, bronchospasm, angioedema, hypotension, tachycardia, altered mental status or gastrointestinal upset [3].Because all penicillin antibiotics share a similar beta-lactam core ring structure, one must consider the potential for cross-allergenicity between different agents in the beta-lactam class. When a beta-lactam is administered, the common core ring structure is metabolized into major and minor antigenic determinants. The major determinant is penicilloyl, and the minor determinants include penicillin, penicilloate, and penilloate [3]. In patients with beta-lactam allergy, the minor antigenic determinants generate IgE-specific responses associated with type I allergy. The major antigenic determinant is more commonly associated with less severe urticarial reactions [3]. In addition to a core ring structure, beta-lactams have various side chains, which can also result in type I allergic reactions [3]. Type I reactions to beta-lactam antibiotics are individualized and have effects of varying severity.Less frequently, beta-lactam allergies may present as other types of hypersensitivity reactions. Type II hypersensitivities are less severe, but can be caused by an IgG response to small molecules like penicillin that become covalently bound to the outside surface of cells. The chemical reaction modifies the structures of human cell surface components, which become perceived as foreign antigens by the immune system. B cells are stimulated to make IgG antibodies against the new epitopes. On binding to their specific cell-surface antigens, the antibodies cause the modified human cells to become subject to complement activation and phagocytosis, resulting in inflammation and damaged tissue [6].Type III hypersensitivities involve small, soluble, immune complexes of antigen and specific IgG, forming deposits in the walls of small blood vessels or the alveoli of the lungs. At these sites, the immune complexes activate complement and an inflammatory response that damages the tissue and impairs its function. Typically, type III hypersensitivities are not associated with beta-lactam exposure [5].Type IV hypersensitivities are caused by CD4 T-cells responding to the epitopes of foreign proteins or to peptides derived from chemically modified human proteins [6]. The result is typically cutaneous reactions such as a maculopapular rash or urticarial eruption. Although rare, severe reactions, such as Stevens–Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) or drug rash with eosinophilia and systemic symptoms (DRESS) may occur as a result of a type IV hypersensitivity. Type IV hypersensitivities are delayed in nature, occurring several days to weeks after the last administered dose, and have been associated with beta-lactam allergy [7].Although data are currently limited in regard to the risk factors for a beta-lactam allergy, there is speculation about a correlation between the clinical and genetic risk factors of self-reported penicillin allergy. A recent case-control study identified factors such as having a family history of penicillin allergy or an IL-4 gene single nucleotide polymorphism as possible risk factors of penicillin allergy. [8] Another recent study found that HLA-DRB1*10:01 predisposed patients to an immediate hypersensitivity reaction [9]. Furthermore, other studies suggest a greater risk of penicillin allergy in females and a greater risk of beta-lactam allergy in those with a history of a prior adverse reaction to penicillin [10,11].As noted earlier, all beta-lactam antibiotics share a similar chemical core ring structure. This core ring consists of a beta-lactam ring attached to a five- or six-membered ring. In addition to this core structure, beta-lactams have side chains (R1 and R2) that vary between the specific agents [12]. Because of the common core ring structure among beta-lactam antibiotics and the potential similarities in side-chains, there is a risk for cross-allergenicity among the different beta-lactam classes. Knowledge and understanding of the risk of beta-lactam cross-allergenicity plays an important role in selecting the appropriate antibiotic therapy for a patient with a beta lactam allergy. The rates of cross-allergenicity between penicillin and other beta-lactams can be found in Table 2.The early studies of penicillin cross-allergenicity with cephalosporin antibiotics cite rates as high as 10%. However, these rates are considered artificially high and are attributed to the contamination of early generation cephalosporins by penicillin because of the early manufacturing processes used to create the cephalosporin ring structure [13]. The actual rate of cross-allergenicity between penicillin and cephalosporins is likely <2% [12,13]. Similarities in the R1 side chains of cephalosporins have been shown to be the most important predictor of cross-allergenicity as compared to a common beta-lactam ring [14,15]. Certain cephalosporins and penicillins have identical side chains, such as cephalexin, cefaclor, and ampicillin, and cefadroxil, cefprozil and amoxicillin. In general, most patients reporting an allergy to beta-lactams can safely receive cephalosporin therapy. However, agents with identical side chains should be avoided in patients with a true IgE-mediated beta-lactam allergy [15].Carbapenems, another group of beta-lactam antibiotics, are generally well tolerated and have a low risk of causing allergic reactions. Studies evaluating the cross-allergenicity between penicillins and carbapenems demonstrate rates of cross-allergenicity of 0%–0.3% in patients with a true IgE-mediated penicillin allergy [16,17]. Typically, carbapenems are considered safe for administration in patients with a reported penicillin allergy [18].Aztreonam is a monobactam antibiotic that differs structurally from beta-lactams because of its monocyclic core ring structure. Because of this major difference, the risk of cross-allergenicity with the beta-lactam class is considered negligible [15]. However, it is important to note that aztreonam and ceftazidime, a third generation cephalosporin, share an identical side chain. Patients with a true ceftazidime allergy may also have an aztreonam allergy, therefore aztreonam should be avoided in these patients [15].The initial management of patients with a history of beta-lactam allergy requires a detailed allergy assessment, given that many patients are inappropriately labeled as beta-lactam allergic. Oftentimes, known adverse effects of a beta-lactam are mislabeled and documented as a beta-lactam allergy. Approximately 90% of patients labeled as allergic can be de-labeled through a detailed allergy assessment or allergy testing [19,20]. The appropriate assessment of beta-lactam allergy includes the evaluation of factors such as the specific agent causing the reaction, the type, severity, and timing of the reaction, and previous tolerability of other beta-lactam agents. The de-labeling of penicillin allergy, based on historical evaluation alone, has been demonstrated to be safe and effective in patients reporting a mild, non-immune-mediated adverse drug reaction, or where subsequent tolerance to the implicated penicillin has been identified through their medical or pharmacy records [19]. Figure 1 includes a list of questions that may be appropriate to ask patients who report a beta-lactam allergy.Additionally, a direct oral challenge may be an effective option in de-labeling patients with low-risk penicillin allergies and has been used in both the inpatient and outpatient setting. Direct oral challenges offer a means to de-labeling penicillin allergies in patients who are low risk or have an inaccurate allergy label without having to pursue traditional allergy testing. Appropriate risk stratification is needed to ensure the safety of a direct oral challenge; however, no standard criteria to determine low-risk allergy patients has been established. Factors such as reaction type, onset of allergy symptoms, and severity have been utilized to determine risk. Validated allergy phenotyping tools may be used to identify low-risk allergy patients and help to facilitate de-labeling when appropriate [21].In patients with ambiguous allergy histories, or those reporting allergies that are severe in nature, additional steps should be taken to determine the nature of their allergy and whether a beta-lactam can safely be administered. Penicillin skin testing (PST) is a safe and effective means of evaluating a penicillin allergy in these patients. It is used to detect the presence of penicillin-specific IgE and predicts the likelihood of a type I allergic reaction to penicillin [22]. PST is indicated in patients with documented or suspected type I clinical penicillin hypersensitivity. Patients with a history of desquamating type IV hypersensitivity reactions, such as SJS, TEN, or DRESS or other non-IgE-related hypersensitivity, are not candidates for PST [21]. Implementing PST requires the consideration of several logistical factors such as the development of policies and protocols, specialized training for staff, storage and preparation of PST components, and determining whether PST should be offered in the inpatient versus outpatient setting [23].PST involves epicutaneous skin testing, followed by intradermal testing, and is conducted using degradation products of penicillin, known as major and minor determinants. PST offers a negative predictive value of 97–99% [24] and a positive predictive value of approximately 50% [25].In patients who have a negative PST, a test dose of the beta-lactam to which the patient reported allergy should be administered in order to confirm the absence of an allergy. The patient should then be observed for 1–2 h after the test dose to confirm no immediate reaction occurs. If no reaction occurs after the observation period, the patient can receive the beta-lactam and should have their allergy information updated accordingly.In patients with a positive PST, beta-lactam therapy may still be administered after desensitization is performed. Avoiding beta-lactam therapy in these patients can lead to the use of suboptimal therapy, as beta-lactams have been demonstrated as superior agents in the management of infections such as methicillin-susceptible Staphylococcus aureus (MSSA), severe Pseudomonas aeruginosa, and syphilis [23].Desensitization may be indicated in patients with a true beta-lactam allergy or in those where the results of the penicillin skin testing is indeterminate, but still require beta-lactam therapy [7]. The process should be performed by a specialist and involves administering incremental increases in beta-lactam doses over time to allow patients to tolerate beta-lactam therapy on a temporary basis. Desensitization is often conducted in a hospital setting or intensive care unit, as patients will require frequent monitoring throughout the procedure. Numerous beta-lactam desensitization protocols exist and often take several hours to days to complete [26].As seen in Table 3, initial management of patients with reported beta-lactam allergies should be decided based on allergy assessment and the suspected reaction. For individuals with low risk of allergy, a direct oral challenge may be considered. For individuals with signs and symptoms of an IgE mediated allergy (type I), PST should be performed. If PST is negative, a beta-lactam challenge dose may be administered. If PST is positive, beta-lactam desensitization may be considered. In patients with signs and symptoms of non-IgE mediated allergy (types II-IV) PST, challenge doses, and desensitization are contraindicated [27].As medication experts, pharmacists are uniquely poised to conduct beta-lactam allergy assessments to assist with de-labeling when appropriate. Several studies have demonstrated the benefit of having pharmacists involved in beta-lactam allergy interviews.One pilot study utilized a pharmacy resident and infectious diseases clinical pharmacist to clarify a beta-lactam allergy, and, where appropriate, recommended a change to the patients’ antibiotic regimen. In total, 32 patients with a documented beta-lactam allergy were interviewed, and 24 were identified as candidates for beta-lactam therapy. The intervention by the pharmacy resulted in over 65% of the patients being changed from a non-penicillin antibiotic to a cephalosporin, carbapenem, or penicillin. Although small, this study demonstrates the efficacy of a pharmacist-driven beta-lactam allergy assessment [28]. In a larger retrospective study, including 418 patients, Holmes and colleagues evaluated the impact of a pharmacy-driven assessment on the prescribing frequency of penicillin or cephalosporin antibiotics in patients with a reported beta-lactam allergy. Pharmacy staff were notified, through an alert system, of any patient with a documented penicillin allergy receiving a non-penicillin antibiotic. Upon notification, the pharmacy staff performed an allergy assessment, which included a review of previous beta-lactam tolerance, and clarification of the reaction type, severity, and timing of the allergy. The pharmacist allergy assessment resulted in increased frequency of beta-lactam prescribing in patients with previously reported beta-lactam allergy, by 12.9%, and a decrease in the days of therapy of non-penicillin antibiotic use, by 123 days of therapy per 1000 patient days, again showing the benefit of pharmacist involvement in penicillin allergy assessment [29].Additionally, a study by Campbell and colleagues evaluated a pharmacist-led penicillin allergy assessment at a community hospital in 380 patients. The assessment included a chart review, patient interview, follow-up antibiotic discussion with the provider, and updating the allergy documentation when appropriate. The pharmacist-led assessment resulted in improved use of guideline preferred antibiotics by 13% and reduced fluoroquinolone use by 11%. [30] Furthermore, the pharmacist-driven allergy assessment has also been associated with cost savings for patients with beta-lactam allergy, with one study estimating cost savings of $21,000 over a 3-month period [31].Pharmacists also play a role in the diagnosis of beta-lactam allergy by participating in PST. When the initial allergy assessment does not provide clear information on whether a patient has a true penicillin allergy, PST can be used as a simple means of diagnosing penicillin allergy. When pharmacists are involved in PST, the state board of the pharmacy should first be consulted to determine whether PST completed by a pharmacist falls within a pharmacist’s scope of practice, as scopes vary between states. If PST is not allowable under the state board of pharmacy, the pharmacist may still serve as part of a collaborative team conducting PST [23].A recent study investigated a pharmacist-driven PST service for adults at a community hospital. Twenty-two patients with documented type I allergies, who were prescribed alternative antibiotics, were identified and underwent PST by trained pharmacists. All of the skin tests were negative, with no type I reactions occurring to the test itself or to the beta-lactam antibiotic administered thereafter. Additionally, 68.2% of the patients were successfully transitioned to a beta-lactam after skin testing, leading to a decreased use of vancomycin and fluoroquinolones. The authors concluded that pharmacist-driven PST can be successfully implemented and is another means for pharmacists to expand antimicrobial stewardship practices [32].Another study describes pharmacist-managed and pharmacist-administered PST incorporation into an antimicrobial stewardship program at a community hospital. Patients who were at least 18 years old, and reported a history of a type I or unknown type of allergic reaction to penicillin, occurring more than 5 years prior, were considered for inclusion. In total, PST was initiated in 90 patients, with 94% completing PST. Nearly 85% of the patients who completed PST were transitioned to a preferred beta-lactam, allowing alternative antibiotics to be avoided for a median of 11 days per patient. This study demonstrates that pharmacist-managed/-administered PST improved the utilization of preferred antibiotics and the avoidance of alternative therapy [33].Additionally, Griffith and colleagues conducted a retrospective cohort study evaluating the impact of an inpatient pharmacist- and pharmacy trainee-administered penicillin allergy assessment and PST service on allergy reconciliation and antibiotic use. There was a total of 161 evaluations conducted, 74% allergy assessments and 26% PST. The reconciled allergies improved from 11% to 75% post-evaluation, and no PSTs were positive. Following PST, antibiotic therapy was optimized in 58% of the patients, the vancomycin use decreased from 44% to 17%, and the use of penicillins increased from 0% to 39% [34].Other studies assessing the role of pharmacists in beta-lactam allergy evaluation and PST also demonstrated similar findings, with increased use of beta-lactams after intervention, decreased use of broader spectrum antibiotics, and no severe adverse drug reactions noted [35,36,37,38].Another area where pharmacists provide valued insight is identifying and managing patients who are candidates for beta-lactam desensitization.Chen and colleagues evaluated the successfulness and the safety of antibiotic desensitization protocols developed by their pharmacy department. Each protocol provided specific instructions regarding reconstitution, administration, time to target dose, reaction, management, storage, and stability. Desensitizations were conducted in an intensive care unit through a multidisciplinary team approach that included a clinical pharmacotherapy specialist. The study reviewed 61 desensitizations, 89% of which were completed without the development of any adverse reactions. The 10% of cases where adverse reactions occurred were managed during desensitization and were able to successfully complete the process. The results suggest that pharmacy-developed antibiotic desensitization protocols are successful and safe [39].Additionally, pharmacists serve as crucial resources when it comes to educating and advising clinicians about the determinants and likelihood of cross-reactivity between penicillin-related compounds. Pharmacists, being well versed in medicinal chemistry, can make adjustments and interventions using this knowledge base. As a result, patients with a history of non-IgE-mediated, non-severe beta-lactam allergies are often able to safely receive beta-lactams that are structurally dissimilar [14]. By taking into consideration these cross-allergenicity rates, pharmacists can directly influence clinicians to determine when patients with reported beta-lactam allergies remain appropriate candidates to receive beta-lactam therapy [14].In a quasi-experimental pre-post study at Aurora BayCare Medical Center, educational efforts and modified workflow were implemented for the purpose of improving allergy histories and documentation, and increasing the use of beta-lactams, when appropriate, among patients with a reported beta-lactam allergy. Pharmacists were educated on allergy mechanisms, beta-lactam cross-reactivity rates and use of the developed reference tools. Prescribers, hospitalists, and intensivists were educated on the efficacy, safety, and economic implications of the consistent avoidance of beta-lactams in patients with reported beta-lactam allergies, and current estimates of cross-reactivity rates between the beta-lactam classes. It was concluded that multidisciplinary education combined with pharmacy-led efforts to confront the challenge of beta-lactam allergies among hospitalized patients may improve allergy documentation and selection in patients with a reported beta-lactam allergy. Although the intervention was not associated with an overall reduction in non-beta-lactam use, it did increase the number of patients who were transitioned from non-beta-lactam to beta-lactam therapy. The authors suggest that the overall reduction in non-beta-lactam use was likely not observed due to the time requirement in ascertaining a sufficient allergy history and allowing for a corresponding intervention to occur, and suggest that more distinct interventions may be required to impact empiric prescribing practices [14].Understanding the beta-lactam allergy is critical to managing patients with infectious diseases. Mislabeled beta-lactam allergies continue to pose a risk to patient care, as patients with a documented allergy are often prescribed suboptimal antibiotic agents to avoid beta-lactam therapy. If a patient reports a beta-lactam allergy, several steps should be taken confirm the allergy. All patients reporting a beta-lactam allergy should undergo a detailed allergy assessment, followed by PST if necessary. In patients with confirmed type I allergies, beta-lactam desensitization may be considered as a means of safely administering beta-lactam therapy on a temporary basis.As medication experts, pharmacists are particularly skilled in managing and offering insight into beta-lactam allergies. Their value has been established in areas including the initial allergy assessment, penicillin skin testing, antibiotic desensitization, and evaluating the risk of cross-reactivity to assist with appropriate antibiotic selection.Conceptualization, N.B. and Y.L.; writing—original draft preparation, N.B., Y.L., D.W.; writing—review and editing, N.B., Y.L., D.W. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Not applicable.The authors declare no conflict of interest.Sample allergy assessment questions.Classification of hypersensitivity reactions [3,5,6,7].* Stevens–Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) or drug rash with eosinophilia and systemic symptoms (DRESS).Beta-lactam cross-reactivity [12,14,15,16,17,18].Initial approach to managing patients with penicillin allergy [21,27].Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+This case report describes a 50-year-old woman who developed an allergic granulomatous reaction after a tattoo touch-up.Tattooing has become increasingly popular in recent years. Adverse cutaneous reactions against tattoo ink compounds have been largely reported and they include eczematous, lichenoid and granulomatous lesions, pseudolymphoma and pseudo-epitheliomatous hyperplasia [1]. Notably, red tattoo pigments are the ones which are associated to adverse events the most [2].A 50-year-old female patient presented to our Department complaining of a cutaneous lesion localized on her tattoo. The patient reported that her tattoo had been placed on her skin using black inks 15 years before and that these body modifications had not been associated with any adverse skin reaction. However, when she decided to have her tattoo touched-up with colored inks (including red) she developed a cutaneous reaction a week later.On physical examination, thick erythematous nodular lesions were present in the areas in which the red pigment was used (Figure 1). Pain and itching were present.Before seeking a dermatological consultation, the patient had tried several local therapies, such as topical steroids and serial intra-lesional corticosteroid injections, with no clinical benefits. Furthermore, she had tried systemic therapies with steroids, antibiotics and antihistamine, which were associated with temporary beneficial effects.We decided to perform a cutaneous biopsy which revealed acanthosis, papillomatosis and focal ulceration of the epidermis, with spongiosis and basal vacuolization; an intense chronic dermal inflammatory reaction with eosinophils was evident. The histological pattern was compatible with an allergic granulomatous reaction which was presumptively attributable to one of the colored pigments that were added to the black tattoo.We also performed SIDAPA and tattoo series patch tests which highlighted a positivity to nickel sulphate at 48 h (+) and 72 h (+++).Considering the severe local pain and because of the inefficacy of topical, systemic and intra-lesional therapies, a surgical excision of the affected areas followed by skin grafting with autograft was made.As mentioned before, tattoo adverse reactions are becoming increasingly common, probably because of the growing popularity of this practice among young people. Allergic reactions are among the most frequent tattoo-related complications and they are usually caused by the chemicals found in tattoo inks: interestingly, the published literature points to the red pigment as the main culprit of allergic adverse reactions on the skin [3,4]. Nevertheless, other pigments like yellow, violet and blue have been reported to be able to cause adverse local reactions as well [4,5].Nickel sulphate is frequently present in red pigments and it can cause various allergic complications in sensitized people [4]. Indeed, the allergic granulomatous reaction described in this report was localized in the red areas of the tattoo, which makes the red ink the most probable one to have triggered the cutaneous side-effects. Moreover, this statement was supported by the result of the patch test that was performed.Chromium, cadmium, iron and titanium have been identified inside of colored tattoo inks and their presence has been linked to environmental factors like contamination [4]. Moreover, there is no strong evidence that supports a role of these metals in the allergic events associated with the application of colored tattoos [4].Unfortunately, we were not able to perform a pigment analysis in our patient.In conclusion, we presented this clinical case to highlight that tattoos can present several adverse effects, which are expected to be increasingly observed in the future due to their growing popularity. Additionally, tattoo ink composition is frequently unknown and it can lead to allergic granulomatous reactions. Further studies to elucidate the composition of tattoo inks are warranted to avoid the occurrence of unwanted adverse reactions. In addition, strict legislation should be developed to regulate the compounds which are used to make tattoo colors [1]. It is advisable for people to seek dermatological advice before changing the color of a pre-existing tattoo, to avoid the onset of adverse reactions. Lastly, we would like to emphasize that typical local and systemic therapies are not always sufficient for the complete resolution of extended cutaneous lesions, especially when they are associated with symptoms like persistent pain and pruritus. In these cases, it is necessary to perform surgical treatments, with results which are not always aesthetically pleasing.Conceptualization: A.T., F.L., G.D.M.; Methodology: A.T., F.L.; Investigation: A.T., A.C., C.I., C.C.; Writing-original draft preparation: G.A.R.A., F.M., F.R.P.; Writing-review and editing: F.M., G.A.R.A.; supervision: A.T., G.D.M. All authors have read and agreed to the published version of the manuscript. This research received no external funding.Not applicable. Informed consent was obtained from the patient to publish this paper. No new data were created or analyzed in this study. Data sharing is not applicable to this article. The authors declare no conflict of interest.Allergic granulomatous reaction on the tattoo of a 50 year-old woman before and after surgical treatment: (a) Thick erythematous nodular lesions at the red-pigment areas of tattoo; (b) Surgically treated tattoo.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Purpose: To elucidate the usefulness of Japanese cedar pollen (JCP)-specific antigen-specific immunoglobulin (IgG) 4 as a biomarker for predicting the efficacy of sublingual immunotherapy for cedar pollen-induced allergic rhinitis. Methods: We divided a total of 105 cases with Japanese cedar pollinosis into three groups: “SLIT Successful,” SLIT Unsatisfactory,” and “SCIT” groups. The SLIT group patients were treated with JCP Droplet (Torii Pharmaceutical Co. Ltd., Tokyo, Japan) for one year from 2015 and were divided into two groups, the SLIT Successful group or the SLIT Unsatisfactory group. The SLIT Successful group (n = 16) were subjects treated by SLIT only, who were able to experience control of their naso-ocular symptoms without the need for antiallergic rescue agents during the peak season of atmospheric pollen. The SLIT Unsatisfactory group (n = 76) comprised subjects treated with SLIT only, who did not respond successfully, and were administered with rescue agents to control their naso-ocular symptoms. The SCIT group had been treated with standardized JCP extract (Torii Pharmaceutical Co., Ltd., Tokyo, Japan) for three years from 2012, and were also able to experience control of their symptoms during the peak pollen season without the need for antiallergic rescue agents. We determined the serum level of JCP-specific immunoglobulin E (IgE), IgG, and IgG4 used in the 3gAllergy-specific IgE assay (3gAllergy). The serum levels of periostin and SCCA2 were measured using established ELISA procedures (clones SS18A and SS17B; Shino-Test, Japan) following the manufacturer’s instructions. We then made ROC curves for each group and assessed which index was best able to predict the efficacy of sublingual immunotherapy. Results: Serum JCP-specific IgE was significantly lower in the SCIT group than in the SLIT Successful group and the SLIT Unsatisfactory group (p < 0.05). Serum JCP-specific IgG was significantly higher in the SCIT group and the SLIT Successful group than in the SLIT Unsatisfactory group (p < 0.05). Serum JCP-specific IgG4 was also significantly higher in the SCIT group and the SLIT Successful group than in the SLIT Unsatisfactory group (p < 0.05). There was no significant difference among serum levels of periostin in the SCIT group, the SLIT Successful group, or the SLIT Unsatisfactory group. There was also no significant difference in SCCA2 among the three groups. In terms of ROC curves, a serum JCP-specific IgG4 value greater than 989.5 UA/mL showed the best sensitivity (93.3%) and specificity (94.7%) (p < 0.05) among other parameters. Conclusions: The serum JCP-specific IgG4 level is significantly correlated with the clinical efficacy of SLIT. Serum JCP-specific IgG4 cutoff levels greater than 989.5 UA/mL were correlated with an effective clinical response to SLIT, with a sensitivity of 93.3% and a specificity of 94.7%.The prevalence of Japanese cedar pollinosis (JCP) is rising rapidly in Japan. Pharmacotherapy is currently the commonest treatment for patients with this condition; however, its clinical benefits are occasionally limited because some patients demonstrate persistent tissue inflammation despite repeated high doses of glucocorticoid (GC) treatment [1]. Allergen immunotherapy (AIT) is the only treatment modality with the potential to both relieve naso-ocular symptoms and improve patients’ quality of life. Sublingual immunotherapy (SLIT), which is regarded as a safe and effective treatment for patients with JCP, has recently been developed [2,3]. However, useful biomarkers for measuring the mechanism and efficacy of AIT have yet to be established [4]. Antigen-specific immunoglobulin (IgG) 4 is a blocking antibody that may serve as a useful marker. House dust mite-specific IgG4 is useful for predicting the effects of subcutaneous immunotherapy (SCIT) [4,5]; however, it has only rarely been applied to investigations of cedar pollen allergy, still less of SLIT. The usefulness of JCP-specific IgG4 and periostin in the management of JCP is still controversial, so to clarify this question, we performed a prospective study to investigate the serum concentrations of JCP-specific IgG4, JCP-specific IgE, squamous cell carcinoma antigen A2 (SCCA2), and periostin in patients with JCP pollinosis who had received AIT.All patients were treated by experienced ENT doctors at Shiga University of Medical Science, Yuta Clinic, and at Tohoku Medical and Pharmaceutical University between October 2014 and August 2015. Informed consent was obtained under protocols approved by the Institutional Review Board (2018-2-139). A retrospective survey of medical records identified 105 patients (50 men and 55 women, average age 41.7). We divided the total of 105 cases with Japanese cedar pollinosis into three groups: SLIT Successful, SLIT Unsatisfactory, or SCIT groups. The details of each group’s clinical background are shown in Table 1. SLIT group patients were treated with JCP Droplet (Torii Pharmaceutical Co., Ltd., Tokyo, Japan) for one year from 2015, and were divided into two groups: the SLIT Successful group or the SLIT Unsatisfactory group. The SLIT Successful group (n = 16) comprised subjects treated by SLIT only, who were able to experience control of their nano-ocular symptoms without any need for antiallergic rescue agents during the peak season of scattered pollen. The SLIT Unsatisfactory group (n = 76) were subjects treated with SLIT only, who did not respond successfully and were therefore administered with rescue agents to control their naso-ocular symptoms. The SCIT group (n = 13) had been treated with standardized JCP extract (Torii Pharmaceutical Co. Ltd., Tokyo, Japan) for three years from 2012, who were also able to experience control of symptoms without antiallergic rescue agents during the peak pollen season. All the serum samples were collected during the peak season of Japanese cedar pollen release in 2015.We measured the serum level of JCP-specific immunoglobulin E (IgE), IgG, and IgG4 used in the 3gAllergy-specific IgE assay (3gAllergy). The serum levels of periostin and SCCA2 were measured using established ELISA procedures (clones SS18A and SS17B; Shino-Test, Sagamihara Japan), following the manufacturer’s instructions [6,7].The effectiveness of SLIT/SCIT was evaluated based on the subjects’ clinical allergic symptoms such as lacrimation, runny nose, and sneezing. We then placed these patients in the SLIT Successful group if patients showed their symptoms to be under control without the use of antiallergic rescue agents.We used one-way analysis of variance (ANOVA) and the Mann–Whitney U test to identify any statistical differences between groups. The analysis was performed using SPSS version 20 statistical software (IBM, Chicago, IL, USA). Differences in corrected p-value of <0.05 were regarded as significant. Whether SLIT was effective or not was assessed by examining the area under the receiver operating characteristic (ROC) curve. Models with an ROC area of at least 0.80 were considered to have predictive value.Serum JCP-specific IgE was significantly lower in the SCIT group than in the SLIT Successful group and the SLIT Unsatisfactory group (p < 0.05; Figure 1A). Serum JCP-specific IgG was significantly higher in the SCIT group and the SLIT Successful group than in the SLIT Unsatisfactory group (p < 0.05; Figure 1B). Serum JCP-specific IgG4 was also significantly higher in the SCIT group and the SLIT Successful group than in the SLIT Unsatisfactory group (p < 0.05; Figure 1C).There was no significant difference among serum levels of periostin in the SCIT group, the SLIT Successful group, or the SLIT Unsatisfactory group (Figure 1D). There was also no significant difference in SCCA2 among the three groups (Figure 1E).Figure 2 shows the sensitivity and specificity obtained by calculating the ROC curves for each serum JCP-specific IgE, IgG, IgG4, periostin, and SCCA2 level and making pairwise comparisons. The area under the curve was 0.653 for serum JCP-specific IgE levels (95% CI, 0.527–0.779), 0.697 for serum JCP-specific IgG levels (95% CI, 0.560–0.835), 0.962 for serum JCP-specific IgG4 levels (95% CI, 0.923–1.000), 0.486 for serum periostin levels (95% CI, 0.355–0.617), and 0.417 for serum SCCA2 levels (95% CI, 0.303–0.531). Our ROC analysis showed the serum JCP-specific IgG4 levels to be the most appropriate for predicting the efficacy of SLIT. Values greater than 989.5 UA/mL showed the best sensitivity (93.3%) and specificity (94.7%).Our study showed serum JCP-specific IgG4 level to be significantly correlated with the clinical efficacy of SLIT. We found that serum JCP-specific IgG4 cutoff levels that exceeded 989.5 UA/mL correlated with an effective clinical response to SLIT, with a sensitivity of 93.3% and a specificity of 94.7%.Our study showed the serum JCP-specific IgG4 level to be significantly correlated with the clinical efficacy of SLIT. We found that serum JCP-specific IgG4 cutoff levels greater than 989.5 UA/mL correlated with an effective clinical response to SLIT, with a sensitivity of 93.3% and a specificity of 94.7%. Studies for determining biomarkers that predict the efficacy of SLIT against Japanese cedar pollinosis are also very helpful and may improve daily QOL, but have not been sufficiently elucidated, especially for JCP-specific immunoglobulins. There have been very few studies that attempt to identify useful biomarkers for evaluating the efficacy of SLIT. Induction of allergen-specific IgG4 has long been regarded as the characteristic immunological feature induced by AIT [8], and several studies have shown that successful use of AIT might induce a substantial increase in allergen-specific IgG4 Abs [9,10]. Antigen-specific IgG4 works as a blocking antibody and is involved in the mechanism of SLIT, and serum Cry j 1-specific IgG4 is elevated after SLIT with JCe pollen extract in association with an increase in IL-10-expressing T cells or B cells [11,12]. Our results were in agreement with these studies: the serum level of JCP-specific IgG4 increased significantly in the SCIT group (p < 0.05) and the SLIT Successful group (p < 0.05). Moreover, the serum level of JCP-specific IgG4 rose significantly higher in the SCIT group than that of each of the SLIT groups (p < 0.05). The immunological effects of SLIT are weaker than those of SCIT [13], so this maybe one of the causes of our result. It is reported that the serum level of JCP-specific IgG4 increased significantly to five times the baseline level after the 2011 JCe pollen season, and this level gradually decreased up to the end of the second season, but still remained higher than at the baseline [3]. It is also reported that JCP-specific IgG4 levels increased significantly after five months of SCIT, with the level gradually increasing if SCIT was continued [14]. There is, therefore, the potential to maintain the serum level of JCP-specific IgG4 at a high level by continuing treatment. AIT has been shown to reduce IgE production [15,16]. It is reported that the JCP-specific IgE level decreased significantly after 17 months of SCIT. However, a 2-year JCe pollen-based SLIT trial showed the JCP-specific IgE titer to be higher after SLIT than the baseline at the end of the first and second JCe pollen season. Similarly, it was found that the serum Cry j1-specific IgE was significantly higher in the SLIT group than in the non-SLIT group after one or two years of SLIT. In our study, the JCP-specific IgE level of the SCIT group was significantly lower than that of the SLIT group (p < 0.05). Moreover, it is reported that the JCP-specific IgE level increased significantly by up to twice the baseline level two months after the start of treatment; the elevated level of JCP-specific IgE persisted until the end of the first pollen season; in the second season, this level decreased but, at the end, it was still higher than at baseline [3]. It is therefore possible that the JCP-specific IgE level might decrease if the trial period were extended. Serum IL-17A, SCCA2, periostin, saliva, and pathogenic Th2 cells have been reported as potentially useful clinical biomarkers for evaluating the efficacy of SLIT for JCP [17,18,19]. Periostin is highly expressed in chronic inflammatory diseases like asthma, chronic rhinosinusitis, atopic dermatitis, and allergic conjunctivitis [20], and has emerged as a useful biomarker of allergic diseases. It is reported that high serum periostin levels (>30.2 ng/mL) are associated with an effective response to house dust mite SLIT, and the degree of improvement as measured by the Rhinoconjunctivitis Quality of Life Questionnaire was correlated with the level of serum periostin [21,22]. In our study, there was no significant difference among serum levels of periostin among the SCIT group, the SLIT Successful group, or the SLIT Unsatisfactory group. SCCA2 is involved in the pathogenesis of several inflammatory diseases: asthma, psoriasis, and atopic dermatitis. It is reported that SCCA2 is useful in aiding diagnosis, estimating clinical severity and disease type, and assessing responses to the treatment of psoriasis and atopic dermatitis. These results suggest that SCCA2 has emerged as a novel biomarker for skin inflammatory diseases [22,23]. The usefulness of SCCA2 as a biomarker for SLIT, however, has hitherto not been evaluated. In our study, there was also no significant difference in SCCA2 among the three groups, as was the case with periostin.Our study has several limitations. First, it was retrospective and contained only a small number of subjects. Our study is based on cases from our own facility and from other facilities, with a total of fifteen; however, it would require more cases, especially in the SLIT Successful group, to evaluate the results more accurately. Second, the amount of pollen released and climatic conditions vary each year. In 2014, the airborne pollen count was lower than usual, possibly resulting in fewer patients in the pre-symptomatic state group and the SLIT Successful group. Third, the duration of the SLIT experiment was markedly shorter than that of the SCIT experiment. The SCIT group was treated for three years, but the SLIT group was treated for only a year because the formulation of SLIT that we used this study had been approved only a year before we embarked on this study. Further studies with serial evaluations of JCP-specific IgE, IgG, and IgG4, including measurements during the JCe pollen release season over several years, are needed to confirm our results. Our results may allow us to speculate that JCP-specific IgG4 plays an important role in the underlying mechanism of SLIT and could act as a predictive marker for clinical remission after SLIT treatment.The roles of each of the authors in the preparation of this manuscript are as follows. S.K., A.Y., Y.O. (Yukiko Ogawa), Y.S., T.S., and H.K. worked on data collection. S.I., J.O., R.I., S.K., and T.E. worked on statistical data analysis. K.I., Y.O. (Yoshitaka Okamoto), and N.O. provided insightful comments and suggestions on the manuscript. N.O. is the guarantor of the integrity of the content of the manuscript. All authors have read and agreed to the published version of the manuscript.This study was funded by JSPS Kakenhi Grant Number JP17K11363 and the Ministry of Health, Labor, and Welfare of Japan.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Tohoku Medical and Pharmaceutical University (protocol code 2016-2-2-054 and date of approval 2 February 2016).Informed consent was obtained from all subjects involved in the study.Data is contained within the article.The authors declare no conflict of interest.Serum concentrations of JCP-specific IgE (A), IgG (B), IgG4 (C), periostin (D), and SCCA2 (E). * p < 0.05, N.S.: not significant (Mann–Whitney U test).ROC curves obtained with serum JCP-specific IgE (A) (decision point, ≤100.3 UA/mL; sensitivity, 66.7%; and specificity), 61.3%, IgG (B) (decision point, ≤11.6 UA/mL; sensitivity, 60.0%; and specificity, 72.0%), IgG4 (C) (decision point, ≤989.5 UA/mL; sensitivity, 93.3%; and specificity, 94.7%), periostin (D) (decision point, 89.5 ≤UA/mL; sensitivity, 28.6%; and specificity, 80.0%), SCCA2 (E) (decision point, ≤0.550 UA/mL; sensitivity, 53.6%; and specificity, 50.7%) by plotting data for them with an effective response to SLIT versus 1-specificity inpatients who showed an ineffective response to SLIT.Clinical characteristics.Chronic rhinosinusitis with nasal polyp, CRSwNP ICS; eastern area of Japan, eastern; western area of Japan, western; ns: not significant.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Nuts are considered healthy foods due to their high content of nutritional compounds with functional properties. However, the list of the most allergenic foods includes tree nuts, and their presence must be indicated on food labels. Most nut allergens are seed storage proteins, pathogenesis-related (PR) proteins, profilins and lipid transfer proteins (LTP). Nut allergenic proteins are characterized by their resistance to denaturation and proteolysis. Food processing has been proposed as the method of choice to alter the allergenicity of foods to ensure their safety and improve their organoleptic properties. The effect of processing on allergenicity is variable by abolishing existing epitopes or generating neoallergens. The alterations depend on the intrinsic characteristics of the protein and the type and duration of treatment. Many studies have evaluated the molecular changes induced by processes such as thermal, pressure or enzymatic treatments. As some processing treatments have been shown to decrease the allergenicity of certain foods, food processing may play an important role in developing hypoallergenic foods and using them for food tolerance induction. This work provides an updated overview of the applications and influence of several processing techniques (thermal, pressure and enzymatic digestion) on nut allergenicity for nuts, namely, hazelnuts, cashews, pistachios, almonds and walnuts.Currently, the main cause of anaphylactic reactions in Western countries is food allergies. It has been estimated that food allergies affect between 1 and 3% of the general population and up to 8% of children. Food allergies cause more than 30,000 anaphylactic reactions in the US [1]. In Europe, food allergies are the leading cause of anaphylaxis, and between 10 and 18% of anaphylactic reactions occur at school [2]. The list containing the 14 most allergenic foods in the European Union includes peanuts and tree nuts. In accordance with Regulation (EU) No. 1169/2011, the presence of nuts must be indicated on food labels [3]. After fruit, peanuts and tree nuts are the most prevalent cause of allergic reactions in Spain (26%) [4]. However, nuts are increasingly consumed in the last years due to their health benefits, which are attributable to their high content of protein, unsaturated fatty acids, vitamins and antioxidants [5].Nut allergen proteins belong to seed storage proteins, such as legumin (11–13S globulin composed of acidic subunits of 30–40 kDa and basic 15–20 kDa), vicilin (7S globulin of approximately 50–60 kDa) and 2S albumin (15 kDa) [6]. Nut allergenic proteins are characterized by their resistance to denaturation and proteolysis [7]. Other nut allergens, such as pathogenesis-related (PR) proteins, profilins and lipid transfer proteins (LTP), are considered to be panallergens because they contribute to the allergenicity of a large group of seeds, pollen, nuts, fruit and other plants [8]. Food processing is used in the industry to ensure safety and to enhance organoleptic properties, in addition to altering food allergenicity. Food processing can modify the structure, properties and function of proteins, and as a result, the IgE-binding capacity of allergens can be affected. As some processing treatments have been shown to decrease the allergenicity of certain foods, food processing may play an important role in developing hypoallergenic foods and using them for food tolerance induction. Other processes, however, can increase the allergenicity of some foods [9]. Heat treatment modifies the structure of proteins, and therefore, epitopes and their immunogenic potential can be affected. This effects depends on both technological parameters and the type of matrix [10,11]. The effect of processing on allergenicity is variable and, as such, new allergenic compounds can be generated, while existing reactive epitopes can also be damaged or destroyed [12,13,14,15]. The structural changes produced using treatments such as boiling, microwave heating and pressure-cooking, and their effects on legume and nut allergenicity, have been evaluated. Importantly, findings indicate that heat- or pressure-based processing reduces IgE-binding capacity [12,13,14,15,16,17,18]. Overall, the effects of processing methods on mitigating or aggravating allergies are largely unknown. This review collected data published between 2010 and 2020 regarding the effects of processing, with and without heating, on allergens from several tree nuts. This review attempts to provide an updated overview on how conventional and novel processing methods influence the immunoreactive potency of allergenic proteins in the most frequently consumed nuts: cashews, pistachios, hazelnuts, almonds and walnuts. Nuts are a rich source of protein and other nutritional compounds with functional properties. This promotes their presence in manufactured foods. In Europe, tree nut allergies are common [19], with a hazelnut allergy being the most prevalent tree nut allergy. In the US, peanuts and tree nuts, such as almonds, walnuts or cashews, seem to be more common allergenic sources [20]. Table 1 summarizes the main nut allergens. Several hazelnut proteins have been described as allergens: Cor a 1 (Bet v 1 homologue), Cor a 2 (profilin), Cor a 8 (lipid transfer protein (LPT)), Cor a 9 (11S legumin), Cor a 11 (7S vicilin), Cor a 14 (2S albumin) and the oleosins Cor a 12, Cor a 13 and Cora a 15 [21]. Ana o 1 (7S vicilin) [22], Ana o 2 (11S legumin) [23] and Ana o 3 (2S albumin) have been identified and characterized as cashew allergens [24]. Pistachios are also well characterized for their allergenic potential and display high cross-reactivity with cashews and mangoes [25]. The five major allergens in pistachios are one 2S albumin (Pis v 1), two 11S legumins (Pis v 2 and 5), one 7S vicilin (Pis v 3) and one superoxide dismutase (Pis v 4) [25,26,27,28]. Most of the epitopic regions of Pis v 1 and Pis v 3 showed a high degree of similarity with the Ana o 1, Ana o 2 and Ana o 3 epitopes. This is considered to be the molecular basis for the IgE-binding cross-reactivity observed between pistachios and cashews [29]. In almonds, six allergenic proteins have been characterized: Pru du 3 (LTP), Pru du 4 (profilin), Pru du 5 (60S ribosomal protein), Pru du 6 (11S legumin), Pru du 8 (antimicrobial seed storage protein) and Pru du 10 (mandelonitrile lyase 2) [30]. Thus far, five allergenic proteins have been identified in walnuts: Jug r 1 (2S albumin), Jug r 2 (7S vicilin), Jug r 3 (LTP), Jug r 4 (11S legumin) and Jug r 5 (profilin) [31]. The major allergen in walnuts is Jug r 4, which is highly homologous with other 11S globulin allergens, such as Cor a 9 (hazelnut), Ana o 2 (cashew) and Ara h 3 (peanut), explaining their IgE cross-reactivity [31].A study reported that the prevalence of tree nut allergies in the US was greater than 1.1% and was more common in individuals under 18 years of age [20]. In 2005, the European Commission funded a large Europe-wide research project (EuroPrevall) designed to evaluate and provide a broad overview of the prevalence, basis and cost of food allergies. For this project, 56 partners from 19 European countries collaborated [32]. Studies involved community surveys, birth cohort studies and clinical studies using double-blind placebo-controlled food challenges (DBPCFC) and SPT. The project provided knowledge about the prevalence of food allergies, as well as the ranking of allergenic foods (food groups) as a function of the number of reactions they provoke both in the overall population and in specific population groups (regarding age and geographic location). In this context, Lyons et al. [33] reported that the highest prevalence of nut allergies was estimated for hazelnut (4%) in accordance with challenge tests and sensitization assessed by SPT. Nut allergies appear to affect adults and adolescents more, probably due to their late introduction into the diet [34]. Hazelnuts are widely consumed in Europe and presented a high prevalence of positive reactions in a double blind, placebo-controlled food challenge (DBPCFC) [19]. In the US, 7.5% of the total of 188 participants were found to be allergic to hazelnuts in a 11-year follow-up study [35]. The effects of variety showed no influence on the allergenicity of hazelnuts, with Cor a 9 and Cor a 1 being the predominant IgE-binding proteins in 13 European varieties [36]. Cashews are the second most allergic nut and a significant health problem in the US [37]. The anaphylactic reactions to cashew are, often, severe clinical manifestations, and even more dangerous than with peanuts. Cashew nuts are consumed all over the world due to their beneficial effects on human health, but they have also been reported to cause allergic reactions in sensitized patients [38]. In a study by Rance et al. [39], it was concluded that 2-year-old infants are more at risk among children sensitive to cashew nuts. The reactions were triggered in three-quarters of cases at first exposure. A possible explanation for this finding is that there is a correlation between earlier exposure to cashew nuts and a greater cashew nut allergy [40]. The symptoms upon the ingestion of cashews were also reported to be more severe compared to those caused due to exposure to other food allergens, such as nuts and peanuts [38]. Pistachio nuts are widely consumed all over the world and primarily produced in Iran and the United States. However, they have also been observed to cause significant allergic reactions in people sensitive to the allergens Pis v 1, Pis v 2 and Pis v 3 [41]. Sicherer et al. [35] estimated an allergy to pistachios in approximately 10% of the individuals evaluated. Almonds can provide many health benefits due to their low glycemic index and being a source of vitamin E and energy, manganese, fiber, protein and various polyphenolic components [5]. Almonds are ranked third after walnuts and cashews in eliciting allergic reactions [35], although some almond-allergic patients tend to pass oral food challenges, probably because many profilin-sensitized patients do not exhibit symptoms [30]. Amandin, or Almond Major Protein, is primarily responsible for IgE-mediated immunoreactivity. Walnuts are considered to be responsible for the highest number of allergic reactions in sensitized subjects [31], but they have health benefits, such as reducing the risk of diabetes and cardio-vascular diseases [5].The reasons for consuming processed foods include ensuring preservation and safety; improving quality, such as flavor, color and taste; convenience; variety; out of season availability; and lack of equipment, time or skills needed for home food preparation of certain foods. A significant quantity of food is processed at home by consumers, in addition to industrial food processing and in institutional settings. The type of processing method can be chosen by product type, scale of processing, available infrastructure, consumer preferences and product sensory qualities. These reasons explain that the same raw food can be often processed differently [42]. As the majority of foods are usually consumed after processing, it was also relevant to understand the protein characteristics which are influenced by processing, such as stability against heat and pressure, as well as mechanical and chemical activities [43]. Thermal processing methods, such as boiling, frying, roasting, baking, pressure cooking or microwave heating, are applied to certain food products before consumption to improve their suitability for specific applications [44]. Non-thermal food processing, such as high hydrostatic pressure (HHP) or enzymatic treatment, can be applied to some foods. These processing treatments can modify the biochemical characteristics of proteins or generate chemical reactions within the food matrix components.Thermal and non-thermal processing methods are applied to foods to improve their preservation, quality, safety and suitability for specific product applications. The processing can affect the solubility, digestibility and other related parameters. Through thermal treatments, proteins can form oligomers, or become aggregated, denatured, fragmented or re-assembled, and these modifications can produce a decreased solubility [45]. The overall IgE-binding capacity of a particular extract can be more or less antigenic or result in new allergens (neoallergens) as a consequence of heat processing [46]. Thermal processing analysis is, therefore, necessary for assessing the allergenicity of existing and newly introduced foods [11]. The influence of heat processing mainly depends on the temperature and time conditions used. In addition, interactions with other food matrix constituents can affect the structure of a protein. Generally, the loss of a secondary structure occurs when the temperature is around 70–80 °C, while at 80–90 °C, rearrangements of disulfide bonds and the formation of new bonds take place. Aggregation occurs at higher temperatures (90–100 °C) [47].The influence of a wide variety of treatments on the allergenicity of nuts has been studied (Figure 1, Table 2) [44,48,49]. Thermal treatments in walnuts [17]; HHP in hazelnuts [50]; and roasting, blanching, autoclaving and microwave heating in almonds [51] have been analyzed, and the results differ depending on the material and the conditions of the treatments studied. Studies with walnuts have indicated that the digestibility of protein probably increases after heat treatment, so the absorption of the protein can also increase in the gastrointestinal tract, and due to this, the possibility of allergens eliciting an allergenic response decreases [52]. However, in some cases, thermal processing may cause some neoantigens that were not originally present to form or the digestibility of a particular allergen may be reduced. These neoantigens may present an additional problem, and these facts can enhance the allergenic manifestation in sensitized patients. The formation of some neoantigens can be produced by the Maillard reaction when sugar residues interact with proteins during heating, generating sugar-conjugated protein derivatives, which enhance the allergenicity of some proteins, such as 2S albumins [53]. However, glycation and aggregation from the Maillard reaction reduced the Ig E binding capacity of legumins and did not affect the IgE-binding capacity of vicilins and nsLTP [43] The IgE recognizes and interacts with epitopes belonging to allergenic proteins. Two types of IgE binding epitopes are possible, either linear or conformational ones. In linear epitopes, amino acid is arranged in linear order in the polypeptide chain, while in the conformational epitopes, amino acids are far apart in the primary sequence but may come together during the folding of the polypeptide chain. Linear epitopes may be more problematic as compared to the conformational ones, as they are mostly resistant to heat treatment. Thermal processing mainly affects conformational epitopes as the bonds can be broken down due to heat. Refolding allows the formation of native conformational epitopes, but few new allergens may be formed, which requires further efforts to minimize the risk associated with neoantigens [54]. Thus, thermal processing can strongly alter the structure, function and allergenicity of foods. Non-thermal processing includes a large number of processing techniques without heating the food to produce modifications in the product. There are a wide variety of processes which induce a change in the conformation structure of the proteins that fall under this category: enzymatic digestion, high hydrostatic pressure, ultrafiltration, fermentation, gamma radiation, pulsed ultraviolet light, ultrasound, etc. (Figure 1). In gamma radiation or pulsed ultraviolet light, the internal energy of molecules can increase when a high dosage of radiation is applied, which can be translated into increased temperatures. The effects of enzymatic hydrolysis on the allergenicity and digestibility of food proteins have been widely reported. Enzymatic hydrolysis under sonication and autoclaving separately resulted in a significant decrease in the IgE-binding capacity of cashew and pistachio nuts (Table 2). Pistachio allergens were more affected by these treatments. However, enzymatic digestion combined with heat was necessary to drastically reduce the IgE-binding capacity of cashew allergens. Highly effective simultaneous processing conditions to abolish the allergenic potency of cashew and pistachio nuts have been proposed [14]. It is important that the evaluation of different enzyme activities to reduce allergenicity is carried out with sera from patients with documented clinical allergy to the source food [64]. However, the use of sera from patients with documented allergies is not enough to address allergenicity. For allergenicity assessment, the serum needs to be combined with assays simulating in vivo allergic reactions (BAT, mediator release assays), although the most reliable assays involve oral food challenge [43]. Moreover, stability under gastric conditions has been regarded as a useful parameter for the identification of allergens [65,66], and in vitro assays for pepsin digestion were included in a 2001 FAO/WHO protocol for the allergenicity assessment of novel food proteins [67]. For the development of special formulations, an alternative to intact proteins is enzymatic protein hydrolysates designed to provide nutritional support to specific population groups, such as infants, elderly, and food-allergic patients. In addition, protein hydrolysates show technological advantages. Extensive enzymatic treatment combined with food processing treatments, such as heat and ultrafiltration, is considered highly effective to obtain protein products with an added high value for human nutrition and decreased allergenicity [14].High pressure alters the tertiary and quaternary structure of most globular proteins without influence on the secondary structure. Thus, high hydrostatic pressure can unfold proteins. The pressure needed for the unfolding range from 100 MPa to 1 GPa, being 500 MPa the most effective, although it varies from protein to protein [68]. The effect of HHP on allergenicity and changes in protein structure of immunoreactive proteins has been investigated in and in nuts such as hazelnut and almond [58]. Hazelnut allergens showed changes in solubility after processing at high pressure (300–600 MPa) for 15 min, although the immunoreactivity was not affected after HHP processing [50,58]. The same HHP conditions of pressure and time did not produce any change on almond immunoreactive proteins [58]. Most plant allergens are pressure stable since pressure processing methods (e.g., HHP) normally contribute to maintain the protein in its native-like state when compared with temperature processing. The IgE-binding capacity of nsLTP, profilins, vicilins and PR-10 is not affected by the application of high pressure, while for 2S albumins and legumins, it can be slightly reduced Combination of pressure-heat and pressure-heat enzymatic hydrolysis treatments is more efficient in reducing the IgE-binding capacity of nsLTP, legumins and vicilins, because pressure changes protein at conformational level making it more susceptible to enzyme activity and temperature [43] Hansen et al. [55] evaluated the effect of processing on hazelnuts, and they observed a reduction in allergenicity after roasting them at 140 °C for 40 min. However, 29% of the subjects showed allergic symptoms upon the consumption of roasted hazelnuts and, therefore, this reduction is not of clinical significance. Thus, for the population suffering from sensitization towards hazelnuts, especially Cor a 1 and Cor a 2, the consumption of roasted hazelnuts cannot be recommended as an alternate method [55]. Worm et al. [56] also reported the impact of roasting hazelnuts at 144 °C on allergic patients. They found that roasting might reduce the risk in most hazelnut-sensitized patients, although the hazelnut allergens are considered to be heat stable and are responsible for causing severe reactions in the sensitive population. Conventional thermal treatments between temperatures of 100 and 185 ° were also evaluated, indicating that hazelnut allergens, especially allergens of lower molecular weight (14 kDa), offer high resistance against thermal treatments [69] (Table 2). Lopez et al. [57] and Cuadrado et al. [58] analyzed the influence of autoclaving and high-pressure processing on hazelnut immunoreactivity. In this study, they concluded that autoclaving, especially at a temperature of 138 °C for 15–30 min decreased the allergens Cor a 1, Cora 8, Cor a 9 and Cor a 11. However, hazelnut allergens showed no reduction in immunoreactivity after processing at high pressure (300–600 MPa), although the protein solubility was affected after HHP processing [50]. The glycation reaction between the amino acid groups of protein with reducing sugars occurring in Maillard reactions was reported to be responsible for reducing the immunoreactivity of Cor a 11 allergen, but Cor a 1 and Cor a 9 were unaffected even after glycation in the presence of glucose at 70 °C [70].The characterization of IgE and IgG immunoreactive proteins from untreated and thermal-treated cashew samples was comparatively studied (Table 2) [14,15,58]. The results indicated that boiling for 60 min did not affect to the IgG-binding proteins from cashews. However, cashews subjected to autoclaving (heat under pressure) showed a reduction in IgG-reactive bands. A band probably corresponding to the Ana o 3 (13 kDa) [10] was especially immunoreactive. In the samples treated with heat and pressure, the IgE reactive bands were drastically reduced. This result cannot be explained by a potential loss in the solubility of proteins due to thermal treatments, since the experiments were carried out under strong protein solubilization conditions [58]. A combination of heat and pressure treatments (autoclaving) was able to decrease the IgE-binding properties of cashews. After autoclaving at 138 °C for 30 min, the IgG immunodetection of Ana o 2 and Ana o 3 was strongly diminished. The influence of other thermal pressured treatments, such as instant controlled pressure drop (DIC), on cashew allergenic capacity was evaluated [62]. The extreme conditions of DIC (7 bar, 120 s) strongly reduced the immunodetection of allergenic proteins when IgE sera from sensitized patients were used for Western blots. The number of IgE-immunoreactive proteins was reduced by 67.2% [62]. Such reduction in immunodetection had a greater effect on pistachios (75%) than cashew nuts, but was not totally eliminated. Therefore, cashew proteins are probably more resistant than pistachio proteins. The observed degradation of proteins after extreme heat/pressure treatments obtained was similar to the degradation produced by some enzymatic treatments in our previous findings [14]. Enzymatic digestion for 2 h under sonication with Protease P 3DS (Amano) reduced the number of IgE-binding protein bands recognized by the sera of cashew-allergic patients. The recognition of Ana o 2 was almost eliminated, but some digestion-resistant proteins were detected by 50% of the tested sera and Ana o 3 was still recognized by IgE in one patient [14]. A more effective method to reduce the allergenic reactivity of cashews was the combination of both enzymatic hydrolysis under sonication and thermal treatment. Although cashew proteins showed high resistance to all processing methods used, autoclaving or a combination of γ-irradiation and autoclaving is able to cause a reduction in allergen detection [60,61]. Mattison et al. [59] found that sodium sulfite and heating treatment can alter the structure of specific cashew allergens, decreasing their IgE-binding potency. The stability of cashew allergens to in vitro digestion has been studied and identified, and Ana o 3 IgE-binding epitopes are the most resistant [71]. The same authors also evaluated the solubility of cashew proteins by SDS-PAGE and LC-MS/MS. They found that it was modified by heat treatment, and the relative amount of peptides from specific cashew allergens was also affected as well as the IgE-binding capacity of the extracts [72]. Oleic acid has been found to reduce the IgE-binding capacity of cashew allergens [73,74]. In vitro studies are important preliminary tests to ensure a possible reduction in IgE cross-linking capacity, before performing further clinical studies. In vivo clinical relevant experiments—such as SPT and mediator release assays (MRA), in which the IgE cross-linking capacity of processed food proteins is analyzed in effector cells of allergy—constitute an essential part on the research of allergenic properties of processed food since an altered ability of food allergens to bind IgE using traditional in vitro immunoassays is not always directly related to a modified allergenic function [15]. The effects of dry roasting and steaming on the allergenicity of pistachio protein were studied (Table 2). The authors reported that the steaming reduced the reactivity of the pistachio allergens compared to dry roasting methods. Pistachios were soaked for 12 h prior to any processing in a solution containing lemon water (pH 3.2–3.2) and sodium chloride (1.6% w/v). They concluded that the ionic strength of the soaking solution in combination with steaming might modify the secondary structure of the protein, resulting in reduced reactivity. They found no significant difference in various sensory attributes, including aroma, color, flavor, taste and overall acceptability [63].Recently, it has been demonstrated that there is an important reduction in 11S and 2S protein detection by autoclave treatment at 138 °C for 30 min. In contrast, LTP was even detected after autoclaving under the same conditions. The IgE binding of pistachio proteins decreased by 73% after boiling, and the lowest detection was found under the hardest autoclave conditions [58]. Similarly to cashews, the IgE immunoreactivity of pistachios was strongly decreased after heat treatment under high pressure (autoclaving at 256 kPa), but not with autoclaving at 120 kPa or boiling, which has been confirmed by SPT and MRA experiments [15], indicating that pistachio autoclaved at 256 kPa for 30 min showed an strong reduction in allergenic potency. These results are also in concordance with our previous findings for other tree nuts and legumes [12,13,15,16,17]. Similarly to cashews and peanuts, the influence of an instant controlled pressure drop (DIC) on pistachio IgE-binding capacity was evaluated, and the data indicated that the IgE immunodetection of allergenic pistachio proteins was markedly reduced under the harshest conditions of DIC (7 bar, 120 s) [62]. Such reduction in immunodetection is more effective in pistachios (75%) than in cashew nuts (67.2%), but is not completely eliminated. According to these findings, instant controlled pressure drop (DIC) can be considered to be an adequate technique for obtaining hypoallergenic pistachio flour for use in the food industry. The effect of enzymatic hydrolysis on pistachio allergens was more effective than that on cashew allergens according to our previous results [14]. Most IgE-binding proteins from untreated and processed pistachio samples were hydrolyzed with Protin (E5) after 1 h of sonication. Such assays included the enzymatic digestion of total protein from whole nut paste (as opposed to soluble extract) under sonication (ultrasound disruption). The whole nut paste was prepared by mixing pistachio defatted flour with distilled water (0.5 g/mL). In this case, the IgE immunoreactivity of pistachios was almost eliminated after the enzymatic digestion of raw and thermally treated samples. The results of protein identification by MS analysis showed that, after the enzymatic treatment, the allergens were degraded due to the thermal treatment and enzymatic digestion. The enzymatic digestion of thermally treated samples produced few resistant peptides, indicating that some fragments of allergenic pistachio and cashew proteins survive even after heat and enzymatic treatments. According to Cuadrado et al. [14], enzymatic hydrolysis has a greater effect on autoclaved cashew and pistachio samples compared to untreated cashew and pistachio samples.Roux et al. [75] investigated the stability of amandin in different almond cultivars, and the results showed a reduction of approximately 40% in reactivity in different blanched and dry roasted cultivars in comparison to unprocessed almonds, but this reduction is not clinically relevant [75]. Venkatachalam et al. [51] also reported the effects of autoclaving, roasting, blanching and microwave processing on amandin. Autoclaving and microwave processing methods were ineffective, which showed that amandin was heat stable towards thermal processing methods, which are normally employed in food industries [76]. Only pulsed UV light treatment produced changes in the surface properties of the protein, which can reduce the viable binding sites for IgE when entering the human body, probably due to protein fragmentation as a result of the photo-thermal effect [77]. Cuadrado et al. [58] demonstrated that after autoclaving at 138 °C for 30 min, the IgG immunoreactivity of Pru du 6, Pru du 2S and Pru du 3 is strongly diminished. They showed that thermal and pressure treatment combined in autoclaving was able to reduce the IgE-binding properties of almonds, but HHP up to 600 MPa did not affect almond immunoreactivity, in agreement with Costa et al. [43], who reported that most plant allergens are pressure stable. (Table 2). Different studies have found that walnut allergenic proteins are highly resistant to processing and do not show any reduction in IgE reactivity (Table 2) [78]. Downs [52] also evaluated the effects of roasting on walnut protein, and the data indicated that the digestibility of 11S legumin and 7S vicilin increased after processing, which can be attributed to secondary structure modifications in the protein and, in some cases, to lower immunoreactivity. Barre et al. [79] concluded that the secondary structure of Jug r 1 allergen is stable under thermal treatment up to 90 °C. Cabanillas et al. [17] evaluated the effect of heat treatments in combination with high-pressure processing on walnuts. It was demonstrated that pressure treatment at 256 kPa and 138 °C effectively reduced the IgE-binding capacity of walnut proteins, whereas pressure treatments (up to 600 MPa) without heating did not affect walnut immunoreactivity, although pressure-treated walnut proteins showed higher susceptibility to digestion. In recent years, an increase in food allergies around the world, especially sensitivity to tree nuts, has been reported. This review provides an updated overview of the effect of processing with and without heating on the immunoreactive potency of allergenic proteins of the most widely consumed nuts. Although some processing methods have achieved promising results in reducing the IgE reactivity of nuts, the relevance of these results at the clinical level is still unclear. It is highly important to understand how processing affects the structural properties of allergenic proteins and its relationship with changes in allergenicity (aggravating or mitigating). The most common feature promoting plant protein allergenicity is molecular stability, related to structural resistance to heat and proteolysis. Therefore, it is critical to analyze how the degree of processing can modify digestibility, solubility and other parameters related to IgE reactivity depending on the type of protein. Legumins and cereal prolamins are less reactive when undergoing protein aggregation, while the same phenomenon leads to an increase in 2S albumin reduction. Certain processing methods can alter some allergenic proteins, resulting in the destruction of the epitopes, but they are unaltered in others. In this way, processing can modify the overall IgE binding profiles of nut proteins. However, as in vitro assays are only an indicator of in vivo allergenicity, further in vitro analysis and in vivo clinical data are required to confirm that these treatments can effectively reduce the in vivo allergenicity of these nuts. These putative hypoallergenic foods could be safely consumed and even utilized as desensitizing food only after such studies.Conceptualization, C.C.; methodology, Á.S. and C.C.; investigation, Á.S., R.L. and C.C.; formal analysis, Á.S. and C.C.; resources, C.C.; data curation, Á.S., R.L. and C.C; writing—original draft preparation, C.C.; writing—review and editing, R.L. and C.C.; supervision, C.C.; project administration, C.C.; funding acquisition, C.C. All authors have read and agreed to the published version of the manuscript.This research was funded by the Spanish Ministerio de Ciencia e Innovación, grant number AGL2017-83082-R. The authors declare no conflict of interest.Summary of food processing methods and their applications in the reviewed nuts.Allergens in nuts (WHO/IUIS Allergen Nomenclature Subcommittee).MW*: molecular weight.Effects of processing on IgE immunoreactivity of nut allergens.* IgE reactivity determined by different techniques and conditions. Pictography: =, ↓, ↑, ~, are a symbolic representation of the global effect of the specific treatment on the IgE reactivity of a given food (=, similar; ↑, increase; ↓, decrease; ~ ↑, slight increase; ~↓, slight decrease).Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Syo-seiryu-to (SST) is a traditional herbal medicine that has been used clinically to treat allergic rhinitis (AR) in Japan. SST improves acute symptoms, such as sneezing and rhinorrhea, as well as chronic symptoms, such as nasal obstruction, in patients with AR. However, its therapeutic mechanisms remain unknown. We examined the effects of SST and eight constituent crude drugs on phorbol 12-myristate-13-acetate (PMA)-induced gene up-regulation of IL-33 and histamine H1 receptor (H1R), which are responsible for the pathogenesis of AR. We found that SST and its crude drugs, except for Pinellia tuber, significantly and dose-dependently suppressed PMA-induced both IL-33 and H1R mRNA up-regulation in vitro. The half-maximal inhibitory concentration values of the seven crude drugs to inhibit PMA-induced IL-33 mRNA up-regulation were correlated with those related to H1R mRNA up-regulation, suggesting that they act on a common signal molecule. These results suggest that SST improves nasal congestion that is induced by IL-33-related eosinophil infiltration and inhibits sneezing and rhinorrhea that are induced by H1R-mediated histamine signaling in the nasal mucosa of AR patients through its inhibition of a common molecule in the gene expression pathways of IL-33 and H1R. The results could explain the advantages of traditional herbal medicine, in which mixing various crude drugs not only acts on a common target to enhance its pharmacological action, similar to the effect of a high concentration of a single crude extract but also has the benefit of reducing the side effects of each crude drug.Syo-seiryu-to (SST) is a traditional herbal medicine that has been used clinically in Asian countries including Japan for the treatment of allergic diseases such as allergic rhinitis (AR) and asthma [1]. SST improves acute symptoms, such as sneezing and rhinorrhea, as well as chronic symptoms, such as nasal obstruction, in patients with AR. The anti-allergic activities of SST include inhibition of histamine release from rat peritoneal mast cells [2], reduction of serum IgE levels in AR patients [3,4], modulation of Th1- and/or Th2-cytokines in CD4+ T cells in mice [5,6,7], and inhibition of airway eosinophil infiltration and blood eosinophil counts in mice [8,9]. Previously, we reported that SST suppressed nasal symptoms and gene expression of histamine H1 receptor (H1R), histidine decarboxylase (HDC), and Th2-cytokines including interleukin (IL)-4 and -5, in the nasal mucosa of AR model rats [10]. However, these pharmacological effects of SST reported are mainly for acute symptoms, and the underlying mechanisms for the chronic symptoms of SST remain to be elucidated.The IL-33 gene was identified as a gene responsible for asthma in two large international joint studies [11,12]. IL-33 is expressed in Th2 cells, mast cells, basophils, and eosinophils. Since IL-33 stimulates these cells to produce Th2-cytokines, such as IL-5 and IL-13, it is suggested that IL-33 plays an important role in allergic reactions [13,14,15,16,17]. Recently, it was reported that levels of IL-33 in the blood and gene expression of IL-33 in the nasal mucosa of AR patients were elevated compared to those in healthy participants [18,19]. Therefore, it is suggested that IL-33 up-regulation is involved in the development of AR [20,21].Histamine is a key chemical mediator of AR. IgE-mediated histamine release from mast cells in response to allergens stimulates H1R on nasal trigeminal nerve endings to cause nasal symptoms including sneezing and watery rhinorrhea in AR patients [22]. Levels of H1R mRNA have been reported to be up-regulated in the nasal mucosa of patients with AR [23,24]. In our previous study, we found that histamine increased the expression of H1R mRNA in HeLa cells [25,26] and that the gene expression levels of H1R in the nasal mucosa were correlated with the severity of nasal symptoms in AR patients [27,28]. Taken together, these results suggest that IL-33 and H1R gene up-regulation in the nasal mucosa leads to the exacerbation of nasal symptoms in patients with AR.In the present study, to clarify the anti-allergic actions of SST, we first examined whether SST suppresses PMA-induced IL-33 and H1R mRNA up-regulation in Swiss 3T3 cells and HeLa cells, respectively, because phorbol 12-myristate-13-acetate (PMA) increases the expression levels of IL-33 mRNA in Swiss 3T3 cells [29] and H1R mRNA in HeLa cells [25]. Since SST is composed of eight crude drugs, we then examined whether each constituent crude drug of SST suppresses PMA-induced IL-33 and H1R mRNA up-regulation. We showed that SST and its crude drugs except for Pinellia tuber suppressed PMA-induced up-regulation of IL-33 mRNA and H1R mRNA. The half-maximal inhibitory concentration (IC50) values of the seven crude drugs to inhibit PMA-induced IL-33 mRNA up-regulation were correlated with PMA-induced H1R mRNA up-regulation.SST was kindly gifted by Tsumura and Co., Tokyo, Japan. Its components: Ephedra herb, cinnamon bark, processed ginger, Asiasarum root, peony root, Schisandra fruit, Glycyrrhiza, and Pinellia tuber were purchased from Tsumura. SST or its crude drugs (27 g each) was added to 375 mL of distilled water and stand for 1 h at room temperature. After that, the mixture was boiled for 2 h and then filtered twice to remove insoluble materials. The supernatant was centrifuged and the obtained supernatant was freeze-dried and kept at −30 °C until used. The freeze-dried extract was re-dissolved in water on the day of the experiments. The yield of freeze-dried SST extract was 59% (w/w) and those of crude drugs were dependent on the crude drugs and around 50–80% (w/w).Swiss 3T3 cells that endogenously expressed IL-33 were cultured in Dulbecco’s Modified Eagle’s medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and antibiotics (10,000 Units/mL penicillin G sodium, 10 mg/mL streptomycin sulfate salt) at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. After reaching approximately 90% confluence, the medium was replaced with starvation medium (FBS 0.5%) and cultured for an additional 24 h before mRNA determination. HeLa cells that endogenously expressed H1R were cultured in MEM-alpha medium (Gibco) containing 8% FBS (Sigma) and antibiotic-antimycotic (Gibco) at 37 °C and 5% CO2 incubator. After reaching about 90% confluence, the media was replaced with FBS-free media and cultured for an additional 24 h before being subject to the mRNA determination.Cells were pretreated with extracts from SST and each crude drug for 12 h before PMA treatment. After treatment with 100 nM PMA for 3 h, the cells were washed several times with Ca2+- and Mg2+-free PBS (PBS [–]) at 37 °C. The cells were harvested with 700 µL of RNAiso Plus (Takara Bio Inc., Kyoto, Japan), mixed with 210 µL of chloroform, and centrifuged at 15,000 rpm for 15 min at 4 °C. The aqueous phase, including RNA, was collected, and the RNA was precipitated by the addition of isopropanol. After centrifugation at 15,000 rpm for 15 min at 4 °C, the resulting RNA pellets were washed with 0.5 mL of ice-cold 75% ethanol at −20 °C. After centrifugation at 15,000 rpm for 15 min at 4 °C, diethyl pyrocarbonate (DEPC)-treated water (DEPC-water) was added to the resulting pellets to prepare RNA solutions. The total RNA concentrations and purity of each sample were measured using the spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Waltham, MA, USA). The RNA solution and DEPC-water were added to the sample tube so that the total RNA was equivalent to 1.0 µg. The total volume of the solution was set to 5 µL. Using the PrimeScript® RT reagent kit (Takara Bio Inc.), a reverse transcription reaction with a thermal cycler (T3000 thermocycler, Biometra, Göettingen, Germany) was performed to obtain cDNA.Reagents containing Premix Taq (Probe qPCR) (Takara Bio Inc.) were mixed to prepare 20 μL of reaction solution per well of a MicroAmp Optical 96-well Reaction Plate (Applied Biosystems, Foster City, CA, USA). The PCR reaction was conducted using a Sequence Detector (Gene Amp 7300 Sequence Detection System, Applied Biosystems), the amplification curve of the PCR product was detected in real-time and analysis and quantification were performed using Sequence Detection software. The sequences of the primers and probe were as follows: a forward primer for human H1R, 5′-CAGAGGATCAGATGTTAGGTGATAGC-3′; a reverse primer for human H1R, 5′-AGCGGAGCCTCTTCCAAGTAA-3′; and probe, FAM-CTTCTCTCTCGAACGGACTCAGATACCACC-TAMRA. Mouse IL-33 primers and a probe kit (Mm00505403_m1, Applied Biosystems) were used for the determination of mouse IL-33 mRNA levels. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard to correct for differences in RNA purity and reverse transcription efficiency, which are the main factors responsible for the fluctuation in quantitative RT-PCR. Theoretically, the expression of GAPDH does not change due to environmental conditions, such as cell activation or proliferation, and the expression level is considered to be always constant. The human GAPDH primer and probe kit (Pre-Developed TaqMan Assay Reagents Control Kit (human GAPDH), Applied Biosystems) was used to determine the human GAPDH mRNA levels. Mouse GAPDH primer and probe kit (TaqMan Rodent GAPDH Control Reagent, Applied Biosystems) was used to determine the mouse GAPDH mRNA levels. Several experiments were conducted using cells with different passage numbers and determined the suitable dose to calculate IC50 values. The final data were obtained from the latest experiments using the cells with the same passage number and used for statistical analysis.The results are shown as means ± SEM. Data were analyzed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). A one-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical analysis. Statistical significance was set at p < 0.05.Stimulation with PMA significantly increased IL-33 mRNA levels in Swiss 3T3 cells (Figure 1A). The hot water extract of SST significantly and dose-dependently suppressed PMA-induced up-regulation of IL-33 mRNA in Swiss 3T3 cells (Figure 1A). H1R gene up-regulation in response to PMA was observed in HeLa cells (Figure 1B). The hot water extract of SST significantly and dose-dependently suppressed PMA-induced up-regulation of H1R mRNA in HeLa cells (Figure 1B).SST is composed of eight crude drugs: Ephedra herb, cinnamon bark, processed ginger, Asiasarum root, peony root, Schisandra fruit, Glycyrrhiza, and Pinellia tuber. Thus, we next investigated whether each constituent crude drug of SST suppresses PMA-induced IL-33 and H1R mRNA up-regulation in Swiss 3T3 cells and HeLa cells, respectively. The hot water extracts from Ephedra herb, cinnamon bark, processed ginger, Asiasarum root, peony root, Schisandra fruit, and Glycyrrhiza significantly and dose-dependently suppressed PMA-induced IL-33 mRNA up-regulation in Swiss 3T3 cells (Figure 2A–G). These extracts also significantly and dose-dependently suppressed PMA-induced H1R gene up-regulation in HeLa cells (Figure 3A–G). However, Pinellia tuber did not suppress PMA-induced up-regulation of IL-33 or H1R mRNAs (Figure 2H and Figure 3H).The IC50 values of Ephedra herb, cinnamon bark, processed ginger, Asiasarum root, peony root, Schisandra fruit, and Glycyrrhiza to inhibit PMA-induced IL-33 and H1R mRNA up-regulation were calculated (Table 1). The IC50 values of Ephedra herb, cinnamon bark, processed ginger, Asiasarum root, peony root, Schisandra fruit, and Glycyrrhiza to inhibit up-regulation of IL-33 mRNA were correlated with those related to up-regulation of H1R mRNA (r = 0.964, p < 0.01; Figure 4). These results suggest that the active compound in each crude drug acts on a common signal molecule involved in the pathways of IL-33 and H1R gene expression.We have shown that the protein kinase Cδ (PKCδ)/heat shock protein 90 (Hsp90)/ERK/poly(ADP-ribose)polymerase-1 (PARP-1) signaling pathway is involved in PMA-induced H1R gene up-regulation in HeLa cells [26]. We also reported that quercetin, (±)-maackiain, U0126, 17-(allyl-amino)-17-demethoxygeldanamycin (17-AAG), and celastrol suppressed H1R gene expression via PKCδ inhibition, disruption of the PKCδ–Hsp90 interaction, MEK inhibition, and the inhibition of Hsp90 ATPase activity, respectively [26,30]. MEK inhibition subsequently inhibited ERK activation. Since IL-33 mRNA was up-regulated in response to PMA, in the present study, we investigated the effect of these compounds on PMA-induced IL-33 gene up-regulation in Swiss 3T3 cells. These compounds suppressed PMA-induced up-regulation of IL-33 gene expression in Swiss 3T3 cells (Figure 5), suggesting that PKCδ, Hsp90, MEK, and ERK, which are involved in H1R gene expression signaling, are also involved in the IL-33 gene expression signal pathway.In the present study, we showed that SST suppressed PMA-induced IL-33 mRNA and H1R mRNA up-regulation in Swiss 3T3 cells and HeLa cells, respectively. We used Swiss3T3 cells and HeLa cells in this study. HeLa cells are not relevant cells to allergic reactions. We have not identified H1R-expressing cells in the nasal mucosa, however, it was reported that the expression level of H1R was marked in patients with nasal allergy than those with non-allergic rhinitis, and H1R-immunoreactivity was found in epithelial cells and vascular endothelial cells [31]. Therefore, HeLa cells are one of the candidates responsible for histamine-induced H1R gene up-regulation, because they are derived from cervical cancer cells arising in epithelial cells. We demonstrated that histamine/PMA stimulation up-regulated H1R gene expression and the PKCδ/ERK signaling pathway was involved in this gene up-regulation [25,26]. The involvement of PKC/ERK signaling in nasal epithelial cells was also reported [32,33]. From these results, it is considered that HeLa cells can be used for the mechanistic studies of H1R gene up-regulation. This is the reason why we used HeLa cells in this study although they are not representative of typical target cells involved in allergic reactions. It was reported that IL-33 was expressed in epithelial cells, endothelial cells, and fibroblasts [34]. Therefore, HeLa cells can be used for the investigation of IL-33 gene expression signaling. Indeed, IL-33 gene up-regulation was induced by PMA stimulation (data not shown). However, we have demonstrated that the expression level of IL-33 mRNA did not relate to that of H1R mRNA in the nasal mucosa of AR patients (29). Thus, it is likely that IL-33-expressing cells are different from the H1R-expressing cells. This is the reason why we used Swiss3T3 cells but not HeLa cells for the IL-33 study. We have shown that activation of the gene expression signaling pathways for H1R and IL-33 gene followed by the elevation of their mRNA levels are responsible for the pathogenesis of AR [27,28]. However, changes in mRNA levels are often not reflected at protein levels. We have reported that histamine or PMA stimulation increased H1R at both mRNA and protein levels in HeLa cells [25]. However, we have no data demonstrating the correlation between IL-33 mRNA level and IL-33 protein level. Further studies are required and are under investigation in our laboratory.Reportedly, IL-33 induces the production of IL-5, which is involved in eosinophil infiltration [13,35]. We previously showed that the expression level of nasal mucosal IL-33 mRNA was correlated positively with the number of blood eosinophils in patients who suffered from Japanese cedar pollinosis [29]. Moreover, Haenuki et al. reported that the infiltration of eosinophils in the nasal mucosa induced by ragweed pollen stimulation was suppressed in IL-33-deficient sensitized mice [19]. Since we previously reported that SST suppressed IL-5 mRNA up-regulation in the nasal mucosa of AR model rats [10], these results suggest that SST suppresses IL-5 mRNA up-regulation through the suppression of IL-33 mRNA up-regulation, leading to inhibition of eosinophil infiltration in the nasal mucosa. Since eosinophil infiltration in the nasal mucosa is associated with nasal congestion symptoms [22], it is further suggested that SST inhibits eosinophil infiltration in the nasal mucosa to improve nasal obstruction in patients with AR [3].SST also suppressed H1R gene up-regulation induced by PMA in HeLa cells. It was reported that nasal mucosal H1R gene expression was up-regulated in AR patients. Furthermore, we have shown that gene expression levels of H1R in the nasal mucosa were correlated positively with the severity of sneezing and rhinorrhea in patients with pollinosis [27]. We also reported that SST suppressed nasal mucosal H1R mRNA up-regulation as well as sneezing in AR model rats [10]. Since IgE-mediated histamine release from mast cells in response to the specific allergens stimulates H1R on nasal trigeminal nerve endings to cause nasal symptoms in AR patients [22], it is suggested that SST suppresses nasal mucosal H1R gene up-regulation to inhibit sneezing and watery rhinorrhea in patients with AR [3]. All these findings suggest that SST not only inhibits sneezing and rhinorrhea induced by histamine signaling in the acute phase reaction with its ability to inhibit the nasal mucosal H1R gene up-regulation but also improves nasal mucosal swelling induced by eosinophil infiltration in the chronic phase reaction with its ability to inhibit the nasal mucosal IL-33 gene up-regulation in AR patients. SST is composed of eight crude drugs, Ephedra herb, cinnamon bark, processed Ginger, Asiasarum root, peony root, Schisandra fruit, Glycyrrhiza, and Pinellia tuber. Among them, seven constituent crude drugs, except Pinellia tuber, suppressed PMA-induced both IL-33 and H1R mRNA up-regulation. As SST and its crude drugs target signaling molecules responsible for IL-33 or H1R gene expression signaling pathways, it is speculated that SST and its crude drugs suppress the basal mRNA level of these genes. In Figure 1A, SST (4.09 mg/mL) showed mRNA level below the control level, suggesting the suppression of basal mRNA expression. In crude drugs, Ephedra herb, cinnamon bark, processed ginger, peony root, Schisandra fruit, and Glycyrrhiza suppressed PMA-induced up-regulation of IL-33 and/or H1R mRNA expression below the control level (data not shown), suggesting these crude drugs also suppress the basal mRNA expression. We think that Asiasarum root also suppresses basal mRNA expression because 3 mg/mL of this crude drug suppressed PMA-induced up-regulation of IL-33 mRNA expression to the control level (1.17 ± 0.098 fold of the control) although we have no experimental data using over 3 mg/mL of extract from Asiasarum root. H1R and IL-33 genes were allergic-diseases sensitive genes and keeping these gene expression levels low are effective for improving nasal symptoms. In this context, this “inverse agonist-like activity” of SST and its crude drugs is very important to suppress basal mRNA levels of IL-33 and H1R.The IC50 values of the seven crude drugs to inhibit the IL-33 mRNA up-regulation were correlated with those related to the H1R mRNA up-regulation. We previously demonstrated that PKCδ/Hsp90/ERK/PARP-1 signaling pathway is involved in the H1R mRNA up-regulation [24,25]. Studies using inhibitors of the H1R gene expression signaling pathway suggest that PKCδ, Hsp90, MEK, and ERK, which are involved in H1R gene expression signaling, are also involved in the up-regulation of IL-33 gene expression signaling. Thus, it is suggested that the suppressive effect of SST and its seven crude drugs was through the inhibition of common signaling molecules involved in both IL-33 and H1R gene up-regulation. All these findings suggest that SST was combined with seven multiple crude drugs with a strategy of mixing their common effects of the suppression of both IL-33 and H1R gene expression, thus leading to additive anti-allergic actions with fewer side effects. According to the data in Table 1, it seems that much higher concentrations of SST are needed to inhibit PMA-induced upregulation of IL-33 and H1R when compared to the individual crude drugs, especially Ephedra herb and cinnamon bark. As the proportion of 8 crude drugs in SST are Ephedra herb (11%), cinnamon bark (11%), processed ginger (11%), Asiasarum root (11%), peony root (11%), Schisandra fruit (11%), Glycyrrhiza (11%), and Pinellia tuber (22%), 2.5 mg of SST contains approximately 0.25 mg each of Ephedra herb, Cinnamon bark. These amounts were the in same order of IC50 values of these crude drugs, suggesting that the contribution of these crude drugs to the suppressive effect of SST is high. Since the IC50 values of other crude drugs are large, it is considered that the contribution of these crude drugs to the suppressive effect of SST is low. In addition, since the hot water extract of the crude drugs contains many compounds that positively or negatively affect IL-33 and H1R gene expression, it is considered that the sum of these complex effects shows the suppressive effect of SST. From these facts, it is difficult to simply compare the suppressive effects of SST and crude drugs using only IC50 values.In addition, it was reported that Schisandra fruit, a crude drug of SST, that improves liver function and Glycyrrhiza, another crude drug, that has an anti-gastric ulcer effect can prevent the development of possible side effects from other crude drugs [36,37]. Pinellia tuber, the remaining crude drug of SST, did not affect the IL-33 and H1R gene up-regulation. However, it is possible that Pinellia tuber has other anti-allergic actions, because it was reported to suppress Th2 cytokines, such as IL-4, -5, and -13, as well as eosinophil infiltration, IgE, and histamine in the airway [38].In conclusion, the present results suggest that SST and its crude drugs alleviate both acute and chronic symptoms of AR by inhibiting both H1R and IL-33 gene expressions, mainly through inhibition of the common molecule in their gene expression pathways responsible for the pathogenesis of AR. In general, traditional herbal medicines are prescribed as mixtures of multiple crude drugs. Although the chemical composition and pharmacological profiles of the crude drugs are still not fully defined at present and, therefore, require further investigations, the results could explain the advantages of traditional herbal medicine, in which mixing various crude drugs not only acts on a common target to enhance its pharmacological action, similar to the effect of a high concentration of a single crude extract but also has the benefit of reducing the side effects of each crude drug.Conceptualization, H.M., H.F. and N.T.; investigation, S.N. (Seiichi Nakano), S.Y., T.E., S.N. (Shiho Naniwa), Y.K. (Yuki Konishi), T.W. and S.K.; data curation, Y.K. (Yoshiaki Kitamura), T.F., H.M.; writing—original draft preparation, S.N. (Seiichi Nakano), Y.K. (Yoshiaki Kitamura), S.K.; writing—review and editing, S.N. (Seiichi Nakano), Y.K. (Yoshiaki Kitamura), N.T. and H.M.; supervision, H.F., N.T. and H.M.; project administration, H.M., H.F. and N.T. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Not applicable.Conflicts of Interest: H.F. is an employee of Medical Corporation Kinshukai. The company has no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All other authors declare no conflict of interest.Effect of SST on PMA-induced up-regulations of IL-33 mRNA in Swiss 3T3 cells (A) and H1R mRNA in HeLa cells (B). The hot water extract of SST was incubated for 12 h before stimulation with 100 nM PMA. After stimulation with PMA for 3 h, the cells were harvested, and total RNA was prepared. IL-33 and H1R mRNA levels were determined using quantitative RT-PCR. Data are expressed as means ± SEM (n = 3–4). ## p < 0.01 vs. control; ** p < 0.01, vs. PMA.Effects of eight crude drugs of SST on PMA-induced up-regulation of IL-33 mRNA. Swiss 3T3 cells were treated with Ephedra herb (A), cinnamon bark (B), processed ginger (C), Asiasarum root (D), peony root (E), Schisandra fruit (F), Glycyrrhiza (G), and Pinellia tuber (H) for 12 h before stimulation with 100 nM PMA. After stimulation with PMA for 3 h, the cells were harvested, and total RNA was prepared. IL-33 mRNA levels were determined using quantitative RT-PCR. Data are expressed as means ± SEM (n = 3–4). ## p < 0.01 vs. control; * p < 0.05, ** p < 0.01 vs. PMA.Effects of eight crude drugs of SST on PMA-induced up-regulation of H1R mRNA. HeLa cells were serum-starved for 24 h and treated with Ephedra herb (A), cinnamon bark (B), processed ginger (C), Asiasarum root (D), peony root (E), Schisandra fruit (F), Glycyrrhiza (G), and Pinellia tuber (H) for 12 h before stimulation with 100 nM PMA. After stimulation with PMA for 3 h, the cells were harvested, and total RNA was prepared. H1R mRNA levels were determined using quantitative RT-PCR. Data are expressed as means ± SEM (n = 3–4). ## p < 0.01 vs. control; * p < 0.05, ** p < 0.01 vs. PMA.The correlation between IC50 values of SST and its seven crude drugs to inhibit the IL-33 and H1R gene expression. A Spearman’s rank correlation test was used for the statistical analysis (r = 0.964, p < 0.01). SST: Syo-seiryu-to; EH: Ephedra herb; CB: cinnamon bark; PG: processed ginger; AsR: Asiasarum root; PR: peony root; SF: Schisandra fruit; G: Glycyrrhiza.Effects of inhibitors of the H1R gene expression signaling pathway on PMA-induced IL-33 gene up-regulation. Swiss 3T3 cells were treated with quercetin (A), (±)-maackiain (B), U0126 (C), 17-AAG (D), and celastrol (E) for 12 h before stimulation with 100 nM PMA. After stimulation with PMA for 3 h, the cells were harvested, and total RNA was extracted. IL-33 mRNA levels were determined using quantitative RT-PCR. Data are expressed as means ± SEM (n = 3–4). ## p < 0.01 vs. control; ** p < 0.01 vs. PMA.Comparison of IC50 values of SST and its crude drugs to inhibit the IL-33 gene up-regulation with those related to H1R gene up-regulation.* no inhibition.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-01-03-00016.txt

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+A 14-year-old male initially presented to the Emergency Department (ED) for a chronic, persistent cough and chest pain with concurrent history of asthma and gastroesophageal reflux disease (GERD). He had been trialed on several medications before this visit for cough, without resolution of symptoms. Despite seeing several specialists after this ED visit and being evaluated for infectious causes and other pulmonary issues, he was eventually found to have eosinophilic esophagitis (EoE). It is not often that EoE is suspected based on cough alone, but with other GERD-like symptoms EoE should be considered.Chronic cough can have a long differential diagnosis. Asthma itself can be a cause. We present a male with chronic asthma who developed a new chronic cough.A 14-year-old white male presented to the Emergency Department (ED) (May 2020) with 8 weeks of persistent cough, chest pain, and difficulty swallowing. He initially was evaluated at several outside facilities (over the 8 weeks before the ED visit) where he had tested negative for COVID-19 and started prednisone, azithromycin (possible pertussis), and codeine cough syrup, and was given a steroid shot (Decadron). He had three previously negative chest X-rays. He works with domestic ducks. He also had a laryngoscopy and was started on esomeprazole (Nexium) and gabapentin for reflux and remained on it for 6 weeks before the ED visit. In addition, he was on cetirizine for seasonal allergies, Symbicort (budeonide/and formoterol fumarate dihydrate combination), montelukast, and albuterol for asthma, clarithromycin (Biaxin as a 2nd round antibiotic for presumed pertussis). Other medications he had tried were benzonatate (Tessalon Perles) and omeprazole (Prilosec), neither of which helped.In the ED, he pointed to the center of his chest when asked to localize the pain and stated the pain was constant. When he coughed, he felt “a shock” to his body and his hands became numb. The cough was disruptive to his sleep. He denied having fever, congestion, rhinorrhea, nausea, vomiting, or diarrhea. His mother voiced concern because of a recent paternal family member who had cardiac issues and died early at 27 years old.His past medical history includes asthma, diagnosed when he was 7 years old, allergy to dust mites, and seasonal allergies. He said this cough was different than his infrequent asthma cough as the new cough was becoming more frequent. Albuterol helped minimally. His sister had asthma. His past surgical history includes adenoidectomy, tonsillectomy, and tympanostomy with tube insertion.His physical exam was unremarkable except for chest tenderness that was not reproduceable on palpation. The rest of the review of systems and physical exam were otherwise negative.Electrocardiogram and chest X-ray were normal. Pseudoephedrine was given in the ED and he was sent home with plans for a pulmonology follow up in one week.He saw Pulmonology via Telehealth (due to living 100 miles from the Pediatric Specialist and limited in-person visits due to COVID precautions at the specialty outpatient clinic). It was noted that he choked when he ate but did not remember aspirating on any food or objects. Review of systems at that appointment was positive for chest pain, cough, heart burn, extremity numbness, and allergies. At that time the differential diagnosis included infection, asthma, gastroesophageal reflux, allergies, foreign body aspiration, and habit cough. At an in-person follow up clinic visit to the same specialist, his spirometry showed normal lung function and no significant response to bronchodilation. His fractional exhaled nitric oxide (FeNO) was 25 ppb (normal range for age <25 ppb). He was instructed to continue current medications with a presumed diagnosis of habit cough.However, due to the persistence of his cough he was then scheduled for a bronchoscopy (4 months after the ER visit). Upper airway inflammation was noted with cobblestoning. The lavage culture grew a small amount of penicillium genus, considered to be a contaminate, and he was put on two weeks of fluconazole. Bronchoalveolar lavage fluid showed a lipid index of 4, not indicating microaspiration. The lavage cell count was mixed neutrophils and mononuclear cells, without eosinophils. The cough improved with fluconazole but was still present and worsened again a month later. Allergy was consulted.He saw Allergy via Telehealth 7 months after the cough had started. It was at this time that the patient revealed he would usually eat meals in his room instead of with the family and he feels food get stuck when eating, especially meat. He also had modified his eating behavior, with small portions and longer chewing. He maintained a proton pump inhibitor, received a TB skin test (negative), and was scheduled with an esophagogastroduodenoscopy (EGD) in 3 weeks (with COVID limitations for numbers of surgical suite patients).The endoscopy showed four eosinophils on a high-powered field in the proximal esophagus and 25 in the distal esophagus with furrowing in three out of four quadrants and mild exudates on macroscopic view (Figure 1). He was prescribed 1 mg of budesonide swallowed BID as a slurry with Splenda with instruction to rescope in 6–8 weeks.Three weeks into therapy, his mother was called for follow up and said the cough was 80% better. The scope 6 weeks after starting budesonide showed total resolution of esophageal eosinophilia and a normal macroscopic examination. His cough remained improved.On a Telehealth visit one year after the start of his cough, he was on budesonide slurry at bedtime. He had stopped his Symbicort (budesonide and formoterol fumarate dihydrate combination). A subsequent in-person visit revealed a normal pulmonary function without bronchodilatation (off all asthma therapy). His cough was resolved totally.Eosinophilic esophagitis (EoE) has had an evolution of definitions over the years. The current criteria for diagnosis include symptoms of esophageal dysfunction with 15 or more eosinophils per high-powered field on esophageal biopsy [1]. The most common presentation of EoE in adolescents includes esophageal dysphagia, food impaction, heartburn, and non-cardiac chest pain [2]. Our patient’s primary complaint was of cough with a past medical history of GERD and asthma. Dysphagia was not a symptom shared on initial presentation or even months into work up. Unknowingly, many of the typical symptoms of EoE were being concealed by compensatory behavioral changes such as chewing his food finely, cutting it up, and drinking lots of water at meals and was accepted as his normal. These behavior modifications are not uncommon and can be developed subconsciously [2,3,4]. For our patient, in addition to these behaviors, he also ate alone in his room and would hide any food he could not chew and spit it back out on his plate.The most common presenting symptoms of EoE in children are reflux, emesis, abdominal pain, and in adolescents, dysphagia and food impaction [5]. Cough is an uncommon presentation of EoE but was the chief complaint for our patient. Evaluation of chronic cough in adolescents contains a long differential, including asthma, GERD, upper and lower respiratory tract infections, pertussis, post-nasal drip, aspiration, and psychogenic cough [6], while less common possibilities of tuberculosis, eosinophilic pneumonia, or eosinophilic granulomatosis with polyangiitis were excluded by chronicity, lack of pulmonary symptoms, and repeated normal chest X-rays. It is reasonable to rule these out in evaluation, as was achieved by the combination of PPI trial, antibiotics, chest X-rays, and adjusting asthma and seasonal allergy controls. When his cough did not persistently improve with extensive interventions, reevaluation of less common causes could have been considered earlier. A case report was written of a 2 year old with a dry cough for 6 months who was also suspected to have GERD but was unresponsive to a PPI and diagnosed with EoE [7]. Few other cases diagnose EoE with a primary complaint of chronic cough [7,8].EoE is more common in males than females and in Caucasians than other races [9] Additionally, asthma and allergic rhinitis are common co-diseases with EoE [10,11].Chronic cough in a patient with other risk factors, such as asthma and allergy, who fits the demographic risk of being a Caucasian male, should prompt consideration of eosinophilic esophagitis, a less common allergic disease. While GERD is a more common cause of cough with reflux and chest pain, without improvement on PPIs other causes should be evaluated. His lack of improvement on a PPI excluded GERD as the primary concern, and his minimization of dysphagia, and the prolonged nature of the processes, despite inhaled asthma corticosteroids and the steroid injection, suggested a slow evolution of his EoE. The budesonide is considered primary therapy for EoE and was the necessary prolonged therapy of benefit [5]. Whether he actually has (had) asthma is debatable, and longitudinal observation should prove helpful.The lack of improvement in the cough prior to the initiation of budesonide but after several different PPI trials is consistent with the EoE diagnosis. A PPI may have benefit as partial EOE medical management, but rarely provides complete benefit. Currently, in pediatric EoE management, a pH probe assessment of GERD prior to a EGD is extremely uncommon. Elements of our decisions on his EoE care are discussed in the AGREE conference proceedings [12].The strength of this case is that most of the care of this patient happened within our facility, so cross-communication between specialists was strong, and this helped us diagnose his EoE despite an uncommon presentation. Another strength to this case is that his history of GERD not successfully treated with a proton pump inhibitor (PPI) was helpful (after the visit to the allergist) in initiating diagnostic evaluation esophagogastroduodenoscopy (EGD). A limitation to this case is that several appointments had to be conducted via Telehealth due to the distance from our clinic and scheduling limitations due to existing COVID precautions. His conflicting asthma diagnosis also delayed the diagnosis. The ability to eventually stop asthma therapy further highlights the complexity of the case, but also the rarity with which EoE presents predominantly with a cough.Conceptualization, V.S., R.H. and A.D.; writing—original draft preparation, V.S.; writing—review and editing, R.H. and V.S.; visualization, A.D.; supervision, H.N. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Ethical review and approval were waived for this study, because it is a clinical report.Written informed consent has been obtained from the patients to publish this paper.The authors declare no conflict of interest.Visualized endoscopy findings at first EGD.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-01-03-00017.txt

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+Background: Diagnosis of allergic rhinitis is achieved by a combination of patient history and different screening tools, followed by specific provocation testing. Screening tools usually involve a skin prick test (SPT), specific serum IgE or a combination of both. Objective: The purpose of this study was to evaluate the correlation of SPT, intradermal testing and specific serum IgE testing in certain allergens and to evaluate sensitization rates, symptom patterns and time of symptoms in a cohort of patients with suspected allergic rhinitis. Methods: Data on 4653 patients with suspected allergic rhinitis were included and divided into five groups: spring bloomers (birch, hazel, etc.), summer bloomers (grasses and rye), autumn bloomers (ribwort and mugwort), mites and mold. Correlation of SPT, intradermal testing and specific IgE test results using Cohen’s kappa and logistic regression were carried out to evaluate the probability of symptoms. Results: Comparison of SPT and specific serum IgE led to kappa coefficients between 0.33 and 0.47, corresponding to a minor to moderate concordance. Comparing the symptoms reported by patients with sensitization diagnosed by SPT, a correlation was only found for spring and summer bloomers with an odds ratio of 1.5 and 2.1, respectively. The most prevalent symptom in the study cohort was rhinitis, followed by others such as asthma, sense of smell and atopic dermatitis. Conclusions: SPT seems to be more sensitive than specific IgE for detection of sensitization. Patients’ symptoms as well as the timing of symptoms, especially for perennial allergies, are not always very pronounced.Common and chronic forms of nasal inflammation include rhinosinusitis, nasal polyposis, nonallergic rhinitis with eosinophilia syndrome (NARES) and allergic rhinitis (AR). Allergic rhinitis is the most frequent manifestation of atopic diseases, with almost 80 million people affected in the United States. It accounts for about 50% of patients with chronic rhinitis [1]. Patients suffer from nasal pruritus and rhinorrhea. Impairment of quality of life, including loss of concentration, loss of sense of smell, headaches and sleep disturbances, are well documented [2]. Diagnosis of allergic rhinitis is achieved by a combination of differentiated patient history and screening tests, followed by specific in vitro testing and nasal provocation to confirm the diagnosis. Many different inhalant allergens are known to cause allergic symptoms, and the patient’s history gives first clues to the relevant allergens [3]. In a screening test (usually skin prick test (SPT) or intradermal testing), the sensitization of a patient to a specific allergen can be evaluated. This can then be further specified or confirmed by in vitro diagnosis of specific serum IgE. However, the above tests sometimes show conflicting results and little correlation. Usually, SPT is considered more sensitive than specific serum IgE testing [4,5]. Other authors report a better correlation of test results depending on the allergen looked at, with persistent allergens such as mite, dog, cat or mold showing a weaker correlation than seasonal allergens [6,7]. The “gold standard” in the diagnosis of allergic rhinitis is nasal provocation [8,9] and should be performed before beginning a time-consuming and expensive therapy, such as subcutaneous or sublingual immunotherapy, in cases with indistinct history and unclear results in the above-mentioned methods.In this study, we wanted to evaluate the correlation of SPT, intradermal testing and specific serum IgE testing in certain allergens. The intention was to evaluate sensitization rates, symptom patterns and time of symptoms in a cohort of patients (from a midwestern German academic hospital) with suspected allergic rhinitis. Furthermore, we wanted to evaluate whether patients’ complaints correlate with the results of SPT. This study was performed in the Department of Otorhinolaryngology, Head and Neck Surgery of the University Medical Center Mainz, Germany. The study was carried out in accordance with the ethical standards established in the Declaration of Helsinki. Patients signed a “Broad consent” at admission which includes using their data and results of diagnostic tests or biomaterials for research as long as they were gathered during clinical routine as designated by the local ethics committee.The database evaluated comprises 4653 patients with suspected allergic rhinitis who visited the allergy clinic between 1997 and 2010. Patient ages ranged from 0 to 88 years.Patients were asked to fill in a standardized questionnaire according to Schultze-Werninghaus. This included history of allergic diseases, affected relatives, questions concerning professional environment and living quarters, animal contact, food and drug allergies. To further specify their symptoms, patients were asked about seasonality and duration of asthma, coughs, rhinorrhea, sneezing, nasal obstruction, eye symptoms such as tearing or itching, urticaria, swelling or itching of the mouth, tongue or pharynx, atopic eczema, gastrointestinal symptoms and sense of smell [10]. Further evaluation to assess severity or more closely define certain symptoms was not used.Allergic rhinitis was diagnosed using patient history, SPT and/or intradermal testing and in vitro testing of patients’ sera for specific compound IgE antibodies. SPT was used as a screening tool in patients with suspected allergic rhinitis with a standard array of allergens (birch, alder, hazel, beech, rye, grass mix, ribwort, mugwort, Alternaria alternate, Cladosporium herbarum, Aspergillus fumigatus, Fusarium roseum, Penicillium notatum, Botrytis cinerea, cat, dog, Dermatophagoides pteronyssinus and Dermatophagoides farinae; ALK Prick SQ 100 SQ-E/mL, ALK-Abelló, Wedel, Germany). Intradermal testing was used in patients with a suspected allergy and negative results in SPT and for all patients with a suspected mite and mold allergy (Allergopharma, Reinbek, Germany). Histamine served as a positive control and saline solution as a negative control. After 20 min, reactions in the SPT and intradermal test were recorded in four degrees of severity according to wheal size (0 for negative; 1 for a wheal of up to 3 mm; 2 for a wheal of 3 mm to 5 mm and 3 for a wheal of more than 5 mm). For evaluation of data, a score of greater than/equal to 1 was deemed to be a positive result.For in vitro testing, which was performed to confirm the result of a positive SPT or intradermal testing in all patients, standard specific serum IgE determination was used (ImmunoCAP, Thermo Fisher Scientific). The measured units were divided into seven groups from 0 to VI as designated by the manufacturer. CAP-classes of ≥ II were considered positive. For polysensitized patients test results were included in the calculation for each category.A standard desktop PC with SPSS 23 software was used for the statistical evaluation. In this explorative analysis, values of p = 0.05 were regarded as significant. Mean values of standard deviation were chosen for graphical representation of the results.For statistical analysis, all patients were divided into five groups. Group one (termed “spring bloomer”) consisted of patients allergic to birch, hazel, alder or beech; group two (termed “summer bloomer”) consisted of patients allergic to rye or grasses; group three (termed “autumn bloomer”) consisted of patients allergic to ribwort and mugwort; group four (termed “mites”) consisted of patients allergic to Dermatophagoides pteronyssinus or farina; and group five (termed “mold”) consisted of patients allergic to Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Fusarium roseum, Penicillium notatum or Botrytis cinerea. If patients were sensitized to more than one allergen they were included in every group they were positive for. Symptoms and their seasonality from the questionnaire were given binominal scores.To evaluate the concordance of SPT, intradermal test and specific IgE test results, Cohen’s kappa was used. For the assessment of kappa, the classification proposed by Landis and Koch [11] was used.For evaluation of the data, two consecutive CAP-classes were taken together so that four classes resulted (0 ≙ ≤ 0.35 KU/L, 1 ≙ 0.36 to 3.49 KU/L, 2 ≙ 3.50 to 52.49 KU/L, 3 ≙ ≥52.50 KU/L).For evaluation of the probability of symptoms, univariate and multivariate logistic regression were performed leading to adjusted odds ratios. Additionally, for evaluation of the point-in-time symptoms reported by patients, logistic regression was performed. Representations of the results of logistic regression were generated at https://www.statstodo.com (accessed on 24 April 2020) using a logarithmic scale.Of the 4653 subjects, 2699 (58%) were sensitized and were divided into the five groups already mentioned (“spring bloomer”, “summer bloomer”, “autumn bloomer”, “mites” and “mold”); 1954 subjects (42%) showed no sensitization (Figure 1).The age distribution of patients showed an almost standard distribution with half aged 28 to 53 years, 25% aged younger than 28 years and 25% aged older than 53 years. Median age was 40.42 years (Figure 2). This group included patients sensitized to birch, hazel, alder or beech. The median age of these patients was 38 years. Patients aged 21 to 40 years constituted the biggest group (44%) followed by patients aged 41 to 60 years (36%). Of these patients, 9.9% were monosensitized. and 90.1% were polysensitized. Comparison of SPT and specific serum IgE in these patients led to a moderate concordance according to Landis and Koch, with a kappa coefficient of 0.418 for birch, 0.474 for alder and 0.449 for hazelnut (p < 0.0001 for all). For beech, no concordance could be found (kappa coefficient = 0.108), probably due to too small a number of subjects (Figure 3). For spring bloomers, this means an overall concordance of 71% for both test methods (Figure 4). When looking at specific symptoms reported by patients sensitized to spring bloomers, data on 3815 subjects could be evaluated. The probability of rhinorrhea was highest, followed by other symptoms such as asthma, atopic eczema, sense of smell and urticaria. Nasal obstruction and coughing did not have a significantly higher probability in subjects sensitized to spring bloomers, as compared to nonsensitized subjects (Figure 5a, Table 1). When looking at the time period of symptoms, patients sensitized to spring bloomers had a higher probability of symptoms in spring and summer, and a lower probability in autumn and winter, compared to nonsensitized subjects (Figure 6a, Table 1).To evaluate whether symptom seasonality reported by patients matched their sensitization pattern, a correlation was performed. This shows that patients sensitized to spring bloomers reported symptoms in spring, with a sensitivity of 30.8% and a specificity of 77% (p < 0.0001). The odds ratio was 1.5, which shows that patients who reported symptoms in spring were sensitized 1.5 times more often.Kappa coefficients when comparing SPT and specific serum IgE.Overall concordance of SPT (intradermal test for mold) and specific serum IgE.(a–e) Correlation of symptoms. (a) Symptoms associated with spring bloomers; (b) Symptoms associated with summer bloomers; (c) Symptoms associated with autumn bloomers; (d) Symptoms associated with mites; (e) Symptoms associated with mold.(a–e) Point in time of symptoms. (a) Time of symptoms associated with spring bloomers; (b) Time of symptoms associated with summer bloomers; (c) Time of symptoms associated with autumn bloomers; (d) Time of symptoms associated with mites; (e) Time of symptoms associated with mold.Patients sensitized to rye or grasses were included in this group. The median age of these patients was 35 years. The biggest group (49% of patients) comprised those aged 21 to 40 years, followed by patients aged 41 to 60 years (31%). Of these patients, 12.2% were monosensitized, and 87.8% were polysensitized.In this group, a minor concordance was found when comparing SPT and specific serum IgE with a kappa coefficient of 0.394 (p < 0.0001) for grasses and 0.375 (p < 0.0001) for rye (Figure 3). The accordance of both tests was 59% in this group (Figure 4).When looking at specific symptoms reported by patients sensitized to summer bloomers, data on 3815 subjects could be evaluated. The probability of being symptomatic was highest for rhinorrhea, whilst other symptoms such as atopic eczema, eye symptoms, asthma and sense of smell followed. Here again, coughing and gastrointestinal symptoms did not have a significantly higher probability in persons sensitized to summer bloomers as compared to nonsensitized persons, and nasal obstruction was associated with a lower probability in sensitized subjects (Figure 5b, Table 2).Patients sensitized to summer bloomers had a higher probability of symptoms in spring and summer, and a lower probability in winter, compared to nonsensitized persons. In autumn, no significant difference could be found between the groups (Figure 6b, Table 2).To evaluate whether the reported symptom seasonality of patients matched their sensitization patterns, a correlation was again performed. This shows that patients sensitized to summer bloomers reported symptoms in summer, with a sensitivity of 35% and a specificity of 80% (p < 0.0001). The odds ratio shows that patients who reported symptoms in summer were sensitized 2.1 times more often.This group included patients sensitized to ribwort and mugwort. The median age of these patients was 38 years. The largest group comprised patients aged 21 to 40 years (45%), followed by patients aged 41 to 60 years (37%). Only 8.7% of these patients were monosensitized, and 91.3% were polysensitized.Comparison of the skin prick test and specific serum IgE showed a kappa coefficient of 0.374 (p < 0.0001) for mugwort (Figure 3). For ribwort, no concordance could be found (kappa coefficient = 0.184), probably due to an insufficient number of analyzed subjects.Looking at specific symptoms reported by patients sensitized to autumn bloomers, data of 3808 persons could be evaluated. Rhinorrhea was the most frequent symptom, followed by other symptoms such as eye symptoms, itching of the mouth or pharynx, atopic eczema and urticaria. Here, again, nasal obstruction and coughing did not have a significantly higher probability in patients sensitized to autumn bloomers, as compared to nonsensitized subjects (Figure 5c, Table 3).Patients sensitized to autumn bloomers were most likely to be symptomatic in spring and summer. In winter, they were less likely to have problems than nonsensitized persons. Interestingly, in autumn itself, no significant difference could be found when comparing sensitized to nonsensitized subjects (Figure 6c, Table 3).In this group, patients sensitized to Dermatophagoides pteronyssinus or Dermatophagoides farinae were included. These patients had a median age of 36 years, with patients aged 21 to 40 years representing the biggest group (49%). Patients aged 41 to 60 years represented 29%. In 22.4% of cases, patients were monosensitized, whereas 77.6% were polysensitized.For mites, a moderate correlation was found between SPT and specific IgE, with a kappa coefficient of 0.432 (p < 0.0001) for Dermatophagoides pteronyssinus and 0.446 (p < 0.0001) for Dermatophagoides farina, which amounts to a concordance of 72% (Figure 3 and Figure 4). In this group, comparison of the results of SPT and intradermal testing was possible with 3405 subjects. For D. pteronyssinus, the kappa coefficient was 0.642—a significant concordance according to Landis and Koch. For D. farina, the kappa coefficient showed a moderate concordance, with a value of 0.585. p-values were 0.0001. The concordance of both tests was 87%.Evaluation of specific symptoms in sensitized patients was possible for 3652 persons. For mite-sensitized patients, again, the probability of rhinorrhea was highest, followed by other symptoms such as atopic eczema, urticaria, eye symptoms and asthma following. As with the other groups, nasal obstruction did not have a higher probability in mite-sensitized as compared to nonsensitized subjects (Figure 5d, Table 4).A difference in the times when symptoms were present could be seen in spring and in summer (Figure 6d, Table 4).This group included patients sensitized to Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Fusarium roseum, Penicillium notatum or Botrytis cinerea. The median age of these patients was 38 years, with patients aged 21 to 40 years representing the biggest group (43%), followed by patients aged 41 to 60 years (34%). Precisely 34.4% of patients were monosensitized, whereas 65.6% were polysensitized.Comparison of intradermal testing and specific serum IgE was possible in a total of 1462 patients sensitized to Alternaria alternata. A low concordance was found with a kappa coefficient of 0.330 (p < 0.0001) and accordance for both tests of 75% (Figure 3 and Figure 4).Specific symptoms in sensitized patients could be evaluated in 3738 subjects. The probability of symptoms of atopic eczema was highest, followed by symptoms of rhinorrhea, urticaria and asthma. Additionally, for mold-sensitized persons, nasal obstruction did not have a significantly higher probability as compared to nonsensitized persons (Figure 5e, Table 5).Subjects sensitized to mold had a higher probability of symptoms in spring and summer as with mites (Figure 6e, Table 5).This study conducted an explorative analysis, comparing different methods of allergological testing in a large cohort of patients from midwestern Germany. The advantage of this study is the large number of patients tested for allergic rhinitis, which provides consistent testing results of skin tests and specific serum IgE, together with data on symptom duration and questionnaire responses.These data found overall sensitization rates to be in line with previously published results [7,12,13,14,15]. Findings confirm that patients with allergies to inhalant allergens in spring (early bloomers such as birch) and autumn (herbs) have the highest polysensitization rates of around 90%, which is a little higher than previously reported [15,16,17], whereas monosensitizations are higher in house dust mites and mold.Comparison of SPT and the results of specific serum IgE testing show a moderate correlation for birch, alder, hazelnut and mites, and only a minor correlation for grasses, rye and mugwort. For mites, Chinoy et al. [4] found a lower concordance of 58.5%, but this might be explained by the use of a Phadebas RAST system, which has a lower sensitivity [18]. Tang and Wu [6] and van der Zee [7] describe a strong concordance of 97% and 93% between intradermal testing and specific IgE testing for mites. As with the above-mentioned studies, SPT in this investigation was more sensitive in detecting sensitization than specific serum IgE testing and shows that additional testing of specific IgE does not necessarily lead to more diagnostic input or a more reliable diagnosis. Pastorello et al. [19], on the other hand, found a lower diagnostic value (sum of sensitivity and specificity) for the SPT than for specific IgE testing.When comparing intradermal testing and specific IgE testing, which was regularly performed in Alternaria alternate, similar results were found with only a moderate correlation and a concordance of 75%. For all allergens, the concordance for negative results was good so that none of the tests missed a significant number of patients with an allergy, but for positive results, the prick test had a tendency to show sensitization earlier than the specific serum IgE.The comparison of intradermal testing and SPT revealed a significant correlation for sensitization to mites. In other studies comparing these methods, percutaneous testing correlated better [20].When comparing symptoms reported by patients with a sensitization diagnosed by SPT, a correlation was only found for spring and summer bloomers. The odds ratio for spring bloomers was 1.5 and for summer bloomers 2.1, showing that patients’ history can give a clue to relevant allergens in these seasonal allergies. On the other hand, only 31% of patients who reported allergic symptoms in spring had a sensitization to spring bloomers. This is less than previously reported when comparing patients with allergic rhinitis versus nonallergic rhinitis [21,22]. Part of this difference in numbers might be explained by the polysensitization of patients to further seasonal or perennial allergens. Additionally, questionnaires evaluating patient symptoms in allergic rhinitis always have difficulties differentiating between allergic and nonallergic rhinitis, as can be seen in the SAPALDIA study [23]. For mite and autumn allergies, no correlation could be found between reported symptoms and allergic rhinitis. This shows that testing only patients with clear symptoms leads to undertreatment, especially in patients with mite allergies, and that more uncommon symptoms and findings need to be evaluated for an underlying allergic disease. One additional reason for false-negative test results could be a possible missing of detecting late reaction after IDT [24].Interestingly, when looking at specific symptoms, the data in this study suggest that nasal obstruction is a symptom that is not associated with sensitization, as there was no difference between sensitized and nonsensitized persons in all groups. This also seems to be true for patients with mite sensitization and contrasts with other publications [25,26,27,28,29]. There may be a certain bias in the acquired data concerning this specific symptom because this study only included patients presenting to an ENT clinic. This might lead to a nonsensitized control group in which patients with nasal complaints, especially nasal obstruction, are overrepresented.The symptom patients reported most frequently was rhinorrhea. Other symptoms such as eye symptoms, atopic eczema and impairment of sense of smell followed. Symptoms of asthma were most prevalent in patients sensitized to grasses or rye, and symptoms of atopic eczema were most prevalent in patients sensitized to mold. Analysis of the time of year when patients were symptomatic found that all sensitized patients, regardless of the type of sensitization, had a higher probability of symptoms in spring and summer. In wintertime, sensitization to spring and summer bloomers seems to have a protective effect, with sensitized patients having a lower probability of symptoms than nonsensitized persons. This was even similar for mite-sensitized patients who did not differ in their probability of symptoms in autumn or winter from nonsensitized persons.In conclusion, this study’s cohort revealed that SPT is more sensitive than specific IgE for the detection of sensitization so that additional testing of specific IgE does not necessarily lead to a more conclusive diagnosis. Sole use of specific IgE as a screening tool might lead to underdiagnosis of sensitization. Using both SPT and compound-specific IgE basically determines the same values with two highly overlapping diagnostic tools. This is, of course, not valid for the determination of monoclonal specific IgE, for which there is a good rationale supporting its use as a tool for confirmation and further diagnostic differentiation and for determining the usefulness and effectiveness of subcutaneous immunotherapy [30,31]. Symptoms reported by patients are very variable especially in a setting with polysensitized subjects where the point in time reported by a patient does not always coincide with the underlying multiple sensitizations.T.H.: acquisition, analysis and interpretation of data, drafting of manuscript. M.D.: acquisition, analysis and interpretation of data, critical revision of manuscript. V.W.-E.: analysis and interpretation of data, critical revision of manuscript. B.R.H.: acquisition, analysis and interpretation of data and critical revision of manuscript. All authors have read and agreed to the published version of the manuscript.The study did not receive funding.Approval by the local ethics committee (Ethikkommission Mainz) in accordance with federal state law.Informed consent was obtained from all subjects involved in the study. Patients signed a “Broad consent” at admission to hospital which included using their data and results of diagnostic tests or biomaterials for research as long as they were gathered during clinical routine.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.We would like to thank Schmidtmann for data analysis and valuable advice in data interpretation.B.R.H. is an employee of Schwarzwald-Baar Klinikum Villingen-Schwenningen GmbH. The company has no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The authors declare that they have no competing interests.Prevalence of sensitization to the different allergen groups; PR = Prick test, IC = Intracutaneous test.Age distribution of whole cohort.Spring bloomers.Summer bloomers.Autumn bloomers.Mites.Mold.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+These authors contributed equally to this paper and should be considered as first authors.Atopic dermatitis (AD) is a chronic relapsing skin disease, associated with impaired skin barrier function and characterized by poorly defined pruritic, erythematous lesions. In this study, the efficacy of a new topical cream (IALUSET VITAL®), containing hyaluronic acid and the extract of Salvia haenkei, in reducing symptoms of moderate AD in adults was investigated. This study was a randomized, double blind, vehicle-controlled clinical study. Treatment efficacy was evaluated considering both objective parameters (Scoring Atopic Dermatitis, SCORAD) and subjective pa-rameters (Patient Oriented Eczema Measure, POEM, and an itching sensation) and through non-invasive bioengineering techniques to measure skin moisturization and Trans Epidermal Water Loss (TEWL). Under the experimental conditions of the study, IALUSET VITAL® significantly reduced AD severity, as shown by the SCORAD index, and was revealed to be effective in alleviating the most common signs and symptoms of moderate AD, suppressing itch and improving skin moisturization, and to have a good safety profile, being well-tolerated by patients. However, statistically significant differences between active and vehicle group were not found in the other parameters analyzed, likely because the basic formulation of IALUSET VITAL® guarantees good emollient properties and the addition of hyaluronic acid and extract of Salvia haenkei as active ingredients results in a great increase in effectiveness.Atopic dermatitis (AD) is a chronic inflammatory skin disease, affecting up to 20% of children and up to 3% of adults [1], characterized by erythematous skin lesions with intense pruritus, a very disabling symptom that considerably impairs a patient’s quality of life. It is widely recognized that AD is a multifactorial disease, involving immune disorders, impaired skin barrier function and environmental factors. Nevertheless, a major debate exists as to whether AD is primarily driven by immune dysregulation (inside-out theory) or epidermal barrier dysfunction (outside-in theory) [2,3]. Common tracts of 10–40% of AD patients are the loss-of-function mutation of the FLG gene, encoding the structural epidermal protein filaggrin, contributing to epidermal barrier dysfunction [4,5] and a reduced content of ceramides, important water-holding molecules in the extracellular space in the horny layer [6]. These events lead to trans-epidermal water loss, a component of a physiological process known as perspiratio insensibilis, and increased permeability to allergens and pathogens and promotes inflammation stimulating the activation of the innate immune response. Cutaneous sensory nerves transmit the increased itch signal to the brain, which leads to further scratching and impairing of skin integrity with the establishment of a self-feeding vicious circle [2,7]. With regard to the treatment of AD, current therapies aim to clear inflamed lesions and reduce itch in order to improve patient’s everyday life. Topical therapies with emollients and anti-inflammatory drugs are the mainstay for mild-to-moderate AD; phototherapy and systemic immunomodulatory drugs can be effective in more-severe AD [8].Hyaluronic acid (HA), a polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is one of the main components of the extracellular matrix [9], especially in the skin that accounts for about 50% of the total content of HA in the body [10]. HA is a key factor in wound healing and tissue repair processes, being involved in proliferation, differentiation, and migration of keratinocytes [11,12,13], as well as in skin aging owing to its ability to retain water and moisturize skin [9]. In addition, evidence from animal studies have shown that HA is also involved in the establishment and homeostasis of epidermal skin barrier, regulating both epidermal differentiation and lipid synthesis/secretion through the interaction with its receptor CD44 [13,14]. In addition, clinical studies support the safety and the efficacy of hyaluronic acid-based emollient foam in treating patients with moderate AD [15,16].Herbal extracts have been used for the treatment of skin diseases, among which AD, for centuries [17]. Recently, both in vitro and clinical studies have shown the efficacy of the extract of Salvia haenkei, a plant native of Bolivia largely used in traditional medicine [18], as an anti-aging agent [19,20]. In addition, an extract of Salvia haenkei has been patented both as re-epithelizing and cicatrizing agent [21] and as an active agent in the treatment of dermatological diseases [22].The aim of this study was to evaluate the efficacy of a novel topical cosmetic product, namely IALUSET VITAL®, composed of a mixture of HA molecules and the extract of Salvia haenkei, in the treatment of pruritus and skin dehydration in moderate AD.This study was a randomized, double blind, vehicle-controlled clinical study conducted at Complife Italia S.r.l., Garbagnate M.se (MI) Italy. All subjects enrolled gave written informed consent. All the study procedures were carried out in compliance with the ethical principles for medical research (Ethical Principles for Medical Research Involving Human Subjects, Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, and its amendment). The study received approval from the Ethics Committee for Non-Pharmacological Clinical Investigations and was registered in the ISRCTN registry (ISRCTN11607227; https://doi.org/10.1186/ISRCTN11607227, accessed on 11 April 2019).The subjects participating in the study (n = 40) were screened starting from a database of 48 subjects and enrolled under the supervision of a board-certified dermatologist from a panel of healthy male and female subjects, of Caucasian ethnicity, aged between 18 and 65 years old (mean age: 42.5 years active group and 45.7 years vehicle group), showing moderate atopic dermatitis (SCORAD between 25 and 40) at baseline. Subjects having a positive history for hypersensitive skin, former history of allergy or sensitivity to cosmetics, toiletries, to solar and/or topical medications, and the history of any confounding inflammatory skin diseases or any other skin disease (e.g., psoriasis, rosacea, erythroderma or ichthyosis), with spontaneously improving or rapidly deteriorating AD, active allergic contact dermatitis, or other non-atopic forms of dermatitis, acute infections, any skin condition that the principal investigator deemed inappropriate for participation or that were pregnant or nursing women were excluded from participation in this study. The study further excluded subjects having (a) oral or intravenous corticosteroids, UVA/UVB therapy, PUVA (psoralen plus ultraviolet A) therapy, tanning booths, non-prescription UV light sources, immunomodulators or immunosuppressive therapies, interferon, or cytotoxic drugs, within four weeks before baseline, and (b) antihistamines, topical antibiotics, topical corticosteroids, topical calcineurin inhibitors, or other topical drug products used for treating AD, within one week before baseline.After the enrollment, a restricted randomization list was generated using PASS 11 (version 11.0.10; PASS, LLC, Kaysville, UT, USA) statistical software running on Windows Server 2008 R2 Standard SP1 64-bit edition (Microsoft, Redmond, WA, USA). The list was created by a biostatistician and stored in a safe place. The randomization sequence was stratified using ‘Efron’s biased coin’ algorithm with a 1:1 allocation ratio. Participants were randomized into two groups: the active group applied the IALUSET VITAL® cream and the control group applied the vehicle cream (Figure 1A). The study adhered to established procedures to maintain separation between the investigator and its collaborators and the staff that delivered the intervention. Investigator and its collaborators who obtained outcome measurements were not informed on the product group assignment. Staff who delivered the intervention did not take outcome measurements. Subjects, investigator, and collaborators were kept masked to products’ assignment.The day before the baseline visit subjects were asked to abstain from any topical product (e.g., sunscreens, lotions, creams) application in the areas to be treated. Reference and active products were applied on the area affected by atopic dermatitis twice a day or more, according to individual needs. Product efficacy was assessed immediately after the first product application (T0′), after 30 (T30min) and 60 (T60min) minutes and after 7 (T7), 14 (T14), and 28 (T28) days of use. Enrolled subjects were intended to use during the entire study period only the product to be tested and to not vary the normal daily routine (Figure 1B).Severity of AD was evaluated by means of objective and subjective methods. Objective evaluation was performed by means of scoring atopic dermatitis index (SCORAD) [23]. Subjective evaluations were performed by means of the Patient Oriented Eczema Measure (POEM), a validated tool used for monitoring atopic eczema severity, focusing on the illness as experienced by the patient [24] and by means of an additional questionnaire about itching sensation with a score scale from 0 (no itching sensation) to 10 (very strong itching sensation). Efficacy of treatment was evaluated also through non-invasive bioengineering techniques. Skin moisturization was measured by means of Corneometer® equipment (Corneometer® CM 825; Courage + Khazaka, electronic GmbH, Köln, Germany), whereas a skin barrier function was evaluated by measuring Trans Epidermal Water Loss (TEWL) using a Tewameter® TM 300 (Courage + Khazaka, electronic GmbH, Köln, Germany).The instrumental data were submitted to a two-way Student t-test while the clinical data were submitted to a Wilcoxon (intragroup comparison) or Mann–Whitney test (intergroup comparison) signed rank test for paired data. Intragroup (vs. T0) or intergroup (active vs. vehicle) variations were considered statistically significant when the p-value was <0.05. For clinical evaluations, the positive effect of the product on the measured parameter was confirmed if more than 50% of the subjects registered an improvement. Finally, for the self-assessment questionnaires, the performance and the pleasantness of the product must be perceived by at least 60% of the subjects.The reference and test cosmetic products were in line with the Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (recast) (Text with EEA relevance) and to its annexes. Test cream (IALUSET VITAL®, IBSA Farmaceutici Italia) contained a mixture of two different molecular weight HA molecules (300 kDa and 800 kDa) and the hydroalcoholic extract from aerial parts of Salvia haenkei. To obtain the extract of Salvia haenkei, plant material was harvested, put in a ventilated stove at 45 °C for 24 h, and then grounded into a fine powder. Dried powdered plant material was placed in a percolator and subjected to six cycles of extraction with water/ethanol (30–70% v/v) mixture. The obtained hydroalcoholic extract was filtered through a filter paper, concentrated under vacuum and dried at 40 °C in an oven under vacuum until complete evaporation of the solvent. Reference cream was identical to the test one, except for HA and the extract of Salvia haenkei. The composition of test cream is reported in Table 1.This study investigated how the cosmetic product IALUSET VITAL® affects the atopic dermatitis symptoms in adults, through a double-blind vehicle-controlled analysis. Caucasian male and female patients who showed a moderate form of AD, but no other comorbidities were selected (for enrolment criteria and characteristics of the patients, see Materials and Methods).Patients were randomized into two demographically equivalent groups: the control group (n = 20, 15 females and 5 males) consisting of patients with a median age of 49 years (range 20–65), applied the vehicle cream; the active group (n = 20, 16 females and 4 males), including patients with a median age of 45 years (range 19–63), applied the IALUSET VITAL® cream. After randomization, control and active group patients also showed similar clinical characteristics at baseline: the median SCORAD index was 30.10 (IQR, Interquartile Range, 25.80–32.25) for the control group and 27.80 (IQR 25.37–31.07) for the active group (Table 2).Objective severity measures of AD symptoms were reduced in a statistically significant manner by IALUSET VITAL®, which induced a continuous decrease of SCORAD index over a 4-week treatment period. At the end of the treatment period, the median and mean changes in the SCORAD index from the baseline were respectively of −11.55 and −12.57 points in the active group compared with −6.35 and −7.42 points induced by the vehicle in the control group (pactive vs. control = 0.003). In addition, in the active group, the variance was reduced by −3.04 points while, in the control group, it was increased by 44.78 points. Overall, these data indicate that IALUSET VITAL® treatment is effective on most AD patients (Figure 2A, Table 3 and Table 4).Subjective measurement tools also revealed that IALUSET VITAL® treatment reduced eczema and pruritus severity from a moderate to a mild degree. In particular, at week 4, the median and mean change in POEM score from baseline were respectively of −2.50 and −3.20 points in the active group (p < 0.01) and of −1.00 and −1.90 points in the vehicle group (p < 0.01). No statistically significant differences were observed between the active and control group (Figure 2B, Table 3 and Table 4).Similar results were observed as concerns the evaluation of itching sensation (Figure 2C, Table 3 and Table 4). Indeed, IALUSET VITAL® significantly reduced itching from a median baseline score of 4.95 (IQR 3.87–6.00) to 4.10 (IQR 2.55–4.80) as it is immediately after application (p < 0.001). This score has been continuously decreased to 1.30 (IQR 0.92–2.27) at 60 min. Total mean change from baseline was −3.15 (p < 0.001). The vehicle-induced effect on patients was also significant, although at T0′ and T30 the effect was more variable. Overall, the vehicle also significantly induced a total reduction in itching sensation from a median baseline score of 5.25 (IQR 4.02–6.32) to 2.10 (IQR 0.95–3.10). Total mean change from baseline was −2.77 (p < 0.001). No statistically significant differences were observed between the two groups.Furthermore, an improvement in moisturization of stratum corneum was observed in the active group as early as 60 min after the initiation of treatment. The median and mean changes in the moisturization index from the baseline were respectively of 4.31 and of 4.87 points (p < 0.001). These results induced by IALUSET VITAL® remained nearly constant during the four weeks of treatment. A statistically significant increase of the skin moisturization index has also been recorded in the vehicle treated group only at 60 min after the first product application. No statistically significant difference was observed between the two groups (Figure 2D, Table 3 and Table 4).Finally, to evaluate if perspiratio insensibilis was affected from IALUSET VITAL® treatment, the trans-epidermal water loss (TEWL) was monitored. Data revealed that both IALUSET VITAL® and vehicles did not alter the water perspiration out of the skin throughout the treatment period (Figure 2E, Table 3 and Table 4).Tolerance and safety were assessed for all the patients during the entire study period. The enrolled subjects did not show neither the occurrence of new physical (erythema, oedema, desquamation, other) and functional signs (burning, itching, other) nor the worsening of basal physical and functional signs. Therefore, both IALUSET VITAL® and vehicle cream were well tolerated by all the subjects during the study duration.The safety of IALUSET VITAL® was also assessed in a previous clinical study on the efficacy of the cream in reducing the effects of skin ageing [20], where the cream was revealed to be highly tolerated with no adverse reactions reported by any of the 50 subjects enrolled.Atopic dermatitis is mainly characterized by dysfunctions of the skin barrier and an uncontrolled inflammatory response. HA has been shown to play an important role in regulating homeostasis of the skin, especially in maintaining selective permeability of the epidermis and controlling inflammatory response. Moreover, because of its hygroscopic property, HA provides a hydrated microenvironment which facilitates the transport of nutrients through the tissue [25]. Finally, HA directly affects the function of skin cells by mediating signaling events that control the proliferation/differentiation of keratinocytes and lamellar bodies production, important mechanisms for maintaining selective permeability and repair of the skin [11,13]. It was also demonstrated that topical application of HA induces keratinocyte proliferation/differentiation and increases epidermal thickness and skin barrier repair [14].Immune response in subjects with AD is dysfunctional, characterized by the release of many pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukins [26]. Several authors have shown that HA reduces inflammatory response by downregulating the expression of pro-inflammatory and upregulating anti-inflammatory molecules [27,28,29,30,31]. Indeed, Kim et al. observed that HA decreases skin lesions in an atopic dermatitis model of DNFB-treated Nc/Nga mice [28].In patients with AD, claudins’ expression levels are reduced [32,33]. These proteins together with the occludin form a family of proteins that are the most important components of the tight junctions (TJs). In turn, TJs are critical in the functioning of the skin barrier because defective TJs increase paracellular permeability, resulting in an enhanced flux of environmental factors such as irritants, microbial products, toxins, and allergens, which, crossing the skin surface, trigger the immune response [34]. Recently, the extract of Salvia haenkei was shown to increase occludin expression as well as to control the expression and localization of filaggrin, a key marker of keratinocytes’ differentiation. Thus, the extract of Salvia haenkei reinforces the adhesion between the cells and favors the maintenance of the barrier integrity [22].Considering the mentioned observations, we performed this clinical study in order to assess on AD patients the efficacy of IALUSET VITAL® cream, a cosmetic containing two molecular weights of hyaluronic acid (300 kDa and 800 kDa) and the extract of Salvia haenkei.This study clearly showed that the regular use of IALUSET VITAL® progressively and significantly reduces AD severity and improves SCORAD, POEM, itch, and stratum corneum moisturization scores. After one week of treatment, a significant decrease of the SCORAD index was already observed in the active group compared to the control group. Interestingly, IALUSET VITAL® treatment improved AD severity from moderate to a mild degree, in a time-dependent manner as shown by the progressive reduction of the mean change of SCORAD index from baseline by 42% compared with 25% induced by vehicles in the control group at week 4.At the end of treatment, 70% and 65% of the patients in the active and control group, respectively, reported a reduction of the POEM score less than the median value at T0. In particular, only the active group showed 10% of patients with total remission of symptoms.As concerns the evaluation of itching sensation, immediately after treatment, 80% and 65% of the patients in the active and control group, respectively, showed a reduction of itch score less than the median value at T0. Moreover, 60 min after the treatment, a reduction of itch score lower than the median value at T0, was recorded in 100% and 95% of the patients in the active and control group, respectively.However, both for POEM and itch score, the differences between the two treatment groups did not appear to be statistically significant.The treatment with IALUSET VITAL® led to a significant increase of skin hydration throughout the treatment period while the vehicle induced a more variable effect in the control group. Paradoxically, although the TEWL analysis showed a positive trend of IALUSET VITAL®, in terms of effectiveness compared to vehicles, no statistically significant difference has been shown. Similar findings have been reported in other studies on moisturizers [35,36,37]. Our results may not be consistent for several reasons. Firstly, the relationship between skin dryness and TEWL is complex, whereby changes in dryness may not necessarily reflect simultaneous changes in TEWL [38]. Then, standardization of TEWL measurements can be technically difficult, while corneometer is an effective and sensitive tool to determine skin moisturization [39]. Therefore, the latter method is more sensitive to measure the skin barrier function than TEWL in AD patients and a larger sample size may be necessary to clarify this discrepancy and achieve a statistically significant trend in TEWL changes.The vehicle used in this study was an emollient base cream, with the same composition of IALUSET VITAL® except for the key ingredients hyaluronic acid and extract of Salvia haenkei. Due to the presence in the formulation of some humectant and emollient agents such as xylitylglucoside, anhydroxylitol, xylitol, isononyl isononanoate, coco caprylate/caprate, and l-arginine, the vehicle cream exhibits some beneficial moisturizing effects that should be taken into consideration when statistically significant differences in effectiveness between the active and control group have not been noted. Indeed, it is well known that emollients make the epidermis softer and more pliable, and they are effective in increasing skin hydration, improving barrier function, and reducing itching in AD [40].Thus, the efficacy of IALUSET VITAL® is guaranteed by the good emollient properties resulting from a well-designed basic formulation and, above all, the addition of active ingredients hyaluronic acid and extract of Salvia haenkei that greatly increase its effectiveness. In addition, IALUSET VITAL® cream was well-tolerated by patients. Overall, this study clearly shows that IALUSET VITAL® has a good safety profile and promotes the relief of the most common signs and symptoms of moderate AD, rapidly suppressing itch and reducing eczema severity.Conceptualization, F.D.V., A.F., G.V. and A.M.G.; Data curation, F.D.V. and A.F.; Formal analysis, V.N.; Investigation, G.V. and V.N.; Methodology, V.N.; Project administration, A.M.G.; Supervision, A.M.G.; Validation, A.M.G.; Visualization, G.V.; Writing—original draft, F.D.V. and A.F.; Writing—review & editing, A.M.G. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee for Non-Pharmacological Clinical Investigations (protocol code Rif. 2019/03 and date of approval 11/04/2019).Informed consent was obtained from all subjects involved in the study.This study was conducted with the scientific support of Gloria Roveda as clinical investigators and Federica Ruggeri for analysis of data.F.D.V., A.F., G.V. and A.M.G. are employees of IBSA Farmaceutici Italia Srl, an Italian pharmaceutical company. V.N. is an employee of Complife Ita. This study has been sponsored by IBSA Farmaceutici Italia Srl. The authors report no other conflicts of interest in this work.Study design. (A) participant flow chart; (B) evaluation of symptom severity in AD patients after treatment with vehicle vs. IALUSET VITAL®, through the parameters shown at the indicated time points.Efficacy outcomes in adults with moderate atopic dermatitis at the indicated time points. (A) SCORAD index was evaluated by the investigator; (B) POEM and (C) itching sensation scores were evaluated by patients; (D) Skin moisturization and (E) TransEpidermal Water Loss (TEWL) were established through non-invasive bioengineering techniques as indicated in Materials and Methods. Dots beyond the bounds of the whiskers denote outliers. Black asterisks indicate significant change from baseline for each group. Red asterisks indicate differences between active and vehicle group. * p < 0.05, ** p < 0.01, *** p < 0.001.Composition of test cream IALUSET VITAL®.Baseline demographics and AD characteristics.Scores of all parameters determined at the indicated time points according to the study design in control and active groups (median, mean, and variance values are shown).Changes from baseline for each parameter analyzed at the indicated times in the two study groups. The median of differences was calculated as the 50th percentile of all individual differences from baseline; the mean of differences was calculated as the average of all individual differences from baseline.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+The vulvar area is a common site of both irritative and allergic contact dermatitis due to the thin skin, easily traversable by irritant and allergic substances. The purpose of this review is to provide an overview of the most frequent allergens causing contact dermatitis in this particular site. A literature search was conducted via PubMed through May 2021. Relevant English language studies are included in this review. Fragrances, preservatives, botanical products, and topical medicaments were found to be the most commonly involved allergens. Contact dermatitis is a very common occurrence that should also be considered in patients with chronic vulvar or anogenital dermatitis who do not respond to appropriate treatments.The vulvar area is a common site of contact dermatitis due to the thin skin, easily traversable by irritant and allergic substances. The nonkeratinized vulvar vestibule is likely to be more permeable than the keratinized portions of the vulva and thus more susceptible to exogenous topical agents [1]. The vulva is an area of occlusion due to both its intrinsic anatomical structure and the frequent use of occlusive napkins or underwear, which increase penetration or absorption of both irritants and allergens. Furthermore, women at different ages, due to urine and feces as children and to vaginal mucosal atrophy and the increase in the vulvar pH in menopause, may have an altered barrier function and, in incontinent elderly subjects, the use of diapers may contribute to increased susceptibility to irritants and allergens.The purpose of this review is to focus on the contact dermatitis of the vulva with particular reference to the most frequent allergens responsible for vulvar allergic contact dermatitis.All the studies dealing with this topic published in English-language literature were analysed in May 2021, with no date limitations. An electronic search was performed using the National Library of Medicine PubMed database. Papers not in English were excluded. The studies identified as relevant, including research articles, controlled studies, guidelines, reviews, case series, and case reports that raised important issues, were included in the present review.The results of this analysis allowed us to provide a comprehensive picture of this issue, based on the available literature.Irritant contact dermatitis (ICD) is the result of a direct damage to the skin by various chemical or physical stimuli. The initiating event is the disruption of the epidermal barrier (i.e., the stratum corneum), with consequent increased skin permeability. This results in an inflammatory cutaneous reaction, caused by proinflammatory mediators released from keratinocytes and by the activation of innate immunity [2,3]. Risk factors for vulvar ICD are multifactorial and include the type of irritant, the length of exposure, the presence of previous dermatoses, and the host’s susceptibility. Women with an atopic diathesis (particularly atopic dermatitis) are more susceptible to ICD as a result of the impaired barrier function of their skin. Furthermore, vulvar skin shows an increased susceptibility to some irritants (maleic acid and benzalkonium chloride) [1]. ICD is more common than allergic contact dermatitis, but the exact prevalence is unknown. The most common vulvar irritants are reported in Table 1.Irritant contact dermatitis from strong irritants (caustics, topical medicaments) can have a rapid onset within minutes or hours of being exposed and presents with erythema, patches, papules, vesicles, bullae, and scaling. In chronic diseases (mostly due to weak cumulative allergens such as detergents or friction), lichenification and fissuring are more typical features. The main symptoms of ICD are burning, stinging, and, less frequently, itching or pain. In most cases the dermatitis is localized to the site of contact. In particular, when due to napkins ICD is located on the convex areas of the vulva, sparing the folds.Avoiding use of the offending agents and providing patients education, together with the prescription of potent topical steroids to reduce inflammation, are crucial to the control of symptoms and signs.Allergic contact dermatitis (ACD) is the consequence of a T-lymphocytes mediated immune reaction to small, molecular weight chemicals (haptens) that penetrate the skin and activate innate immunity and then the adaptive immunity [3]. During the sensitization phase, naive T cells are activated in a process that involves Langerhans cells and dermal dendritic cells; in the elicitation phase, T cells migrate into the skin and induce skin damage through the release of proinflammatory cytokines and by killing hapten-loaded keratinocytes.The sensitization phase of ACD results in the expansion of skin-homing hapten-specific T cells that, upon subsequent hapten challenge, migrate into the skin and induce the skin damage through the release of proinflammatory cytokines and by killing hapten-loaded keratinocytes. Vulvar ACD may occur as a primary disorder or may complicate an underlying vulvar dermatosis. The risk of ACD increases in the case of pre-existing ICD and with the use of multiple topical treatments.Vulvar ACD may develop as an acute eczema where the allergen was applied. In that case an itching vesicular or exudative eczema develops on previously healthy skin. However, the onset is often a complication of previous different cutaneous dermatoses (lichen sclerosus, psoriasis, atopic dermatitis, etc.) presenting as a local aggravation or exacerbation of symptoms and signs (Figure 1). The diagnosis may not be easy because of the confounding clinical aspects related to the preexisting dermatosis. In this case, history and the clinical aspect are very important in differentiating a lichen flare-up with an allergic dermatitis. The appearance of acute inflammatory lesions as erythema, edema, and vesiculation suggests contact sensitization. Furthermore, a poor response to an appropriate topical corticosteroid therapy could be indicative of contact sensitization to these molecules. The prolonged contact with the allergen can cause lichenification. (Figure 2).ACD can often severely affect quality of life for women who already suffer from a debilitating vulvar disease.Sometimes the area of involvement spreads over the borders of the vulva not only due to the spread of inflammation but also because of the modality of contact with the allergens [4]. (Figure 3).Distant localizations may also develop due to inadvertent hand transfer or rubbing of the adjacent areas (e.g., thighs). The contamination of clothing or napkins may lead to persistent dermatitis. Furthermore, a rapid spread to distant sites (auto-eczematization) may also result from the absorption and diffusion of allergens inducing a sort of id-dermatitis.Clinically, vulvar irritation and allergic dermatitis can be difficult to distinguish, and diagnosis is made on the basis of history, clinical investigation, and patch testing.A particular situation in vulvar ACD may be represented by the so-called “Connubial dermatitis”, a dermatitis that occurs because of contact with substances transferred to the patients’ skin by her partner. The diagnosis of connubial dermatitis should be considered in cases of probable allergic contact eczema when patch test results are apparently inconsistent with the patient’s clinical history. In these situations, it may be necessary to extend medical investigation to the patient’s partner as well, in order to clarify the source of allergic contacts when no obvious exposures can be found.Vulvar ACD is frequently linked to direct contact with cosmetics and detergents or medicaments. Less frequently textiles or dyes are described. Sometimes unsuspected allergens such as nail varnish (ectopic contact dermatitis) can be the cause.A review of the studies concerning vulvar ACD is reported in Table 2 [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19].It is not surprising that a high level of sensitization (39–78%) is found testing patients affected by different vulvar disorders (vulvar symptoms, vulvar dermatoses, or anogenital symptoms). In selected conditions as well, like lichen simplex chronicus [20], similar percentages can be found. The reported incidence of clinically relevant patch test results for patients presenting with vulvar complaints are likewise high, ranging from 16% to 54% [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19].Fragrances, preservatives, and topical medicaments (especially corticosteroids, neomycin, and topical anesthetics) are the relevant allergens usually found. Although some authors consider nickel a relevant positivity for the disease, in the majority of cases it is considered a non-relevant allergen that simply reflects the high level of sensitization in the general population [9,15,20].Sensitizing fragrances have been identified in a large number of products, including cleansers, douches, toilet paper, personal hygiene sprays, sanitary or incontinence pads, and topical medications such as hemorrhoid creams. Fragrances are an important cause of vulvar ACD (Table 3) (Figure 4).Most standard patch test series include fragrance mix I, Myroxylon pereirae (balsam of Peru), and fragrance mix II. Fragrance mix I is composed of amyl cinnamal, cinnamyl alcohol, cinnamal, eugenol, geraniol, hydroxycitronellal, isoeugenol, oak moss absolute, and sorbitan sesquioleate. Fragrance mix II is composed of lyral, citral, citronellol, farnesol, hexyl cinnamic aldehyde, and coumarin. Balsam of Peru is a naturally occurring compound derived from the South American tree, Myroxylon pereirae. It includes benzyl acetate, benzyl alcohol, cinnamic acid, cinnamic alcohol, cinnamic aldehyde, eugenol, and isoeugenol [21].Sensitivity to fragrance mixes I and II, and M. pereirae has been reported in numerous studies [5,8,11,14,17].A retrospective, cross-sectional study including 347 patients (both male and female) with anogenital diseases found fragrances to be the most frequent allergens [18]; M. pereirae, fragrance mix I, and cinnamal were positive, respectively, in 26.7%, 20.6%, and 5.5% of patients with anogenital dermatitis.Many studies found a significant number of patients with genital ACD to be allergic to fragrance mix I or balsam of Peru [11,14,17,22,23,24].Together, these two fragrance screening agents were found to be clinically relevant in up to 20% of patients with genital ACD [11]. In other studies, fragrance sensitization was less frequently detected but almost always clinically relevant [15].Preservatives are additive substances with biostatic or biocidal power; they are added in topical products, especially emulsions and solutions, to prevent microbial proliferation. Preservatives are often used in synergistic mixtures; the most used are the esters of paraoxybenzoic acid (effective against fungi and molds) and formaldehyde releasers (more effective against bacteria).Numerous studies confirm that preservatives are a frequent cause of vulvar ACD [6,14,15,18,21]. In a retrospective study, the preservatives most associated with anogenital ACD were quarternium-15 (13.7%), paraben mix 12% (8.2%), methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) (6.9%), ethylenediamine dihydrochloride (5.7%), and 2-bromo-2-nitropropane-1,3-diol (5.5%) [18].The role of the ethylenediamine as an allergen is confirmed by a study in which its positivity reached 8% [6].Benzoic acid is reported as a frequent cause of iatrogenic ACD (particularly associated with antifungal medication) in the genital area [25]. Isothiazolinones seem to play a predominant role in the etiology of genital ACD [26,27,28,29]. They are found in intimate cleansers, cleansing wipes, and detergents [30,31]; in Europe their use has been restricted because of the high rates of sensitization.A recent study on the association between vulvar sensitization to MI and vulvodynia showed that repeated MI exposures can provoke allergy-driven genital pain [32].Preservatives must be considered as a possible cause of vulvar ACD as they are always present in topical products; it is often very difficult to trace the preservative involved.A variety of topical medications are used, often empirically or for self-prescription, in the management of vulvar symptoms. Corticosteroid creams/ointments, anti-itch creams, antibiotics, antifungal creams, and hormonal creams have been implicated in vulvar ACD.In 2008 the North American Contact Dermatitis group determined that the most clinically relevant medicaments in anogenital ACD were local anesthetics, antibiotics, and corticosteroids. Clinically relevant reactions to anesthetics among patients with anogenital ACD were high, with 12.5% of the patients reacting to benzocaine and 13.5% to dibucaine [18]. The “caine” anesthetics are widely recognized causes of topical medication-induced genital ACD [10]. Esther anesthetics (benzocaine, procaine, and tetracaine) are known allergens and cross-react with sulfa drugs, para-aminobenzoic acid, paraphenylenediamine, and thiazide diuretics [14,33].In patients with vulvar disease, a history of sulfa allergy or hair dye allergy can provide diagnostic clues to the culprit allergen [14]. Unfortunately, benzocaine is frequently present in over-the-counter topical anti-itch preparations, such as Vagisil, or antihemorrhoidal creams [19,34]. Amide anesthetics (bupivacaine, lidocaine, dibucaine, mepivacaine, and prilocaine) have also been reported but they seem to be less potent and less common sensitizers [33].As reported by several studies, neomycin is the most common allergen among topical antibiotic preparations. Drugs that cross-react with neomycin include framycetin, streptomycin, bacitracin, kanamycin, gentamicin, and tobramycin [4,5,8]. In a study by Al-Niaimi et al., topical antibiotics (specifically neomycin and framycetin), along with anesthetics, were the most common cause of sensitization among the 49% of clinically relevant patch test results [15]. In a retrospective analysis, 9.7% of patients with anogenital ACD had clinically relevant reactions to neomycin and 5.5% reacted to bacitracin [18].Topical corticosteroids may be sensitizers, due to the steroid molecule itself or its vehicle components. Genital contact sensitization rates for topical corticosteroids range from 1.1% to 3.3% [17]. Positive patch tests to clobetasol and tixocortol pivalate were found in patients using topical steroids for pre-existing dermatoses such as lichen sclerosus or vulvar eczema [5].Corticosteroids were one of the most common relevant allergens in patients with anogenital ACD; reactions to hydrocortisone-17-butyrate (13.5%), clobetasol-17-propionate (10.8%), budesonide 0.1% (9.7%), and tixocortol-21-pivalate (6.9%) were noted. Sensitization to topical corticosteroids should be considered in patients with vulvar dermatoses that respond poorly to the appropriate corticosteroid therapy.Antimycotics can cause ACD as well. Imidazoles are the most frequently prescribed topical antifungal drugs. ACD caused by them is regarded as rare considering their high prescription rates [35]. Among the imidazoles, miconazole is the most frequent contact sensitizer [36]. A study showed that 1.8% of patients with positive patch test results were sensitized to clotrimazole [10]. Clotrimazole is a synthetic, broad-spectrum, phenethyl imidazole, and a cross-reaction between clotrimazole and miconazole was observed in some case reports [35,36].Terconazole was shown to affect over twice the number of patients as other antifungals, with up to 7% sensitization [14].Rare cases of sensitization to nystatin have also been reported [37].Botanical products are very popular especially due to the belief that they are safer than synthetic products [38,39]. These products are contained in creams, vaginal suppositories, and plugs as active principle/raw material, additives, or excipients. They are known allergens [40,41,42,43]. Many cases of ACD due to the use of botanical extracts are described in the literature concerning the vulvar area [13,44,45].In an Italian study, about 60% of patients with chronic vulvar complaints reported use of botanical products, among which 16.7% reported adverse effects to them [39].The most commonly used medicinal plants were chamomile, Aloe vera, and Calendula officinalis.It must be emphasized that a considerable rate of patients who complained of cutaneous side effects due to natural products was not sensitized to the natural active principle. Among the patients referring cutaneous side reactions to botanical products, only 57.1% showed at least one positive reaction, and in testing the patients with integrative botanical series only 28.7% relevant positivities were found. These results also suggest that the botanical integrative series does not improve diagnostic accuracy; they should be tested only in particular cases of strong clinical-anamnestic suspicion.Sometimes fragrances and preservatives may be the true responsible allergens in cases of suspected ACD attributable to herbal components.A peculiarity of botanical extracts is the possibility of causing allergic sensitization after excretion via urine or feces after ingestion [13].In patients with chronic vulvar or anogenital dermatitis, the possibility of an ACD should be taken into consideration early in the course of the disease, especially in patients who do not respond to appropriate treatments. Prompt and accurate diagnosis of vulvar ACD in patients with a pre-existing vulvar disease is important because their symptoms will not improve until this diagnosis is made and appropriately managed.To take a detailed history in patients with vulvar dermatitis and to discover relevant allergens is crucial. We suggest initially performing patch tests with the European baseline series and then with additional series with fragrances, preservatives, medicaments, and botanical extracts. We also strongly recommend testing with the patients’ own products as well [25].This research received no external funding.The authors declare no conflict of interest.A case of psoriasis complicated by allergic contact dermatitis due to topical medications.Lichenification following persistent allergic contact dermatitis.A case of allergic contact dermatitis in which the area of involvement spreads over the borders of the vulva.Allergic contact dermatitis due to fragrances contained in a soothing cream.List of the most common irritants responsible for vulvar ICD.Review of the studies concerning vulvar ACD.List of the most common fragrances and medicaments responsible for vulvar ACD.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Background: Microbial infection or exposure to endotoxin later in life exacerbates established asthma. Mast cells are involved in the exacerbation of asthma. This exacerbation involves a toll-like receptor (TLR)–mediated response of mast cells. In the clinical practice of otolaryngology, otolaryngologists experience an exacerbation of nasal congestion when infectious rhinitis develops in patients with allergic rhinitis, but the mechanisms are unknown. Therefore, this study investigated the effect of lipopolysaccharide (LPS) on allergic rhinitis using a mouse allergic rhinitis model. Methods: Female BALB/c mice, TLR4 gene mutant C3H/HeJ mice or mast cell–deficient WBB6F1-W/Wv mice were sensitized intraperitoneally with ovalbumin (OVA)/alum, and were intranasal challenged with OVA and/or LPS. Nasal symptoms and histologic changes were examined. Cytokines in nasal tissue were examined by Western blot. The effects of LPS on degranulation and cytokine production of bone marrow–derived mast cells (BMMCs) were investigated. Results: Nasal administration of LPS together with the antigen exacerbated nasal symptoms, eosinophil infiltration of the nasal mucosa, and increased IL-5 production in the nasal mucosa. It was not observed in C3H/HeJ mice and WBB6F1-W/Wv mice. The addition of LPS increased the production of IL-5 from BMMCs in a dose-dependent manner, but no effect on degranulation was observed. Conclusions: Intranasal administration of LPS exacerbates allergic rhinitis through Th2 cytokine production from mast cells. This observation provides clues to the mechanism of exacerbation of allergic rhinitis caused by an infection in daily clinical practice.Microbial infection exacerbates established asthma or contributes to the initial development of the clinical onset of asthma [1,2]. In particular, microbial infection or exposure to endotoxin early in life is considered to protect from the later development of asthma by stimulating the immune system toward a T-helper lymphocytes type 1 (Th1) response from Th2 response [3,4]. This is well known as the hygiene hypothesis. On the other hand, microbial infection or exposure to endotoxin later in life exacerbates established asthma [2].In the last 20 years, it has been extensively elucidated that toll-like receptors (TLRs) are mammalian homologues of the Drosophila toll receptor and have a role in the innate recognition of bacteria. Furthermore, TLR2 and TLR4 are reported to be implicated in the recognition of various bacterial cell wall components [5].Systemic administration of lipopolysaccharide (LPS) before sensitization inhibits Th2 response and suppresses the development of airway inflammation in murine asthma models [6]. On the contrary, administration of LPS with antigen in the reaction phase exacerbates asthma. Administration of LPS has been shown to enhance the Th2 response through activation of mast cells in the reaction phase [7].Otolaryngologists experience an exacerbation of nasal congestion when infectious rhinitis develops in patients with allergic rhinitis. The exacerbation of asthma caused by microbial infection has been well studied, but none for allergic rhinitis.Therefore, the mechanism of exacerbation of allergic rhinitis caused by microbial infection was investigated using the mouse allergic rhinitis model.Six-week-old female BALB/c mice, C3H/HeJ, C3H/HeN, WBB6F1-Kitw/Kitw-v, and WBB6F1-+/+ mice were purchased from CLEA Japan (Meguro, Tokyo, Japan). C3H/HeJ mice are a nonresponder strain to LPS. WBB6F1-Kitw/Kitw-v mice are deficient in mast cells. These mice were maintained under specific pathogen-free conditions and received an ovalbumin (OVA)-free diet at the laboratory of the animal research center of Shimane University. All mice were 6 to 7 weeks of age at the beginning of individual experiments. Animal care and experimental procedures were approved by the Animal Research Committee of Shimane University (approval code: IZ27-150, IZ30-76, and approval date: 1 March 2016–31 March 2021) and conducted according to the Regulations for Animal Experimentation at Shimane University.Mice were intraperitoneally sensitized with 100 μg OVA mixed with 1 mg alum on day 0 and day 7. On days 21, 22, 23, 24, 25, 26, 27, and 28 after the first sensitization, sensitized mice were intranasally challenged with 400 µg OVA together with or without 4 µg LPS from Pseudomonas aeruginosa (Sigma-Aldrich, St. Louis, MO, USA) dissolved in phosphate-buffered saline (PBS) into the bilateral nostril.Just after the final intranasal challenge with OVA with or without LPS on day 28, the mice were placed into an observation cage (one animal/cage) for about 10 min for acclimatization. The mice were placed into the observation cage again, and the number of sneezes was counted for 5 min by the method of Sugimoto et al. [8].Mice were killed 12 h after the final intranasal challenge with OVA with or without LPS on day 28. The heads were removed and fixed in 10% formaldehyde solution for 24 h at room temperature. The heads were decalcified in 5% formic acid for 36 h at room temperature and neutralized in 5% sodium sulfate solution for 12 h at room temperature after fixation. Coronal nasal sections were then stained with hematoxylin and eosin.The nasal mucosal tissue was collected, frozen in liquid nitrogen, and then crushed with a homogenizer in PLC lysis buffer. Protein G    beads was added with each primary antibody (anti-IL-5, anti-IL-10, or anti-IL-10 antibody, Pharmingen, Franklin Lake, NJ, USA) and incubated at 4 °C for 24 h using a rotator. It was washed with RIPAI buffer and Western blot was performed according to the manufacturer’s instructions.Bone marrow cells were obtained by flushing the femurs of BALB/c mice. The bone marrow cells were cultured at 37 °C in a RPMI-1640 medium, supplemented with 5 ng/mL IL-3, 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin, 50 μg/mL streptomycin, and 50 mM 2-mercaptoethanol. After changing the medium every week for 4 weeks, the recovered populations were composed of >95% mast cells, as judged by flow cytometry of the expression of FcεRI and c-kit.Bone marrow–derived mast cells (BMMCs) were cultured for 24 h with various concentrations of LPS (1 ng/mL–1000 ng/mL). As a positive control, BMMCs (1 × 106 cells/mL) were sensitized by incubating for 2 h at 37 °C with 0.5 μg/mL anti-DNP IgE antibody in complete RPMI medium with 10% FBS. The cells were washed and then stimulated with 10 μg/mL DNP-HSA. After culturing for 24 h, IL-5, IL-10, and IL-13 in the supernatant were measured by the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions.Degranulation rate was evaluated by β-hexosaminidase release assay. BMMCs were cultured for 30 min with various concentration of LPS (1 ng/mL–1000 ng/mL). As a positive control, BMMCs (1 × 106 cells/mL) were sensitized by incubating for 2 h at 37 °C with 0.5 μg/mL anti-DNP IgE antibody in complete RPMI medium with 10% FBS. The cells were washed and then stimulated with 10 μg/mL DNP-HSA for 30 min.β-Hexosaminidase assay was conducted according to the method of Razin et al. [9]. Supernatant (50 μL) and pellet samples were incubated with 50 μL 1 mM p-nitrophenyl-N-acetyl-β-d-galactosaminide, dissolved in 0.1 M citrate buffer, pH 5.0, in a 96-well plate at 37 °C for 1 h. The reaction was stopped with 200 μL/well 0.1 M carbonate buffer, pH 10.5. The plate was read at 405 nm by a plate reader. The net percentage of β-hexosaminidase release was calculated as follows: β-hexosaminidase in supernatant/(β-hexosaminidase in supernatant + β-hexosaminidase in pellet) × 100.RNA was isolated with Trizol from BMMCs stimulated with LPS for 24 h. Northern blots with 5 to 10 μg of RNA were hybridized to a GATA3 cDNA probe. Normalization of RNA loading was done with a probe for GAPDH.Statistical analysis of the primary data was made using JMP. Data were presented as mean ± standard deviation. The one-way ANOVA and parametric independent samples “t” test were applied to evaluate the number of sneezes. Dunnet’s test was applied to evaluate the degranulation rate of BMMCs and cytokine production from BMMCs to compare with negative or positive control A value of p < 0.05 was taken as significant.To determine the role of LPS in the effector phase of nasal allergy, OVA-sensitized BALB/c mice were intranasally challenged with OVA together with or without LPS on days 21, 22, 23, 24, 25, 26, 27, and 28. The number of sneezes by each mouse after the final nasal challenge was counted. The number of sneezes was significantly greater in mice administrated LPS intranasally together with OVA (Figure 1A). Eosinophil infiltration into the nasal mucosa was increased in mice receiving LPS intranasally (Figure 1B). Nasal instillation of LPS increased the production of IL-5 in the nasal mucosa (Figure 1C).To prove whether the aggravation of nasal allergy because of nasal administration of LPS is mediated by TLR4, similar experiments were performed using C3H/HeJ mice, which are TLR4 gene mutant mice, and C3H/HeN mice, which are their wild type. The number of sneezes (Figure 2A), eosinophil infiltration into nasal mucosa (Figure 2B), and IL-5 production in nasal mucosa (Figure 2C) were aggravated in C3H/HeN mice, not in C3H/HeJ mice, after the final intranasal administration of LPS together with OVA.To find out whether mast cells are involved in this reaction, similar experiments were performed using WBB6F1-Kitw/Kitw-v mice, which are deficient in mast cells, and WBB6F1-+/+ mice, which are their wild type. Eosinophil infiltration into nasal mucosa (Figure 3A) and IL-5 production in nasal mucosa (Figure 3B) were aggravated in WBB6F1-+/+ mice, not in WBB6F1-Kitw/Kitw-v mice after the final intranasal administration of LPS together with OVA.Whether LPS affects its degranulation was investigated using BMMCs. Addition of LPS did not cause mast cell degranulation (Figure 4A). When LPS was added to mast cells, Th2 cytokine production was observed (Figure 4B). When the expression of GATA3 gene having a binding capacity to the promoter region of the IL5 gene was examined by northern blotting, the expression of GATA3 gene was enhanced in a dose-dependent manner of LPS (Figure 4C).The relationship between the onset or exacerbation of asthma and infectious diseases or environmental endotoxin (LPS) is well researched [8]. Childhood asthma and allergies are called the epidemic of the 21st century, because of a sharp rise in prevalence from the middle of the 20th century in high-income countries. Microbial infection or exposure to endotoxin early in life is considered to protect from the later development of asthma [3,4]. Asthma rates are rapidly rising in urban areas of low- and middle-income countries [9]. In mice experiments, farm dust or lipopolysaccharide treatment induced the expression of A20, a regulator of TLR-induced inflammation in respiratory epithelium, which is instrumental in reducing allergic airway inflammation [10]. Microbial infection or exposure to endotoxin later in life exacerbates established asthma. The causes of this exacerbation are suggested as follows: (1) by increasing the severity of the airway inflammation; (2) by increasing the susceptibility to rhinovirus-induced colds; and (3) by causing chronic bronchitis and emphysema with the development of irreversible airway obstruction after chronic exposure of adults [2]. Respiratory tract infections and common colds account for more than half of the causes of fatal asthma in Japan. In the clinical practice of otolaryngology, otolaryngologists experience an exacerbation of nasal congestion when infectious rhinitis develops in patients with allergic rhinitis. Unlike asthma, it is not fatal, so the cause of the exacerbation of allergic rhinitis because of infection is unknown.The mammalian TLR family consists of 13 members and recognizes specific patterns of microbial components, called pathogen-associated molecular patterns. TLR2 recognizes lipoprotein, lipopeptide, peptidoglycan, and lipoteichoic acid from Gram-positive bacteria. TLR4 recognize LPS from Gram-negative bacteria [5]. Generally, the cause of acute rhinosinusitis is that the virus first infects and then develops into bacterial inflammation. Most of the causative bacteria are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis [11]. Since S. pneumoniae is a Gram-positive bacterium, its cell component is recognized by TLR2. Since H. influenzae and M. catarrhalis are Gram-negative bacteria, its cell component is recognized by TLR4.Murakami et al. demonstrated that LPS activates mast cells to exacerbate asthma in a mouse asthma model [7]. LPS activated mast cells and exacerbated allergic rhinitis in the reaction phase using a mouse allergic rhinitis model in the present study. However, there is a difference in terms of expressions of TLR in upper and lower respiratory tracts. Acute nasal inflammatory diseases are infectious, have a bacterial etiology, and cause inflammatory responses elicited by nasally pathogenic exposure. In these responses, nasal epithelial cells are thought to play important roles as the initial point of contact with pathogens. The same is true for lower respiratory tract infections. Both TLR2 and TLR4 are expressed in the epithelial cells of the lower respiratory tract, but only TLR2 is expressed in the epithelial cells of the nasal mucosa [12]. TLR4 is reported to be expressed in the nasal epithelial cells of mice [13] or humans [14]. Neutrophil inflammation occurs when TLR2 agonist is administered to the nose of mice [15], but intranasal administration of LPS does not cause nasal inflammation (Supplementary Figure S1). LPS causes neutrophil inflammation in the lungs when administered intratracheally [16]. Xu et al. reported that intranasal administration of TLR4-short hairpin RNA (shRNA) inhibits murine allergic rhinitis by regulating the NF-κB pathway [13]. TLR4 in the nasal mucosa is thought to have a mechanism that enhances allergic rhinitis.Mast cells play an important role in type 1 allergic reactions, but they have a protective function against enterobacterial infections. BMMCs express TLR 2, 4, 6, and 8 but not TLR5, and produce inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-13) by LPS stimulation [17]. The data of the present study showed BMMCs produced Th2 cytokines via TLR4 stimuli. It has been reported that stimulation of LPS derived from E. coli does not induce degranulation of BMMCs [17]. Our study using LPS derived from P. aeruginosa showed a similar result. Mast cells migrate to the nasal epithelium from the mucosal connective tissue in allergic rhinitis patients [18]. It is possible that allergic rhinitis was exacerbated by LPS, activating mast cells migrated to the nasal epithelium and enhancing Th2 cytokines.Cytokine production from mast cells is mediated by MAP kinase, both via FC ε receptor and TLR [19]. Cytokine production by LPS stimulation of BMMC, the production of IL-5, IL-10, and IL-13, was suppressed by the p38 inhibitor, and the production of IL-10 and IL-13 was suppressed by the JNK inhibitor. We investigated the gene expression of GATA3, which has the ability to bind to the promoter region of the IL5 gene [20]. LPS stimulation enhanced GATA3 gene expression in a dose-dependent manner. The present study showed that the mechanism of IL5 production from mast cells stimulated with LPS depended on p38 and GATA3.The major effects of TLR4 are activation of antigen-presenting cells and enhancement of antigen presentation; it determines the direction of naive T-cell differentiation. In fact, vaccines containing TLR ligands as adjuvants are in clinical use. In allergic rhinitis, drugs containing TLR ligands are used clinically in subcutaneous immunotherapy [21], and clinical studies are being conducted in sublingual immunotherapy [22]. The mechanism of immunotherapy for allergic rhinitis is the induction of regulatory T cells. A preliminary experiment of this study was conducted using 40 µg LPS, which is the limit dose that can be dissolved, but in that case, the symptoms of allergic rhinitis were alleviated and the exacerbation of mucosal eosinophil infiltration was not observed. Depending on the amount of antigen and LPS, there may be effects like immunotherapy. This study focuses on mast cells, not antigen presentation or T cells. Further studies are needed on antigen presentation or T-cell function.This study data explained one of the mechanisms by which bacterial acute rhinosinusitis exacerbates allergic rhinitis. When patients with allergic rhinitis develop bacterial acute rhinosinusitis in daily clinical practice, it may be necessary to step up the treatment of allergic rhinitis.Intranasal administration of LPS exacerbates allergic rhinitis through Th2 cytokine production from mast cells. This observation provides clues to the mechanism of exacerbation of allergic rhinitis by infection in daily clinical practice.The following are available online at https://www.mdpi.com/article/10.3390/allergies1040020/s1, Figure S1: H&E stain of coronal section of head of mice, intranasal administrated with 4 µg LPS for 7 days.N.A., I.M. and T.F. have done all experiments together and produced data. H.K. made this project and supervised all experimental processes with his immunological background. T.S. supervised all experiments from a clinical point of view. All authors have read and agreed to the published version of the manuscript.This study was carried out using Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.Animal care and experimental procedures were approved by the Animal Research Committee of Shimane University (approval code: IZ27-150, IZ30-76, and approval date: 1 March 2016–31 March 2021).We thank Ryotaro Ishimitsu and Akemichi Murata for their excellent technical advice.We have no conflict of interest to submit this manuscript.Allergic symptom, eosinophil infiltration, and cytokine production in nasal mucosa after nasal challenge with PBS, OVA, or OVA and LPS in OVA-sensitized BALB/c mice. (A), The number of sneezes after challenge with PBS, OVA, or OVA and LPS. (B), Nasal coronal sections stained with hematoxylin and eosin. The black bar indicates 10 µm. The arrow heads indicate infiltrating eosinophil. (C), Cytokines in nasal mucosa evaluated by Western blot. In each experiment, seven mice were used. * p < 0.5.Allergic symptom, eosinophil infiltration, and cytokine production in nasal mucosa after nasal challenge with PBS, OVA, or OVA and LPS in OVA-sensitized C3H/HeN or C3H/HeJ mice. (A), The number of sneezes after challenge with PBS, OVA, or OVA and LPS. (B), Nasal coronal sections stained with hematoxylin and eosin. The arrow heads indicate infiltrating eosinophil. (C), Cytokines in nasal mucosa evaluated by Western blot. In each experiment, seven mice were used. * p < 0.5.Eosinophil infiltration and cytokine production in nasal mucosa after nasal challenge with PBS, OVA, or OVA and LPS in OVA-sensitized WBB6F1-+/+ or WBB6F1-Kitw/Kitw-v mice. (A), Nasal coronal sections stained with hematoxylin and eosin. The arrow heads indicate infiltrating eosinophil. (B), Cytokines in nasal mucosa evaluated by Western blot.The effect of LPS on mast cell degranulation, Th2 cytokine production, and expression of GATA3 gene. (A), Degranulation rate of BMMCs stimulated with LPS evaluated by β-hexosaminidase release assay. (B), Cytokine productions of BMMCs stimulated with LPS evaluated by ELISA. (C), Effect of MAP kinase inhibitor on cytokine production of BMMC stimulated with LPS (1000 ng/mL) evaluated by ELISA. curcumin: JNK inhibitor (50 mMol), PD98059: ERK inhibitor (30 mMol), SB203580: p38 inhibitor (30 mmol). (D), Expression of GATA3 gene of BMMCs stimulated with LPS evaluated by Northern blot. * p < 0.5.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Many ingredients found within nail cosmetic products are capable of sensitizing patients’ immune systems and causing contact dermatitis (CD). These include but are not limited to tosylamide, (meth)acrylates, and formaldehyde. A clear temporal relationship between nail cosmetic procedures and an eczematous outbreak on the hands, face, or other ectopic body regions can be a key indicator of CD secondary to nail cosmetic exposure. Once an inciting allergen is identified through patch testing, elimination and avoidance becomes a mainstay of treatment alongside the use of emollients and topical anti-inflammatory therapies. Patients should be counselled to approach future nail cosmetic products and procedures with caution and careful attention to ingredients, regardless of whether or not it has a “hypoallergenic” label.Nail cosmetics have been a mainstay of grooming and beauty since the 17th century, with the earliest findings of nail coloring and enhancements dating back to ancient civilizations in Egypt and China [1]. Today, millions of people worldwide utilize nail cosmetic products in order to achieve aesthetically pleasing nails, with the nail cosmetic industry growing from a USD 3 billion industry in 2007 to a USD 45 billion industry by 2012 [2]. In the USA alone, spending on nail salon services exceeds USD 8.5 billion yearly [3]. While many of the products used to achieve this look have been shown to be relatively safe, there are several complications associated with their use. These complications include mechanical and traumatic complications, infection, exposure to carcinogenic ultraviolet (UV) radiation (used to cure and set gel nail polish), and contact dermatitis (CD) [4]. The latter complication will be the focus of this narrative review article.CD, which can be further divided into allergic (ACD; 20% of cases) and irritant CD (ICD; 80% of cases), is an immune reaction that occurs in response to external compounds. This causative relationship between nail cosmetic products and CD has been well documented for over 50 years [5,6,7]. Specifically, a 1987 study showed that 4.2% of CD cases was due to the use of a cosmetic product, with approximately 8% of those cases attributed to nail cosmetics [6]. Since then, the number of patients diagnosed with CD secondary to use of a nail cosmetic product has grown along with the nail cosmetics industry, likely due to a combination of increased use and sensitization to the included ingredients [8,9,10,11]. In fact, the prevalence of contact allergies to one or more (meth)acrylates was shown to have significantly increased by more than 20% between 2009–2013 and 2014–2019 among nail technicians and product users [12]. A retrospective analysis of 38,775 patients who had previously undergone patch testing found that 769 (2%) had an allergic or irritant patch test reaction to a common ingredient in nail cosmetics [9]. This study was consistent with prior estimates that 1–3% of the population is sensitized to at least one ingredient found in nail cosmetics [5]. CD secondary to nail cosmetics is most prevalent among women aged 26–46 and nail technicians within their first year of work; however, it can develop within any demographic if nail products are used.In order to consolidate the current body of literature on this growing problem and provide suggestions for improved diagnosis, management, and education, PubMed searches were conducted using the key phrases “nail contact dermatitis”, “nail contact allergy”, “nail cosmetics allergy”, and “acrylate allergy”. In the current narrative review article, we will discuss the most common causative ingredients, clinical presentation, evaluation, and management of contact dermatitis secondary to nail cosmetics.According to modern beauty standards, aesthetically pleasing nails have a glossy, smooth surface, no overhanging or ragged cuticles, a tip extending beyond the nail plate, an oval contour to the nail plate, and a gentle curve when visualized from the side [1]. A variety of products can be used to achieve these looks, including liquid nail polish, nail wraps, gel nail polish, dipping powders, and acrylics (often referred to as “tips”) [4]. Other nail care products include nail strengthener, cuticle softener, and nail hydrating polishes, oils, and serums. More detailed descriptions of these products can be found in Table 1. Regular manicures using nail polish and gel manicures are the two most popular nail cosmetic procedures in the USA [3].While the products discussed above are certainly useful in enhancing nail appearance, various ingredients within these products can produce both ACD and ICD. Table 2 displays the most common allergens and irritants in nail cosmetic products and which products/procedures they are found in. While the list of these ingredients is extensive, a recent retrospective analysis of patch testing results performed in those diagnosed with CD secondary to nail cosmetic ingredients most frequently revealed positive reactions in response to hydroxyethyl methacrylate (56.6%), tosylamide (36.2%), methylmethacrylate (27.8%), ethyl acrylate (25.2%), and ethyl-2-cyanoacrylate (6.9%) [9]. We discuss these and other common ingredients below:Tosylamide/formaldehyde resin (TSFR) has long been known to be the most common cause of ACD related to nail polish, with data from the North American Contact Dermatitis Group (NACDG) suggesting that 4% of all positive patch tests involved sensitivity to TSFR [13]. It has been found to sensitize both nail components as well as produce ectopic ACD in areas of touching, scratching, or rubbing [14]. Because of this, many nail polish brands have switched formulation to include tosylamide epoxy resin instead; however, this has also been shown to sensitize both locally and ectopically [15].Methacrylates (powder and liquid) are mixed to form a flexible polymer for acrylic manicures. These ingredients can also be found in smaller quantities in both regular and gel nail polish. The three most common allergenic forms of these ingredients are 2-hydroxyethyl methacrylate (2-HEMA), 2-hydroxypropyl methacrylate (2-HPMA), and ethylene glycol dimethacrylate (EGDMA) [16,17]. These ingredients are most sensitizing in their liquid form (during application) and rarely cause reactions once hardened and cured. In addition, methacrylates are extremely cross-reactive with one another, so people are often allergic to multiple [15].Acrylates are mainly used in gel manicure systems. These include 2-hydroxyethyl acrylate (2-HEA), 2-ethylhexyl acrylate (2-EHA), 2-hydroxypropyl acrylate (2-HPA), ethyl acrylate (EA), and triethylene glycol diacrylate (TREGDA) [18]. With the increasing popularity of and consumer access to “at-home” gel manicure kits, sensitization to acrylates has increased, and those that use such kits are also more likely to be sensitized [19,20].Dibutyl phthalate (DBP) is considered a “plasticizer” that increases nail polish flexibility [21]. It has not only been seen to cause ACD, but it has also been shown to interfere with male reproductive development in animal models [22].Benzophenone is an additive to regular and gel nail polishes that protects cosmetic products by absorbing UV light and preventing product degradation prior to use. Cases report both ACD and photocontact dermatitis as a result of contact with benzophenone [23].Formaldehyde (also listed as formalin or methylene glycol) is the main ingredient in nail strengthening products and has been shown to cause ACD [14].Solvents such as ethyl acetate and isopropyl alcohol have been shown to cause ICD and ACD, albeit rarely. However, more recent studies have led many to believe that reactions to isopropyl alcohol are more common than once thought [24]. These ingredients can be found in both nail polishes as well as nail dehydrators.Ethyl Cyanoacrylate (ECA) is an ingredient in nail glue that has been associated with ACD, paronychia, and nail dystrophy [25,26]. While similar in name to (meth)acrylates (see below), evidence shows that there is no cross-sensitization between the two ingredients.Methacrylic Acid (MAA) is an ingredient in acidic nail primer that is known to be an extremely corrosive chemical. It can cause ACD if it accidentally comes in contact with the skin or cuticle on application [10]. Non-acid primers, those that do not contain MAA, have become more popular in order to avoid this adverse reaction; however, these still contain other allergens and irritants.Tosylamide/formaldehyde resin (TSFR) has long been known to be the most common cause of ACD related to nail polish, with data from the North American Contact Dermatitis Group (NACDG) suggesting that 4% of all positive patch tests involved sensitivity to TSFR [13]. It has been found to sensitize both nail components as well as produce ectopic ACD in areas of touching, scratching, or rubbing [14]. Because of this, many nail polish brands have switched formulation to include tosylamide epoxy resin instead; however, this has also been shown to sensitize both locally and ectopically [15].Methacrylates (powder and liquid) are mixed to form a flexible polymer for acrylic manicures. These ingredients can also be found in smaller quantities in both regular and gel nail polish. The three most common allergenic forms of these ingredients are 2-hydroxyethyl methacrylate (2-HEMA), 2-hydroxypropyl methacrylate (2-HPMA), and ethylene glycol dimethacrylate (EGDMA) [16,17]. These ingredients are most sensitizing in their liquid form (during application) and rarely cause reactions once hardened and cured. In addition, methacrylates are extremely cross-reactive with one another, so people are often allergic to multiple [15].Acrylates are mainly used in gel manicure systems. These include 2-hydroxyethyl acrylate (2-HEA), 2-ethylhexyl acrylate (2-EHA), 2-hydroxypropyl acrylate (2-HPA), ethyl acrylate (EA), and triethylene glycol diacrylate (TREGDA) [18]. With the increasing popularity of and consumer access to “at-home” gel manicure kits, sensitization to acrylates has increased, and those that use such kits are also more likely to be sensitized [19,20].Dibutyl phthalate (DBP) is considered a “plasticizer” that increases nail polish flexibility [21]. It has not only been seen to cause ACD, but it has also been shown to interfere with male reproductive development in animal models [22].Benzophenone is an additive to regular and gel nail polishes that protects cosmetic products by absorbing UV light and preventing product degradation prior to use. Cases report both ACD and photocontact dermatitis as a result of contact with benzophenone [23].Formaldehyde (also listed as formalin or methylene glycol) is the main ingredient in nail strengthening products and has been shown to cause ACD [14].Solvents such as ethyl acetate and isopropyl alcohol have been shown to cause ICD and ACD, albeit rarely. However, more recent studies have led many to believe that reactions to isopropyl alcohol are more common than once thought [24]. These ingredients can be found in both nail polishes as well as nail dehydrators.Ethyl Cyanoacrylate (ECA) is an ingredient in nail glue that has been associated with ACD, paronychia, and nail dystrophy [25,26]. While similar in name to (meth)acrylates (see below), evidence shows that there is no cross-sensitization between the two ingredients.Methacrylic Acid (MAA) is an ingredient in acidic nail primer that is known to be an extremely corrosive chemical. It can cause ACD if it accidentally comes in contact with the skin or cuticle on application [10]. Non-acid primers, those that do not contain MAA, have become more popular in order to avoid this adverse reaction; however, these still contain other allergens and irritants.The classic clinical presentation of CD associated with nail cosmetic use (typically ACD) includes proximal nail fold, hyponychial or paronychial tenderness, edema, erythema, fissuring, and paresthesia [3]. The exact location of the reaction within the nail complex depends on where the aggravating product was applied. More severe cases of ACD, typically associated with gel and acrylic manicures, may also include paronychia, thickened and dry nail plates, and onycholysis, including hemorrhagic onycholysis (Figure 1) with total nail detachment. Nail technicians who are more frequently exposed to these ingredients can present with eczematous hand dermatitis [17].In addition to the nails, CD may develop on ectopic body sites that the nails frequently come in contact with through touching, itching, or rubbing. This includes the face, neck, eyes and presents similarly to ACD and ICD caused by other triggers—erythematous, indurated, scaly plaques with possible vesiculation, fissuring, and edema. Some ingredients, such as acrylates, are also capable of becoming airborne, producing a more generalized, symmetrical airborne contact dermatitis [27].A recent NACDG retrospective analysis of data from 2001 to 2016 found that the face was actually more commonly involved in nail-cosmetic-related CD compared to the hands and nails themselves (43% vs. 27.6% of cases, respectively) [9]. Tosylamide allergy alone was more associated with face involvement, whereas (meth)acrylate allergy was more highly associated with hand involvement. In addition, 14.4% of cases involved the eyelids, and 12.1% of cases were considered to have scattered/generalized distribution [9]. A separate retrospective review of European data from 2013 to 2015 found greater involvement of the hands (88.9%) than the face (36.8%, including eyelids, lips, and cheeks) [8].The diagnosis of CD secondary to nail cosmetic use can often be made through a thorough patient history and physical exam, with special attention paid to the temporal relationship of nail cosmetic use/procedures to the onset of skin findings [28]. ACD will typically present 7–10 days after first/current exposure to the allergen, whereas ICD will present much sooner, often immediately or within a few days. Additionally, ICD tends to occur more on the dorsal plane of the hands, compared to the palmar aspect in ACD, and less frequently presents with vesicles and/or pain [29]. The differential diagnosis may also include atopic dermatitis and dyshidrotic eczema. The diagnosis of ACD can be confirmed by patch testing, although treatment and counselling may begin in the interim with a strong clinical suspicion of CD.Patch testing is the standard diagnostic and confirmatory test for determining causative allergens in ACD and has both a sensitivity and specificity around 70–80% [30]. Up to 10% of all patients with ACD have a positive result for one or more ingredients in nail cosmetics [31]. However, as many commercially available patch testing kits do not include many of the antigens found in nail cosmetic products, additional customized testing using diluted versions of these ingredients should be performed for the most thorough results [3]. In fact, close to one-fifth of nail care product-associated allergens may be missed without additional screenings beyond the NACDG screening [9]. Suggested patch testing protocols for these ingredients can be found in Table 3. While broad-spectrum testing is useful for both identifying the current causative agent and future planning, suspected causative agents can be narrowed based on the procedure experienced and its included products (Table 2).Causative substance identification, removal, and avoidance is a mainstay of treatment for CD of any etiology. Ingredients that are more specific to certain products and/or nail procedures are likely easier to eliminate and, if desired, substitute. However, caution must still be taken. Patients must be aware that most polishes labeled “hypoallergenic” do not contain TSFR; however, these polishes typically still contain (meth)acrylates [38]. Additionally, some allergic to (meth)acrylates may find regular nail polish (that contains (meth)acrylates in much lower concentrations) to be a suitable substitution, while others continue to react. Some brands, such as CND Shellac and BRISA, claim to have created “acrylate-free” gel polishes, which have anecdotally been nonreactive in some with proven acrylate allergies. However, since cosmetics do not have ingredient regulating bodies, anyone with a history of CD secondary to nail cosmetic use should try new products with extreme caution [16].For nail technicians and beauticians who occupationally come in contact with these allergens, latex gloves should be avoided as (meth)acrylates have been shown to penetrate through this material [39]. Rather, nitrile gloves should be used and switched every hour, as after this time frame, protection can diminish. Trilaminated polyethylene gloves are the most effective at protecting underlying skin from (meth)acrylates and other ingredients, but these are typically impractical [40].For immediate treatment of CD skin lesions, topical steroids can be prescribed. Caution should be taken to prescribe a steroid of appropriate potency for the affected region, such as higher potency steroids for hand lesions and lower potency steroids for the more delicate the face [41]. Ointments may be chosen over creams as they are less likely to contain allergenic preservatives [28]. Alternatively, topical calcineurin inhibitors, such as pimecrolimus, tacrolimus, and cyclosporine, can be useful for ACD of the eyelids [35].Emollients and barrier creams, such as petrolatum, dimethicone, and paraffin, should be recommended in both treatment and prevention of CD by providing an extra layer of protection from allergens above the stratum corneum [42]. These creams have also been shown to have some anti-inflammatory properties as well as the ability to restore the skin barrier [41]. Hyaluronic acids can enhance intracellular lipid production, while ceramides help to retain moisture [33]. Occlusive dressings with emollients placed over affected areas (when practical) may help emollient adherence to skin and decrease subsequent exposure to allergens prior to healing.As the popularity of nail cosmetic products and procedures continues to increase, nail-cosmetic-related CD has increasingly become a significant burden to patients, clinicians, and nail technicians. The high number of potentially allergenic ingredients in combination with the lack of a cosmetic regulatory body makes identification and elimination of triggering substances particularly challenging. As such, careful diagnosis, causative agent identification, treatment, and counseling are pertinent for positive outcomes.All authors were equally responsible for the conceptualization, methodology, resources and data curation, writing, revisions/editing, and visualization of this project. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.Zoe M. Lipman has no conflict of interest to declare. Antonella Tosti has served as a consultant or advisor for DS Laboratories, Monat Global, Almirall, Thirty Madison, Lilly, Leo Pharmaceuticals, Bristol Myers Squibb, and Procter & Gamble.Hemorrhagic onycholysis in patient after a gel manicure.Description of common nail cosmetic products.Common allergens and irritants in nail cosmetic procedures.Patch testing protocols for nail cosmetic ingredients [3].T.R.U.E. (thin-layer rapid use epicutaneous) test contains 36 allergens, and the ACDS (American Contact Dermatitis Society) core series contains 80 allergens.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Gly m 7, a novel soybean allergen, was recently reported. In this study, we attempted to detect Gly m 7 in various soybeans and processed soybean foods using raised anti-Gly m 7 antibodies and enzyme-linked streptavidin, specifically binding to the biotin moiety of Gly m 7. There was a large difference in Gly m 7 levels in various soybean-processed foods. When Gly m 7 levels were determined, all cultivars contained this allergen almost completely, but the biotin moiety detected by streptavidin varied, suggesting that biotinylated levels of Gly m 7 might differ among cultivars. The thermal stability of Gly m 7 was determined by heating soybean extracts. During detection using anti-peptide antibodies, detectable intact Gly m 7 was gradually reduced by heating. Gly m 7 was not detected by peptide or biotin detection in worm-wounded soybeans. Soybeans were immersed in distilled water as a pretreatment step for germination, and Gly m 7 levels were compared by immersion time (4–96 h). Intact Gly m 7 was rapidly degraded in detection by both peptide and biotin moieties. This suggested that Gly m 7 was degraded by some protease(s) during germination. These results would be useful for understanding the properties or risk assessment of Gly m 7, a newly discovered soybean allergen.Soybeans (Glycine max L.) are a common ingredient in many foods, including fermented foods, in both traditional Asian and Western cuisines. In addition, properties of soybean protein make it an excellent choice for food processing widely used as an additive in various processed foods. Soybeans also have various unique physiological effects, including lowering serum lipids [1,2,3]. Soybean protein has received a great deal of attention in recent years as a material for “veggie meat” [4,5].However, consumption of soybeans can cause allergic reactions in some individuals, and various allergens have already been identified in soybeans [6]. The major allergens of soybeans are the Kunitz soybean trypsin inhibitor [7]; Gly m Bd 30 K [8,9]; Gly m Bd 28 K [10]; Gly m 5 [11,12], which has been identified as 7S globulin (β-conglycinin); and Gly m 6 [13], which has been identified as 11S globulin (glycinin). Gly m 8 (2S albumin) has been recently identified as a clinically important allergen in soybeans [14]. These allergens are thought to cause systemic reactions in patients with soybean food allergies. In contrast, oral allergy syndrome and severe allergic reactions, including anaphylaxis, caused by soybean protein-containing foods have recently been reported in patients with allergies to birch pollen (pollinosis). Starvation-associated message 22 (SAM 22: Gly m 4: PR-10), which has a molecular weight (MW) of 17 kDa, has been detailed to be the major causative allergen of this pollen-related food allergy [15,16]. In addition to Gly m 4, Gly m 3 has been identified as another pollen-related soybean allergen. This molecule is homologous to the Bet v 2 allergen, which has been identified as a profilin, a type of cytoskeletal cellular protein [17].Recently, John J. et al. have reported Gly m 7, a seed biotinylated protein, as a new allergen in soybeans [18]. The amino acid sequence of Gly m 7 (Allergome 10,214, Gly m 7.0101) is shown in Figure 1. It is known that the MW of this molecule is about 70 kDa, the lysine residue at the 125 amino acid position may be biotinylated, and the function of this molecule is speculated to be a biotin source or carrier during plant germination [18,19]. The reactivity of this molecule with subjects allergic to soybeans and peanuts have been examined, and 18 out of 23 were positive [18]. In patients with a peanut allergy, it has been suggested that soybean Gly m 7 shows stronger basophil activation ability than Gly m 5. In addition, despite the low content of Gly m 7 in soybean, it has been reported that its reactivity in soy allergy patients is high [18].Gly m 7 is abundant during late embryogenesis in soybean seeds and is proposed to belong to the late-stage embryogenesis accumulating (LEA) protein family [18,19]. Generally, the LEA protein is structurally stable, and it is considered that Gly m 7 also shows thermal resistance to soybean cooking and processing, but the details are unknown.Therefore, in this study, we produced antibodies against Gly m 7 to detect peptide moieties. In addition, we used the strong and specific binding between biotin and avidin to detect the biotin moiety of the biotinylated Gly m 7 protein in various soybeans and processed soybean foods under various conditions.Horseradish peroxidase (HRP)-labeled anti-rabbit IgG and HRP-labeled streptavidin were obtained from Thermo Scientific (Waltham, MA, USA). ECLTM western blotting reagent and Hyperfilm-MP TM X-ray films were obtained from GE Healthcare (Piscataway, NJ, USA). The polyvinylidene fluoride (PVDF) membrane (Immobilon-P TM) was obtained from Millipore (Billerica, MA, USA).Rabbit polyclonal antibodies against Gly m 7 were obtained by immunizing rabbits with Gly m 7. Briefly, two synthesized peptides, “DITAGKDTPQGSI” (A) and “KGNKDRPELKTRT” (B), corresponding to the sequences of Gly m 7 (Figure 1), were conjugated to keyhole limpet hemocyanin and mix-immunized to rabbits. These sequences were determined as antigen peptides based on the hydrophobic/hydrophilic information of the protein and the sequence information presumed to be the surface of the molecule. After three immunizations, the final antigens were injected and boosted. Anti-serum was obtained from the rabbits. Antibody production was performed at Scrum Inc. (Tokyo, Japan).Various kinds of soymilk and processed soybean foods (kinu-tofu, momen-tofu, thick fried tofu, thin fried tofu, boiled soybeans, kome-miso (salty miso made from soybean and rice), shiro-miso (sweetened miso), and natto) were purchased at supermarkets near the university. The samples were diluted with distilled water, and the experiment was conducted at a constant dilution ratio. Solid processed soybean foods were weighed at a fixed amount (5 g), and distilled water (20 mL) was added and homogenized for 30 s with a food processor with blades and squeezed with quadrupled gauze to obtain an extract. By this operation, the food sample was finely crushed, mixed, and homogenized. Experiments were conducted under constant conditions of equal original sample weights. To compare relative levels of Gly m 7 in various soybean cultivars, 17 major soybean cultivars were obtained from a food company in Japan.Approximately 3 g of mature dry soybeans was immersed overnight in 30 mL of distilled water, and soaked soybeans were then homogenized for 30 s in 30 mL of distilled water using a commercial food mixer. The homogenate was filtered through four layers of gauze. The filtrate was used to determine protein concentrations, and subsequently analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or enzyme-linked immunosorbent assay (ELISA) after dilution. The protein concentration of the extracted soybeans was determined using the Bradford method [20].A mouse monoclonal antibody against Gly m Bd 30 K [6] was kindly provided by Dr. Tadashi Ogawa (Professor Emeritus at Kyoto University). Rabbit polyclonal antibodies against Gly m 5 (7S globulin; β-conglycinin; α′, α, and β subunits) were obtained as previously described [21]. Mouse polyclonal antibodies against Gly m 6 (11S globulin; glycinin) were obtained by immunizing mice with purified 11S globulin in our laboratory. Rabbit polyclonal antibodies against Gly m 4 were obtained by immunizing rabbits with recombinant Gly m 4, as described previously [22]. Rabbit antibodies against soybean trypsin inhibitors were obtained from Rockland (Gilbertsville, PA, USA). The specific reactivity of these antibodies was confirmed in our previous studies.Extracted soybean proteins were subjected to SDS-PAGE [23]. Proteins on the gel were stained with Coomassie Brilliant Blue (CBB R-350, GE Healthcare) to visualize the total protein patterns. Western blotting analysis was conducted by transferring the SDS-PAGE gel onto an Immobilon-PTM PVDF membrane (Millipore) using a semi-dry blotting method [24]. The membrane was incubated in 10 mM phosphate-buffered saline (PBS) (pH 7.5) containing 0.1% Tween-20 (PBST) and 5% skim milk for blocking (blocking solution). The membrane was then incubated for 1 h at room temperature in a blocking buffer containing allergen-specific antibodies. After washing the membranes four times with PBST for 10 min, the bound primary antibodies were detected using HRP-conjugated goat anti-rabbit or anti-mouse IgG and an ECLTM western blotting kit (GE Healthcare). The resultant chemiluminescent signals were detected on an X-ray film (HyperfilmTM MP, GE Healthcare). Western blotting experiments were carried out three times, and band densities were determined using the Alpha EaseTM software (Alpha Innotech, San Leandro, CA, USA).HRP-labeled streptavidin was used to detect the biotin moiety of Gly m 7. After SDS-PAGE, gels were blotted onto a PVDF membrane, and the membrane was blocked with blocking solution, as described above, and incubated with HRP-labeled streptavidin (1:1000 dilution) for 1 h. After incubation, the membrane was washed with PBST three times for 5 min with shaking. The ECL reagent was used for signal generation. The chemiluminescent signal was exposed to X-ray films (GE Healthcare).ELISA was carried out to evaluate titers of the antibodies produced. Briefly, soybean extracts were coated on an ELISA plate by sequential dilution. After sample coating and blocking, diluted antibodies (antisera) were added to wells. After reacting for 1 h at 37 °C and washing with PBST five times, HRP-labeled secondary antibodies were added to wells. The bound HRP-labeled secondary antibodies were detected by reacting with a tetramethylbenzidine peroxidase substrate (KPL, Gaithersburg, MD, USA) for 5–15 min. The reaction was stopped by adding 100 μL of 1 M phosphoric acid to amplify the signal. Absorption was measured at 450 nm using an ARVOsx-1 1420 multilabel counter (PerkinElmer Life Sciences, Boston, MA, USA). Measurements were performed three times, and the mean values were plotted.Results were expressed as the mean ± standard deviation (SD). Data were analyzed using the Tukey–Kramer or Dunnett’s method with the Stat View v. 5.0 (SAS Institute, Tokyo, Japan). For the comparison of two groups, Student’s t-test was used to determine significant differences. All experiments were performed at least three times. Statistical significance was defined as p < 0.05, indicated by different letters or asterisks.Based on the amino acid sequence of Gly m 7, two highly antigenic peptide sequences, “DITAGKDTPQGSI” (A) and “KGNKDRPELKTRT” (B), were selected, and synthetic peptides were prepared (Figure 1). The synthesized peptide was conjugated to keyhole limpet hemocyanin as a carrier protein and subsequently administered to rabbits. The antisera obtained were used to assess the titer and reactivity. ELISA (Figure 2a) and western blotting (Figure 2b) were performed using soybean homogenates. As a result, a dose-dependent increase in the absorbance of the soybean extract was confirmed by ELISA. The increase in absorbance using preimmune antisera was obviously lower than that using the obtained antisera (Figure 2a). In addition, we detected immunoreactive protein bands at approximately 70 kDa, which is consistent with the MW of Gly m 7 (Figure 2b, lane 1). This band was not detected when preimmune antisera were used (data not shown). These results indicated that the obtained anti-Gly m 7 antibodies containing antisera were able to react with Gly m 7 in soybean. Depending on the type of sample to be analyzed, some reactive protein bands were detected in addition to the position considered to be Gly m 7 when using anti-Gly m 7 polyclonal antibody raised. Therefore, the specificity of this antibody used in this study might be not very high (data not shown).HRP-labeled streptavidin was used to detect the biotin moiety in Gly m 7. When concentrations and blocking conditions of HRP-labeled streptavidin were optimized, it reacted specifically with an approximately 70 kDa protein band, which appeared to be in a position equal to the anti-Gly m 7 antibody-reactive band (Figure 2b lanes 1 and 2). This result indicated that the biotin moiety of Gly m 7 could be detected using HRP-labeled streptavidin under these conditions.The resulting antibodies were used to detect and compare the relative levels of Gly m 7 in various processed soybean foods (Figure 3a). The presence of a biotin moiety was also detected using HRP-labeled streptavidin (Figure 3b). Food samples were collected in aliquots, crushed, and subjected to western blotting in the same amount, and Gly m 7 was detected in kinu-tofu, momen-tofu, fried tofu (thick-type), fried tofu (thin-type), and boiled soybeans. The presence of Gly m 7 was particularly prevalent in tofu (lanes 2 and 3) and thick fried tofu (lane 4). In thin fried tofu and boiled soybeans, Gly m 7 levels were lower than in the previous three products, and it was not detected in fermented foods, such as kome-miso, shiro-miso, and natto. In thin fried tofu and boiled soybeans, Gly m 7 may have been degraded by factors, such as heating and pressurization. In miso and natto, both fermented foods, Gly m 7 might be degraded by proteolytic degradation during fermentation. Therefore, the allergen risk associated with this allergen might be low in these processed soybean foods. Detection of the biotin moiety using HRP-labeled streptavidin (Figure 3b) was also similar to the results obtained with anti-peptide antibodies for detecting the peptide moiety (Figure 3a).Relative levels of Gly m 7 were compared between commercially available soy milk beverages (Table 1). More Gly m 7 levels were detected in dense soymilk (samples No. 1, 2, 5, 6, 8, and 9; protein concentration > 30 mg/mL) than in other soymilks (samples No. 3, 4, and 7; protein concentration < 30 mg/mL) (Figure 4a(1)). Relative Gly m 7 levels and protein concentrations of these soymilk-related beverages were positively correlated, suggesting that concentrations of Gly m 7 were higher at higher protein concentrations (Figure 4a(2)). Therefore, it can be said that the risk of this allergen is also high in soymilk beverages with high protein concentrations. The detection of the biotin moiety using HRP-labeled streptavidin was also similar to results obtained with the anti-peptide antibodies (Figure 4b(1,2)). Therefore, biotin conjugation may show the presence of Gly m 7 in soymilk beverages.Relative levels of Gly m 7 in various major soybean cultivars were compared (Figure 5). In the detection of peptide moieties, total proteins or Gly m 7 were equally detected in any soybean cultivar (Figure 5a,b)), whereas differences were identified in the detection of Gly m 7 and biotin moieties (Figure 5c). Differences in the ratio of peptide to biotin moieties were also identified in each cultivar (data not shown). It was considered that the biotin bound to this molecule in each cultivar might not necessarily be the same amount.The thermal stability of Gly m 7 was determined by heating soybeans. The purpose of this experiment was to investigate the stability of this allergen when soybeans (extracts) as foods were heated. Soybean extract was prepared as described in Section 2.4. Obtained soybean extract (10 mg/mL in distilled water) was added in micro-tubes and heated at 100 °C using heat block. No obvious precipitates were observed, so the mixed samples were subjected onto SDS-PAGE and western blotting. In detection using anti-peptide antibodies, the detectable intact Gly m 7 around 70 kDa was gradually reduced by heating (100 °C) (Figure 6a,b)). The half-life of the apparent degradation was approximately 45 min. Similar results were obtained for the detection of biotin sites (Figure 6c). Therefore, this molecule showed heat stability during heating, and it was considered that biotin might not be separated by heating. Similar results have been reported in the previous study (18). They showed that Gly m 7 is a boiling-soluble protein since it did not precipitate when the extract was heated for 10 min. In addition, when compared with other major soybean allergens, Gly m 6 showed the highest thermal stability, followed by Gly m 4, Gly m 5, and Gly m 7, which showed similar thermal stability under the present conditions (Figure 6d).Gly m 7 levels of uninjured and worm-wounded soybeans were compared (Figure 7). In this experiment, the worm was identified to be Etiella zinckenella based on bite appearance (data not shown). The bite periods were not clear because the soybeans were wounded during storage time (half year). It was confirmed that Gly m 7 was not detected either by peptide detection (Figure 7c) or by biotin detection (Figure 7c) in worm-wounded soybeans. From this result, it was considered that the Gly m 7 peptide was degraded, rather than only the biotin being liberated from Gly m 7. In contrast, Gly m 4, a soybean allergen related to pollinosis, was clearly increased by worm wounding (Figure 7c). This result was consistent with that of our previous reports [23]. Total protein patterns of the two samples were quite similar, suggesting that the bulk soybean proteins did not change (Figure 7b).Soybeans were immersed in distilled water as a pretreatment step for germination, and Gly m 7 levels were compared by immersion time (4, 24, 48, 72, and 96 h). In this case, bulk proteins did not change (Figure 8a). It was confirmed that the longer the immersion time, the intact Gly m 7 was degraded more in detecting both peptide and biotin moieties (Figure 8b,c)). This suggested that Gly m 7 was degraded by some protease(s) during soybean germination. Interestingly, Gly m 7 was significantly reduced, but no significant reduction was observed in patterns of major proteins detected by CBB staining (Figure 8a) or in Gly m 4, Gly m 5, or Gly m 6 of other major allergens (Figure 8d). Therefore, this degradation may be a phenomenon specific to this allergen. Degradation intermediates were also detected at positions of approximately 50 kDa, and other MWs, such as that of intact Gly m 7, were degraded (Figure 8b,c).Gly m 7, recently reported as a new soybean allergen, undergoes modification (biotinylation) by biotin in molecules as a characteristic property. Taking advantage of this property, this molecule can be detected using HRP-labeled streptavidin. In this study, we generated anti-peptide antibodies against Gly m 7. The antibodies produced were able to detect putative intact Gly m 7 molecules (approximately 70 kDa) by western blotting. Using these multiple detection tools, we sought to characterize Gly m 7 in various soybeans and processed soybean foods. In some experiments, two close bands were detected at near 70 kDa by both methods. At this time, the substance and reasons for these two protein bands are unknown. However, since both of the two bands are also detected by biotin detection, we believe that they are both Gly m 7-related molecules. The reason of these putative molecular polymorphisms is unknown, but limited degradation and post-translational modifications are presumed. In the densitograph, the lower band of the major darker one is measured as Gly m 7.When this molecule was detected in various processed soybean foods (Figure 3) and soy milk (Figure 4), food rich in proteins, such as tofu, showed higher levels of this molecule per constant weight. Similarly, the higher the protein concentration, the more abundant the presence of this molecule was for soymilk. The purpose of this experiment is to investigate whether this allergen level changes in various soymilks. Since there are various products with different concentrations of soymilk, in order to evaluate the risk of allergens when taking a sip as a “food”, it was necessary to evaluate with a constant dilution rate without adjusting the protein concentration. In both cases, the pattern of detection of the peptide portion of this molecule using anti-peptide antibodies and the pattern of biotin detection were similar, indicating that biotin might not be released in these processed foods and remained bound. This suggests that methods, such as adsorption of this allergen using avidin, may also be feasible in processed foods. For example, the biotin-bound Gly m 7 molecule may be specifically adsorbed and removed by a passing-through to an avidin column. In case of detection or capture of this allergen using ELISA or avidin column, the free biotin contaminated in food sample could interfere the processes. It is important to consider this possibility when using these procedures. In addition, Gly m 7 was not detected in fermented foods, such as natto and miso, where the protein content was lower than that of tofu, possibly because the protein was degraded by microbial proteases during fermentation. Other major soybean allergen levels have also been reported to significantly reduce in miso [25]. Therefore, it has been suggested that the risk of soy allergens is generally low in fermented foods, such as miso and natto.When the relative levels of Gly m 7 in various soybean cultivars were compared, it was almost equally detected in any soybean cultivar in terms of peptide moieties, whereas biotin moieties identified differences in Gly m 7 detection (Figure 5). Differences in the ratio of peptide to biotin moieties were also identified for each cultivar. Therefore, it is suggested that the biotin bound to this molecule in each variety might be different. It suggests that different soybean cultivars may have different levels of biotin binding. In other words, it is possible that biotin is not bound to all Gly m 7 molecules, suggesting that the proportion may vary depending on the cultivars. It is unclear whether such differences result from differences in cultivars or from subtle differences in soybean seed maturity. The effect of biotin contained in this molecule on allergenicity is unknown, but if biotin modification affects allergen risk, it might vary between cultivars. In connection with this, we have recently reported that Gly m 7 levels in GM soybeans are comparable to those in non-GM soybeans [26].As shown in Figure 6, Gly m 7 was heat-stable, and it degraded gradually upon heating for up to approximately 2 h. Since biotin detection results were similar, biotin was not easily separated by heating in this case. Since biotin was bound to this molecule in many processed foods, heat processing does not seem to dislodge biotin. Although there were some differences, the thermal stability of this molecule was similar to that of other major soybean allergens. The aim of this study was to investigate the degree of degradation of various allergen molecules contained in soybean (extract) as a complex system, when heated, rather than to precisely test the thermal stability of isolated allergen proteins. Soybeans also contain high levels of stored protein, fiber, and oil, etc. These ingredients may exhibit matrix effects and affect the stability of allergen molecules. In addition, in terms of food safety assessment, we believe that it is beneficial to evaluate the stability of these allergens under unfractionated and crude conditions.Previously, we have reported changes in various allergens in soybean and edamame (young soybeans) when they suffered worm wounding. Results have shown that two allergens related to pollinosis (Gly m 3 and Gly m 4) are significantly increased by worm damage (22). In particular, Gly m 4 belongs to the PR (pathogenesis-related)-10 family, and its increased expression in pests and worm wounding is reasonable. It is well-known that pathogen-related (PR) proteins are upregulated when plants are stressed by pathogens and worms. As a result of examining the effect of worm wounding on Gly m 7, it became clear that the protein disappeared (Figure 7). Under the same conditions, it was confirmed that it increased with respect to Gly m 4. Thus, variations in the content of different allergen types at the time of insect or worm damage are of interest in terms of the relationship between soybean cultivation management and allergenicity. However, the physiological significance of Gly m 7 disappearances due to worm damage is unknown.When dried soybeans were immersed in water, it was hypothesized that Gly m 7 was specifically degraded by some proteases (Figure 8). In this process, degradation intermediates were detected at positions of approximately 50 kDa and other MWs. In this study, the dried soybeans were soaked in water for a long time. As a result, specific degradation of Gly m 7 was observed. Therefore, this process is slightly different than at the germination process. It is unclear whether this degradation is a physiologically meaningful phenomenon that occurs during germination or an artificial phenomenon. However, given that Gly m 7 is a biotin-binding protein, and its suggested role includes providing biotin during germination, it could be a physiological phenomenon. As the other soybean allergens examined in this study were not remarkably degraded by immersion alone, this simple method could potentially be applied as a Gly m 7-specific reduction method. This specific degradation of Gly m 7 during germination may be related to the physiological roles of this molecule in soybean.In conclusion, in the present study, we conducted a variation analysis of levels of this allergen in foodstuffs and various treatments using a detection system that could detect the peptide and biotin parts of Gly m 7, a recently identified soybean allergen, and clarified some characteristic properties. This study allowed us to update the allergenicity of soybean and soybean foods. In the future, the clinical significance and risk evaluation of this allergen should be advanced. The development of an ELISA method for accurate quantification of Gly m 7 is also expected.Conceptualization, T.M.; methodology, T.M. and E.Y.; software, A.F.; validation, A.F. and E.Y.; formal analysis, T.M.; investigation, N.T., A.F. and A.M.; resources, T.M.; data curation, A.F. and N.T.; writing—original draft preparation, N.T.; writing—review and editing, A.F., N.Z. and T.M.; visualization, N.T. and A.F.; supervision, N.Z.; project administration, T.M.; funding acquisition, T.M. All authors have read and agreed to the published version of the manuscript.This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (C)) Grant Numbers JP25450187 and JP16K07756 to T.M. This study was also supported in part by a grant from the Agricultural Technology and Innovation Research Institute (ATIRI), Kindai University.Not applicable.Not applicable.The data presented in this study are available upon request from the corresponding author.The authors acknowledge Ryouhei Masaki for their experimental support.The authors declare no conflict of interest.Amino acid sequence of Gly m 7. Selected peptide antigen regions for antibody production are indicated as box A and box B. The putative biotin binding Lys (k) residue is indicated by * at 125 aa. The sequence data was referred from UniProtKB-C6K8D1 (C6K8D1_SOYBN) (Allergome 10214, Gly m 7.0101).Detection of Gly m 7 using raised anti-Gly m 7 antibodies and horseradish peroxidase-labeled streptavidin in soybean extracts. (a) Enzyme-linked immunosorbent assay (ELISA) results for detection of Gly m 7 using raised anti-Gly m 7 antibodies. Soybean extracts were immobilized on ELISA plates, and immunoreactions were performed using raised anti-Gly m 7 antibody (×1000 diluted) (solid line) and preimmune sera (×1000 diluted) (dashed line). (b) Detection of Gly m 7 using raised anti-Gly m 7 antibodies (lane 1) and horseradish peroxidase-labeled streptavidin (lane 2).Relative levels of Gly m 7 allergens in processed soybean food samples. (a) Gly m 7 levels were detected by anti-Gly m 7 antibodies (b) Relative levels of Gly m 7 in various processed soybean food samples using horseradish peroxidase-labeled streptavidins. Processed soybean food samples (10 µg (a) or 100 µg (b) protein/lane) were separated via sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and the Gly m 7 levels were detected. There are significant differences (p < 0.05) between different lowercase letters. (lane 1, standard soymilk; lane 2, kinugoshi-tofu; lane 3, momen-tofu; lane 4, thick fried tofu; lane 5, thin fried tofu; lane 6, boiled soybeans; lane 7, kome-miso; lane 8, shiro-miso; lane 9, natto).Relative levels of Gly m 7 allergens in various soymilks. Soymilk samples were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis using anti-Gly m 7 (a) or western blot-like analysis using horseradish peroxidase -labeled streptavidin (b). Experiments were performed three times, and the data are presented graphically, with bars representing means and error bars representing standard deviation (SD). There are significant differences (p < 0.05) between different lowercase letters. The representative band images are also shown in (1). The relations of sample concentrations and relative Gly m 7 levels are indicated in (2). The relative intensity of Sample 1 was calculated as 100. Sample numbers (1–9) correspond to the sample numbers in Table 1.Relative levels of Gly m 7 allergens in various soybean cultivars (1–17). (a) Coomassie Brilliant Blue staining of various soybeans. (b) Relative Gly m 7 levels detected by anti-Gly m 7 antibody. (c) Relative Gly m 7 levels detected by horseradish peroxidase-labeled streptavidin. *: p < 0.05, **: p < 0.01. (samples; 1, Tachinagaha; 2, Enrei; 3, Fukuyutaka; 4, Ryuuhou; 5, Ootsuru; 6, Yukihomare; 7, Toyokomachi; 8, Toyohomare; 9, Yukisizuka; 10, Oosuzu; 11, Nanbushirome; 12, Miyagishirome; 13, Tanrei; 14, Nagomimaru; 15, Tamahukura; 16, Toyoharuka; 17, Murayutaka).Heat stability of Gly m 7 and other soybean allergens. Soybean extracts were heated for the indicated time and subsequently subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis. (a) Coomassie Brilliant Blue staining of heated soybeans (b) Gly m 7 levels detected using anti-Gly m 7 antibodies (c) Gly m 7 levels detected using horseradish peroxidase-labeled streptavidin. The relative intensity at Time Point 0 was set at 100 %. Signal intensities were measured as described in the Materials and Methods section. Experiments were performed three times, and the data are presented graphically, with bars representing means and error bars representing the SD. *: p < 0.05, **: p < 0.01. (d) Time-dependent changes of other soybean allergens. (1) Gly m 4, (2) Gly m Bd 30K, (3) Gly m 6 (basic subunit), (4) Gly m 5.Effect of worm wounding on the Gly m 7 and Gly m 4 allergen levels. Appearance (a) and protein profiled by Coomassie Brilliant Blue (CBB) staining (b), Gly m 7 and Gly m 4 levels detected by antibody or horseradish peroxidase (HRP)-labeled streptavidin of non-wounded (1) and worm-wounded mature soybeans (2) (c). Total proteins were extracted from mature soybeans (non-wounded and worm-wounded) and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (60 μg/lane). Total protein was stained with CBB G-350, and Gly m 7 was detected by anti-Gly m 7 or HRP-labeled streptavidin as described in the Materials and Methods. Similarly, Gly m 4 levels were detected by anti-Gly m 4 antibody. Detected band intensities were expressed. Lane 1, non-wounded mature soybeans; Lane 2, worm-wounded mature soybeans. ***: p < 0.0001, **: p < 0.01, student’s t test.Effect of soybean immersion time on Gly m 7 and other allergens. Dried mature soybeans were immersed in dH2O for the indicated time (4, 24, 48, 72, and 96 h), and the immersed soybeans were extracted to test proteins or allergen levels. (a) Total protein pattern detected by Coomassie Brilliant Blue staining; (b) Gly m 7 levels detected by raised anti-peptide antibody; (c) Gly m 7 biotin moiety detected by horseradish peroxidase-labeled streptavidin; (d) other allergen levels detected by the specific antibodies. (1) Gly m 4, (2) Gly m Bd30K, and (3) Gly m 5. **: p < 0.01.Protein concentrations of tested soymilks.Protein concentrations of the samples referred to the product information.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Official website of The Microbiome Centre: https://microbiome-center.nl/en/.We set up this preliminary study to begin to evaluate one main question: could strengthening the microbiome have potential benefits for the skin condition of patients suffering with adverse effects after stopping long-term topical steroid use? We aim to turn it into a much larger study if the results show the interventions might help. After commonly being prescribed for eczema, cessation of topical steroid use, especially after long periods of inappropriate use, can leave lasting adverse effects on the body and skin, known by some as topical steroid withdrawal (TSW). This preliminary study involved seven human participants suffering with skin problems associated with TSW who approached Dr. Anja Gijsberts-Veens of their own volition because they were interested in more natural recovery methods. Five completed the study in full. Progress in skin condition was tracked by self-assessed symptom severity questionnaires filled out at the beginning and end of the five-month study. The skin microbiome was addressed by using a 100% natural product shown in previous work to significantly increase skin microbiome biodiversity. Three participants implemented dietary changes and supplementation in response to guidance after fecal sample analysis, with the aim of improving gut microbiome health. The average improvement in skin symptoms for all participants was 40%, and average symptom improvement ranged from 14% for Patient 5 to 92% for Patient 1. On average, the participants saw an improvement in 85% of their symptoms and stagnation or regression in 11% and 4%, respectively. Our results suggest that the interventions used might improve the skin condition of TSW patients, but the small sample size and the lack of a control group mean that more definitive conclusions should be reserved for our follow-up work, which addresses these issues. We also aim to swab the skin of participants to assess the effect on the skin microbiome from skin and gut treatments, as well as including a more in-depth analysis of skin and gut microbiomes.This preliminary study was motivated by one main question: could strengthening the microbiome have potential benefits for the skin condition of people suffering with adverse effects after stopping long-term topical steroid use? If the results from this very small sample group suggest it might, we aim to expand the study to use a larger sample size and a control group to allow us to draw more reliable conclusions. This study is very small and will only be used to justify the possibility of larger follow-up work, so the results are not definitive. We also aim to perform more in-depth analyses such as sequencing the skin microbiome of participants and a full gut microbiome analysis, which were beyond the remit of this study. A small cohort of seven patients was recruited who were suffering with symptoms commonly associated with topical steroid withdrawal (TSW), after having stopped the long-term use of topical steroids. The treatment was split into two parts: skin and gut microbiome. For the gut microbiome, dietary changes were implemented following advice from Dr. Anja Gijsberts-Veens after a gut microbiome analysis was performed by taking fecal samples. Additionally, all five participants who completed the study used a 100% natural cosmetics product, shown to significantly increase the biodiversity of the skin microbiome [1], on their skin for 5 months. To investigate the effect of these interventions, a symptoms questionnaire was given to them at the beginning and the end of the 6-month study.This study was believed to be important due to the prevalence of eczema continuing to rise at an alarming rate in the western world [2]. It is important to find ways of helping those with severe eczema and TSW, especially as this problem could become even more prevalent after a year of altered lifestyles due to the COVID-19 pandemic [3]. Eczema and TSW are both believed to be from alterations to the immune system, and so harnessing the microbiome may emerge as one of the most promising areas to treat them.A damaged microbiome, low in biodiversity [4], has been linked to the majority of common skin problems, including eczema [5,6], and the rise in chronic allergies in the western world [7]. Sufferers of atopic dermatitis also suffer from systemic problems such as food allergies, asthma, and allergic rhinitis [8]. As an integral part of the immune system, the microbiome is now thought to be crucial for protecting against whole-body systemic problems, not just those in the immediate vicinity of the skin or gut [9,10,11,12]. It is for this reason that treating the skin and gut microbiome together could pose a treatment for such issues.Methods of treatment for chronic skin conditions such as eczema and psoriasis so far have been centered on the use of topical steroids [13]. At their inception around 60 years ago, they were a breakthrough for treating dermatoses due to their immunosuppressive and anti-inflammatory effects [14,15]. This introduction of modern medicine and drugs has transformed global health in a way unparalleled in human history.However, there is a growing challenge: the overuse of potent drugs intended only for acute illnesses and short-term use, where they are used for long-term, chronic conditions [16,17]. Medical guidelines state that very high potency topical steroids should not be used for more than three weeks continuously [18,19]. Despite managing the symptoms very effectively in the short term, and helping many people with severe skin problems get on with their daily lives, long-term and inappropriate use of high potency versions can lead to worrying adverse effects [14].Partly due to the rise of reports of the condition on social media, studies, including one by the National Eczema Association, have confirmed that discontinuation after overuse can result in whole-body problems [20]. This is often called topical steroid withdrawal (TSW), or red burning skin syndrome (RBSS) [21,22,23]. These can last for long periods of time and cause significant psychological distress [24]. The mechanisms and reasons are not fully understood, but many factors are thought to contribute [22]. Topical symptoms include widespread red, sensitive skin, oozing sores, bone-deep itch, and ‘elephant-like’ looking skin [23,25,26,27]. Although it is often not accepted as a condition in the medical profession, in this study, we refer to the short-term adverse effects and the long-term damage left behind as TSW.This preliminary study investigated seven human volunteers who came to Dr. Anja Gijsberts-Veens of their own volition due to her status as a doctor of functional medicine. They did not want to continue topical steroid use and were interested in investigating more natural ways of helping their bodies recover. Each participant had suffered with eczema before using topical steroids on their skin over long periods of time; this ranged from 15 to 48 years. All the patients had made the decision to stop using topical steroids themselves and had reported a regression in their skin condition that showed symptoms associated with TSW. Six were women, and one was a man. We note that only Patients 1–5 finished the skin microbiome stipulations for the study and completed the final questionnaire. The participants were made aware of the conditions for the study at the beginning. The participants were required to fill out a questionnaire at the start and end of the study to keep track of the change in symptoms and their severity on the skin.All participants provided verbal consent prior to enrollment in the study. Results and data from this study cannot be linked to a certain individual due to anonymous reporting and data handling. The process was agreed on by Dr. Anja Gijsberts-Veens of the Microbiome Centre and the Centre for Functional Medicine, the Netherlands.The gut microbiome of the participants was addressed under the supervision of Dr. Anja Gijsberts-Veens. Patients 3, 4, and 7 had their gut microbiome analyzed by taking a fecal sample; the laboratory work was done by BIOVIS, and the Microbiome Centre in the Netherlands wrote up a report using the data and created individualized probiotic supplements to help the gut microbiome of the patients who opted for it. The following were included in the report: species, diversity, dysbiosis index, pH, digestion (of sugar, fat, proteins), zonulin (used as a biomarker of impaired gut barrier function for certain diseases [28]), inflammation markers (calprotectin, alpha 1 antitrypsin), and secretory IgA.The results of the fecal analyses and advice for each participant who opted for it are shown in the Supplementary File S1. The advice from Dr. Anja Gijsberts-Veens is available on request from the authors. A gut microbiome analysis was not performed again at the end of the study after the skin and gut interventions. It was beyond the remit of this study to perform a full gut microbiome analysis; if the results of this study show promise, this will be done in future work. For this reason, the sample collection, DNA extraction, and analysis methodology are included in Appendix A, as they are not the main focus of this study.Human fecal microbiome samples were taken noninvasively and handled with approval by and in accordance with the professionals at BIOVIS and the Microbiome Centre in the Netherlands. They stated that no ethical concerns were raised by the methods applied and approved the procedures in this study. Informed verbal consent was obtained from each person prior to the study. Samples were treated anonymously, and human material was not the focus point of this study. Microbial samples or data derived cannot be linked to a certain individual. The process of the experimentation was agreed upon by Dr. Anja Gijsberts-Veens, the Microbiome Centre, and BIOVIS.Dietary and lifestyle changes were then implemented depending on the results of the microbiome analysis, along with the use of personalized probiotics. The changes implemented were done with the goal of increasing gut microbiome biodiversity, which has been shown to positively impact the skin [11,29]. In our 2017 work, we noticed that biodiversity is the only current reliable indicator of ecosystem health [7,30,31,32,33,34,35,36]. This was inspired by research that found the healthiest gut ever recorded in infants in Burkina Faso who displayed ‘unprecedented’ levels of diversity [37]. Previous work has postulated that probiotics are implemented incorrectly without personalization [38]. The dietary advice consisted mainly of cutting out inflammation-inducing foods, including lots of sugar, processed foods, coffee, and alcohol; increasing the amount of pure, unprocessed, organic, and diverse whole foods; and drinking plenty of water. The lifestyle changes included a mindset shift towards a long-term lifestyle change, rather than a quick-fix solution. In addition, exercise, sun, time in nature, meditation, and a positive attitude were advised.The products used to address the skin microbiome were a 100% natural face and body wash shown to significantly increase skin microbiome biodiversity [1]. All information on the product can be found in our previous work, where we investigated its effect on the skin microbiome of human participants [1]. The guidelines for use of the product for the skin are listed below:Use the product on the skin at least 1× per day for a minimum of 4 months.To use the product, mix with a small amount of water to form a solution, and gently massage onto the body.Use no other cosmetics products and try to stick to this as rigidly as possible. Exemptions in extreme cases, or where it was unavoidable were allowed, e.g., wearing some makeup for an important business meeting.In the beginning, introduce the product slowly to the body by using it mixed with a small quantity of water once every couple of days, and slowly build up to using it 1× per day by the end of the first month.Use the product on the skin at least 1× per day for a minimum of 4 months.To use the product, mix with a small amount of water to form a solution, and gently massage onto the body.Use no other cosmetics products and try to stick to this as rigidly as possible. Exemptions in extreme cases, or where it was unavoidable were allowed, e.g., wearing some makeup for an important business meeting.In the beginning, introduce the product slowly to the body by using it mixed with a small quantity of water once every couple of days, and slowly build up to using it 1× per day by the end of the first month.The participants were asked to fill out a questionnaire on their symptoms the day before they started the microbiome strengthening plan and as soon as they finished. It asked them to rate the severity of each symptom they were experiencing by giving them a number out of ten. Zero meant the symptom was nonexistent, and ten meant it was the worst possible. Microsoft Excel was used to turn the answers into the symptom severity graphs in Section 3.2.We also performed bivariate linear correlation analysis on some of the variables in this study using the Pearson correlation coefficient method [39,40] to investigate whether a relationship existed between the amount of time topical steroids was taken and the average improvement of symptoms. We did the same for time elapsed between stopping the use of topical steroids and the start of the study and the average improvement in symptoms.Microsoft Excel was used to calculate the Pearson correlation coefficient, or ‘r’ number. If this number exceeds rreq, or p, a statistically significant correlation can be reported. This is calculated using the table of correlation coefficients displayed in Table 1 [41], where rreq, or p, needed for correlation is shown at different degrees of freedom and probability levels. This table was turned into the graph shown in Figure 1, where a formula for rreq was obtained. ‘Degrees of Freedom’ is the number of data points on any given graph, minus two. Therefore, for this study, the ‘r’ number required to statistically show a correlation rreq = 0.807 at the p = 0.05 level because there are five participants. The working is displayed below.
+        y = −0.196ln(x) + 1.0219(1)
+        y = −0.196ln(3) + 1.0219
+        rreq = 0.807
+        y = rreq or p;x = degrees of freedom.y = rreq or p;x = degrees of freedom.Table 2 collates the important information regarding each participant involved in the study. The participants were required to fill out a questionnaire on the symptoms of their skin at the beginning and end of the study. They were asked to give each symptom a score out of ten based on the severity, with ten being the worst possible, and zero meaning nonexistent. Figure 2A–E shows the severity of symptoms before and after the study for Patient 1, 2, 3, 4, and 5 respectively. Figure 2F shows each participant’s average percentage improvement in symptoms, along with the overall average, which was +40%. Figure 3 shows the percentage of symptoms that improved, regressed or did not improve for all participants. Table 3, Table 4, Table 5, Table 6 and Table 7 show the percentage improvement for Patient 1 to Patient 5.On average, the participants in this study saw an improvement in 85% of their symptoms, no change in 11% and 4% of symptoms regressed. Figure 3 shows this. The participants in this study had all used topical steroids regularly for 15 to 48 years, which puts them in the severe end of the spectrum.We note Patient 2’s pregnancy during the study could have done so, which turned out to be 49% on average. Pregnancy was shown to worsen the eczema in 75% of women who were previous sufferers and cause other skin problems [42,43]. Also commonly reported in previous TSW patients is the occurrence of secondary fungal or bacterial infections, which implies the microbiome is out of balance. Patient 2 used two courses of antifungal creams during the study, which could also have set back progress because the creams could kill off fungi that are important constituents of healthy skin, subsequently leaving the skin microbiome more damaged and open to infection.Patient 3 had only stopped using topical steroids one week before starting the study. Previous work has shown that TSW symptoms appear days to weeks after discontinuation of topical steroid use [21]. Therefore, it is likely in this scenario that the symptoms could get worse in at least the first few weeks of the study, regardless of other factors.In Figure 4A, we plotted the length of time topical steroids were used by each participant against the average improvement in symptoms, which shows a statistically significant positive correlation (r = 0.98). The table in Figure 4B shows the values used to create Figure 4A and the correlation coefficient, or ‘r-number’, between the variables. In Figure 4C, we plotted the length of time between stopping steroids and starting the study against the average improvement in symptoms for each participant. There was no statistically significant correlation observed between these two variables (r = 0.57). The table shown in Figure 4D displays the values used to create the graph in Figure 4C and the correlation coefficient, or ‘r-number’, between the variables. The ‘p-value’ needed to be surpassed by r for a statistically significant correlation to be seen was p = 0.8065. The calculation of this was explained in Section 2.3 of the Materials and Methods. The sample size in this study is too small to use these results as definitive evidence that a relationship does or does not exist between these variables.The discussion is split into two parts to answer the questions posed in the Introduction (Section 1).This study aimed to rebalance and increase the diversity of the microbiome. There appeared to be improvements in the participants’ skin condition related to the interventions. We note that the use of five patients is too small to make conclusions with certainty and that an improvement in skin health could be influenced by other factors, including a reduction in exposure to synthetic ingredients and pollution in our western environment [1], time after quitting steroids [26], the seasons [44,45], exposure to the sun, and reduction in stress [46].Therefore, we cannot be certain the microbiome interventions were major factors in skin condition improvement, but we can use certain indicators as a guide. Having used topical steroids for many years, Patients 1, 2, 4, and 5 had come to a standstill with their skin condition, with their skin cycling with the seasons. The end point of the study was during the winter months when it is common for the skin condition to get worse for some people with severe eczema and TSW [44,45,47]. However, despite small, expected fluctuations, a large, exaggerated seasonal regression was not seen for these participants as it had been in previous years. For example, in the previous four years, Patient 1 had been through a cycle of skin improvement in the summer due to sun exposure and deterioration in the winter. During this study, this was not seen. In fact, the improvements continued throughout the winter, and some symptoms disappeared.Could some improvements be due to time being the ‘best healer’? Although our correlation analysis is inconclusive, it is a possibility; previous work suggested time is a factor, but the duration of topical steroid use was much shorter than this study [26,48]. We stress the importance of ‘expectation management’ when dealing with TSW. Previous work has shown even a few months of topical steroid use can result in years of damage left behind [26]. Symptoms can even get much worse within the first few months [21], so after suffering with TSW and eczema for many years with minimal improvement, even an improvement of 20% could signal positive signs. Rebuilding the microbiome will be a long-term process, not an overnight one, especially if the western environment is degrading it [1,7].The gut microbiome advice consisted of dietary improvements, supplementation, and ingesting personalized probiotic supplements, all of which have been shown in previous work to positively affect skin health [49,50,51,52,53]. In addition, cutting out certain ‘triggering’ foods was advised. This can reduce the severity of itching and burning, commonly associated with severe eczema and TSW [52,53]. However, this may manage symptoms more than solving the underlying problem.The skin health product used in this study was shown to significantly increase skin microbiome diversity in previous work [1]. It is not a topical probiotic product; if implemented incorrectly, these could decrease skin microbiome biodiversity [38]. Instead, the product tries to create the right conditions on the skin for biodiversity to flourish [1,7]. As healthier skin is characterized by an increase in biodiversity [7], this intervention could also have influenced skin condition. A damaged skin microbiome, low in biodiversity, is linked to most common skin problems, including eczema [5,6], psoriasis [54], skin cancer [55], and many more [11,56,57,58,59,60,61,62,63,64,65]. Stopping or drastically reducing the use of other cosmetics containing synthetic ingredients was also an integral part of the skin microbiome plan. Studies have explained how exposure of the skin to 21st-century chemicals, such as those in modern cosmetics, steroids, and cosmetics, is thought to have contributed to skin microbiome damage [1,7,66,67,68,69,70,71,72,73,74] and a large rise in allergies [4,65,67,75,76,77,78,79,80,81,82,83,84,85,86,87].We believe these preliminary results imply an improvement in skin condition might be attained by using the interventions described, but because of the small sample size and lack of a control group, it should be investigated more thoroughly in future work. The use of a control group will aim to minimize the effect of placebo and other factors. A much larger sample group will allow us to investigate whether the improvements in this study are anomalous and will help us to make predictions of the microbiome’s potential for helping similar conditions with more confidence. To thoroughly investigate the effect of the described microbiome interventions, we will take skin swab samples from the participants to assess any changes to the skin microbiome, primarily in biodiversity, by sequencing the skin’s microbes before and after the study. To do this, we will use our discovery of the ‘first reliable skin health measuring mechanism’, recently updated [4], where a relative increase in microbial biodiversity is associated with healthier skin [7]. We will also conduct an in-depth analysis of the gut microbiome before and after the study, analyzing primarily the change in biodiversity.We should also run correlation analysis on the variables to see if any relationships exist, including both Pearson [39,40] and Spearman rho methods [39,88], preceded by a Shapiro–Wilk normality test [89,90,91].It is well known that a gut–skin axis exists [49,92,93,94], but often researchers and medical practitioners underestimate the influence of the skin microbiome on the gut. For example, as the body’s first line of defense, a damaged skin microbiome could be a major cause of food allergies [8,11,95]. We believe that addressing both skin and gut microbiomes simultaneously may impact whole-body health more than addressing either in isolation. Therefore, we will observe effects on both gut and skin.This small, preliminary study explored the idea of using the skin and gut microbiome for helping people suffering with severe skin problems after the long-term use of topical steroids, initially prescribed for eczema. Techniques shown to improve the skin and gut microbiome were used with the aim of creating the right environment for a healthy diversity of microbes to thrive. The average symptom change of all five participants who finished the study was +40%, and the individual averages ranged from +14% to +92%. On average, 75% of symptoms improved, 11% stayed the same, and 4% got worse. Partly because the study ended during the winter months when skin health normally deteriorates, we believe these preliminary results suggest that the interventions used might improve the skin condition of TSW patients, but the small sample size and the lack of a control group mean that more definitive conclusions should be reserved for our follow-up work, which addresses these issues. We will also sequence the skin microbiome of participants to see if changes in skin condition are linked to changes in diversity and alterations in the microbial community. Future studies on this topic are increasingly important, especially as eczema prevalence is rising quickly among adolescents, even before the COVID-19 pandemic.The following are available online at https://www.mdpi.com/article/10.3390/allergies2010001/s1, Supplementary File S1: Gut microbiome analyses and corresponding advice for all patients who opted for it (Patients 3, 4, & 7).Conceptualization, C.W.-R., A.G.-V. and S.W.-R.; methodology, C.W.-R. and A.G.-V.; validation, A.G.-V.; formal analysis, C.W.-R. and S.W.-R.; investigation, C.W.-R., A.G.-V. and S.W.-R.; resources, A.G.-V.; writing—original draft preparation, C.W.-R. and S.W.-R.; writing—review and editing, C.W.-R., S.W.-R. and A.G.-V.; visualization, C.W.-R., S.W.-R. and A.G.-V.; supervision, C.W.-R. and A.G.-V.; project administration, C.W.-R. and A.G.-V. All authors have read and agreed to the published version of the manuscript.This research received no external funding.The Microbiome Centre stated that no ethical concerns were raised by the methods applied and approved the procedures used in this study.Informed consent was obtained from all subjects involved in the study.The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.We would like to thank Linda Russell and Nick Wallen for their support, without whom our work would not be possible. BIOVIS and the Microbiome Centre in the Netherlands also deserve special mention for their work on gut microbiome analyses and advice. We would also like to mention that we used the library and information services of University College London (UCL) and the University of Notre Dame, where the authors were alumni and researchers, respectively.Christopher Wallen-Russell and Sam Wallen-Russell are employees of research and development company Pavane Consultants Ltd., and directors of JooMo Ltd. As license holder for the JooMo Ltd. range of skin health products, Pavane Consultants Ltd. is interested in determining how skin health can be measured and which environmental factors have caused the huge increase in skin allergy problems in the past 75 years. The company has no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All other authors declare no conflict of interest.This section details the methods used by BIOVIS which will be used more in the follow-up, larger study. For the participants who opted for gut microbiome analysis, a fecal sample was collected in a clean container and then sent to the BIOVIS laboratories. To provide for anaerobic conditions within the container, they were filled to at least 50% [96]. Because stool samples must be fresh, otherwise they do not reflect the microbial communities of the gut, they were analyzed within two days of being taken.In the microbiome analysis, a region encompassing the V3 and V4 hypervariable regions of the 16S rRNA gene is targeted for sequencing, utilizing the Illumina MiSeq instrument. The sequence information of the sequenced regions enabled taxonomic classification of microbial communities from stool specimens collected within the study.For sequence library preparation, primers were designed that contain an overhang adapter sequence, index sequence, spacer sequence, and the locus-specific sequence of the V3 and V4 regions. Bacterial genomic DNA extracted from stool specimens was amplified, fluorimetrically quantified and normalized, indexed (barcoded) for sequencing using the Nextera XT DNA Library Preparation Kit (Illumina, Eindhoven, the Netherlands), and pooled prior to sequencing on the MiSeq platform. The amplicon pools were prepared for sequencing with MagSi-NGSPREP Plus—Magnetic Beads for NGS (MagnaMedics GmbH, Aachen, Germany), and the size and quantity of the amplicon library was assessed via the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Darmstadt, Germany), respectively. The PhiX Control library (Illumina) was combined with the amplicon library (at 20%). The library pool was clustered to a density of approximately 500–750 K/mm2. The prepared libraries were sequenced on 300PE MiSeq runs. The resulting sequencing data matches the specifications when Q30average >75%. The image analysis, base calling, data quality assessment, and demultiplexing were performed on the MiSeq instrument. Utilizing the QIIME (V.1.8.0) software package, the sequences were quality trimmed (FASTQ) and paired-end aligned (PEAR). Taxonomic classification of the microbial communities was conducted by assigning the resulting sequences to operational taxonomic units (OTUs) using the USEARCH algorithm with a 97% threshold of pairwise identity and classified taxonomically (species classifier RDP), referring to both the Greengenes database and the Human Intestinal Tract database (HITdb).Graph showing rreq, or p, probability needed for the correlation coefficient, r, to be equal or more than for a statistically significant correlation to be observed.Change in severity of symptoms seen by the participants. The bars in black show the severity of each symptom at the start of the study, and the bars in white show the severity at the end of the study. Severity was measured in a self-assessed value out of ten, where ten was the worst possible. Graph (A) is Patient 1, Graph (B) shows Patient 2, Graph (C) shows Patient 3, Graph (D) shows Patient 4, Graph (E) shows Patient 5, and Graph (F) shows the average percentage change in symptom severity for each participant. A positive percentage indicates an improvement, and a negative percentage change indicates a worsening of symptoms.Percentage of symptoms that improved, regressed, or did not improve. This was done as a total of all the symptoms that the patients who finished the study (1–5) were dealing with.(A) Graph showing the length of time the participants used topical steroids for before quitting, against the average improvement in symptoms for the participants in this study (r = 0.98). (B) Table showing the figures used to create the graph in (A), including the correlation coefficient between the two variables, ‘r’. (C) Graph showing length of time elapsed between stopping the use of topical steroids and starting the study against the average improvement in symptoms for the patients. (D) Table showing the numbers used to create the graph in (C), including the correlation coefficient, ‘r’, between the two variables.Table of correlation coefficients at varying degrees of freedom.Participant information.Severity scores for Patient 1 before and after completing the study. The numbers in the table are out of ten, with ten being the worst severity, and zero meaning the symptom has ceased to exist. The percentage change in the severity of each symptom is shown in the bottom row.Severity scores for Patient 2 before and after completing the study. The numbers in the table are out of ten, with ten being the worst severity, and zero meaning the symptom has ceased to exist. The percentage change in the severity of each symptom is shown in the bottom row.Severity scores for Patient 3 before and after completing the study. The numbers in the table are out of ten, with ten being the worst severity, and zero meaning the symptom has ceased to exist. The percentage change in the severity of each symptom is shown in the bottom row.Severity scores for Patient 4 before and after completing the study. The numbers in the table are out of ten, with ten being the worst severity, and zero meaning the symptom has ceased to exist. The percentage change in the severity of each symptom is shown in the bottom row.Severity scores for Patient 5 before and after completing the study. The numbers in the table are out of ten, with ten being the worst severity, and zero meaning the symptom has ceased to exist. The percentage change in the severity of each symptom is shown in the bottom row.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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+Plants such as the Toxicodendron species, consisting of poison ivy, poison oak, and poison sumac, largely contribute to allergic contact dermatitis with itch as a predominate symptom. Many individuals are affected by this skin condition, with approximately 50% to 70% of adults in North America demonstrating a degree of clinical sensitivity to this species of plants. In this review, we discuss the prevalence, pathophysiology, and clinical features of this contact dermatitis, as well as both treatment and prevention directed towards alleviation of itch. Updated research is emphasized throughout this review, although it is evident that this field is evolving, and more research is necessary to enhance treatment.The most common cause of allergic contact dermatitis in the US is exposure to plants, specifically the Toxicodendron species, which include poison ivy, poison oak, and poison sumac. Contact with this species of plants causes a weeping rash that is largely characterized by significant pruritus. Allergic contact dermatitis is a common skin condition that affects millions of people per year, with anywhere between 10 to 50 million cases yearly, and it is a significant medical condition that occurs frequently [1]. Urushiol is the major allergen that elicits the response in the Toxicodendron species and it is dispersed throughout the plant including the leaves, stems, and roots [2]. The reaction occurs via direct contact with any part of the plant, as well as from indirect exposure to contaminated sources, such as clothing, shoes, and pets.Pruritus is a significant manifestation of rash; other characteristics include an eruption of delineated erythematous vesicles, papules, and edema. For many years, the treatment of allergic contact dermatitis has not changed but recent understanding of the underlying mechanism of itch can contribute to both the treatment and prevention. In this review, we will discuss the prevalence, pathophysiology, clinical features, and treatment of allergic contact dermatitis to the Toxicodendron plant species, highlighting the advancements made in understanding the underlying mechanism of itch and its potential in therapeutic relief.Toxicodendron dermatitis affects millions of individuals yearly. In the US adult population, it has been reported that approximately 50% to 75% of people demonstrate a reaction to urushiol, the allergenic component of oleoresin [3]. Most geographical locations in the US contain the plant species and subsequent allergic contact dermatitis affects individuals of all ages, ethnicities, and skin types [4]. Studies have shown that sensitization to urushiol typically occurs in early adolescence, between the ages of 8 and 14, with findings suggesting that infants are not as susceptible to sensitization [5]. In 2012, the US Centers for Disease Control and Prevention reported that emergency department visits for allergic contact dermatitis due to poison ivy was 929,290, compared to 472,000 visits in 2002 [6]. As a result, dermatitis from the Toxicodendron species contributes greatly to medical morbidity and these figures are increasing.Outdoor activities in the wilderness, rural areas, and sub-rural areas, as well as occupational exposure from farming, construction, landscaping, and forest firefighting increase susceptibility. For instance, the US Forest Services reported that dermatitis from Toxicodendron resulted in 10% of lost time due to injury related to rash [7]. Additionally, in areas with dense forestry, such as California, Oregon, and Washington, one third of firefighters are disabled with dermatitis from poison ivy and related species each fire season [8]. The medical burden is significant in these occupations, with the cost of treatment accounting for 1% of the budget of yearly workers’ compensation in California, for which the total was 11.4 billion dollars in 2014 [8].The allergenicity of poison ivy, poison oak, and poison sumac is due to urushiol, a catechol ring [9]. The composition of the catechol ring with variations in unsaturated to saturated bond ratios in the side chain components distinguishes urushiol found in poison ivy, poison oak, and poison sumac [3]. The antigenicity of urushiol can be attributed to changes in the aliphatic chain composition. Although slight variations occur in the chemical structure of urushiol found amongst the Toxicodendron species, cross reactivity is common and sensitization to one often yields susceptibility to an allergic reaction to other plants in the species [3].When the epidermis encounters an antigen, the antigen-presenting cells found within the epidermis, Langerhans cells, will uptake the antigen, travel to the nearest lymph node, and present it to T lymphocytes [10,11]. A similar process occurs once exposure to urushiol occurs, and following the migration of Langerhans cells, T lymphocytes are activated and travel to the site of exposure. Several cytokines are released at the site of exposure that propogate the inflammatory response.Additionally, Langerhans cells have CD1a molecules expressed on their surface. CD1a is a class I major histocompatibility complex molecule that is sensitive to lipids that presents antigens to T lymphocytes and is strongly associated with the reaction to urushiol breakdown molecules, specifically [12,13]. These antigen-presenting cells bind the substrate and present them to CD4 T-helper lymphocytes found in draining lymph nodes, initiating the immunological pathway. The activated CD4 T-helper lymphocytes activate both T-effector cells and T-memory lymphocytes, propogating the cytotoxic immune response against urushiol.In addition to the adaptive immunological response, local cells found in the epidermis and dermis also contribute to the inflammatory response and subsequent itch. Keratinocytes and monocytes secrete cytokines, prostaglandins, leukotrienes, and other immunomodulators that mediate additional immunochemical responses to urushiol [10,14]. Studies have found that keratinocytes initiate the release of interferon-γ and tumor necrosis factor α (TNF-α) as an early response [3,15]. The delayed response by keratinocytes produces the release of cytokines interleukin 1, interleukin 6, interleukin 8, and granulocyte-macrophage colony stimulating factor, continuing the immune response [3,13].The immunological pathway of allergic contact dermatitis to the Toxicodendron species has been largely studied; however, the mechanism of its itch has rarely been investigated until recently. New developments have suggested that IL-33 plays a major role in plant itch. Liu et al. employed the use of transcriptome microarray analysis and found that the cytokine IL-33 was upregulated in mice exposed to urushiol [16]. Cytokine IL-33 binds to receptor ST2, an Interleukin 1R receptor that is most commonly expressed in small, dorsal root ganglia. The binding of IL-33 to ST2 leads to the influx of Ca2+, eliciting a sensory reponse of itch. Injection of the IL-33 cytokine intensified the itch induced behavior such as scratching and corresponding inflammation, demonstrating a strong relationship between the upregulation of IL-33 and the pruritic mechanism provoked by urushiol. In addition, the use of molecules that attenuated IL-33 showed decreased behavioral itch responses, further indicating the association between the IL-33/ST2 cascade and pruritus, seen in Figure 1.Furthermore, the spinal IL-33/ST2 pathway was found to exacerbate chronic itch by increasing astrocytic Janus Kinase 2 (JAK2) binding to the signal transducer and activator of transcription 3 (STAT3) and upregulating the release of TNF-α [17]. TNF-α activated the release of gastrin releasing peptide (GRP), which binds to gastrin releasing peptide receptor (GRPR), mediating an increased itch response. Neutralization of IL-33, ST2, and JAK2 provided alleviation to chronic itch responses by diminishing the GRP/GRPR signaling cascade.A similar model employed the use of whole transcriptomes and measured itch mediators and found that urushiol induced a TH2 immune response and upregulated the synthesis of cytokine thymic stromal lymphopoeitin (TSLP) [18]. TSLP is a cytokine that serves as a T-cell regulator and facilitates a pruritic response. In addition to TSLP, other molecules associated with itch-related behavior were serotonin (5-HT) and endothelin (ET-1). The use of anti-TSLP, 5-HT inhibitors, and ET-1 inhibitors reduced behaviors associated with itch, such as scratching, in the mouse model when exposed to urushiol, signifying a correlation between TSLP, 5-HT, and ET-1 and pruritus that may be translational to human exposure to Toxicodendron plant species.Given that these reponses are largely histamine independent, blockage of IL-33/ST2 signaling pathways and TH2 dependent immunomodulators, such as TSLP, can provide therueptic relief to the itch response that is frequently observed in individuals sensitive to Toxicodendron dermatitis.Individuals who are sensitized to poison ivy, poison oak, and poison sumac will develop an acute response in response to re-exposure. Classically, the dermatitis that develops is described as a pruritic eczematous eruption that is often in the form of delineated streaks where contact with the plant brushed the surface of the skin [19]. The sharp demarcated eruptions consist of erythematous papules and vesicles that typically present within 24–48 h following exposure, however this can range from 5 h to 15 days in some individuals [20]. The clinical presentation of linear markings and sharp borders is a key feature that aids in the identification of this plant dermatitis.Initially, individuals may experience erythema, edema, and an eruption of papules followed by vesicles and bullae. In more mild cases, vesicles and bullae may not occur. Fluid from vesicles and bullae have not been found to contain antigen load and therefore do not contribute to the dissemination of the disease. Variations can occur in an individual due to differing concentrations of antigen and time of exposure. Occupational workers can experience additional sequelae of generalized dermatitis and respiratory tract inflammation due to aerosolization of urushiol in wildfire smoke [3]. The heightened response of the reaction can occur anywhere from 1–14 days after initial exposure. Without treatment, Toxicodendron dermatitis can last for 3 weeks and up to 6 weeks in highly sensitized individuals, significantly affecting individuals and their quality of life.A rare manifestation of allergic contact dermatitis from poison ivy, poison oak, and poison sumac is black spot dermatitis [21]. The sap from the plants deposits on the skin and forms black lesions preceding an eruption of erythematous papules and vesicles. Additionally, there have been reports of erythema multiforme following severe reactions to allergic contact dermatitis [22]. In these cases, individuals did not have prior history of herpes simplex virus suggesting that erythema multiforme may be an underreported reaction in severe cases. These patients presented with generalized itchy papules and widespread target lesions on the torso and extremities.Long-term complications are not common; the most prevalent are hyperpigmentation and secondary infection superimposed to the areas affected. Transient hyperpigmentation may occur following the localized inflammation and it is more common in individuals with darker skin tones [4]. Typically, hyperpigmentation can persist for a few months. One study analyzed the occurrence of secondary infection following allergic contact dermatitis to poison ivy and found that half of a total of 33 subjects developed infection. Isolates that were found from the areas of infection included Staphylococcus aureus, Group A β-hemolytic strep, Prevotella, Porphyromonas, and Fusobacterium [23]. A very rare complication of allergic contact dermatitis to the Toxicodendron species is the development of nephrotic syndrome [24].The treatment and prevention of Toxicodendron dermatitis has not changed for many years. The main goal of treatment is therapeutic relief aimed at alleviating many of the symptoms that individuals experience, predominately pruritus. Baths with baking soda and colloidal oatmeal and the use of cold compresses can help improve the itch. Additionally, over the counter topical treatment includes the use of cooling agents, such as calamine lotion, which aids in relieving dryness and reducing itch with menthol and phenol [25,26].The mainstay of treatment has been corticosteroids. Specifically, high potency topical steroid clobetasol has been found to be most effective during the early reaction. Alternatives to higher potency steroids are mid-potency topical steroids, such as triamcinolone and betamethasone, which may be a better alternative due to lower cost. In children, the use of low potency topical corticosteroids such as hydrocortisone can be used to prevent side effects such as atrophy of the skin [27]. Systemic corticosteroids may be used in severe and widespread cases. Cases that can benefit from the use of systemic steroids include individuals with greater than 20% body surface area affected, extensive vesicles, bullae, blistering, and itch, as well as involvement of sensitive areas such as the face or genitals [28]. Oral prednisone can be initiated at 1 mg/kg/day with a maximum dose of 60 mg/day for severe cases and should be continued for 2–3 weeks with tapering [29]. Alternatively, the use of intramuscular injection of triamcinolone for 3 weeks has been found to be therapeutic in severe cases and demonstrated increased compliance. One consideration when using triamcinolone intramuscular injections is the risk of rebound if the course of treatment is not sufficient [30]. Furthermore, the rebound dermatitis appears to be more steroid-resistant, so management of systemic steroid treatment must be closely followed [31]. The use of long-term systemic corticosteroids is limited by side effects such as risk of infection, hyperglycemia, and hypertension amongst other systemic effects.The use of antihistamines has limited efficacy, considering the histamine-independent cascade that underlies the mechanism of itch [32,33]. While the effect is limited, antihistamines are still considered one of the treatments for allergic contact dermatitis from the Toxicodendron species. Therefore, the newer studies on the IL-33/ST2 signaling cascade and TSLP show that it can be useful in treating the underlying mechanism of itch in unresponsive patients undergoing treatment with steroids and antihistamines or when contraindications are present.Use of topical immunomodulators that reduce itch, such as tacrolimus and pimecrolimus, was reported in a few case reports. Tacrolimus and pimecrolimus are calcineurin inhibitors that diminish the immunological cascade by decreasing cytokine production as well as the activation of T cells and Langerhans cells in the dermal skin [34,35]. One study analyzed the use of topical tacrolimus ointment 0.1% in patients with orbital allergic contact dermatitis and found a significant improvement in symptoms and a positive trend in reduction of itch [36]. A randomized controlled trial compared the efficacy of tacrolimus ointment 0.1% to vehicle ointment and found it superior at minimizing dermatitis and that it significantly reduced pruritus [37].We have also used a compounded Topical JAK/STAT inhibitor that reduced poison ivy itch. JAK/STAT pathway inhibition can decrease many cytokines that are involved in inflammatory processes [38]. As an emerging treatment, JAK/STAT inhibitors have been approved for use in rheumatoid and psoriatic arthritis, and are undergoing investigation for treatment of atopic dermatitis, dermatomyositis, and numerous other skin conditions. The use of topical JAK/STAT inhibitors requires further evaluation; however, there is evidence that shows promising efficacy in reducing itch in the treatment of allergic contact dermatitis.Prevention can be achieved by various modalities, such as limiting exposure to Toxicodendron species, washing of affected areas, pretreatment with topical barriers, and desensitization. Upon exposure, washing the affected areas immediately can breakdown the oily sap containing urushiol and prevent a reaction. Significant water flushing can effectively remove urushiol from the skin; flushing within 10 min can remove 50%, flushing within 15 min can remove 25%, and flushing within 30 min can remove 10% of urushiol substance [3]. Following 30 min, breakdown of urushiol and penetration of skin is likely to occur. Additionally, there is some evidence that suggests the use of chemicals that inactivate urushiol and soap as effective methods to remove urushiol from the skin. The chemical inactivator Tecnu, the oil remover Goop, Dial Ultra dishwashing soap, and Zanfel soap have all been found to significantly remove urushiol from the skin [39,40]. Additionally, pretreatment with topical barriers such as quarternium-18 bentonite, linoleic acid, Hollister Moisture Barrier, and Hydropel have also demonstrated efficacy at preventing or limiting the extent of reaction to urushiol [41,42,43]. One longstanding practice implemented by Native Americans is desensitization to urushiol by ingesting poison ivy leaves; however, this mechanism is controversial [44]. Previous findings have shown that ingestion or parenteral intake of urushiol demonstrates hypo-sensitization rather than desensitization. However, further studies did not find hypo-sensitization to be statistically significant in human models [45,46]. Moreover, there have been reports of increased pruritus and urticaria with ingestion or injection of urushiol [3].In addition to these treatments, there are currently clinical trials investigating the use of a vaccine injection to prevent poison ivy, oak, and sumac-derived contact dermatitis [47]. The name of the immunomodulating injection is 3-pentadecyl-1,2-phenylene bis (4-(4-aminophenyl)butanoate) (PDC–APB). A recent animal study, published in 2018, revealed that administration of intramuscular injection PDC–APB resulted in a very mild or nonexistent skin reaction following urushiol exposure in the experimental animal group when compared with the control animal group [48]. Currently, there is a phase I trial for the use of PDC–APB that will explore the efficacy and safety of its use against urushiol [49].Overall, the landscape of treatment for allergic contact dermatitis from poison ivy, poison oak, and poison sumac is directed at eliminated or diminishing itch and has not substantially changed over the last few years. However, developments in understanding the primary mechanisms of itch can impact upcoming treatment mechanisms and decrease pruritis in affected individuals.Toxicodendron dermatitis is one of the most common causes of allergic contact dermatitis and affects millions of individuals in the US yearly. Exposure to poison ivy, poison oak, and poison sumac in sensitized individuals evokes a weeping erythematous eruption of papules and vesicles that is highly pruritic. Many of the treatments for allergic contact dermatitis target the symptom of itch. There have been advancements in understanding the pathophysiology of itch in allergic contact dermatitis from urushiol that highlight the IL-33/ST2 pathway and cytokine TSLP. While management of urushiol-mediated allergic contact dermatitis has been unchanged, the effective targeting of underlying itch mechanisms can provide innovate treatments.Conceptualization, G.Y.; resources, A.L., G.Y.; writing—original draft preparation, A.L.; writing—review and editing, A.L., G.Y.; supervision, G.Y. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.No new data were created or analyzed in this study. Data sharing is no applicable to this article.G.Y. is a consultant and a board member of TREVI, Pfizer, Galderma, Novartis, Sanofi Regeneron, Eli Lilly, Kiniksa, Bellus, and LEO GSK. G.Y. is supported by grants by Sanofi Regeneron, Pfizer, Novartis, Kiniksa, and LEO. Angelina Labib has no conflict of interest.In response to urushiol, IL-33 is upregulated and binds to the ST2 receptor in dorsal root ganglia, mediating the transcription of TNF-α and subsequent inflammatory cascade that ultimately increases itch behavior.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/allergies/allergies-02-01-00003.txt

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+Allergic rhinitis (AR) is an important public health issue worldwide due to its increasing prevalence and impact on quality of life, school performance, and work productivity. Subcutaneous immunotherapy (SCIT) is used to treat AR and involves repeated injections of allergen extracts. SCIT is used for cases of severe AR with symptoms that are not adequately controlled by medication, when the side effects of medication limit treatment options, or where the aim is to cure rather than symptomatically treat. Although SCIT is effective, it is not necessarily curative. Furthermore, there is also a low but present risk of systemic allergic reactions, with systemic side effects occurring in less than 0–1% of treated patients. Sublingual immunotherapy (SLIT) has emerged as an effective and safe alternative to SCIT. SCIT and SLIT are the only immunotherapies currently available for AR. In addition to sublingual administration as an alternative to SCIT, other routes of antigen administration have been attempted with the goal of increasing safety while maintaining efficacy. This review discusses the efficacies of SCIT and SLIT, their mechanisms, the utility of intralymphatic immunotherapy (ILIT) as an alternative route of antigen administration, and the potential for immunotherapy using other routes of antigen administration.The parenteral administration of allergens as immunotherapy has been used for nearly a century as a disease-modifying therapy for allergic respiratory diseases [1,2]. In contrast to pharmacotherapy for allergic rhinitis (AR), which focuses on symptom control, the main aim of allergen-specific immunotherapy (SIT) is to modify the immune system and cure AR. SIT also effectively reduces symptoms and the need for medication over time and prevents sensitization to new allergens and the development of asthma [3,4].Subcutaneous administration was the sole effective route for administering allergens for more than 100 years [5].During this time, extensive efforts were made to improve and standardize the quality of allergen extracts, with subcutaneous immunotherapy (SCIT) ultimately being established as a safer and more effective treatment [4,6]. Sublingual administration was developed in the 1990s and has become the standard route of administration. A drop of a solution is placed under the tongue and kept in the mouth for two minutes before being swallowed [7].Other routes of administration were also considered, and various teams have investigated the efficacy of intranasal administration, with some reports [8,9,10]. Oral administration has failed to demonstrate efficacy against respiratory allergies but is expected to be effective against food allergies [11]. A previous study on grass pollen reported that transdermal administration using a patch test (applied to the skin for one to several days) reduced the symptoms of AR [12]. Intralymphatic allergen injections have recently been investigated as a promising new route of administration [13]. Intralymphatic immunotherapy (ILIT), consisting of three injections into the lymph nodes, has reduced the symptoms of rhinoconjunctivitis in humans [14].Three main approaches are employed to increase the efficacy and safety of antigen-specific immunotherapy: improving the antigen administered, developing a route of antigen administration, and improving the administration schedule [13].This review focuses on the effects of improving the route of antigen administration on the efficacy of antigen-specific immunotherapy.SCIT is used to treat AR and involves repeated injections of allergen extracts. SCIT is used for cases of severe AR with symptoms that are not adequately controlled by medication, when the side effects of medication limit treatment options, or where the aim is to cure rather than symptomatically treat.Although SCIT is effective, it is not necessarily curative. Furthermore, there is also a low but present risk of systemic allergic reactions, with systemic side effects occurring in less than 0–1% of treated patients [15]. It also needs to be administered under the supervision of a skilled physician who is trained to adjust the immunotherapy dosage, and patients need to be observed for 15 to 30 min after the injection due to the risk of serious systemic side effects [16].The efficacy of SCIT for seasonal allergic rhinitis (SAR), which was confirmed by greater reductions in seasonal symptoms and rescue medication than with a placebo, was recently reported [3]. The findings of a large, randomized trial also supported the dose-dependent efficacy of SCIT for SAR [17].Since the effects of SCIT and regularly administered medication have not yet been directly compared in detail, future studies are warranted. Limited evidence is currently available to support the efficacy of the subcutaneous administration of allergens for perennial AR and there is a paucity of up-to-date systematic reviews or meta-ensembles. There are also no systematic reviews or meta-analyses. However, individual studies have demonstrated efficacy. Varney et al. [18] reported a 58% reduction in symptoms and a 20% reduction in the use of rescue medication with house dust mite (HDM) immunotherapy despite a large placebo effect. In a randomized controlled trial (RCT) on the efficacy of HDM immunotherapy for rhinitis, significant decreases were observed in a clinical index derived from symptom and drug scores, visual analog scores, the results of nasal challenge and skin prick tests, and also in each parameter examined [19].SLIT is effective for adults and children, and monitoring by physicians is only required for the first dose [20,21]. SLIT is considered to be safer than SCIT because side effects are generally confined to the upper respiratory tract and gastrointestinal tract [22]. The clinical and immunological effects of SLIT, similar to those of SCIT, have been suggested to persist after 3 years of continuous use [23].Furthermore, localized changes occur in the oral cavity that are specific to SLIT [24].Although further studies are needed on the persistence and concordance of SLIT, particularly in children [23], we consider it to be an effective treatment and preventive method for asthma [25,26].Intralymphatic immunotherapy (ILIT) for some allergens, such as cat dander and pollen, was found to be effective for AR [14,27,28,29,30]. ILIT induces tolerance to a specific allergen more rapidly than SCIT and SLIT after only three injections of allergen extracts and does not induce serious adverse effects. Antigens administered into the lymph nodes may be efficiently delivered to antigen-presenting cells, followed by the activation of T and B cells. Therefore, ILIT may be advantageous because only a few injections of a small amount of an allergen extract are needed to achieve clinical effectiveness.We previously demonstrated the clinical effectiveness of ILIT against Japanese cedar pollinosis and investigated whether the clinical effectiveness of ILIT, in which pollen extracts were intralymphatically injected three times before the pollen season, persisted in two subsequent pollen seasons [31].SCIT and SLIT are the only immunotherapies currently available for AR. Although there are many unanswered questions regarding the underlying mechanisms of action, antigen epitopes fragmented by antigen-presenting cells may be taken up by the affiliated lymph nodes, leading to an immune non-response. However, in comparison with the amount of antigen administered subcutaneously or sublingually, the amount of antigen taken up by the lymph nodes with ILIT appears to be markedly smaller (Table 1). The direct administration of an antigen into the lymph nodes may induce immune tolerance with a very small amount of the antigen and this has been the focus of research in a number of clinical studies.The basic treatment of AR is considered to be antigen avoidance and drug therapy, with non-sedating antihistamines and intranasal corticosteroids being the mainstay of pharmacotherapy [32,33,34]. Inadequate responses to these drug treatments are often attributed to poor compliance. Immunotherapy may be considered when antigen avoidance, medication, and intranasal corticosteroids do not effectively control symptoms. Since the induction phase of AR cannot be altered by pharmacological treatment, medication must be repeated based on the symptoms being presented [24,32]. In contrast to pharmacotherapy, immunotherapy is regarded as a curative treatment that alters the induction phase. Immunotherapy, whether subcutaneous or sublingual, has the potential to cure the disease, in contrast to pharmacotherapy, and its efficacy has been reported in a number of RCTs [35,36], which may be maintained for 2–3 years after the completion of immunotherapy [24,37,38,39,40,41].The criteria for selecting immunotherapy are inadequate responses to pharmacotherapy and the goal of a cure. In cases in which pharmacotherapy is inadequate, immunotherapy may be applied in addition to pharmacotherapy, which switches the main focus of treatment from pharmacotherapy to immunotherapy, with drug administration as a rescue.The efficacy of immunotherapy differs between SAR and perennial AR, with SCIT being more effective for the former and less effective for the latter [4,42]. SLIT is also highly effective for SAR; however, evidence to support its efficacy for perennial AR, particularly in children, is currently limited [43].Antigen-specific immunotherapy involves altering the route of administration of an antigen that induces nasal allergic symptoms and administering it subcutaneously or sublingually such that allergic reactions are induced locally. These local allergic reactions are of little importance as side effects because subcutaneous administration merely causes redness and itching of the skin and sublingual administration results in itching of the mucous membrane of the oral floor under the tongue.In SLIT, the reported frequency of adverse reactions widely varies. Some studies identified mild local itching as an adverse reaction, whereas others did not recognize minor local symptoms as an adverse reaction. Although there is currently no information on fatal adverse reactions associated with SLIT, it is safer than SCIT [43].Among antigen-specific immunotherapies, the frequency of adverse reactions differs between SCIT and SLIT. The incidence of systemic adverse reactions to SCIT was previously reported to be 0.025% in terms of the number of inoculations [15], while that of serious anaphylactic reactions to SCIT for SAR was 5.4 per million injections (0.0005%), with an increased incidence (46%) during the period of high pollen counts [44,45]. Anaphylactic reactions due to SLIT have been reported in 11 cases, indicating that anaphylaxis occurred once in approximately 100 million injections, and no fatal cases have been reported to date [45]. In a study on systemic reactions in 66 trials with 4378 patients receiving approximately 1,181,000 doses of SLIT, systemic adverse reactions were detected for 169 out of 314,959 doses (0.056%), while severe systemic reactions occurred for 14 out of 1,181,000 doses (0.0014%) [46]. The most common systemic reaction was asthma, which required hospitalization in one case [46].Senti et al. [14] reported that there were significantly fewer adverse reactions with ILIT than with SCIT, with 18 mild grade 1 or 2 adverse reactions and 2 cases of asthmatic reactions requiring treatment in a medical facility during the first 4 months. In contrast to SCIT, there were 18 mild adverse reactions and 2 asthmatic reactions requiring institutional care in the first 4 months, and ILIT only caused 6 mild adverse reactions and no systemic anaphylactic reactions requiring hospitalization. Due to differences in the number of antigens administered within the first four months, the two groups were not comparable; however, the lack of anaphylactic reactions attributable to antigen administration into the lymph nodes is noteworthy.The causes of anaphylaxis may be associated with prior systemic reactions, concomitant immunodeficiency, poor compliance with treatment, re-administration after treatment interruption, severe or poorly controlled asthma, a high pollen count, the use of non-standardized extracts, and the lack of dose escalation periods.Adverse reactions to antigen-specific immunotherapy may be broadly divided into excessive local reactions and systemic adverse reactions. In SLIT, oral symptoms, such as oral itching, swelling of the lips, and pharyngeal irritation, are common local reactions [47]. This corresponds to redness and swelling at the injection site in SCIT. Systemic adverse reactions to SLIT are less common but are mostly asthma attacks or gastrointestinal symptoms. Other severe systemic reactions include abdominal pain, vomiting, edema of the palate, and urticaria that persists for 48 h [46,47].A more detailed understanding of the balance between the efficacy of SCIT and SLIT is needed to control symptoms and the frequency and severity of side effects. A comparison of a large double-blind study on SCIT by Frew et al. and a double-blind study on SLIT by Dahl et al., both of which had similar methodologies and sample sizes, showed the similar efficacies of SCIT and SLIT for reducing nasal and ocular symptoms (26 to 36% reduction in symptoms) [17,37,38]. The adverse effects of SLIT included oral pruritus in 46% and lip swelling in 18% of patients, with 4% of 634 patients withdrawing from the study, while those of SCIT included grade 2 systemic adverse reactions in 17.2% and grade 3 systemic adverse reactions in 4.4%. Comparisons of the symptom suppressive effects of SCIT and SLIT in the literature provided inconclusive findings, with some studies suggesting that SCIT was more effective [48,49] and others showing equivalent scores of −0.92 (p < 0.0001) for SCIT and −0.40 (p = 0.0008) for SLIT in the form of sublingual tablets, concluding that SCIT is more effective. A large-scale direct comparison of the efficacy and incidence of side effects between SCIT and SLIT is warranted in the future.SCIT requires frequent visits during the dosage increase period, whereas 12 visits per year, namely once a month, are sufficient for maintenance therapy. Six visits per year are necessary for SLIT prescribed for two months. The number of antigen doses for SCIT is 12 per year, whereas SLIT requires daily dosing 365 days per year. SCIT also requires more frequent hospital visits and may lead to poorer adherence. However, patients may decide whether daily dosing in SLIT or 12 visits and subcutaneous administration in SCIT is more suitable.Adherence to SLIT with fewer hospital visits may not always be the better option. In a study performed in the Netherlands [50], the adherence rates of SCIT and SLIT at 1 and 3 years were 80 and 23% and 38 and 7%, respectively, with significantly higher adherence rates being observed for SCIT. The social background and preferences of patients clearly play an important role in retention rates.Durham et al. [51] demonstrated that SCIT and SLIT both effectively reduced symptoms and the need for rescue medication in patients with AR, with these effects being stronger for SAR than for perennial AR and in adults than in children. SLIT tablets were also found to be effective in patients with perennial AR caused by HDM [7,52,53,54,55].Three years of treatment with both SCIT and SLIT has been shown to provide long-term clinical benefits for at least two years after the discontinuation of treatment. The findings of indirect comparisons of the relative efficacy of SCIT and SLIT are controversial, with two studies favoring SCIT [49,50] and a third finding no difference [56].In terms of tolerability and safety, SLIT was shown to be superior to SCIT, albeit in an indirect comparison [7,52,53,54,55]. SCIT may cause anaphylaxis and, thus, requires close observation, whereas SLIT is more tolerable than SCIT based on information in a large database of clinical trials and post-marketing surveillance. A large database of clinical trials and post-marketing surveillance showed that systemic side effects were rare, anaphylaxis was extremely rare, and SLIT may be safely self-administered [7,52,53,54,55]. Local side effects, such as itching and swelling of the mouth, are common but generally mild and resolve without treatment; therefore, withdrawal due to local side effects is rare.In contrast to SLIT, for which the maintenance dose is fixed, the maintenance dose for SCIT may be set according to the individual thresholds of patients. An increase in the interval between the maintenance doses of the antigen from once a month to once a week just before the pollen season may increase symptom suppressive effects during the pollen season (unpublished data). The ability to tailor the intensity of treatment to individual patients may be an important feature of SCIT that differentiates it from SLIT.The efficacy and safety of three ILIT have been investigated in a double-blind, randomized study using a group of patients treated with SCIT for more than three years as controls [14]. Senti et al. [14] randomized 99 patients to receive SCIT for more than 3 years and with 54 subcutaneous injections (total antigen dose: 4,031,540 SQ-U) and 66 patients to receive a total of 3 doses of intralymphatic injections (3000 SQ-U) over an 8-week period. Efficacy and changes in antibody titers were assessed at the start of treatment and after 4 months, 1 year, and 3 years.When isotope-labeled IgG antibodies were subcutaneously administered into the right lymph nodes and to the left side of the abdomen and examined 24 h later, the antigen administered directly into the lymph nodes remained in the lymph nodes and also accumulated in the surrounding lymph nodes. In contrast, the antigen administered subcutaneously dispersed, with only a very small amount of uptake in the regional lymph nodes [28].The mechanisms by which the administered antigen is utilized by T and B lymphocytes in the lymph nodes as a source of immunity have not yet been elucidated; however, the direct administration of the antigen into the lymph nodes has been shown to achieve long-lasting, highly concentrated antigen retention in the lymph nodes.Regarding adverse reactions, there were no cases of swelling of antigen-treated lymph nodes [31]. In a few cases, redness and itching were observed at the injection site; however, this appeared to be a subcutaneous reaction to the small amount of antigen that leaked into the skin. Anaphylactic reactions are not expected to occur in the lymph nodes because they are essentially free of mast cells and poorly vascularized.Although SCIT and SLIT both specifically suppress allergic reactions to the antigen administered, their mechanisms of action remain unclear. The route of administration of the antigen differs; however, the antigen itself is essentially the same, and the mechanisms of action of SLIT and SCIT appear to be similar in terms of the induction of immune tolerance in the affiliated lymph nodes.The induction of regulatory T cells and their production of inhibitory cytokines, such as IL-10 and TGF-b, which suppress allergic inflammation and increase the IgG4/IgE ratio, are considered to be the main mechanisms of action of immunotherapy [57]. There are several subsets of regulatory T cells, including Foxp3-positive T cells, Foxp3-negative Tr1 cells, and Th3 cells, and antigen-specific immunotherapy is considered to mainly induce Foxp3-positive T cells and Foxp3-negative Tr1 cells. In an autologous study on SCIT, IL-10-producing Foxp3-negative Tr1 cells were found to be significantly induced [58], while in a study on SLIT, Foxp3-positive T cells and Foxp3-negative Tr1 cell numbers both increased, and the VAS score for symptom improvement positively correlated with the number of Foxp3-positive T cells [59]. Furthermore, a positive correlation was noted between the VAS score for symptom improvement and the number of Foxp3-positive T cells [59].Although antigen-specific immunotherapy increases antigen-specific IgG4 antibodies, their involvement in the mechanism of action of this treatment remains unclear. Matsuda et al. [58] indicated that SCIT resulted in a significant increase in antigen-specific IgG4 antibodies 1 year after treatment, whereas SLIT did not. However, a significant increase was observed in antigen-specific IgG4 antibodies 3 years after SLIT, although it was not as large as that with SCIT [58].IgG antibodies have been suggested to function as blocking antibodies that inhibit the binding of the antigen to IgE antibodies, but this has not been shown to correlate with symptom suppression. The effects of antigen-specific immunotherapy on B cells include the promotion of IgG4 antibody production and the induction of IL-10-producing regulatory B cells [58].The co-binding of FceRI, a high affinity IgE receptor, with FcgRIIb, a low affinity IgG receptor, has been shown to inhibit degranulation reactions in mast cells and basophils [60,61,62,63,64]. The chimeric antibody, gamma-Fel d1, is a genetically engineered antibody that combines Fel d1, a major antigen responsible for cat allergy, with the Fc site of human IgG RIIb receptors and suppresses effector functions by inhibiting signal transduction [64,65].The IgG4 antibody produced by antigen-specific immunotherapy may form an antigen–antibody complex with the antigen, which then binds to the IgE antibody on FceRI, while the IgG4 antibody in the complex binds to FcgRIIb, thereby suppressing the chemical mediator release signal from IgE-FceRI in mast cells and basophils.However, it currently remains unclear whether the IgG4 antibody, which is increased by antigen-specific immunotherapy, suppresses effector function in a dose-dependent manner or through co-binding to FceRI and FcgRIIb by antigen–antibody complexes.Antigen-specific immunotherapy has also been shown to induce IL-10-producing regulatory B cells [66]. We previously reported that SCIT for cedar pollinosis significantly induced CD19-positive IL-10-producing B cells [58].IL-10 is efficiently produced by regulatory T cells and regulatory B cells induced by antigen-specific immunotherapy, and IL-10 is considered to suppress various allergic reactions and induce the production of the IgG4 antibody from B cells.IL-10-producing regulatory cells are crucially involved in the clinical therapeutic mechanisms underlying immunotherapy. A previous study on SCIT using HDM extract in patients allergic to HDM showed that SCIT induced reductions in the proliferation of peripheral blood mononuclear cells (PBMC) and the production of IFN-γ, IL5, and IL13 by PBMC stimulated with Der p 1 (a major allergen of HDM) 70 days after treatment from the levels measured prior to treatment initiation [67].IL-10 production levels after 3 years of SLIT were found to correlate with the amelioration of clinical symptoms assessed using forced expiratory flow between 25 and 75% (FEF25-75) [68].The use of other antigen administration routes beside sublingual administration as an alternative to SCIT has been attempted, with the goal of increasing safety while maintaining efficacy. Two clinical trials showed that the inhalation route was effective but limited by bronchospasms [69,70]. Although the oral route was also examined, the findings obtained were not promising [14,71,72]. In contrast, several well-designed clinical trials demonstrated the effectiveness of nasal immunotherapy [73]. Although encouraging, intranasal administration is less acceptable to patients, mainly because of persistent local side effects, such as sneezing and a runny nose, which require topical nasal premedication, and its therapeutic modalities are limited. Intranasal immunotherapy has been limited to AR and its long-term efficacy and preventive effects currently remain unknown. The transdermal use of allergen-containing patches has generated considerable interest; however, data are still preliminary [12].Conceptualization, T.T. and R.K.; original draft preparation, T.T.; review and editing; T.T. and R.K.; review and editing, R.K. All authors have read and agreed to the published version of the manuscript.This research received no external funding.Not applicable.Not applicable.All data generated or analyzed during this study are included in this published article.The authors declare no conflict of interest.Comparison among SCIT, SLIT, ILIT.Note: AE: Adverse event.Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Med-MDPI/antibiotics/antibiotics-01-01-00001.txt

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).By studying the literature about tetracyclines (TCs), it becomes clearly evident that TCs are very dynamic molecules. In some cases, their structure-activity-relationship (SAR) are well known, especially against bacteria, while against other targets, they are virtually unknown. In other diverse fields of research—such as neurology, oncology and virology—the utility and activity of the tetracyclines are being discovered and are also emerging as new technological fronts. The first aim of this paper is to classify the compounds already used in therapy and prepare the schematic structure that includes the next generation of TCs. The second aim of this work is to introduce a new framework for the classification of old and new TCs, using a medicinal chemistry approach to the structure of those drugs. A fully documented Structure-Activity-Relationship (SAR) is presented with the analysis data of antibacterial and nonantibacterial (antifungal, antiviral and anticancer) tetracyclines. The lipophilicity and the conformational interchangeability of the functional groups are employed to develop the rules for TC biological activity.The number of articles published on tetracycline drugs has reached the threshold of 50,000 papers since 1948. Over the last 10 years, technological fields are emerging in bacteriology and cellular physiology of eukaryotic cells. However, chemical mechanisms of tetracyclines are not completely understood in terms of their function in human cells and, to this day, no (Q)SAR model is validated without doubts. Tetracyclines were first discovered by Dr. Benjamin Dugger of Lederle Laboratories in the mid 1940s as the fermentation product of an unusual golden-colored soil bacterium aptly named Streptomyces aureofacians [1]. Tetracyclines (TCs) are a class of antibiotics able to inhibit protein synthesis in gram positive and gram negative bacteria by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site [2]. This mechanism has been confirmed by X-ray crystallography [3]. TCs bind specifically to the bacterial ribosome and not specifically with eukaryotic ribosomes. TCs belong to a notable class of biologically active and commercially valuable compounds. This fact may be simply illustrated by mentioning the most important clinical application of TCs, their employment as broad antimicrobial-spectrum antibiotics for human and veterinary use [4]. While they were initially developed as antibiotics, they also hold promise as non-antibiotic compounds for future study and use. Tetracyclines, as dynamics entities, possess unique chemical and biological characteristics that may explain their ability to interact with so many different cellular targets, receptors and cellular properties [5]. The discovery of new uses for tetracyclines and their novel biological properties against both prokaryotes and eukaryotes is currently under investigation by numerous scientists throughout the world. TCs as drugs show only few side effects: one is chelation of calcium and subsequent intercalation in bones and teeth while the other is somewhat like photosensitizing drugs, given their phototoxic action on keratinocytes and fibroblasts. The mechanisms of phototoxicity in vitro and in vivo are not yet entirely clear [6].Structure-Activity-Relationship (SAR) of Tetracyclines (TCs). Shaded: Contact region with 30S rRNA. In blue polygonal: same anthracycline region.The therapeutic uses are as follows: antibacterial and non-antibacterial. In the literature, these uses fall into five main categories, namely: (I) newer and more potent tetracyclines used in anitibacterial resistance [7], (II) the nonantibacterial uses of tetracyclines targeted toward inflammation [8] and arthritis [9,10,11,12]; (III) in neurology: (a) In tissue destructive diseases acting like antifibrilogenics [13]; (b) Inhibiting caspase-1 and caspase-3 expression in Hungtington’s disease [14]; (c) Ischemia [15]; (d) Parkinson’s [16] and other neurodegeneration diseases; (IV) antiviral and anticancer [17,18,19]; (V) Tet repressor controlled gene switch [20].Currently, as a consequence of their overuse, bacteria have developed TC resistance (efflux pump type) as opposed to the oldest compounds. Medicinal chemists with the intention to optimize structure and improve the antibacterial power have successfully introduced an alkaline group on C-9 of minocycline skeleton, starting as a compound from total synthesis: Tigecycline (a patent of Pfizer and Wyeth, available in therapy from 2005). Searching for new molecules, it is not only important to study the binding of drugs specifically to bacterial ribosomes, but also to understand how the tetracycline skeleton can act as a chelator and ionophore [21]. Moreover, the next generation of antibacterial tetracyclines is currently in progress and will be highly specific for bacterial species and will contain new groups and new rings on the classical skeleton [22]. Mechanism of action of TCs is divided into two categories: “Typical”, if they act as bacteriostatic; “atypical”, if they act as batericidic. Typical TCs bind specifically to the bacterial ribosomal subunits. All of them that do not have ribosomes as their primary target are considered atypical. Moreover, these atypical mechanisms of action are very toxic both for prokaryotes and eukaryotes (even for mammalian cells). Until now, all TCs used in therapy are broad-spectrum against microbial agents, but researchers are developing a platform to introduce in therapy only novel TCs with a narrow-spectrum for infectious diseases [23].Both laboratory and clinical studies have investigated the anti-inflammatory properties of tetracyclines. These include: Acting as an inhibitor forlymphocytic proliferation [9], suppression of neutrophilic migration [10], inhibition of phospholipase A2 [11] and accelerated degradation of nitric oxide synthetase [12].In recent times, starting from the end of the 1990s [24], TCs have showed to be anti-caking of β-amyloid protein and are therefore useful in the treatment of neurodegenerative diseases like Alzheimer’s and the Prion Diseases [25,26]. In particular, Minocycline reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window [27]. Also, Minocycline inhibits caspase-3 expression and delays mortality in a transgenic mouse model of Huntington Disease [28]. Researchers focused on the mechanisms of intracellular pathway communication and genetic control leading to the attenuation of microglia activation [29] and protection of Schwann cells [30].In the same way that tetracyclines act as pro-apoptotics in neuronal cells, they also act in peripheral metastasis of generalized tumor cells. Experimental data using various carcinoma cell lines and animal carcinogenesis models showed that doxycycline, minocycline and chemical modified tetracyclines (CMTs) inhibit tumor growth by inhibiting matrix metalloproteinase (MMPs) and by having a direct effect on cell proliferation [18,19]. The first use of tetracyclines in viral infections was reported by Lemaitre in 1990 [31]. In 2005, Zink [32] documented the first anti-inflammatory and neuroprotective activity of an antibiotic against a highly pathogenic viral infection. Minocycline is also significantly effective against West Nile virus replication in cultured human neuronal cells and subsequently prevented virus-induced apoptosis [33].The tetracycline-controllable expression system offers a number of advantages: Strict on/off regulation, high inducibility, short response times, specificity, no interference with the cellular pathway, bioavailability of a non-toxic inducer, and dose dependence. The tet-off system [34], which uses the tetracycline-responsive transcriptional activator (tTA), and the tet-on system [35], which uses the reverse tetracycline-responsive transcriptional activator (rtTA), provides negative and positive control of transgene expression.Historically, tetracyclines are considered First generation if they are obtained by biosynthesis such as: Tetracycline, Chlortetecycline, Oxytetracycline, Demeclocycline. Second generation if they are derivatives of semi-synthesis such as: Doxycycline, Lymecycline, Meclocycline, Methacycline, Minocycline, Rolitetracycline. Third generation if they are obtained from total synthesis such as: Tigecycline. However, some researchers consider Tigecycline to be distinct from other tetracyclines drugs and are considered as a new family of antibacterials called Glycylcyclines.The present paper introduces a new schematic point of view about the denomination and the classification of Tetracycline-Structure-Based drugs. In the years to come, new TCs, which now are advanced in the clinical trials (Phase III of Pharmaceutical Trials Protocol), will be available in therapy. TCs obtained via total synthesis, such as Tigecycline, are considered members of the Third generation if they show wide spectrum activities (both Gram+ and Gram−) Aminomethylcycline derivatives are considered in the same way as Glycylcycline (Scheme 1). In the last five years, thousands of medicinal chemists around the world have synthesized, tested and patented more than 310 tetracycline similar compounds, in particular in the USA [36]. Harvard University [37] and Tetraphase [38] made available pentacycline antibacterials (a structural modification of Doxycycline with five rings), azatetracycline and flurocycline (heteroatoms insert into the D ring, as show in Figure 1) and alkylaminotetracycline antibacterials.Wide-spectrum of tetracycline activities as antibiotic drugs. TCs are subdivided in: Antibacterial (with typical and atypical mechanism of action), antifungal and antineoplastic.All these compounds are the “logical results of modification around the four rings of tetracyclines that historically started with the master work of Golub and McNamara [39,40] when, thirty years ago, the first eight compounds called chemically modified tetracyclines (CMTs) were introduced in the literature. In 1983, Golub and McNamara [39,40] introduced a new concept concerning the therapeutic usefulness of tetracyclines. They proposed two main ideas. First, tetracyclines, but not other antibiotics, can inhibit the activity of collagenase—a specific collagenolytic metallo-neutral protease produced by host tissues which has repeatedly been implicated in periodontal destruction. Second, this newly discovered property of the drugs could provide a novel approach to the treatment of diseases, such as periodontal diseases, but also certain medical disorders (e.g., non-infected corneal ulcers), which involve excessive collagen destruction. In these cases, TCs appear to inhibit collagenase activity by a mechanism unrelated to the drug’s antibacterial efficacy. In fact, all CMTs have been modified by the removal of the dimethylamino group from the C4 position on the A ring. To better classify the new compounds it is very important to understand which chemical properties make it possible for tetracycline-structure-based drugs to act as a “chameleonic” entity. As discussed in the previous paragraph, TCs can be considered as wide-spectrum antibiotics versus bacteria, fungi, virus and cancer cells. In this view TCs can be considered an optimum example of multi target drugs and the first well documented in the literature. Moreover, Doxorubicin and all the other anthracyclines are structurally correlated to tetracyclines and it is appropriate to classify them both in the same scheme because of their chemical similarities, chemical physics properties and their use as anti-cancer drugs.Figure 1 shows the TCs rigid skeleton with the numeration of the four rings, groups and the upper and lower sides of the molecule such as they are commonly called. Many of the chemical modifications of both the first and second generation tetracyclines produced variably active or inactive compounds. An active tetracycline (antibacterial activity) must possess a linearly arranged DCBA naphthacene ring system with an A-ring C1-C3 diketo substructure and an exocyclic C2 carbonyl or amide group. All TCs that act as inhibitor of protein synthesis in bacteria need the amino group in position C4 and keto-enolic tautomers in position C1 and C3 of the A ring. The amino group in the C4 position is pivotal for the antibacterial activities (Scheme 2). A C4-dimethylamino group with its natural 4S isomer is required for optimal antibacterial activity, while epimerization to its 4R isomer decreases Gram–negative activity [41]. It also requires a C10-phenol and C11-C12 keto-enol substructure in conjunction with a 12a-OH group (Scheme 2) outlining a lower peripheral region. All those substituents, with the respective tautomeric equilibrium, are indispensable for recognition and bonding in ribosomal subunits, where chemical modification abolishes bioactivity. Modification of the amide in C2 is possible but with loss of potency. Positions C5 to C9 can be chemically modified to affect their bioactivity and they are designed for the upper peripheral regions, generating derivatives with varying antibacterial activity. Groups R1, R2 and R3 are modifiable to give more selectivity to the biological target in antifungal TCs, but not for the antibacterial activity (Scheme 2). The D ring is the most flexible to change. All modifications of group R4, R5 and R6 are allowed to give highly bacterial specificity and deep changing in pharmacokinetics as result of modifying log P (Table 1).Structure Activity Relationship of Tetracycline family drugs. Since their introduction in therapy, into the early 1950s, tetracyclines have constantly been modified according to the capabilities of the pharmaceutical laboratories in a given time. Starting as antibacterials, tetracyclines have demonstrated antifungal, antiviral and antitumor properties as well. Nowadays, the new tetracyclines are the most powerful drugs for serious skin infection and the future prospective goal is to separate their anti-inflammatory properties form the antibiotic properties.Experimental data of TCs and their pharmacokinetics values (adapted from www.drugbank.ca).Hydroxyl groups are a source of radical oxygen species (ROS) that irreversibly damage macromolecules such as DNA, RNA and proteins. All these effects are considered as fundamental to in cellular death for oxidative stress. In the case of antitumor anthracyclines and CMT-3, these act to block the enzymatic transcriptional complex formation on DNA and then induce apoptotic events. Antifungal and antitumor TCs act in a different way from antibacterial TCs. Once passed into the eukaryotic cells, TCs change the electronic balance equilibrium sequestering divalent ions (e.g., Ca2+) much more then monovalent ions (e.g., K+). It is known that TC forms complexes in different positions with calcium and magnesium ions that are available in the blood plasma [42,43]. Most tetracycline acted as bacteriostatic or typical, as protein synthesis inhibitors against bacteria. But it was found that more lipophilic tetracyclines were atypical, with a bactericidal mechanism that relied on membrane damage (as ionophores). Now, medicine is showing the tetracyclines as a family are chemically and biologically dynamic, with multiple mechanisms of activity and capable of interacting with multiple targets, either ribosomal or cellular membranes.TCs have different acid groups in their structure and the possibility to adopt different conformations. The different proton-donating groups of this molecule offer several possibilities for metal ion substitution. The complexation with metal ions increases the stability of the various TC derivatives. In 1999, Duarte [44] developed a computational and experimental study to evaluate the weight of the various chemical tautomeric behaviors of tetracyclines in solution. The degree of protonation of TC depends upon the specific tautomers that in aqueous solution are more stable than others. It is important to analyze all the possible tautomers of this molecule in their different degrees of protonation and conformations to understand the role of tautomerism in the chemical behavior of TC. Duarte [44] optimized the structures of all 64 tautomers and calculated their heats of formation (ΔHf°). There are different tautomers in equilibrium in each degree of protonation of TC. They have similar stabilities and they are present in considerable amount in the medium. All the substituent groups contribute with steric effects and polar induced vectors to the geometrical shape of each compound (Figure 2).In this work a new framework is introduced for the classification of old and novel TCs and their Structure Activity Relationship. The possibility to open access of large chemical data base has changed enormous. All data reporting in this paper has been verified and compared with the National Health Institution Public Library (Bethesda, MD) using PubChem Project. The computational analysis has been performed on a data set of 1325 TCs with a similarity score of 90%, starting from more than 322,000 compounds recorded in PubChem (this paper is in preparation). From that set were chosen the best in class TCs with a similarity score major of 95% (Scheme 1). For each TC selected, PubChem shows at least 112 tautomers and conformers structure with “rule of five” data. The new classification proposed is based on a medicinal chemistry approach due to the complexity of the novel TCs drugs, e.g.,: Chemical modified tetracycline (CMT), Anthracycline drugs (Doxorubicin) and structurally-correlated-tetracycline (Pentacyclin, Glycylcyclin, Flurocyclin, Aminomethylcyclin and Azatetracylin). 3D geometrical shape of Tetracycline (A), Tigelcycline (B) and SF2575 (C). TCs show different conformation and biological activities due their functional groups on the same rigid molecular skeleton.The role of lipophilicity of TCs is the main factor to explain the biological potency and dynamics of this old family of drugs. It is important to understand the conformational flexibility and the affinity of TCs of metals ions due to the several functional groups present in the molecular skeleton to obtain a significant SAR study. After more than 60 years of scientific investigation, TCs are considered a master example of the pleitropic family of compounds with promising therapeutic properties. Due to the wide use in therapy and their low toxicity, TCs can be considered the first Multi-Target Drugs fully and well discussed in the history of pharmacology.Newer and more potent antibacterial compounds can be expected to fight the tetracycline-resistant pathogens. New, third-generation derivatives have been designated to be more potent, especially against bacteria possessing ribosomal protection and efflux mechanisms. TCs have increased in potency over time compared to other structural classes of antibiotics. TCs have shown a complex mechanism of action towards various targets due to their conformations under physiological conditions. Such conformations, depending on pH and metal ion concentrations, allow TCs to act in a “chameleonic-like” manner in a large number of diseases. As already suggested, the future of TCs will have increased utility as one of the best anti-inflammatory drugs. This review is dedicated to my wife and to my family-in-law for their unconditional support to my work. The author wishes to thank Karina Mastronardi for her comments in the editing of this paper.The author declares no conflict of interest. 

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The miracle of antibiotics is hard to exaggerate. Each day, in every corner of the world, antibiotics improve, or could be improving outcomes in the septic neonate, the child with pneumonia, the new mother after a complicated delivery, the patient undergoing surgery, the nursing home resident with a urinary tract infection, the patient being treated of cancer, or the trauma patient on life support. The miracle also keeps our animals healthy for effective food production.But the miracle of these ‘wonder drugs’ is under threat and may be short lived: antimicrobial resistance is relentlessly increasing, especially for Gram negative organisms, prompting the oft expressed concern that we are plummeting head-long back into the pre-antibiotics era where clinicians and families once again will have to stand by and watch patients and loved ones die from once easily-treated infections. One thing is clear: there is no single, simple, magic bullet that will solve this problem. Clearly, multidisciplinary, creative responses are required, making antibiotics one of the most exciting, challenging, and fulfilling fields to be researching. To name but a few areas, we need more urgent and clearer focus on: Biomedical innovation, capitalizing on the tantalizing promises of genomics and personalized medicine for new agents that are better targeted to individual host and pathogen characteristics to achieve improved clinical outcomes.Better diagnostics, especially those that are useful at the point of care to guide clinical decision making about whether and what agent to prescribe. These diagnostic tests should be affordable and feasible also in resource-poor settings.Enhanced understanding of how antibiotics are processed in the body, their effect on an individual’s microbiological ecology, and studies of treatment efficacy and effectiveness.Enhanced surveillance on the incidence of infections, the way they are currently treated, clinical outcomes, and the influence of antimicrobial resistance, so we can better know where we are headed and model the effect of any possible changes in practice. Data will need to be clinically useful and better used in informing clinical decision-making, clinical guidelines and policy development.Associated costs and cost effectiveness.Improved ways of achieving translating new, robust evidence in clinical care in a wide range of settings internationally.Improving prevention of infections through changed lifestyle of individuals and communities, better farming methods, improved immunization and reduced opportunities for transmission.Enhanced access to effective antibiotics for those who will benefit and better ways of curtailing use where they are not effectiveHow different classes of antibiotics, infection related strategies, and antibiotic use in humans and animals interact to produce both beneficial and unwanted outcomes. We need to see the world in an integrated, systemic way.To achieve these broad aims of biomedical innovation and antimicrobial stewardship, we will need the integrated perspectives of the clinical, mathematical, social, veterinary, economic, marketing, and policy sciences to first better understand the complexity of the culture and consequences of antibiotic use and human behavior in relation to infections, and then to develop, implement and evaluate the required innovations. So Antibiotics aims to bring together the relevant disciplinary perspective, including user perspectives, from around the world to contribute additional fresh impetus within the field. We will publish not only traditional research but also innovative, speculative perspectives, provided the criterion of scientific rigor is always met. We will welcome a broad range of research methods, so long as these are rigorous and appropriate to the research question. We will be nimble, fair and effective. We aim to become the leading and most vibrant open access journal in the field. Humanity faces a complex future. The challenges around preserving and developing antibiotic effectiveness are a microcosm of the challenges to humanity’s very existence; how do we maximize benefit from human ingenuity without destroying our future through failing to address the consequences of our current hubris? Good science and its timely, effective dissemination will contribute to this broad mission. With your support, this exciting new journal will rise to the challenge! Antibiotics therefore warmly welcomes researchers, users and readers, from every corner of the globe, who care about preserving and enhancing the miracle of antibiotics.

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).It was shown that 5-chloro-8-hydroxyquinoline, an antituberculosis agent, gels aqueous alcohol solutions efficiently. Thermal stability and gel-to-sol transition temperature of 1% gel in CD3OD/D2O (2:1) was studied by 1H-NMR. Fibrous structures of four xerogels have been characterized by scanning electron microscope.Tuberculosis (TB) is one of the most serious infectious diseases caused by a single pathogen. One-third of the human population is reported to be latently infected with Mycobacterium tuberculosis, and millions of lives are lost every year [1].In addition to many known antituberculosis drugs in use [2], 5-chloro-8-hydroxyquinoline or 5-chloroquinol-8-ol (Cloxyquine) has shown to incur in vitro activities against Mycobacterium tuberculosis [3]. Also, 8-hydroxyquinoline itself is effective against Mycobacterium tuberculosis [4] and some derivatives are shown to act as antibacterial agents that target intra- and extracellular Gram-negative pathogens [5]. Further, a novel polymorph of 5-chloro-8-hydroxyquinoline with improved water solubility and faster dissolution rate has been reported which can improve the bioavailability of the drug [6]. In addition, tacrine-8-hydroxyquinoline hybrids have been shown to have potential as multifunctional agents for the treatment of Alzheimer’s disease and have copper(II) complexing properties [7]. It has also been recently shown that some related mixed ligand transition metal complexes have antituberculosis activity [8]. The importance of mixed ligand transition metal complexes in drug development is further, more generally, described [9,10] Drug resistance in Mycobacterium tuberculosis was shown to be related to pharmacokinetic/pharmacodynamic (PK/PD) factors [11]. Therefore the gelation ability of this potential drug in aqueous solution can be of extreme importance owing to its usability in controlled release applications [12]. In conjunction with our interest in low molecular weight supramolecular (self assembling) gelators [13] we are now reporting our study on the gelation behavior of 5-chloro-8-hydroxyquinoline in aqueous alcohol solutions. These gelation properties can probably be related also with the supramolecular synthon pattern in solid cloxiquine, reported recently [14]. We consider that these results can help in tailoring better drug delivery and pharmaceutical formulation combating tuberculosis. Figure 1 shows the structure and numbering of 5-chloro-8-hydroxyquinoline.The structure and numbering of 5-chloro-8-hydroxyquinoline.Table 1 lists the gelation data in various aqueous alcohol solutions at 295 K. The gel formation was observed by the test tube inversion method. Systematic temperature studies of all gels were not performed. However, for 1% 5-chloro-8-hydroxyquinoline gel in CD3OD/D2O (2:1) the gel-to-sol temperature was between +28 °C and +30 °C observed by 1H-NMR.Gelation data of 5-chloro-8-hydroxyquinoline (cloxiquine).As can be seen, increasing the concentration of 5-chloro-8-hydroxyquinoline results in a better stability of the formed gels. However, 5-chloro-8-hydroxyquinoline is not a real hydrogelator because increasing the water molar ratio generally results in a precipitate formation.In addition, the topography of the gels was characterized by the electron microscopy. Four xerogels have been characterized by scanning electron microscopy (SEM), as described in Figure 2, Figure 3, Figure 4, Figure 5. SEM image of xerogel from 1:1 EtOH/water 1% gel.SEM image of xerogel from 2:1 MeOH/water 1% gel.SEM image of xerogel from 1:1 1-PrOH/water 2% gel.SEM image of xerogel from 1:1 1-PrOH/water 1% gel.As can be seen, SEM images 2–5 revealed the presence of long rod-like structures with variable rod diameters. However, there exists some variation in the width and the length of the rods. In xerogels from 1:1 EtOH/water 1% gel (Figure 2) and from 1:1 1-PrOH/water 1% gel (Figure 5) the width variation is clearly larger and the average widths is greater than in the others (Figure 3 and Figure 4) which also show less bundled structures. In all xerogels, rods reveal no or very weak branching and the overall structure can thus be considered as an open-type network. The fractures of the network seen in Figure 5 are probably due to the faster evaporation of the solvent.The gel-to-sol transformation was studied by variable temperature NMR. This technique was successfully used recently in gel-to-sol transformation studies [15]. Figure 6 describes 500 MHz 1H-NMR spectra of 1.0% 5-chloro-8-hydroxyquinoline in CD3OD/D2O (2:1) from +18 °C (bottom) to +30 °C (top) in 2 °C steps. As can be seen, a clear increase in the signal intensity happens between +28 °C and +30 °C. This means that the number of motionally limited or NMR “silent” molecules of the gel [16] decreases significantly between these two temperatures owing to the gel-to-sol transformation where the molecular motion of 5-chloro-8-hydroxyquinoline is no longer restricted. Although this gel-to-sol change happens below the physiological conditions this finding suggests that the gel stability could be improved by other modifications of solvent systems. 5-Chloro-8-hydroxyquinoline (95%) was purchased from Sigma-Aldrich and used without purification because its 1H-NMR spectrum did not reveal any impurity signals [17]. All solvents were also from commercial sources and used as obtained. Deionized water was from our own laboratory. The samples were prepared by weighing an accurate amount of 5-chloro-8-hydroxyquinoline in a known volume of the solvent system in a test tube. After that the mixture was heated in a water bath until the solute dissolves. Then the test tube was closed and allowed to cool to room temperature. The gel formation was detected by the tube inversion technique as non-mobility of the sample and by visual inspection.Variable temperature 500 MHz 1H-NMR spectra of 1.0% 5-chloro-8-hydroxy-quinoline in CD3OD/D2O (2:1) from +18 °C (bottom) to +30 °C (top) in 2 °C steps. The clearincrease in the signal intensity between +28 °C and +30 °C is due to the gel-to-sol transformation.SEM images have been taken with Zeiss EVO 50 scanning electron microscope. In Figure 2, Figure 3, Figure 4, Figure 5 are also given the working distance 12.0 mm and acceleration voltage 19.99 kV.VT 1H-NMR spectra were run with Bruker Avance DRX 500 FT NMR spectrometer in CD3OD/D2O 2:1-mixture using 30° flip angle and 4 scans. The chemical shift scale is referenced to the trace signal of CHD2OD at 3.31 ppm from internal TMS.It was shown that 5-chloro-8-hydroxyquinoline, an antituberculosis agent, efficiently gels aqueous alcohol solutions. A variable temperature 1H-NMR study reveals that the gel-to-sol transition of 1.0% 5-chloro-8-hydroxy-12-quinoline in CD3OD/D2O (2:1) happens between +28 °C and +30 °C. Although this gel-to-sol transition takes place below the physiological conditions, this finding suggests that the gel stability could be improved by other modifications of solvent systems. This finding can be useful for the drug delivery and preparation of pharmaceutical formulations of 5-chloro-8-hydroxyquinoline.We are grateful to Esa Haapaniemi for his help in VT NMR runs.The authors declare no conflict of interest. 

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The synthesis of N-heterocyclic carbene (NHC) silver(I) acetate complexes with varying lipophilic benzyl-substituents at the 1 and 3 positions starting from 4,5-diphenylimidazole, opened a new class of antibiotic drug candidates. These NHC-silver(I) acetate derivatives exhibit interesting structural motifs in the solid state and proved to be soluble and stable in biological media. The leading candidate, SBC3, which was known to exhibit good antibacterial activity in preliminary Kirby-Bauer tests, was tested quantitatively using minimum inhibitory concentrations. NHC-silver(I) acetate complexes were found to have MIC values ranging from 20 to 3.13 μg/mL for a variety of Gram-positive, Gram-negative and mycobacteria tested. These values represent good antibiotic activities against potential pathogens when compared to clinically approved antibiotics. Most striking is the fact that SBC3 is active against methicillin-resistant Staphylococcus aureus with a MIC value of 12.5 μg/mL.Staphylococcus aureus (S. aureus or SA) is a common type of bacteria found in about 30 percent of healthy people, primarily on the skin or in the nose. Most of these individuals are healthy, but some people become infected with SA indicating that the bacteria can penetrate through a break in the skin causing skin and tissue infections. SA can also cause more serious illnesses such as surgical wound infections, bloodstream infections, bone infections, and pneumonia typically in hospital settings. In the past few decades, a more dangerous form of SA has emerged, which is known as methicillin-resistant S. aureus (MRSA). What sets MRSA apart is that it is resistant to the entire class of commonly prescribed β-lactam antibiotics including methicillin, amoxicillin and oxacillin among others; an effect firstly documented in 1964 [1]. In the meantime MRSA has grown to a major health problem leading to significant mortality worldwide [2]. The same is true for Mycobacterium tuberculosis, the causative agent of TB in humans, which is developing increasing resistance to front-line antibiotics [3], demanding that new lead compounds are urgently needed for the drug development pipeline. This emergence of resistant bacteria has triggered interest from industry and academia to synthesise and evaluate new antibiotic drug candidates like carbene-silver acetates derived from N-heterocyclic (NHC) carbenes [4,5,6]. The lead compound from our group (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(I) acetate (SBC3) showed strong inhibition of E. coli and S. aureus in preliminary Kirby-Bauer tests [7] and its molecular structure is shown in Figure 1. Molecular structure of SBC3.SBC3 is therefore investigated in this paper for its antibacterial activity against a variety of bacterial strains using minimum inhibitory concentration (MIC) tests. For the antibiotic testing of SBC3 the following seven bacterial strains were chosen: Mycobacterium bovis BCG Pasteur, Mycobacterium smegmatis, Salmonella typhimurium, Staphylococcus aureus BH1CC, a methicillin-resistant S. aureus (MRSA) strain, BH1CC ΔSCCmec, an isogenic methicillin sensitive (MSSA) derivative of BH1CC, Pseudomonas aeruginosa and Escherichia coli (NCIB strain 9485). The results of the minimum inhibitory concentration testing of SBC3 against the full bacterial panel after an incubation period of 20 h delivered MIC values between 20 and 3.13 μg/mL; the complete experimental results are shown in Table 1.Minimum inhibitory concentration (MIC) of SBC3 against different bacterial strains.SBC3 shows significant activity against the M. bovis BCG Pasteur and M.smegmatis with MIC values of 20 and 5 μg/mL. Similar activities resulting in MIC values of 12.5 μg/mL are also found for Salmonella typhimurium and S. aureus; the latter bacterium is inhibited at the same concentration independent of its methicillin susceptibility. The best activities of SBC3 are found against E. coli and P. aeruginosa with MIC values of 6.25 and 3.13 μg/mL.This MIC value of around 3 μg/mL for SBC3 with a molecular weight of 567 g/mol is already quite impressive since conventional antibiotics like Imipenem (β-Lactam Antibiotic/Carbapenem, 299 g/mol), Ceftazidime (β-Lactam Antibiotic/Cephalosporin, 547 g/mol) and Piperacillin + Tazobactam (β-Lactam Antibiotic, 518 g/mol) inhibit at the same concentrations of 3–6 μg/mL against the pathogen P. aeruginosa.The following bacterial strains were used for the antibiotic testing: Mycobacterium bovis BCG Pasteur, Mycobacterium smegmatis, Salmonella typhimurium, Staphylococcus aureus BH1CC, a methicillin-resistant S. aureus (MRSA) strain, BH1CC ΔSCCmec, an isogenic methicillin sensitive (MSSA) derivative of BH1CC, Pseudomonas aeruginosa and Escherichia coli (NCIB strain 9485).25 mg of the compound SBC3 was dissolved in 2.5 mL of 100% DMSO, giving a concentration of 10 mg/mL in each case. In order to progress, 0.02 mL of this primary solution was added to 1.98 mL Mueller Hinton II broth (MHBII) and 0.2 mL placed in wells in the first row of a microtitre plate. The concentration of SBC3 is 100 μg/mL in row 1 wells containing it at this stage. 0.1 mL MHBII was placed in all other wells of the microtitre plate and 0.1 mL of the material in row 1 was transferred to wells in the next row (already containing 0.1 mL of MHBII broth), where mixing took place by repeatedly pipetting up and down, and this was continued down the plate so that a dilution series was set up. Each well was inoculated with 0.1 mL of MHBII containing ~1 × 105 bacterial cells, and the plates incubated overnight at 37 °C. With the addition of the inoculum, the concentration of SBC3 in the first row became 50 μg/mL; for the two Mycobacteria an analogues protocol was used and the highest concentration of SBC3 was 40 μg/mL. Tests were performed in duplicate for each strain, and one DMSO control without SBC3 for each strain was included in the test. After an incubation period of 20 h, the absorbance of the wells was read using a plate reader set to measure at 600 nm. An OD600 absorbance reading <0.1 was interpreted as no growth of the bacterial strain; the plates were examined visually for growth as well.SBC3 is a low molecular weight and lipophilic drug-like molecule, which combines high stability and low light-sensitivity in the solid state with good stability and antibacterial activity in the biological medium. It shows significant activity against the M. bovis BCG Pasteur and M.smegmatis as well as against Salmonella typhimurium, MSSA and MRSA. The best activities are found for E. coli and P. aeruginosa, which makes SBC3 already as active as conventional β-lactam antibiotics against these two bacterial strains. Breaking the resistance in MRSA is also a good argument for the further development of silver-based antibiotic drug candidates like SBC3.This project is funded by the UCD School of Chemistry and Chemical Biology.The authors declare no conflict of interest.

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Helicobacter cinaedi causes infections, such as bacteremia, diarrhea and cellulitis in mainly immunocompromised patients. This pathogen is often problematic to analyze, and insufficient information is available, because it grows slowly and poorly in subculture under a microaerobic atmosphere. The first-choice therapy to eradicate H. cinaedi is antimicrobial chemotherapy; however, its use is linked to the development of resistance. Although we need to understand the antimicrobial resistance mechanisms of H. cinaedi, unfortunately, sufficient genetic tools for H. cinaedi have not yet been developed. In July 2012, the complete sequence of H. cinaedi strain PAGU 611, isolated from a case of human bacteremia, was announced. This strain possesses multidrug efflux systems, intrinsic antimicrobial resistance mechanisms and typical mutations in gyrA and the 23S rRNA gene, which are involved in acquired resistance to fluoroquinolones and macrolides, respectively. Here, we compare the organization and properties of the efflux systems of H. cinaedi with the multidrug efflux systems identified in other bacteria.Helicobacter cinaedi is a motile, Gram-negative, spiral bacterium belonging to the enterohepatic group of Helicobacter species of genus Helicobacter (the other group consists of gastric Helicobacter species, whose most well-known representative is the infamous H. pylori) [1]. During the last two decades, this bacterium has increasingly been recognized as a human pathogen that causes infections such as bacteremia, diarrhea and cellulitis in mainly immune-compromised patients and occasionally in immunocompetent ones with a high potential for recurrence [2,3]. A possible association between H. cinaedi and atrial arrhythmias and atherosclerosis was also suggested [4]. This pathogen grows slowly over several days on blood agar, even at its optimal conditions, such as a wet microaerobic atmosphere at 37 °C, and often appears as a swarming thin film that is difficult to observe [1,5]. Therefore, it is often problematic to isolate, detect and sub-culture [5,6]. Antimicrobial chemotherapy has been used successfully to treat such infections, but prolonged courses of multiple antimicrobials for at least 2–3 weeks may be required [1]. Recently, molecular epidemiological analysis in Japan showed that all H. cinaedi isolates since 2000 had acquired resistance to clarithromycin (macrolides) and ciprofloxacin (quinolones), for which the MIC90 (μg/mL) was >128 and 128, respectively, and contained typical mutations in gyrA and the 23S rRNA gene, respectively [7,8]. Unlike H. pylori, enteric Helicobacter species, such as H. cinaedi, are intrinsically resistant to amoxicillin (penicillin) [9]. High-level resistance and intrinsic resistance often require the presence of endogenous multidrug efflux pumps [10,11], which have not yet been analyzed in H. cinaedi. Multidrug efflux transporters are fundamental antimicrobial resistance mechanisms in Gram-negative bacteria [12]. Multidrug efflux transport has been studied extensively in bacteria, including ε-proteobacteria, such as H. pylori and Campylobacter jejuni, but not H. cinaedi [e.g., 13,14]. Most bacterial multidrug efflux pumps function as secondary transporters coupled with the proton-motive force (e.g., AcrB of Escherichia coli [15], MdfA of E. coli [16], and EmrE of E. coli [17]) and, although very rare, the sodium-motive force (e.g., NorM of Vibrio parahaemolyticus [18]), while some pumps hydrolyze ATP (e.g., MacB of E. coli [19] and VcaM of Vibrio cholerae [20]). Multidrug efflux transporters can be single component transporters that act at the cytoplasmic membrane (e.g., MdfA and EmrE of E. coli and NorM of V. parahaemolyticus) in both Gram-negative and -positive bacteria or three component transporters that span the entirety of the Gram-negative cell envelope (e.g., AcrAB-TolC of E. coli and MexXY-OprM of Pseudomonas aeruginosa [21]), i.e., cytoplasmic membrane transporter component (e.g., AcrB and MexY), outer membrane factor (OMF) component (e.g., TolC and OprM) and periplasmic component belonging to the membrane fusion protein (MFP) family (e.g., AcrA and MexX) [13,14]. Although bacterial multidrug efflux transporters fall into five families, i.e., resistance nodulation cell division (RND) (e.g., AcrB of E. coli and MexY of P. aeruginosa), major facilitator (MF) (e.g., MdfA of E. coli), small multidrug resistance (SMR) (e.g., EmrE of E. coli), multi-antimicrobial and toxic extrusion (MATE) (e.g., NorM of V. parahaemolyticus) and ABC (ATP binding cassette) (e.g., VcaM of V. cholerae)), the RND family is the most clinically relevant in Gram-negative bacteria [14].Very recently, we announced the complete genome sequence of H. cinaedi PAGU 611 isolated from a case of human bacteremia in Japan [22]. The clinical microbiological aspect of this strain was described as H. cinaedi-case 1; strain 923 [3]. Three months after our original report, another group published the sequence of the strain ATCC BAA-847, which was isolated in the 1980s in the USA [23]. The genome sequence of H. cinaedi CCUG 18818, although just a whole genome assembly and not complete, is also available from the Human Microbiome Project [24]. H. cinaedi PAGU 611 had a threonine to isoleucine mutation at position 84 of GyrA and adenine to guanine at position 2060 in PAGU 611 and ATCC BAA-847 (position 2018 in CCUG 18818) in the 23S rRNA gene, both of which are the same mutations identified in recent ciprofloxacin- and clarithromycin-resistant H. cinaedi isolates in Japan [7,8]. In addition to the slow, poor and, sometimes, failed growth described above, genetic tools for H. cinaedi are not sufficiently developed to take full advantage of the wealth of information generated by genome sequencing and to elucidate the function of unknown genes identified through sequencing. Fortunately, gene replacement via homologous replacement in H. cinaedi is possible by electroporation; however, no complementation system, e.g., a plasmid vector, is currently available for this organism [25]. We identified 10 putative drug transporter genes (2 RND, 1 MF, 2 MATE, 1 ABC, 4 SMR) in the genome of H. cinaedi PAGU 611 [22] (Figure 1). All transporters have homologues in H. hepaticus ATCC 51449, while only two-fifths are in H. pylori 26695 (Table 1). Interestingly C. jejuni subsp. jejuni NCTC 11168 has, rather, the most homologues (Table 1). Here, we compare the organization and properties of the multidrug efflux systems of H. cinaedi with the characterized and uncharacterized pumps available in the database.We identified two open reading frames (ORFs) belonging to the hydrophobe/amphiphile efflux-1 (HAE1) sub-family [26] of the RND family (locus-tags HCN_0595 and HCN_1563) encoded in the 2.08 Mbp chromosome of H. cinaedi PAGU 611 (Figure 1). One consists of three genes (HCN_0593-HCN_0594-HCN_0595) that encode OMP, MFP and RND, respectively, and the other consists of two genes (HCN_1564-HCN_1563) that encode MFP and RND, respectively. The ORFs were obtained from the chromosomes of ATCC BAA-847 and CCUG 18818. Both a three-gene operon (MFP, RND, and OMF) and a two-gene operon (MFP and RND) are genetically common as a multidrug efflux operon, while the latter is functionally associated with an OMF component that is encoded by a separate gene that is physically unattached to the other two members on the chromosome. For example, in P. aeruginosa PAO1, mexAB-oprM and mexXY encode two multidrug efflux pumps (MexAB-OprM and MexXY-OprM, respectively) and contribute to natural antimicrobial resistance [27]. However, three-gene RND-type multidrug efflux operons (e.g., mexAB-oprM of P. aeruginosa [28] and cmeABC of C. jejuni [29]) are usually in the order MFP-RND-OMF, unlike H. cinaedi, H. pylori and H. hepaticus [9,30].Drug efflux genes encoded in the genome of H. cinaedi PAGU 611. Chromosomal positions of drug efflux genes coding for putative inner membrane efflux transporters (red), outer membrane proteins (green), membrane fusion proteins (orange), and cytoplasmic proteins (light blue) are indicated by the kb (kilobase pair) in the H. cinaedi PAGU 611 genome [22]. Arrows correspond to the lengths and directions of the genes.The RND components of RND-type tripartite multidrug efflux pumps determine their substrate specificity [31,32]; therefore, we focused on the structure and function of the RND components of H. cinaedi (HCN_0595 (YP_006638872) and HCN_1563 (YP_006235870)), rather than the OMPs or MFPs. BLAST analysis [33] showed that HCN_0595, with a calculated molecular mass of 112 kDa, exhibited strong sequence homology to the uncharacterized RND component of the HH0222 pump (NP_859753, 86% (94%) identity (positive)) of HH0224-HH0223-HH00224 (named HefABC after those of H. pylori [9]) of H. hepaticus ATCC 51449 and significant homology to the HefC (NP_207402, 58% (78%) identity (positive)) RND component of the HefABC pump of H. pylori 26695 [30] (Table 1)and uncharacterized pumps (49%–58% (70%–78%) identity (positive)) of various other Helicobacter species (e.g., H. acinonychis, H. cetorum, H. mustelae, H. bizzozeronii, H. bilis, H. suis, H felis, H. pullorum, H. winghamensis, and H. canadensis) and other ε-proteobacteria (e.g., Wolinella succinogenes). The HefC pump of H. pylori was shown to play a critical role in resistance to bile salts and ceragenins, non-peptide mimics of antimicrobial peptides [30]. This pump might also be involved, to some extent, in antimicrobial resistance, including metronidazole [e.g., 34], although genetic evidence for the HefC pump has not been provided [35]. In C. jejuni subsp. jejuni NCTC 11168, the best-studied organism for efflux systems in ε-proteobacteria, CmeF (YP_002344428), the RND component of the CmeDEF pump [36], but not CmeB (YP_002343803), the RND component of the CmeABC pump, showed significant similarity (38% (59%) identity (positive)) to HCN_0595. The contribution of CmeDEF to intrinsic resistance is likely to be small or secondary compared with that of the major multidrug efflux system CmeABC [36,37]. We could not find any other characterized pumps that were significantly similar to HCN_0595.Homologues in the other representative ε-proteobacteria for the putative drug efflux transporters of H. cinaedi PAGU 611. Homologues in H. hepaticus ATCC 51449, H. pylori 26695 and C. jejuni NCTC 11168 for the putative efflux transporters of H. cinaedi PAGU 611 are shown using BLAST analysis.HCN_1563, with a calculated molecular mass of 113 kDa, exhibited strong sequence homology to uncharacterized RND pumps of enterohepatic Helicobacter species, including HH0174 (NP_859705, 88% (95%) identity (positive)) of the HH0175-HH0174 pump (named CmeAB after those of C. jejuni [9]) of H. hepaticus ATCC 51449 and HRAG_01727 (ZP_04580572, 80% (90%) identity (positive)) of H. bilis ATCC 43879, but not of gastric Helicobacter species, such as H. pylori (Table 1). Actually, HCN_1563 exhibited high similarity with major RND multidrug efflux pumps (CmeBs) of Campylobacter species (e.g., CmeB of C. jejuni subsp. jejuni NCTC 11168 (YP_002343803, 53% (73%) identity (positive)) (Table 1). The genome of H. cinaedi was the most similar to that of H. hepaticus [22], which exhibits a unique combination of features mainly from H. pylori and C. jejuni [38]. HCN_1563 might be a pump required to survive in the gut environment, but not the gastric environment. The contribution of the CmeABC efflux pump to acquired resistance of C. jejuni with target mutations to macrolides and fluoroquinolones has been described [10,39,40,41], which is similar to the ciprofloxacin- and clarithromycin-resistant H. cinaedi clinical isolates identified in Japan since 2000 [7,8]. With the exception of ε-proteobacteria, the BepE (NP_697326, 55% (73%) identity (positive)) and BepG pumps (NP_699529, 43% (65%) identity (positive)) of Brucella suis 1330 are taxonomically classified within α-proteobacteria [42], the TtgB pump (YP_006536083, 47% (68%) identity (positive)) of Pseudomonas putida DOT-T1E [43] and the AheB pump (YP_857414, 43% (65%) identity (positive)) of Aeromonas hydrophila subsp. hydrophila ATCC 7966 [44] are noteworthy as very similar pumps. HCN_1563 also exhibited significant similarities, as judged from phylogenetic distance, with well-studied multidrug efflux pumps (AcrB/AcrD/AcrF pumps (41% (61%–64%) identity (positive)) of E. coli and MexB/MexF/MexD/MexY pumps (39%–43% (61%–65%)) of P. aeruginosa [13,14]. COBALT analysis [45] of representative RND pumps in Gram-negative bacteria, including all RND pumps from P. aeruginosa PAO1 and E. coli K12, characterized their relationships, and we focused on two branches containing the two RND pumps of H. cinaedi (Figure 2). The branch belonging to the HCN_0595 pump only includes HefC of H. pylori and CmeF of C. jejuni, while the branch containing the HCN_1563 pump includes not only CmeB of C. jejuni, but also the BepE/G pumps of B. suis and TtgB of P. putida (Figure 2). Taken together, we assume that the HCN_0595 pump of H. cinaedi plays a similar role to HefC of H. pylori and CmeF of C. jejuni, while the HCN_1563 pump has a similar role as CmeB of C. jejuni. In addition, the two pumps of H. cinaedi must play very similar roles to those of H. hepaticus, which is not surprising, according to their biological and genomic similarities. Recently, HefA (HH0224), the OMF component of HefABC of H. hepaticus ATCC 51449, was shown to be involved in resistance to amoxicillin and some antimicrobials, as well as bile acids [9]. As the authors failed to isolate a mutant RND pump (HH0222 (HCN_0595 orthologue) and HH0174 (HCN_1563 orthologue) in Figure 2), we do not know if the resistance to amoxicillin and bile acids is caused by HH0222 or HH0174, because the HH0174 gene is a two-gene operon, like the HCN_1563 gene [9]. It is noteworthy that the HefC pump of H. pylori played a role in cholesterol-dependent resistance in the bile salt-rich enterohepatic environment [30]. Cholesterol enhanced H. pylori resistance to various antibiotics, such as clarithromycin, amoxicillin and ciprofloxacin, as well as bile salts (e.g., deoxycholate) [30,46]. It is intriguing to determine if H. cinaedi resistance is enhanced by cholesterol and if the RND pumps of H. cinaedi play a role in cholesterol-dependent resistance. Actually, hefABC of H. hepaticus and H. pylori and cmeABC of C. jejuni were inducible by bile acids [9,30,47]. It is of note that CmeABC of C. jejuni plays a critical role in colonization in vivo [48].Phylogenetic trees for RND pumps of various bacteria. According to the COBALT program, the trees were constructed using the Fast evolution method and rendered with (A) Rectangle and (B) Radical. The accession numbers are shown in parentheses. The branches belonging to HCN_0595 and HCN_1563 of H. cinaedi PAGU 611 are shown in red and named “a” and “b”, respectively. The proteins are abbreviated (e.g., “AcrB_ECOL” stands for “AcrB of E. coli”). Abbreviations; PAER, Pseudomonas aeruginosa; CJEJ, Campylobacter jejuni; HPYR, Helicobacter pyroli; HCIN, Helicobacter cinaedi; ECOL, Escherichia coli; BSUI, Brucella suis; PPUT, Pseudomonas putida; AHYD, Aeromonas hydrophila.Although cognate regulators (e.g., repressors, activators, or two-component systems) located upstream of the RND efflux genes often exist, no cognate regulator was found upstream or downstream of the RND efflux operons of H. cinaedi, H. hepaticus and H. pylori. In C. jejuni, cmeR, which is a transcriptional repressor located immediately upstream of the cmeABC operon, encodes a 210 amino-acid protein that shares sequence and structural similarities with the members of the TetR family of transcriptional repressors [49]. BLAST analysis did not identify a homologue of CmeR in the genomes of Helicobacter species. Actually, H. cinaedi possesses only a small set of genes encoding transcriptional regulators, very similar to H. hepaticus [22,38].Very recently, CosR, an oxidative stress responsive global regulator essential for viability [50], was shown to regulate the cmeABC operon negatively by binding directly upstream of cmeABC in C. jejuni NCTC 11168 [51]. CosR homologues are found mostly in ε-proteobacteria [51]. BLAST analysis showed that a quite similar CosR homologue (HCN_1079, YP_006235418.1) exists in H. cinaedi PAGU 611 (74% (86%) identity (positive)) and the strains ATCC BAA-847 and CCUG 18818. This homologue might also be involved in the expression of an efflux gene in H. cinaedi. In Gram-negative bacteria, oxidative stress responses are linked to the development of antimicrobial resistance, resulting from the activation of a resistance mechanism in which the RND multidrug efflux system is an important component [52]. For example, exposure to reactive oxygen species, such as peroxide, leads to MexXY-dependent aminoglycoside resistance in P. aeruginosa [52,53]. We point out that the putative start codon of all CosR homologues (HCN_1079, HCBAA847_0895, and HCCG_01220) of the H. cinaedi strains is TTG, which is a minor start codon [54], and found that an ATG codon located 3 codons before this TTG is also a possible start codon that is preceded by ribosome binding site-like sequences [55].CmeG homologues (e.g., HCN_0741 (YP_006235115) of PAGU 611) found in the three H. cinaedi strains showed significant homology (43% (64%) identity (positive)) (Figure 1, Table 1). BLAST analysis showed that HH1614 of H. hepaticus ATCC 51449 is a strong homologue (80% (90%) identity (positive)) (Table 1). CmeG was shown to function as a multidrug efflux transporter of the MF family that contributes to antimicrobial resistance and oxidative defense (hydrogen peroxide) in C. jejuni [56]. Mutations of cmeG significantly reduced resistance to various classes of antimicrobials, including ciprofloxacin, tetracycline, gentamicin, ethidium bromide and cholic acid, and overexpression of cmeG in the wild-type background increased resistance to fluoroquinolones [56]. CmeG shows significant homology to well-known MF-type multidrug efflux transporters of Gram-positive bacteria, such as NorA of Staphylococcus aureus (27% identity) and Bmr of Bacillus subtilis (27% identity) [56]. Finally, we discuss other probable drug efflux systems found in H. cinaedi, although the clinical significance and natural function of their homologues in other characterized bacteria remain unknown. Two MATE family multidrug efflux family transporters (HCN_0708 and HCN_0807) were found in H. cinaedi PAGU 611 and the other two H. cinaedi strains (Figure 1). BLAST analysis showed strong homologues of HCN_0708 and HCN_0807 are HH0167 (NP_859698) and HH0031 (NP_859562) of H. hepaticus ATCC 51449 (81% (90%) and 76% (87%) identity (positive)), respectively (Table 1). COBALT analysis with MATE pumps characterized in other bacteria showed that HCN_0708 was close to HP1184 of H. pylori [57], followed by VmrA of Vibrio parahaemolyticus [58], while HCN_0807 seemed unique, but comparably close to BexA of Bacteroides thetaiotaomicron [59] and VcmH of Vibrio cholerae [60] (Figure 3). Both BexA and VcmH gave resistance to hydrophilic quinolones (e.g., norfloxacin and ciprofloxacin) when expressed in an E. coli mutant lacking an acrB gene encoding the major RND multidrug efflux pump [59,60].Phylogenetic trees for the MATE pumps of various bacteria. According to the COBALT program, the trees were constructed using the Fast evolution method and rendered with (A) Rectangle and (B) Radical. The proteins are abbreviated (e.g., “NorM_ECOR” stands for “NorM of E. coli”). The accession numbers are shown in parentheses. HCN_0708 and HCN_0807 of H cinaedi PAGU 611 are shown in red. Abbreviations; HCIN, Helicobacter cinaedi; BTHE, Bacteroides thetaiotaomicron ; VCHO, Vibrio cholera; ECOL, Escherichia coli; VPAR, Vibrio parahaemolyticus; PAER, Pseudomonas aeruginosa; ABAU, Acinetobacter baumannii; NGOR, Neisseria gonorrhoeae; SAUR, Staphylococcus aureus; HPYR, Helicobacter pyroli. Two putative SMR family efflux systems (HCN_2017-HCN_2016 and HCN_1600-HCN_1599), both of which encode two SMR components, were found in H. cinaedi PAGU 611 and the other two H. cinaedi strains (Figure 1). BLAST analysis showed that strong homologues of HCN_2017-HCN_2016 and HCN_1600-HCN_1599 are HRAG_00571-HRAG_00572 (ZP_04582237-ZP_04582238) of H. bilis ATCC 43879 (94% (96%) and 98% (99%) identity (positive)) and HH509-HH508 (NP_860040-NP_860039) of H. hepaticus ATCC 51449 (59% (74%) and 67% (86%) identity (positive)), respectively (Table 1). Of note, a strong homologue of HCN_2017-HCN_2016 was HH1451-HH1452 (NP_860982-NP_860983) of H. hepaticus ATCC 51449 (56% (69%) and 61% (73%) identity (positive)) (Table 1). Both components appear to be necessary for pump activity, e.g., EbrAB and YkkCD of Bacillus subtilis [61,62]. BLAST analysis with E. coli K12, P. aeruginosa PAO1, B. subtilis 168 and Staphylococcus aureus N315 suggested that HCN_2017-HCN_2016 showed significant similarity to YkkCD (NP_389192 and NP_389193; 43% (60%) and 48% (68%) identity (positive), respectively) of B. subtilis, while HCN_1599-HCN_1600 showed significant similarity to MdtJI (NP_416117 and NP_416116; 38% (57%) and 38% (63%) identity (positive), respectively) of E. coli K12. YkkCD is a multidrug efflux pump that gives rise to broad specificity, including to cationic (e.g., streptomycin, tetracycline and ethidium bromide), neutral (e.g., chloramphenicol), and anionic compounds (e.g., phosphonomycin), when expressed in E. coli [62]. In addition, MdtJI overexpression conferred resistance to deoxycholate when expressed in an E. coli mutant lacking acrB, a major RND multidrug efflux pump [63], and rescued cell toxicity and growth inhibition due to the over-accumulation of spermidine in a spermidine acetyltransferase-deficient E. coli mutant [64].One ABC family efflux system was found in H. cinaedi PAGU 611 (Figure 1). It consists of four genes (HCN_0962-HCN_0963-HCN_0964-HCN_0965) encoding an inner membrane transporter, ATP binding protein, MFP and OMF, respectively, which means that it is an ABC transporter that spans the entirety of the Gram-negative cell envelope. The same efflux system was observed in the two other H. cinaedi strains. BLAST analysis showed that HH1856 (NP_861387) of H. hepaticus ATCC 51449 was a strong homologue of HCN_0962 (87% (93%) identity (positive)) (Table 1). BLAST analysis with E. coli K12 and P. aeruginosa PAO1 suggested that HCN_0962 was significantly similar to the inner membrane domains of both MacB (NP_415400; 34% (56%) identity (positive)) of E. coli K12 and PvdT (33% (54%)) of P. aeruginosa PAO1. MacB and PvdT are inner membrane components of the macrolide-specific ABC transporter MacAB of E. coli [19] and of the de novo synthesized pyoverdine secretion system PvdRT-OpmQ of P. aeruginosa, respectively [65].The genome of H. cinaedi possesses probable uncharacterized drug efflux systems consisting of two RND pumps, one MF pump, two MATE pumps, two SMR pumps and one ABC pump, all of which are very similar to those of H. hepaticus. Because multidrug efflux pumps have roles in not only bacterial drug resistance, but also in other systems, including virulence and the stress response [52,63], characterizing the multidrug efflux pumps of H. cinaedi should lead to the understanding of various physiological aspects of this organism and, ultimately, conquering H. cinaedi infections. To do so, it is necessary to develop genetic tools and improve the culture method for this organism, while we can also use multiplex technologies, such as real-time PCR, DNA microarrays, proteomics and metagenomics. In the meantime, each pump can be cloned and characterized in organisms that lack a homologue, such as E. coli, C. jejuni and H. pylori, but some uncertainties will remain. Interestingly, H. cinaedi PAGU 611, but not ATCC BAA-847, possesses one plasmid, pHci1 (~23 kbp, 29 predicted coding sequences, of which 27 are hypothetical proteins) [22,23]. As such, it may represent a diamond in the rough that can be developed into a stable shuttle vector, although no replication protein or origin of replication have yet been found in this plasmid. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (KAKENHI 23790106) from the Japan Society for the Promotion of Science and a research grant from the Institute of Pharmaceutical Life Sciences, Aichi Gakuin University. The authors declare no conflict of interest. 

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+Current address: Division of Pulmonary Medicine, Kantonsspital St. Gallen, Rorschacherstrasse 95, CH-9007 St. Gallen, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Background: In 14 randomized controlled studies to date, a procalcitonin (PCT)-based algorithm has been proven to markedly reduce the use of antibiotics along with an unimpaired high safety and low complication rates in patients with lower respiratory tract infections (LRTIs). However, compliance with the algorithm and safety out of controlled study conditions has not yet been sufficiently investigated. Methods: We performed a prospective international multicenter observational post-study surveillance of consecutive adults with community-acquired LRTI in 14 centers (Switzerland (n = 10), France (n = 3) and the United States (n = 1)). Results: Between September 2009 and November 2010, 1,759 patients were enrolled (median age 71; female sex 44.4%). 1,520 (86.4%) patients had a final diagnosis of LRTI (community-acquired pneumonia (CAP), 53.7%; acute exacerbation of chronic obstructive pulmonary disease (AECOPD), 17.1%; and acute bronchitis, 14.4%). Compliance with the PCT-guided therapy (overall 68.2%) was highest in patients with bronchitis (81.0% vs. AECOPD, 70.1%; CAP, 63.7%; p < 0.001), outpatients (86.1% vs. inpatients, 65.9%; p < 0.001) and algorithm-experienced centers (82.5% vs. algorithm-naive, 60.1%; p < 0.001) and showed significant geographical differences. The initial decision about the antibiotic therapy was based on PCT value in 72.4%. In another 8.6% of patients, antibiotics were administered despite low PCT values but according to predefined criteria. Thus, the algorithm was followed in 81.0% of patients. In a multivariable Cox hazard ratio model, longer antibiotic therapy duration was associated with algorithm-non-compliance, country, hospitalization, CAP vs. bronchitis, renal failure and algorithm-naïvety of the study center. In a multivariable logistic regression complications (death, empyema, ICU treatment, mechanical ventilation, relapse, and antibiotic-associated side effects) were significantly associated with increasing CURB65-Score, CAP vs. bronchitis, multilobar pneumonia, but not with algorithm-compliance. Discussion: Cultural and geographic differences in antibiotic prescribing affected the compliance with our PCT-guided algorithm. Efforts to reinforce compliance are needed. Antibiotic stewardship with PCT is possible, effective and safe without increasing the risk of complications in real-life conditions.The efficacy and safety of procalcitonin (PCT)-guided antibiotic stewardship in lower respiratory tract infections (LRTIs) has been documented in several randomized controlled trials (RCTs) [1,2,3,4,5,6,7]. In these studies the start or discontinuation of antibiotics was recommended or discouraged based on PCT cut-off values. The total antibiotic exposure was reduced by 25%–75% depending on diagnosis and site of care without an increase in morbidity or mortality. The greatest reduction was achieved by fewer initiations of antibiotic therapy in viral bronchitis in ambulatory patients. In sicker hospitalized patients with CAP, the most marked reduction was achieved mainly by early discontinuation and thus shortening of antibiotic courses.Results from RCTs may not unconditionally be extended to daily use because of the possibility of unexpected negative effects in the implementation of the study concept, selective in- and exclusion of study patients, limited validity or the lack of practicability, respectively. Except for a single center post study surveillance at the cantonal hospital of Aarau, Switzerland [8] and a study in an ambulatory setting in Germany [7], there are no data about the real life efficacy of PCT-guided antibiotic stewardship for LRTI. In this context, we performed a prospective, observational, multi-center, international quality control survey to assess the PCT-guided antibiotic stewardship in in-and outpatients with LRTIs and to show that the results can be generalized [9]. In this paper we provide insights in this study with the focus on safety and compliance with the prespecified algorithm. Between September 2009 and February 2011 we enrolled consecutive patients with LRTI presenting to emergency rooms or practitioner’s offices in centers in Switzerland (n = 10), France (n = 3) and the United States (n = 1) in a prospective, observational, international, multi-center, quality control survey. Three of the Swiss hospitals had previous experience with the PCT algorithm (“algorithm experienced centers”), all others were considered algorithm naive (“PCT naive centers”). LRTIs (acute bronchitis, acute exacerbation of chronic obstructive pulmonary disease (AECOPD), community acquired pneumonia (CAP) as well as the severity of the COPD were defined according to guidelines [10].Diagnostic workup and treatment were left to the responsibility of the treating physicians. Measurement of PCT levels was recommended in all patients on admission and for inpatients every 2 to 3 days as long as they received antibiotic treatment. PCT measurement was available daily around the clock using highly sensitive immunoassays according to the different centers availability (Kryptor®, BRAHMS AG, Hennigsdorf, Germany or Vidas®, BioMérieux, Marcy l'Etoile, France). These results were available in approximately one hour. All patients were registered by the physician on duty (or in the US center by study nurses) on a password-secured website. The website displayed the recommended and previously published PCT algorithm and the cut-off values (Figure 1) [9]. Algorithm for procalcitonin (PCT)-guided antibiotic therapy. ARDS, acute respiratory distress syndrome; BOOP, bronchiolitis obliterans with organizing pneumonia; CAP, community-acquired pneumonia; COPD GOLD, chronic obstructive pulmonary disease Global Initiative for Chronic Obstructive Lung Disease; CURB-65, confusion, serum urea nitrogen, respiratory rate, blood pressure, and age 65 years or older; HIV, human immunodeficiency virus; ICU, intensive care unit; IMC, intermediate care unit; MOF, multiple organ failure; PSI, Pneumonia Severity Index; SCLC, small-cell lung cancer; SIRS, systemic inflammatory response syndrome; and TB, tuberculosis. Study personnel (in the USA) and physicians were instructed in initial face-to-face 1-hour seminars about the quality control survey, the website and the treatment algorithm. Throughout the study, weekly reminder e-mails were sent to study personnel and physicians.According to the predefined overruling criteria antibiotic therapy was permitted by the algorithm despite of low PCT levels but not compulsory. These included the following: admission to the intensive care unit, life-threatening comorbidity, complications of LRTI (abscess or empyema), difficult-to-treat organisms (e.g., Legionella) or high clinical severity scores (Pneumonia Severity Index (PSI), CURB65) (Figure 1). The primary end point of the study was the total duration of antibiotic treatment within 30 days [9]. In this analysis we investigated the reasons for non-adherence to the PCT algorithm, the duration of antibiotic therapy during the initial presentation and the risk of complications within 30 days. Discrete variables were expressed as counts (percentage) and continuous variables as medians and interquartile ranges (IQR), unless stated otherwise. Frequency comparison was done by the Chi-square test. Two-group comparison of normally distributed data was performed by Student’s t-test. For data not normally distributed, the Mann-Whitney-U-test was used. In order to assess independent risk factors for complications we used a generalized linear model including 16 variables with a binomial distribution and a logit link, which represents a logistic regression model.The Swiss and French local ethics committees considered this study as a quality control survey; only at the US site was an informed consent necessary from all the patients. 1810 patients were registered on the website. Of those, 1759 had complete data sets from the index visit (Switzerland: 1361; USA: 295; and France: 103). Of 1520 patients (86.4%) with a final diagnosis of LRTI, 1425 (93.8%) had sufficient follow-up information at day 30 after enrolment and constitute the main analysis population. 44.4% of patients were female, the median age was 71.0 years. The final diagnosis was in 53.7% CAP, in 17.1% AECOPD and in 14.4% acute bronchitis. 173 (11.4%) patients were enrolled as outpatients. 71.3% had at least one comorbidity [9].Of 1520 patients with LRTIs, 1208 (79.5%) received antibiotics with a mean duration of antibiotic therapy of 6.9 days (IQR: 2–10 days). Algorithm compliance was higher in algorithm experienced (82.5%) than algorithm-naive centers (60.1%; p < 0.0001). It was highest in patients with bronchitis and influenza (81%), followed by AECOPD (70.1%) and CAP (63.7%). There were remarkable differences between countries and treatment sites. The algorithm compliance in outpatients in France (85.1%) and outpatients in Switzerland (87.6%) was similar (p = 0.63). In inpatients the compliance showed a great variability with 33.5% in the USA, 66% in France and 74.5% in Switzerland (p < 0.001 for USA vs. France or Switzerland; p = 0.06 for France vs. Switzerland) [9]. Antibiotic therapy strictly followed PCT cut-off ranges on initial presentation in 72.4% of patients. In 8.6% of patients predefined overruling criteria were applied (Figure 1), resulting in an overall algorithm compliance of 81.0% (Figure 2). The most important overruling reasons were high clinical severity (CURB65, PSI by patients with CAP and GOLD class by patients with AECOPD) in 3.9% and respiratory instability (respiratory rate ≥30/min or O2 saturation <90% with 6 L O2/min) in 2.3% (Figure 2). In 19.0% of patients antibiotic therapy was prescribed due to clinical judgment only despite of low PCT levels and without prespecified overruling reasons. During the entire index presentation (practitioner’s office visit, emergency room visit or entire hospitalization by inpatients), overall algorithm compliance was 68.2%.The overall mean duration of antibiotic therapy was 6.9 days (IQR: 2–10) with significant differences between diagnoses (acute bronchitis: 3.5 days, AECOPD: 4.1 days, CAP: 8.8 days; p ≤ 0.001 for all comparisons). The duration of antibiotic therapy was significantly shorter in case of algorithm adherence than non-adherence (6.2 days vs. 8.4 days; p < 0.001). After stratification for diagnosis this trend was found as well in patients with bronchitis and AECOPD, but in patients with CAP and influenza the trend was not significant (Figure 3). Algorithm compliance was an independent significant predictor for a shorter duration of antibiotic therapy in a Cox proportional hazards model (Table 1). In contrast, CAP (vs. bronchitis), treatment in France (vs. Switzerland), in-hospital (vs. ambulatory) treatment, renal insufficiency, as well as treatment in a PCT naive center were associated with longer antibiotic therapy duration. Compliance and overruling reasons. Mean duration of antibiotic therapy, diagnosis and algorithm compliance.Predictors of antibiotic therapy duration within 30 days.HR > 1 denotes shorter, HR < 1 longer antibiotic duration.In a multivariate logistic regression analysis algorithm compliance was not associated with a higher risk for complications (death, pleural empyema, ICU, mechanic ventilation, relapse, side effects of antibiotics) within 30 days. Significant risk factors for complications were an increasing CURB-65 score, the diagnosis of CAP (vs. bronchitis) and multi-lobar pneumonia, while a previous stroke was associated with a lower risk (Table 2). Risk factors for complications within 30 days.Compliance with the algorithm was not associated with risk of complication (p = 0.26).Since the majority of centers and patients were in Switzerland with only a single US center and a small fraction of outpatients, the generalizability is limited. In this international multi-center post study survey of PCT-guided antibiotic therapy for LRTI, being the largest to date, we show that after short introductory seminars, our recommended algorithm was suitable and had a high compliance-rate for daily use in real-life conditions [9]. This fact was previously only shown in small studies [4,7,8]. Antibiotic therapy can be individualized and notably reduced (bronchitis, AECOPD) or significantly shortened (CAP) [11]. Good algorithm compliance was essential and led to a significantly shorter antibiotic therapy duration. The algorithm compliance was remarkably high with 81% on initial presentation and with 68% during the entire index presentation. In contrast, in the EDCAP study for patients with low PSI scores on admission, outpatient treatment was recommended. However, the compliance with these recommendations was only 37% [12,13]. The use of antibiotics [14] and the algorithm compliance were strongly affected by cultural and geographic factors. Not surprisingly the compliance was best in Switzerland, where antibiotic use and antibiotic resistance are considerably lower than in France and in the USA [15]. A clear correlation between the use of antibiotics and antibiotic resistance was confirmed in an earlier study [16]. The algorithm compliance was the greatest in patients with bronchitis and in outpatients, i.e., patients with particularly high probabilities for viral infection. Importantly, algorithm compliance did not lead to higher complication rates, which could serve as an important argument for the safety of our concept. The predefined criteria permitting antibiotic therapy despite low PCT levels, certainly contributed to the safety of our algorithm. Limitations of clinical and laboratory tests and especially biomarkers have to be considered. In daily practice it is important to be well aware of the kinetics of PCT as well as situations with false-high and false-low results (Figure 1) [17]. While highly elevated PCT levels were found in patients with pneumococcal CAP [18], the same was not true in CAP due to atypical organisms such as mycoplasma [19], where the impact and necessity of antimicrobial therapy is debated. Furthermore antimicrobial pre-treatment, parapneumonic effusion, loculated infection (empyema), early phase of infection and most severe immunosuppression may lead to lower PCT levels [20]. Unspecific elevations of PCT levels in the absence of a bacterial infection can typically be seen in situations of massive stress, for example in severe trauma, postoperative, severe SIRS, shock, acute respiratory distress syndrome, but also in patients with tumor (e.g. medullary thyroid cancer, small cell lung cancer) or fungal infections and malaria [21,22,23].However, there is no other biomarker including CRP with such an extensive safety and efficacy record as PCT, notably in patients with LRTI and sepsis [11,24,25]. In a meta-analysis, the superiority of PCT over CRP has already been shown [26]. We are grateful to the whole study team as well as the medical and nursing teams of the following hospitals and practitioner’s offices: Switzerland: Kantonsspital Aarau, Bürgerspital Solothurn, Kantonsspital Olten, Spital Uster, Kreisspital für das Freiamt Muri, Spital Menziken, Kantonsspital Liestal, Klinik Barmelweid, Institut für Arbeitsmedizin, Baden.USA: Plant Hospital, Clearwater, Florida, Harvard School of Public Health, Boston, Massachusetts.France: Hôpital Pitié-Salpétriêre, Paris, Eric Carre, MD, Baume-les-Dames, Centre Hospitalier de la Région d’Annecy.BioMérieux provided PCT kits and testing devices and additionally a small financial compensation per enrolled patient. Drs Albrich and Mueller have received support from Thermofisher Brahms AG to attend meetings and fulfilled speaking engagements. Drs Albrich and Mueller have received support from bioMérieux to attend meetings. Dr. Mueller has served as a consultant to and received research support from Thermofisher Brahms AG and bioMérieux.No sponsor had a role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of this manuscript.

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+This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division) family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i) with itself, (ii) with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii) with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter β-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.One of the most common nosocomial human pathogens, Staphylococcus aureus can cause a variety of hospital- and community-acquired infections and intoxications. Treatment of this Gram-positive bacterium has become difficult due to its ability to rapidly develop resistance against virtually all currently used antibiotics. Genes potentially involved in cell wall synthesis, a pathway unique to bacteria, may represent novel targets for the therapy of staphylococcal infections.The main component of the bacterial cell wall is a three-dimensional peptidoglycan meshwork whose backbone consists of the alternating saccharides N-acetylglucosamine and N-acetylmuramic acid (MurNAc). The characteristic pentapeptide branching off the MurNAc unit is synthesized in S. aureus by three non-ribosomal peptidyl-transferases; FemABX. Using Gly-tRNA as donor and the peptidoglycan precursor lipid II as substrate, they add in a sequential fashion five glycines to form a characteristic pentaglycine interpeptide (Gly5) [1,2,3,4]. Cross-linking of adjacent peptidoglycan strands and anchoring of surface proteins, contributing to the virulence of S. aureus, occurs via this Gly5-interpeptide [5]. An incomplete Gly5 interpeptide leads to aberrant growth, requiring compensatory mutations to assure survival [6,7], while a complete lack is lethal [2]. Importantly, methicillin resistant S. aureus (MRSA) depend for high-level resistance on the correct formation of the peptidoglycan precursor, including a complete Gly5 chain [8,9,10]. After transport across the cellular membrane, the peptidoglycan precursor is incorporated into the existing cell wall by the PBPs (for penicillin binding protein), exoplasmic enzymes catalyzing transglycosylation of the sugar moiety and transpeptidation of the Gly5 chain. β-lactams, such as methicillin, inhibit the latter reaction by irreversibly binding to the active site of the transpeptidase domain. The orf down-stream of femX, sa2056, encodes a putative 114.7 kDa protein with 12 predicted transmembrane domains belonging to the resistance-nodulation-cell division (RND) family. RND proteins are ubiquitous and have diverse biological functions, ranging from multidrug exporters, such as AcrB in Escherichia coli to morphogen receptors in Drosophila melanogaster, as found for patched (Ptc) (reviewed in [11]). SA2056 is annotated as a hydrophobic/amphiphilic exporter-1 (HAE1) family protein (subclass 2.A.6.2 in the Transporter Classification database; TCDB [12]), with 93% of the SA2056 amino acid sequence matching AcrB/AcrD/AcrF family motifs (Kyoto Encyclopedia of Genes and Genomes database; KEGG [13]).The genetic organization femX-sa2056 is conserved among all published annotated staphylococcal species. Previous attempts to knock-out sa2056 had been unsuccessful, and Northern blot analyses had indicated co-transcription of femX and sa2056 [14], suggesting sa2056 to be essential and to have a cell wall-related function associated with femX. Both femX and sa2056 lie on the negative strand of the S. aureus chromosome and are separated by a 117 bp segment. Rho-independent transcription terminators are predicted by TransTermHP downstream of both femX and sa2056 [15]. Apart from the promoter upstream of femX, the program softberry identified an additional putative promoter in the intergenic region between femX and sa2056 [16]. Microarray analyses had shown sa2056 to be slightly upregulated in response to daptomycin, peracetic acid and chlorination [17,18,19]. On the other hand, sa2056 is downregulated by mupirocin and mitomycin and in a graRS and clpP mutant background [20,21,22]. These alterations are paralleled by femX only in the case of daptomycin or mupirocin challenge and in the clpP mutant, suggesting that transcription of femX and sa2056 can occur simultaneously or autonomously, depending on the conditions. Interestingly, SA2056 was found to harbor single-nucleotide polymorphisms (SNPs) in an in vitro generated ceftobiprole-resistant mecA-negative COL variant [23]. In this strain, additional SNPs were present only in two other genes: in pbp4 encoding the only low-molecular-weight PBP of S. aureus, PBP4 and in gdpP, influencing the levels of the second messenger c-di-AMP [24]. Both PBP4 and GdpP directly or indirectly play a role in cross-linking of peptidoglycan and β-lactam resistance [25,26,27,28,29,30], further supporting the hypothesis that also SA2056 could play a role in peptidoglycan synthesis.In this study, analysis of the markerless sa2056 knock-out mutant CQ33 [31] was extended to various growth and stress conditions. In addition, SA2056 was tested for interaction with peptidoglycan synthesis enzymes in a bacterial two hybrid system and in pull-down experiments. Although we could not find a phenotype for the mutant, we could show that SA2056 interacted with some of the FemABX factors and the PBPs, suggesting SA2056 to play a subsidiary role in peptidoglycan synthesis.The transcriptional profile of sa2056 was determined by Northern blot analyses with specific DIG-labeled probes against femX or sa2056 (Figure 1). sa2056 was transcribed mainly during exponential growth and partially co-transcribed with femX, as a 4.55 kb-transcript could be detected with both probes. Transcriptional start site determination by primer extension did not identify a promoter initiating a sa2056-specific mRNA (data not shown), suggesting that the mRNA of approximately 2.9 kb hybridizing only with the sa2056 probe might result from processing of the 4.55 kb transcript. However, we cannot exclude the presence of an alternative promoter that could be active under different conditions than used here. The hairpin structure between femX and sa2056 might function as a transcriptional or translational attenuator, further regulating SA2056 levels. Of interest, femX transcription (1.5 kb) was not altered by the deletion of sa2056.Specific antibodies against a recombinant hexahistidine-tagged SA2056 protein were produced and used for Western blot analyses. SA2056 production was highest in late and post-exponential phase (Figure 1) and could be detected in the membrane part of fractionated wild-type cells and not in the sa2056 mutant. Thus, S. aureus expressed SA2056 during growth, suggesting that it has a function in dividing cells.Expression of sa2056 in strain Newman and its sa2056 mutant. Genetic organization of the femX-sa2056 region in (a) the wild-type and (b) the sa2056 mutant. Construction of the sa2056 mutant is detailed in supplementary figure S1. (c) Growth curves from Luria-Bertani broth (LB) cultures monitored during 9 h. (d) Northern blot analyses of RNA samples taken after 1, 3, 5 and 7 h of growth. Digoxigenin (DIG)-labeled probes for femX (left panel) and sa2056 (right panel) were used. Relevant bands are indicated. Ethidium bromide-stained 16S rRNA is shown as an indication of RNA loading. Bands that might be caused by interference of bulk 16S and 23S rRNA are designated by asterisks. Specific antibodies against SA2056 (114.7 kDa) were used for Western blot analyses of (e) cell wall (CW), cell membrane (CM) and cytoplasmic (CP) fractions isolated from exponentially growing cells and (f) membrane preparations from samples taken after 1, 3, 5 and 7 h of growth.Bacteria grew in LB during 7 h or 4 days without any apparent difference between wild-type and mutant concerning optical density or colony forming units as reported before [31]. Increasing or decreasing the temperature to 43 °C or 17 °C and addition of salt (1.5 M NaCl) or sucrose (1 M) to test osmotic stress conditions did not lead to any growth difference that might have indicated altered cell envelope stability. Biofilm formation was determined, but was found to be similar as in the wild-type. Both autolysis and ultrastructure of the cells, as determined by electron microscopy, were unchanged (data not shown).Resistance levels were tested for different antibiotic classes, including β-lactams, glycopeptides or substances affecting peptidoglycan precursor synthesis and a variety of RND-substrates (Supplementary Table S1). Also, daptomycin was included, because this membrane-active antibiotic had been shown to induce sa2056 transcription 2.04-fold [17]. However, MICs were virtually identical in wild-type and mutant. The sa2056 mutant was found to be only moderately more resistant to hypochlorite compared to the parent (growth at 5 mM respectively 2.5 mM hypochlorite). No change in resistance was found for mitomycin, mupirocin, peracetic acid or puromycin (data not shown).To see whether the presence of the methicillin resistance-mediating PBP, PBP2a, had any influence, we transformed Newman and Newman sa2056 with the plasmid pME2, encoding mecA controlled by its promoter [32]. However, there was no difference in the expression of homogeneous resistance as deduced from population analysis profiles on oxacillin (data not shown).These data suggested that, although expressed, sa2056 is not required for S. aureus growth or stress tolerance under the conditions tested.The protein SA2056 is predicted to have 12 transmembrane (TM) domains and two large exoplasmic loops, displaying a typical RND topology with an internal symmetry [11]. Using the TMHMM program, putative TM regions were determined, and fragments containing increasing numbers of TMs (Supplementary Figure S2) were fused to the N-terminus of the E. coli alkaline phosphatase PhoA [33]. PhoA is widely used in topology studies, because it folds only in the exoplasm into an enzymatically active conformation [34,35,36]. Expression of the fusion proteins was confirmed by Western blot analyses (data not shown), and PhoA activity was determined (Figure 2). Topology was confirmed, except for fragments ending after TM2 and TM8. Sequence analyses with other membrane prediction programs (DAS, SOSUI, HMMTOP, MEMSAT) revealed ambiguities regarding the end of TM2 and the start of TM3, for which additional F3 constructs were made (F3a–e; S2) by adding up to five amino acids. However, all of the constructs directed PhoA to the exoplasm, suggesting that the short stretch between TM2 and TM3 might not allow PhoA to protrude into the cytoplasm. Similarly, the same might be true for TM8 and TM9, which are separated by 3–6 aa, depending on the program. Taken together, we could confirm the overall topology and show that SA2056 covers cytoplasmic, membrane and exoplasmic spaces. Thus, SA2056 has the potential to interact with proteins present in these different subcellular locations. Analysis of SA2056 topology. (a) Model of SA2056 topology depicting cytoplasmic (white), membrane (black) and exoplasmic (blue) segments. The N- and C-terminus of the protein are both predicted to be located in the cytoplasm. (b) SA2056 fragments F1–14 cloned to the N-terminus of PhoA. (c) Activity of fusion proteins was measured in biological and technical triplicates; mean values for each clone are given, and the standard deviation is indicated. SA2056 fragments directing PhoA to the exoplasm were expected to produce values at least five times higher than the background levels (dashed line) measured in the phoA-negative E. coli strain CC118 (control). To monitor the localization of SA2056 in the cell, the green fluorescent protein (GFP) was fused to the C-terminus of SA2056, and expression of SA2056-GFP was visualized in exponentially growing cells (Supplementary Figure S3). Quantification of the fluorescence signal of the hemispherical and septal membrane showed that the signal from the septum was approximately twice as high, suggesting that the increased brightness was caused by the presence of two instead of one plasma membrane at the septum and not by a preferred localization of SA2056 to the septum. However, because of the relatively high background signal in the cytoplasm, no ultimate conclusion about the localization of SA2056 could be drawn. Discrete fluorescent patches, which could reflect a heterogeneous distribution of SA2056 in the membrane, were also observed in the membrane in dividing and non-dividing bacteria and were not influenced by the addition of methicillin (data not shown). In parallel to the construction of a sa2056 deletion mutant, interaction studies were performed using a bacterial two-hybrid system (BACTH) developed by Karimova et al. [37]. To test whether there was a physical link to peptidoglycan synthesis besides the transcriptional coupling to femX, interactions with the FemABX factors and the PBPs were examined. As the E. coli RND protein AcrB had been reported to form trimers [38], SA2056 was also tested for interaction with itself. Candidates were fused to the Bordetella pertussis adenylate cyclase CyaA domains T18 and T25, as described in Materials and Methods. pKT25 and pUT18-vectors encoding the fusion proteins were co-transformed into the cya negative E. coli reporter strain DHM1. Co-transformants were plated on MacConkey agar containing lactose as the only carbon source. Interacting partners bring the T18 and T25 domains close enough together to allow them to regain their catalytic function, i.e., the conversion of ATP into cAMP. Production of cAMP was monitored on indicator plates, where the expression of cAMP-dependent enzymes, such as β-galactosidase, leads to the degradation of lactose, acidification of the medium and color formation. To estimate the strength of interaction, β-galactosidase activity was determined. As negative controls, vectors encoding only T18 or T25 were combined with candidate proteins.For pUT18-pbp4/pKT25-sa2056 co-transformations, no viable clones were obtained; the same plasmids were used successfully in other transformations, suggesting that this particular combination was unfavorable for the cells and that the plasmids themselves were not toxic. The interaction of PBP4 with SA2056 was therefore tested only in one orientation. For every combination, three representative clones were analyzed (Figure 3). The highest value of all negative controls was multiplied by five to set the threshold for significant interactions. SA2056 was found to strongly interact with itself, suggesting that, although not extruding RND substrates, this protein has the potential to form homotrimers like other RND proteins. Possibly due to an unfavorable conformation of the T25-SA2056 protein, SA2056 was able to interact with FemB, PBP1 and PBP2 only when fused to the T18 domain.SA2056 interactions determined using the bacterial two-hybrid system. (a) SA2056 interactions with itself and the FemABX factors. (b) SA2056 interactions with penicillin binding proteins (PBPs). Three representative co-transformants containing the plasmids indicated were analyzed regarding β-galactosidase activity, which was determined by measuring the formation of o-nitrophenol (top). Means of three technical replicates and their standard deviation are shown. The threshold corresponding to the highest negative control value multiplied by five is indicated by a dashed line. Alternatively, the ability of co-transformants to degrade lactose to lactate was tested on MacConkey agar (bottom), where acidification of the medium leads to pink colonies.To exclude that endogenous E. coli proteins were mediating or hindering interactions, pull-down experiments with purified recombinant proteins were performed. Proteins were tagged with glutathion-S-transferase (GST) or a hexahistidine (His6) peptide. SA2056-GST or GST alone was bound to glutathion-sepharose, blocked with BSA and incubated with His6-tagged interaction candidates. After washing, bound proteins were detached with sample buffer and separated on denaturing polyacrylamide gels (Figure 4). Similar amounts of GST-tagged bait proteins were present in the reactions, as determined by Coomassie staining (Figure 4a). SA2056 was confirmed to interact with itself and FemB and was found to very weakly interact also with FemA and FemX under these conditions (Figure 4b). Pull-down experiments with recombinant PBP-His6 proteins were unsuccessful, possibly requiring further optimization of assay conditions allowing PBPs to interact with SA2056. For instance, co-factors might be required that are only present in the cell or a membrane environment.Pull-down experiments. Recombinant glutathion-S-transferase (GST)-tagged SA2056 and FemABX proteins were incubated with SA2056-His6 and aliquots of bound proteins were separated on denaturing polyacrylamide gels for Coomassie staining (a) or (b) transferred to a polyvinylidene fluoride (PVDF)-membrane for Western blot analysis using specific antibodies against SA2056. Relevant bands are indicated. GST (26 kDa), GST-FemA (74.1 kDa), GST-FemB (74 kDa), GST-FemX (74.3 kDa), SA2056-His6 (115 kDa). Probably due to degradation, an additional band (~70 kDa; GST-SA2056’) was visible in the preparation of GST-SA2056 (147.2 kDa) and detected with the antibodies. For appropriate separation, a 10%-(a) and a 7.5%-polyacrylamide gel (b) were used. GST-tagged proteins had a tendency to run slightly faster than expected from their predicted mass.Since FemB was found in both E. coli and in vitro experiments to interact with SA2056, a femB sa2056 double mutant was constructed to determine whether the lack of sa2056 in the compromised femB single mutant leads to a phenotype. A femB transposon mutation was transduced into the Newman sa2056 mutant, and the resulting femB sa2056 double mutant was tested for any alterations in β-lactam or lysostaphin resistance compared to the femB mutant, which is known to have a reduced β-lactam and an increased lysostaphin resistance [39,40]. However, the femB sa2056 mutant showed similar resistance to cefoxitin and lysostaphin, as did the single mutant femB (data not shown), suggesting that under the conditions tested sa2056 does not play a major role in S. aureus, and thus, a major function in the last steps of peptidoglycan precursor synthesis is unlikely. This was also supported by testing of recombinant SA2056 in the previously described in vitro peptidoglycan synthesis assay, where no alterations in pentaglycine interpeptide synthesis was found upon addition of SA2056 (data not shown) [4].In S. aureus, two additional RND proteins are present: SecDF and SA2339, a homologue of MmpL that may be involved in lipid transport. While the importance of SecDF for S. aureus resistance and expression of virulence factors has been recently described, the function of SA2339 has not yet been identified. Like sa2056, deletion of sa2339 leads to no phenotype regarding growth or resistance [31]. It is possible that SA2056 and SA2339 share functional redundancy, similar to the S. aureus LytR-CpsA-Psr proteins [41] and that deletion of just one of the genes does not impair S. aureus sufficiently to produce a phenotype. Alternatively, SA2056 might be part of a complex network involving several players, where the absence of just one factor has little impact on S. aureus under the conditions tested here. Strains and plasmids used in this study are listed in Supplementary Table S2. Bacteria were grown aerobically at 37 °C in Luria-Bertani broth (LB), where nothing else is mentioned. Good aeration for liquid cultures was assured by vigorously shaking flasks with an air-to-liquid ratio of at least 4. For growth curves, strains were grown in triplicate, and means with standard deviations were determined.Antibiotic susceptibilities were determined using Etest strips (AB-Biodisk, Solna, Sweden), containing exponential gradients of active components, on MH agar plates with an inoculum of a 0.5 McFarland standard, corresponding to 108 cells/mL. Minimal inhibitory concentrations (MICs) were read after 24 h of incubation. Alternatively, broth microdilution methodology was used. For qualitative susceptibility determination, bacterial 0.5 McFarland suspensions were swabbed across agar plates containing appropriate concentration gradients of test substances.To sample RNA and protein, cells from overnight cultures were used to inoculate prewarmed LB to an optical density at 600 nm (OD600) of 0.05, corresponding to 107 cells/mL.The plasmid pME2 containing the mecA promoter and gene from strain COLn [32] was introduced into strains of interest, as described in [31].Total RNA was isolated as described previously [42] by using a FastRNA kit and a Fastprep reciprocating shaker (Bio 101). For Northern blots, 5 to 10 μg of total RNA per lane was separated on a 1.5% agarose-20 mM guanidine thiocyanate gel and transferred overnight onto a positively charged nylon membrane (Roche, Rotkreuz, Switzerland). The blots were hybridized with specific digoxigenin-labeled DNA probes, which were produced using a PCR DIG probe synthesis kit (Roche). Primers used are listed in Supplementary Table S3. Data shown were confirmed in at least two independent experiments.Genes encoding FemABX, SA2056, PBP1-4 and 2a were amplified from genomic DNA using primers listed in supplementary table T3. Amplicons were cloned into pET24b(+) using NheI/XhoI or BamHI/XhoI. In the case of pGEX-2T, PCR products were inserted into BamHI/EcoRI digested plasmids.His6-tagged and GST-tagged FemABX factors were purified as described in [43]. For the membrane proteins SA2056 and the PBPs, the E. coli BL21 derivative CE43 was used, since it had been selected for increased membrane protein production [44]. Transformants were grown in LB at 37 °C. At an OD600 of 1.5, the expression of the recombinant proteins was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Bacteria were then grown for 20 h at 25 °C and were collected by centrifugation.For purification of His6-tagged proteins, pellets were resuspended in 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazol and 0.5% N-lauroylsarcosine. Protease inhibitors (Complete EDTA-free, Roche) were added as recommended by the manufacturer. Cells were lysed on ice for 30 min with 2 mg/mL lysozyme, 0.15 mg/mL DNase and 0.075 mg/mL RNase. The cleared lysate was gently mixed for 2 h at 4 °C with Ni-NTA beads (Qiagen, Hombrechtikon, Switzerland). Beads were collected and washed with 50 mM Tris/HCl pH 7.5, 500 mM NaCl, 20 mM imidazol, followed by a second wash with the same buffer containing 50 mM imidazol. Proteins were eluted with 50 mM Tris/HCl pH 7.5, 500 mM NaCl, 1 mM n-dodecyl-β-D-maltoside (DDM), 100 mM imidazol followed by a second round of elution with the same buffer containing 200 mM imidazol. Proteins were stored at 4 °C or, supplied with 20% glycerol, at −20 °C.GST-tagged proteins were isolated similarly with minor changes: Resuspension was done in 50 mM Na2HPO4 pH 7.8, 150 mM NaCl, 10 mM DDM. Glutathion (GSH)-Sepharose 4B (GE Healthcare) was used for binding of the GST-tagged proteins, which were washed once with the same buffer and then eluted with 10 mM GSH, 50 mM Tris-HCl pH 8. Ten mM DDM was added only in the case of the membrane proteins SA2056, PBP1-4 and 2a.Based on the method used by Schneewind et al. [45], cell wall, cell membrane and cytoplasmic fractions of bacteria grown until an OD600 of 1 were prepared, as described in [31]. Representative data from two independent experiments are shown.Recombinant His-tagged SA2056 was prepared as described above and used for the production of specific rabbit antibodies (Davids Biotechnology, Regensburg, Germany). For Western blots, 10 μg protein was loaded and separated by SDS-10% polyacrylamide gel electrophoresis. Page Ruler (Thermo Scientific, Waltham, MA, USA) was used as a molecular size marker. Gels were either stained with Coomassie or transferred onto nitrocellulose (Hybond; Amersham Biosciences, Glattbrugg, Switzerland) or polyvinylidene fluoride (PVDF; Immobilon-P, Millipore, Zug, Switzerland) membranes. Membranes were blocked with skim milk and preincubated with 40 μg/mL human immunoglobulin G (Calbiochem, Darmstadt, Germany) to saturate any immunoglobulin-binding proteins and, thereby, prevent cross-reactivity of antigen-purified rabbit antibodies against SA2056. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., Suffolk, UK) was diluted 1:10,000 and detected with SuperSignal West Pico solutions (Thermo Scientific). The gene fragments of interest were cloned into the 5' XhoI and 3' KpnI sites of plasmid pHA-1, which carries a phoA gene lacking both the 5' segment coding for the signal sequence and the first five residues of the mature protein [36]. Between the SA2056-fragment and the PhoA moiety, an 18 amino acid linker was present. The constructs were transformed into the phoA-negative E. coli strain CC118, which is not able to use arabinose and galactose. For each construct, three transformants were used for the determination of PhoA activity.Three mL LB medium was inoculated with 30 μL overnight culture and grown at 37 °C to an OD600 of 0.5. The protein expression was induced with 0.2% arabinose during 1 hour at 37 °C. One mL of the culture was centrifuged at maximal speed in a microcentrifuge, washed with 1 mL ice cold 1 M Tris-HCl (pH 8.0) and resuspended in 1 mL ice cold 1 M Tris-HCl (pH 8.0). After measuring the OD600, 3 aliquots (0.05, 0.1, 0.2 mL) were adjusted to a total volume of 0.5 mL with ice cold 1 M Tris-HCl (pH 8.0). Cells were permeabilized by adding 20 μL chloroform and 10 μL 0.01% SDS and incubation at 37 °C for 5 min. Addition of 0.5 mL of 2 mM p-nitrophenyl phosphate (pNPP, Sigma-Aldrich, St. Louis, MO, USA) in 1 M Tris-HCl (pH 8.0) initiated the reaction. After 10 min incubation at 37 °C, the reaction was stopped by adding 0.2 mL 0.5 M K2HPO4 (pH 8.0). The cell debris was removed by centrifugation, and the absorption was measured at 550 and 420 nm to determine residual cell debris, respectively, and color formation by pNPP degradation. Miller Units were calculated using the following formula:
+		
+          
+          
+		Five topology prediction programs were used: TMHMM [46], DAS [47], HMMTOP [48], MEMSAT [49], SOSUI [50]. Candidate genes were amplified from genomic DNA using primers listed in Supplementary Table S3 and cloned into pUT18 and pKT25 vectors [37] using the restriction sites PstI and KpnI. Fifty μL of RbCl-competent DHM1 cells were co-transformed with 20 ng of each plasmid and plated on Difco MacConkey agar containing lactose (BD, No. 212123), 25 μg/mL kanamycin (Km) and 100 μg/mL ampicillin (Ap). Transformants were grown at 30 °C, and three representative clones from a minimum of 50 clones were restreaked for further experiments. One mL LB with 0.5 mM IPTG, 25 μg/mL Km, 100 μg/mL Ap was inoculated with one clone and grown at 30 °C for 16 h. Cells were centrifuged and stored at −20 °C until use. For spotting, 0.5 mL LB was inoculated with 5 μL overnight culture, of which 0.1 mL were transferred into microtiter dishes and used for spotting on MacConkey agar plates. For determination of β-galactosidase activity, frozen pellets were resuspended in 1 mL Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 pH 7) and OD600 was determined. 1 mL Z buffer was used as control and treated like the samples. For each clone, measurements were made in triplicate. One hundred μL aliquots were added to 900 μL Z buffer, 35 μL chloroform, and 35 μL 0.1% SDS was added. Cells were vortexed during 10 s for permeabilization, followed by an incubation step at 28 °C for 5 min. 0.2 mL of a 0.4% o-nitrophenol-β-galactopyranoside (ONPG) solution in buffer Z was added and incubated for 5 min at 28 °C. The reaction was stopped by adding 0.5 mL of a 1 M Na2CO3 solution. Cell debris was removed by centrifugation during 1 min at full speed. The supernatant was used to measure the absorption of the β-galactosidase product o-nitrophenol at 420 nm and to determine light scattering of any remaining particle at 550 nm. Activity, one unit corresponding to the hydrolyzation of 1 nmol of ONPG per min at 28 °C, was calculated using the following formula and is given in nmol OD−1 min−1 cm−1:
+		
+          
+          
+		Per reaction, 2 μg recombinant GST-tagged protein was bound to 10 μL of GSH-sepharose 4B slurry by mixing 1 h at 4 °C in binding buffer (50 mM Na2HPO4 pH 7.8, 150 mM NaCl, 1 mM DDM). Sepharose was washed four times with binding buffer and blocked with 5 mg/mL bovine serum albumin (BSA) in binding buffer for 0.5 h at 4 °C. Sepharose was washed four times with interaction buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DDM, 0.2% BSA). Two μg recombinant His6-tagged protein was added, and the reactions were mixed for 1 h at room temperature. Sepharose was washed four times with wash buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DDM, 50 mM NaCl). Bound material was detached by adding 20 μL of sample buffer (4.4 M urea, 2.7% non-idet P-40, 2.7% β-mercaptoethanol, 0.16 M Tris-HCl pH 6.8, 6.2% SDS, 4.5% glycerol, bromophenol blue) and heated for 5 min at 65 °C. 7 μL were separated on a denaturing 7.5 or 10% polyacrylamide gel. 10%-gels were stained with Coomassie to visualize GST-tagged proteins and to exclude that any other E. coli proteins were present that might have mediated interactions. 7.5%-gels were blotted onto a PVDF-membrane and used for Western blots with specific rabbit anti-SA2056 or rabbit anti-His6 (Abcam) antibodies, which were detected as described above. Representative data from two independent experiments are shown.Under standard laboratory conditions, the yet uncharacterized RND protein, SA2056, is expressed in S. aureus and, thus, must be assumed to have a function. SA2056 can interact with itself, suggesting that it could form a trimer and work as an efflux pump; the substrate is likely to be very specific, as deletion of sa2056 had no influence on resistance against typical RND substrates or a range of antibiotics. The interaction found between SA2056 and FemB using two different methods hints at a possible, subsidiary role in peptidoglycan synthesis or cell division, but could also be of a more general nature in coordinating processes occurring at the membrane. This yet uncharacterized role could represent a novel aspect of the functional diversity of RND proteins. In the case of S. aureus, the function of SA2056 might be redundant with a second uncharacterized RND protein, SA2339, or be of importance only under certain conditions that were not tested here and remain to be identified. We are grateful to D. Sjöstrand and T. Urbig for kindly providing pHA-1(yedZ). We are grateful to Ursula Lüthy (Center for Microscopy and Image Analysis, University of Zurich) for the TEM analysis. CQ was funded by the Olga Mayenfisch and the Gottfried und Julia Bangerter-Rhyner foundation. ALK was supported by a FEMS Research Fellowship. DA, TS, IW and HGS were supported by the German Research Foundation (DFG; Wi-1912/2-1 to 2-2, SA 292/13-1 and SCHN1284/1-2). MMS was funded by the Bonizzi-Theler foundation.The authors declare no conflict of interest. 

Med-MDPI/antibiotics/antibiotics-02-01-00046.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Due to the continuous release of antimicrobials into the environment, the aim of this study was to compare the frequency of detection of sulfamethazine, sulfamethoxypyridazine and trimethoprim in surface water collected from urban and rural areas in Northwestern Spain. A monitoring study was conducted with 314 river water samples analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry. The results indicated that 37% of the samples contained residues of at least one of the investigated antimicrobials, and every sampling site yielded positive samples. At sites located near the discharge points of wastewater treatment plants and near the collection point of a drinking-water treatment plant, more than 6% of the samples were positive for the presence of antimicrobial residues. In the last decade, interest in the occurrence and fate of pharmaceutical compounds in the aquatic environment has grown significantly due to the exponential increase in the global production and consumption of these compounds [1]. The main concern is that regardless of what drug is administered, once the drug has been metabolized, part of the initial dose is excreted in feces and urine in its original form and/or as metabolites. Through the domestic wastewater system, pharmaceutical compounds used in human medicine are conducted to wastewater treatment plants (WWTPs), where they should be removed. However, studies have demonstrated the presence of these compounds in the final effluents discharged by WWTPs and, consequently, their introduction into the aquatic environment [2]. Veterinary drugs used in animal production may be excreted directly into the environment or accumulated in manure pits. The application of manure to agricultural land as fertilizer may be another route through which active compounds are introduced into the environment [3,4,5]. The environmental persistence, rate of spread and bioaccumulation ability of biologically active substances differ depending on their chemical properties and on the environmental conditions. The continuous input of these compounds into the environment may lead to ecotoxicological effects [4,6,7]. In particular, antimicrobials are one of the most important groups of pharmaceuticals, employed in both human and veterinary medicine. These compounds have been used in large quantities for decades, and the emergence of antimicrobial resistance has prompted researchers to investigate their presence in the environment [8,9]. Such active compounds are frequently detected in environmental water samples. Data collected from different countries (USA, UK, Belgium, Croatia, India, Japan, Spain, Portugal) have revealed concentrations of pharmaceuticals in various aquatic environments ranging from the ng·L−1 to the mg·L−1 [10,11,12,13,14,15,16,17,18]. These differences in drug concentrations may depend on the matrix, sampling site, date and weather conditions [19]. Importantly, resistant bacteria might also reach the food chain and affect human health, especially via drinking water [20].Sulfonamides are the most commonly used antimicrobial group in human and veterinary medicine. The main advantage of this family of compounds, aside from their relatively low cost, is that they provide a broad spectrum of action, affecting a variety of micro-organisms (Gram-positive and Gram-negative bacteria, Chlamydia and some protozoa), interrupting folic acid synthesis and preventing micro-organismal multiplication. These antimicrobials are commonly combined with trimethoprim, which inhibits protein synthesis, due to their synergistic effects [21]. This study focuses on the occurrence of two sulfonamides (sulfamethazine and sulfamethoxypyridazine) and trimethoprim in surface water. Previous studies have investigated the presence of these drugs in the aquatic environment [5,11,14,22,23,24,25]; however, the aim of the present study was to compare their frequency of detection in urban areas and rural areas (with and without farming activities) in the largest Galician river, the Miño River, in Northwestern Spain. A total of 314 river water samples were analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The limits of detection (LOD) and limits of quantification (LOQ) of the method and the maximum, minimum and mean concentrations and detection frequencies of the investigated drugs in the 314 surface water samples are summarized in Table 1. Site numbers and characteristics are specified in Table 2 and Figure 1.Chemical properties, limits of detection (LOD), limits of quantification (LOQ) of the method, maximum, minimum and mean concentrations and detection frequencies of the investigated drugs in surface water samples.MW: Molecular Weight.Site number (Figure 1) and type, number of samples analyzed and number of positive sample of each sampling site.Graphic representation of the study area.The results indicated that 37% of the samples collected were positive for the presence of at least one of the three drugs investigated. Among these positive samples, 34% were collected from rural areas dedicated to farming activities, and 66% were collected from urban areas or rural areas with no farming activities. Figure 1 indicates the location of the urban sampling sites (points 3, 12, 13 and 14), rural with not much farming activity sites (points 1, 2, 4 and 5) and rural farming sampling sites (points 6, 7, 8, 9, 10 and 11) selected for this study, which had the mean percentages of positive samples of 10, 6 and 5%, respectively. Although more positive samples were expected in agricultural areas, because large quantities of veterinary pharmaceuticals are used in food production [26], these results indicated that human pharmaceuticals are concentrated in areas near WWTPs. The opposite effect was observed for veterinary pharmaceuticals, which appeared to be dispersed directly into the environment in the study area. The selected drugs, sulfamethazine, sulfamethoxypyridazine and trimethoprim, are antimicrobials commonly used in human and veterinary medicine due to their price and broad spectrum of activity. Besides that these three antimicrobials are employed to the treatment of different bacterial infections, between 60%–80% of the initial dose is excreted via urine in both animal and human, explaining their high detections after the discharge of the WWTPs.Positive samples were detected at all sampling sites (Table 2); indeed, more than 3% of the samples collected at each site were positive for at least one pharmaceutical. Overall, sampling sites located downstream from WWTP discharge points, sites number 3 and 13, yielded the largest number of positive samples, 17 and 22, respectively; more than 14% of the samples were positive. The literature provides evidence that WWTPs are point sources of pharmaceuticals in the aquatic environment due to the inefficient removal of these compounds during wastewater treatment processes [27,28,29,30,31]. Samples obtained from a tributary of the Miño River (point 6) had the lowest percentage of positive samples (3%). No clear explanation of this result was found; samples obtained from brooks and streams that discharge into this tributary yielded more than 5% of positive samples. Notably, samples obtained near the collection point of a drinking-water treatment plant (point 5) were positive for the presence of at least one of the analyzed antimicrobials. Seven positive samples were detected from this point (6% of the samples collected) (Table 2), with a maximum concentration of 56.3 ng·L−1 for trimethoprim. If the drinking-water treatment plant does not fully remove these antimicrobial residues, antimicrobials may be present in the water supplied to the human population, possibly leading to food-safety problems due to the long term exposure to low concentrations. Therefore, the presence of sulfamethazine, sulfamethoxypyridazine and trimethoprim should be investigated in the drinking water supply in the study area. Sulfamethazine, sulfamethoxypyridazine and trimethoprim were detected in 54%, 32% and 37% of the positive samples, respectively. Sulfamethazine, the most frequently detected drug—62 samples—had a maximum concentration of 63.6 ng·L−1 (Table 1), but trimethoprim had the highest maximum concentration of any drug measured in this study—85.4 ng·L−1—detected in 43 samples (Table 1). Sulfamethoxypyridazine was detected in 37 samples with a maximum concentration of 11.2 ng·L−1 (Table 1). The detection frequency and maximum concentration of trimethoprim obtained here were similar to those reported by Conley [19] in the Tennessee River; however, the mean concentration obtained in that study was half that measured here. Other studies that have investigated the presence of trimethoprim in the aquatic environment have obtained similar results of concentration and detection frequencies [13,14,32].García-Galán [25] reported lower concentrations of the two sulfonamides in the Ebro River compared to those observed here, possibly due to a dilution effect resulting from higher river flow in the Catalonian samples [33,34], to the effect of sunlight [35], to lower consumption or to greater removal efficiency. However, Díaz-Cruz [22] detected sulfamethazine and sulfamethoxypyridazine concentrations of up to 12 µg·L−1 in surface water from the Llobregat River, collected at a sampling point downstream from agricultural areas.A standardized bias or standardized kurtosis outside the range of −2 to +2 for the compared factor levels indicated significant non-normality in the data. Thus, one-way ANOVA could not be employed. Therefore, the Kruskal-Wallis test was used to compare the median values instead of the means. There were statistically significant differences with a 95.0% confidence level (p-values less than 0.05) between the median concentrations of sulfamethazine and sulfamethoxypyridazine by sampling date, between the March and May sampling dates for sulfamethazine and between the November and December sampling dates for sulfamethoxypyridazine. Sulfamethoxypyridazine concentrations also differed significantly with solar irradiation, temperature and humidity during November and December. This observation was already described by Vieno [29], who concluded that winter conditions increased the detected levels of pharmaceuticals due to the lower temperatures, which reduce biodegradation. Although sulfamethazine concentrations also differed significantly with solar irradiation, temperature and humidity, only solar irradiation and sampling date corresponded to the same sampling period. Trimethoprim concentration did not differ significantly with weather conditions or sampling date, in contrast to the results of Hua [30], who reported higher concentrations of this compound in early spring. On the other hand, the trimethoprim and sulfamethoxypyridazine concentrations showed significant differences based on the physical and chemical parameters analyzed (nitrites, ammonium, conductivity, turbidity and pH). Sulfamethazine concentrations did not show significant differences based on ammonium and nitrite levels.The concentrations of trimethoprim and sulfamethoxypyridazine differed significantly among sampling sites; the points located downstream from WWTPs discharges had the greatest number of positive samples. The study area was located in the Miño River and one of its tributaries. The Miño River is the largest river in the Galician region (Northwestern Spain), with a length of 340 km and a wide drainage basin of 17,026 km². This river has a high flow rate and is fed by many small rivers that traverse both livestock farming areas and urban areas [36]. The study area encompassed the upper basin, which includes the metropolitan area of Lugo, with approximately 98,000 residents [37], and areas dedicated to agriculture and farm production.A total of 14 sampling points were selected for this study, focusing on the section of the Miño River between the villages of Rábade and Chantada in the province of Lugo. Eight sampling points were located at different points along the Miño River and six along its tributary, the Asma River and other streams and brooks. The study area is shown in Figure 1.The sampling strategy was based on the locations of dairy farms. Because slurry generated by these livestock farms is generally used as fertilizer for field crops and grazing pastures in the same area, river sites near these farms were selected to investigate their impact in the Galician environment. Other sites along the Miño River were chosen to evaluate the influence of the human population, which consumes large amounts of pharmaceuticals. Thus, samples were collected at sites downstream from the discharge points of two WWTPs.The surface water samples were collected over three seasons (late autumn, winter and spring), from November until May. This period covers the portion of the year when common crops are grown in Galicia, such as corn, potatoes, horticultural products, vineyards and forage crops. In this region, these crops are normally fertilized with slurry and manure. Antimicrobial compounds are more frequently used on livestock farms during the same period. Between November and May, animal infections become more frequent due to inclement weather conditions; consequently, prophylactic antimicrobial use is common.The method was developed and validated using river water samples collected from the Miño River, located in Northwestern Spain. To monitor the presence of drug residues in this Galician water, 157 surface-water samples were collected in 1-L polyethylene vessels, each sample divided in duplicates of 500 mL, over a seven-month period. After collection, the samples were filtered and stored at 4 °C until extraction, which took place within 48 h after sample collection.All water samples were taken to the lab within six hours after sampling and filtered under vacuum using a 0.47-µm glass-microfiber filter (Filter-Lab, La Rioja, Spain) to remove suspended solids. The filtered samples were acidified with 0.1 N HCl to a pH of 3. Solid-phase extraction (SPE) was carried out using Strata®-X cartridges previously conditioned with 4 mL of methanol and 4 mL of Milli-Q water. The cartridges were eluted with 8 mL of methanol, which were collected in conical glass Pyrex® tubes and later evaporated to dryness in a nitrogen stream at 45 °C. The extracts were reconstituted with 200 µL of 0.1% formic acid in methanol and stored at −18 °C until further analysis by HPLC-MS/MS. The extracts were analyzed within one week following extraction.The samples employed for the determination of physical and chemical parameters were stored at 4 °C in the laboratory until analysis, which was carried out within one day of sampling. The physical and chemical parameters were determined following the manufacturers’ instructions for the respective kits and equipment. Sulfamethazine, sulfamethoxypyridazine and trimethoprim (>98% purity) and the internal standard (IS) sulfadoxine-d3 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The therapeutic and chemical properties of the selected drugs are presented in Table 1. Methanol and acetonitrile (HPLC-grade, ≥99.9%) were obtained from Scharlau Chemie (Barcelona, Spain), and formic acid (>99%) was purchased from Acros Organics (Geel, Belgium). Hydrochloric acid solution (0.1 N) was purchased from Merck (Darmstadt, Germany). Purified water was prepared in-house using a Milli-Q water system from Millipore (Bedford, MA, USA) and nitrogen gas (>99.98% purity) was generated by an in-house nitrogen generator from Peak Scientific Instruments Ltd. (Chicago, IL, USA).Each compound was accurately weighed (±0.0001 g) on an analytical balance (Ohaus® GA200, Nänikon, Switzerland) to prepare individual stock solutions at a concentration of 0.6 mg·mL−1 in methanol. These stock solutions were mixed with 0.1% formic acid in methanol to obtain stock solutions of 1 µg·mL−1, which were further diluted with 0.1% formic acid in methanol to obtain standard mixtures at 12.5, 25, 50, 75, 100 and 150 ng·mL−1. The stock solution of the IS (sulfadoxine-d3) was prepared at 0.6 mg·mL−1 and was diluted with 0.1% formic acid in methanol to obtain a working solution of 1 µg·mL−1. All of the standard solutions were stored in the dark at −18 °C for a maximum of six months.Samples were analyzed on an HPLC-MS/MS system consisting of an HPLC model 1100 from Agilent Technologies (Waldbronn, Germany) equipped with a quaternary pump, a degasser and an auto-sampler and coupled to a mass spectrometer (MS) model API 4000™ from Applied Biosystems/ MDS Sciex (Toronto, Canada) with an integrated TurboIonSpray® for molecule ionization. The software Analyst 1.4.1, also from Applied Biosystems/MDS Sciex (Toronto, Canada), was employed to acquire the data and to control the system. The chromatographic analyses were performed by injecting 10 µL of extract into a Synergi 2.5-µm Polar-RP 100A column (50 × 2.0 mm) connected to a Polar-RP security-guard cartridge (4.0 × 2.0 mm), both obtained from Phenomenex (Macclesfield, UK). An MS2 Minishaker vortex mixer from IKA® (Staufen, Germany), a vacuum station manifold with Strata®-X solid-phase extraction (SPE) cartridges (60 mg, 3 mL), both from Phenomenex (Macclesfield, UK), and a Turbo Vap® II evaporator from Zyrmark (Hopkinton, MA, USA) were employed for sample preparation and extraction.Physical and chemical parameters (nitrites, ammonium, conductivity, turbidity and pH) were measured for each collected sample. These analyses utilized the following equipment and kits: Visocolor® ECO Nitrite test (0.02–0.5 mg·L−1 NO2−) and Ammonium 3 (0.2–3 mg·L−1 NH4+), both from Macherey-Nagel GmbH & Co. KG (Düren, Germany), a conductivity meter model CON6/TDS6 (Hand-held Conductivity/TDS Meter) from Eutech Instruments Pte. Ltd/Oakton Instruments (Vernon Hills, IL, USA), a turbidimeter model TN-100 from Eutech Instruments Pte. Ltd. (Singapore) and a pH meter model MicropH 2000 from Crison (Barcelona, Spain). Analytical determination was performed according to a previously reported method [38] based on solid-phase sample extraction, detection and quantification by HPLC-MS/MS. Analytes were separated using a gradient mixture of two components, A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water). The flow rate was 0.15 mL·min−1 throughout the run.Mass-spectrometry measurements were performed using positive electrospray (ESI+) and pharmaceutical compounds were identified using two multiple reaction monitoring (MRM) transitions and their retention times (tR).The results were analyzed using the software Statgraphics Centurion XVI (StatPoint Technologies, Inc., Warrenton, VA, USA) to identify statistically significant trends in the antimicrobial concentrations. Sulfamethazine, sulfamethoxypyridazine and trimethoprim were detected at sufficiently high frequencies to be analyzed individually (Table 1).The effects of the weather conditions, sampling site characteristics (rural and urban areas), sampling date and physical and chemical parameters (nitrites, ammonium, conductivity, turbidity and pH) were tested using one-way ANOVA (p = 0.05).The occurrence of antimicrobials in the environment, especially in aquatic systems, has recently become a matter of concern. This study monitored sulfamethazine, sulfamethoxypyridazine and trimethoprim residues in river water samples. The results confirmed the presence of these drugs in the Galician aquatic environment; positive samples were detected at all sampling sites. The compounds were present at the ng·L−1 level, with a maximum concentration of 85.4 ng·L−1 for trimethoprim. Of the total samples analyzed (n = 314), 37% were positive. The sampling sites located downstream from WWTPs discharge points yielded the highest numbers of positive samples, most likely due to a concentration effect. At the site located near the collection point of a drinking-water treatment plant, 6% of the samples collected were positive for the presence of at least one of the analyzed antimicrobials, with a maximum concentration of 56.3 ng·L−1 for trimethoprim. The relationships between the concentrations of the selected pharmaceuticals in the Galician surface water and various environmental factors were statistically tested. The results showed that the concentration of sulfamethoxypyridazine depended on the sampling date and weather conditions (temperature, humidity and solar irradiation). However, this relationship was not observed for trimethoprim. The most important issues of concern related to the presence of these compounds in the environment are the possibility that they may exert ecotoxicological effects on non-target organisms and that they may possibly enter into the human food supply via the water cycle. The authors wish to thank the Fondo Europeo Agrícola de Desarrollo Rural (FEADER) and the Consellería de Medio Rural for funding this study through the Project FMR331A. The authors declare no conflict of interest. 

Med-MDPI/antibiotics/antibiotics-02-01-00058.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources and energy providing enzymes, such as ATPase, and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for the treatment of these dreaded diseases, which are claiming more and more lives every year throughout the world. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered to patients suffering from specific bacterial infections. Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp) has been found to be synergistic with penicillin and chlorpromazine (CPZ); in addition, some antibiotics are also synergistic. Along with the antibacterial action described in this review, many phenothiazines possess plasmid curing activities, which render the bacterial carrier of the plasmid sensitive to antibiotics. Thus, simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.Antibiotics have been found to be one of humankind’s most imperative weapons in combating microbial infections. Although there are highly effective antibiotics to cure nearly all major infectious diseases, such health benefits have come under threat, not only because many of these possess toxicity but also due to emergence of antibiotic-resistant bacteria. Therefore, the medicines required to cure major diseases threaten to erode the medical advances of recent decades. New antibacterial molecules and new therapeutic approaches are needed to overcome multi drug resistant (MDR) and extreme drug resistant (XDR) states in severe infectious diseases [1,2,3,4]. Thus, there is an indispensable need to explore newer molecules with lesser degrees of resistance [5]. Since the 1970s, several groups of workers independently undertook a systematic study to determine antimicrobial action of drugs belonging to various pharmacological classes not recognized as antimicrobials. This resulted in the accumulation of a large amount of evidence on many types of drugs possessing moderate to powerful antimicrobial action. All such drugs with antimicrobial activity are collectively termed as non-antibiotics (Kristiansen [6]). After the discovery by Paul Ehrlich [7] of the antimicrobial action of methylene blue, the search for drugs with antimicrobial property began. Ultimately the neuroleptic phenothiazine chlorpromazine (CPZ) was synthesized in 1950s. With global use of chlorpromazine, reports showed that patients receiving chlorpromazine had a lower incidence of bacterial infections [8]. After this, there was a boom in search for drugs, such as, antihistamines, anti-inflammatory agents, antihypertensives, cardiovascular drugs, antipsychotics and neuroleptics with possibilities of potent antimicrobial properties [9]. However, the antihistaminic and antipsychotic agents have been studied most extensively for their antimicrobial action both in vitro and in vivo [10,11,12,13]. Phenothiazines proved to be a unique class of compounds with prominent antibacterial activity against most of the pathogenic bacteria (Table 1). The MIC values of CPZ (chlorpromazine); Pr (promazine); Pz (promethazine); Pc, (prochlorperazine); Md (methdilazine); Fz (fluphenazine); Tm (trimeprazine); Tf (trifluoperazine); Tp (triflupromazine); Tz,(thioridazine); and Fp (flupenthixol). CPZ, Pr, Md, Fz, Tm, Tf, and Fp with respect to most of the Gram positive bacteria were from 10 µg/mL level, a few organisms could be inhibited by Md and Fz at 2 to 5 µg/mL level. The compound Tf was highly active against Gram positive bacteria as several of them revealed MIC as low as 2 µg/mL. Among Gram negative organisms, vibrios were most sensitive to many of the phenothiazines. However, several strains of Salmonella spp. and Shigella spp. exhibit greater sensitivity than others of the same genera. Klebsiellae, pseudomonads and acenetobacters were highly resistant to almost all of these drugs. Many of these phenothiazines were bacteriostatic, while some others were able to kill a pathogen within 6 to 18 h. Antibacterial activity of synthetic phenothiazines by in vitro screening.Please note: The bactericidal effect can be reached with multiple of the MICs. CPZ, chlorpromazine; Pr, promazine; Pz, promethazine; Pc, prochlorperazine; Md, methdilazine; Fz, fluphenazine; Tm, trimeprazine; Tf, trifluoperazine; Tp, triflupromazine; Tz, thioridazine; Fp, flupenthixol.The mechanism by which the phenothiazines act on bacterial cells in vitro has been studied by several researchers during the past few years. In 1979, Kristiansen [14] observed that CPZ was bacteriostatic to S. aureus at low level, but as the doses of CPZ were increased, it produced bactericidal action on the same organism. It was shown further that CPZ was involved in bacterial haemolysins, as the erythrocytic membranes of animals were altered in such way that haemolysis of the membrane was affected. Therefore, at low concentrations CPZ possibly interfered with the transport of potassium through the bacterial membrane much in the same way as it is occurs in mammalian tissue [14]. In 1986, Galeazzi et al [15] observed that CPZ was a competent cell permeabilizer and was capable of conducting microbial peroxidase and peroxidase like reactions. Whenever studied, CPZ increases the permeability of the bacterium to antibiotics, as evident from the items presented in this herein review. In 1991, Amaral and Lorian [16] observed that when E. coli was grown at the sub-MIC level of CPZ, the cells became elongated and filament-like in 5 h, but reverted to rod-like shape after 24 h. It was found that the electrophoretic pattern of proteins extracted from the cell envelopes of all forms of CPZ treated cells was distinctly different from those of both the untreated cells of E. coli.In 2000, Amaral et al [17] observed that CPZ failed to produce any inhibitory effect on cell proliferation of Salmonella that were allowed to remain in the sub-inhibitory state of agglutinability with the specific O antibody. Thus, the resistance to CPZ was dependent upon changes induced by CPZ in the cell wall. It was postulated that CPZ probably was able to bind with 55 KDa protein in the cell wall and interfered with the recognition of O antigen by the specific antibody.According to Radhakrisnan et al [18], phenothiazine thioridazine (TZ) proved to be a unique drug, as it could induce complete destruction of different Gram positive bacteria within a span of only two hours; however, with respect to all the different Gram negative organisms it was observed that although there was a gradual decrease in the number of viable cells after addition of Tz in a highly multiplying state of the organisms, the cells remained viable up to 18 h, revealing the bacteriostatic nature of Tz on such bacteria. It was suggested that the drug was possibly able to penetrate quite easily the peptidoglycan layer of the cell wall of Gram positive bacteria, but was unable to have any negative effect on the components of the outer membrane of Gram negative cell envelope such as lipoprotein or the lipopolysaccharide.Since there is no specific drug to cure the sleeping sickness caused by Trypanosoma brucci, Page and Lagando [19] investigated the action of Tz on the pellicular membrane complex of the infective bloodstream form of the parasite. Although Tz could induce rapid changes in cell shape but failed to affect structural integrity of the microtubular complex. However, the drug was successful in damaging both the nuclear and the cytoplasmic membranes. In this way like CPZ Tz was also found to have action on cell envelopes. Investigations on the structure activity relationships of the phenothiazines containing halogen atoms showed that their antimicrobial properties were possibly linked to the methyl-thio substituent at position 10 and a halogen moiety at position 2 of the basic phenothiazine ring [20]. As the thioxanthene skeleton is similar to phenothiazine except for the absence of a tertiary nitrogen atom at position 9, the presence of a trifluoromethyl group at position 2 of the tricyclic ring may be possible for rendering the antibacterial property to Fp, producing a structure similar to the anti-inflammatory antibacterial agent diclofenac sodium [10]. All these studies revealed that both CPZ and Tz have different kinds of action on the cell envelopes of both Gram positive and Gram negative bacteria.To evaluate the efficacy of phenothiazines in animal systems, a series of studies were conducted with a Swiss strain of male white mice weighing 18–20 g each were taken (Table 2). The naturally mouse virulent bacterium Salmonella enterica serovar Typhimurium NCTC 11 and NCTC 74 obtained from London served as the challenge strains. Both these strains were simultaneously sensitive to many antibiotics and the phenothiazines. Virulence of strains was significantly increased with repeated mouse passages and the median lethal dose (MLD or LD50) was determined following standard technique [21]. Protective capacity of each phenthiazine was determined by injecting a definite dose of the drug followed by challenge with 50 LD50 dose of the virulent salmonella to groups of mice. Toxicity levels of the compound were determined at the same time. In a separate experiment, the actual bacterial load in various organs was determined in treated and untreated mice. While evaluating the effects of phenothiazines in challenged mice it was noted that Pr was the best drug since it could offer protection at the level of 2–8 µg/mouse, and Tm was the next in order. However, Pr was much less toxic than Tm since the latter produced severe convulsion followed subsequently by death when the doses were greater than 16 µg/mouse. The drugs Md, Tf, Tp and Fp were much less toxic and offered statistically significant protection at the levels of 15–30 µg/mouse. Since the in vitro MIC of Tz in Salmonella enterica 74 was 500 µg/mL 200 µg/mouse was required to protect the challenged mice. Higher amounts of Tz also produced convulsion in animals (Table 2).Anti-salmonella activity of phenothiazines in vivo.Pr, promazine; Md, methdilazine; Fz, fluphenazine; Tm, trimeprazine; Tf, trifluoperazine; Tp, triflupromazine; Tz, thioridazine; Fp, flupenthixol.It is known that phenothiazines are concentrated by macrophages almost up to 100-fold of its original amount in a medium in which macrophages are maintained in the laboratory [22,23]. These increases of intracellular concentration take place in the lysosome [13,22,23] resulting in reaching the bactericidal level of the compound [22,23,24]. According to Amaral et al [17] a phenothiazine may promote loss of 55 KDa virulence protein and hence there is a great possibility that viable cells of salmonella lose their virulence inside the phagolysozome. Although a very large number of viable cells of S. enterica are retrieved from untreated animals 18h after challenge, there was always statistically significant reduction in the number of viable cells recovered from treated animals. From such data, however, a definite conclusion cannot be made regarding loss of virulence proteins in the phagocytosed salmonellae until the exact mechanism is unveiled and determined. Nevertheless, it is now known that a phenothiazine such as TZ affects the activity of genes that play a role in the survival of the Gram-negative bacterium [24,25]. The main genes affected by exposure to a phenothiazine such as thioridazine are those that code for plasma membrane based proteins that regulate the permeability of the cell envelope [25]. In a study of determination of antimicrobial potentiality of different plant extracts Dastidar et al. [26] observed that a prenylflavonone labeled as YS06 procured from the root of Sophora plant was active both in vitro and in vivo (Table 3). The in vitro MIC values were between 25 and 200 µg/mL level of the pure compound; it was bactericidal and could ably protect mice infected with S. enterica at doses of 40–80 µg/mouse. Such a phenomenon was further confirmed by determining reduction in the number of viable cells in mice receiving both prenylflavonone and the challenge when compared to the set of animals that were given the challenge only. Subsequently an isoflavonoid compound (YS19) derived from the same plant revealed that this was a bacteriostatic agent and could inhibit bacterial growth at 25–200 µg/mL level and successfully protected mice at an amount of 30–60 µg/mouse [27]. Subsequent animal experiments showed that much like YS06, YS19 could also reduce number of viable salmonellae in spleen, liver and heart blood of mice receiving both the agent and organism.Antibacterial action of plant derived compounds.Mazumder et al. [28] observed that the flower extract of M. ferrea possessed potent in vitro bactericidal action on salmonellae, and that the extract was able to offer significant protection to mice challenged with virulent salmonellae. In 2008, Mishra et al. isolated a flavonone from the bark of Butea frondosa and detected powerful antibacterial action both in vitro and in vivo. Many other antimicrobial compounds have been isolated [29,30,31,32,33]. Thus microorganisms are not the only source of antibacterial agents like antibiotics, but various other studies further strengthen the possibilities of procuring and securing from many types of natural sources.The majority of medicinal compounds in use today owe their origin to a given phenothiazine [34]. This is not surprising since these compounds have activities on the plasma membrane of bacteria [35,36,37], protozoa [38], eukaryotes [39]; in short, all living cells. The following sections discuss specific aspects of phenothiazine activities inasmuch as these activities have potential for the development of new medicinal compounds for therapy of infections and cancers. The reader is encouraged to visit reference 32 for a comprehensive presentation of the evolution of phenothiazines as antimicrobial agents.In general, phenothiazines are electron donors and bind by charge transfer complexes (CTC) formation to target molecules when an electron is supposed to go from the highest filled molecular orbital to the lowest empty orbital of the acceptor molecule on the target. When the phenothiazine acts as an electron donor at the surface of the plasma membrane of the cell or within the lipid bilayer of the plasma membrane, then the electron transfer on the outside will result in depolarization of the membrane. Because this depolarization reduces the activity of the plasma membrane (conductivity, etc.), the phenothiazine has been referred to as a membrane-stabilizing agent. However, when the phenothazine acts as an electron donor on the cytoplasmic side of the plasma membrane, hyperpolarization results and membrane-linked processes are inhibited. If the biological activity is actually due to charge transfer complex formation, we expect pharmacological activity from electron donation by the phenothiazine (there are some exceptions to this rule: CPZ- sulfon- or, sulphoxydes and methylene blue, where due to the asymmetric distribution of charge distribution main cause for ineffective activity). In general, one may say that the activity of the phenothiazine on the medial side of the plasma membrane is dependent upon a very high concentration of the compound. These concentrations are clinically irrelevant since they cannot be safely achieved in the patient but can be readily achieved in vitro. Therapeutically, a phenothiazine such as CPZ is administered at far lower concentrations that limit the activity of the agent to the surface of the plasma membrane (i.e., electron acceptor). It should be noted that a variety of agents can obviate the surface activity of CPZ such as caffeine [40]. In vitro caffeine forms precipitates with CPZ therefore reducing the neuroleptic effects of the agent. At the level of the plasma membrane they can disperse CPZ from its binding sites of the neuron hence patients who are managed with CPZ must take care not drink excessively caffeine rich liquids such as tea and coffee.The main mechanism of action of most phenothiazines that have a variety of effects on the activity of the plasma membrane involves the inhibition of calcium binding to calcium dependent enzymes [41]. However, because of the differentiation of cells, the constituents on the surface of the plasma membrane determine whether a specific phenothiazine will have activity on that given cell type [42]. This means that various members of the phenothiazine group may present with specific activities; e.g., neuroleptics chlorpromazine and flupentazine and the phenothiazine derived antihistamines methdilazine and trimeprazine the tranquilliser promethazine. Nevertheless, although the major mechanisms may differ, whenever studied, most phenothiazines have activity against bacteria albeit at in vitro concentrations which are clinically irrelevant Among the activities reported for phenothiazines are those that affect the activity of efflux pumps of bacteria, mycobacteria and cancer cells that express a multi-drug resistant phenotype [43]. Efflux pumps extrude noxious agents that penetrate into the cell and therefore afford protection from those agents. To the bacterium or cancer cell, antibiotics and anticancer agents are noxious agents that must be expelled prior to reaching their intended targets. Although all living cells have these efflux pumps at a basal level, they can be rapidly over-expressed when the concentration of an agent is increased [44,45,46,47,48]. Moreover, other proteins that regulate permeability of the cell envelope such as porins, are down-regulated [45,46].With respect to bacteria, serial exposure to increasing concentrations of an antibiotic results in progressive increases in resistance to the given antibiotic. Serial exposure of pansusceptible Mycobacterium tuberculosis to progressive increases of isoniazid (INH) increases resistance to the drug [49]. Similar exposure of antibiotic susceptible Escherichia coli to increasing concentrations of tetracycline promote progressive increases of resistance to the antibiotic that is accompanied by increased expression of genes that regulate and code for various efflux pumps of the organism [50]. If at any one point during the latter study the last concentration of tetracycline is serially maintained, further increases in the expression of efflux pump genes takes place and accompanied with accumulation of mutations in genes that code for proteins sensitive to beta-lactams, streptomycin and gyrase A. As prolonged exposure to a constant concentration of tetracycline, the expression of efflux pump genes is reduced to base-line levels [51,52]. These results have been interpreted to indicate that the organism follows the 2nd law of thermodynamics inasmuch as the energy needed for maintenance of an over-expressed efflux pump system is great, and given the unchanging environment containing a high level of the noxious agent (antibiotic), it can conserve energy by activating a mutator gene that promotes mutations in essential proteins, as predicted by Chopra et al. [53]. In all studies so far conducted, including those involving other Gram-negatives [25] and Gram-positives [54,55,56] and mycobacteria [49,57,58,59] phenothiazines such as chlorpromazine and thioridazine reverse the antibiotic induced resistance. The mechanism by which a phenothiazine reverses efflux pump mediated resistance to an antibiotic appears to be indirect. Firstly, depending on the environmental pH, the source of energy that drives the efflux pump differs. At pH lower than 7, the phenothiazine does not inhibit the efflux of a noxious agent whereas at pH above 7, inhibition of efflux results from exposure to a concentration of the phenothiazine that is devoid of antibacterial activity [60]. These results are interpreted to indicate the possibility that the phenothiazine inhibits the generation of hydronium ions from the hydrolysis of ATP by ATP synthase activity, and therefore, the maintenance of the proton motive force is affected. Because at low pH of the environment, the hydronium ions that are bound at the surface of the cell envelop [61,62] create the proton motive force, the needed energy for efflux is independent of metabolism and therefore not affected by then phenothiazine. Lastly, phenothiazines are well known inhibitors of the proton motive force at pH ca. 7 [63,64], therefore the interpretation of the pH dependent effects of the phenothiazine on the efflux pump of bacteria receives support.Since the 1950s, as a consequence of extensive use of chlorpromazine for therapy of psychosis, sporadic reports appeared suggesting that this neuroleptic could cure a pulmonary tuberculosis infection [8]. However, it was the advent of multi-drug resistance world-wide during the late 1980’s that the use of chlorpromazine for therapy of tuberculosis was seriously considered and immediately dismissed due to the severe toxicity produced by this neuroleptic. Moreover, the concentrations of chlorpromazine needed were in the range of 15 to 30 mg/L, and this was far greater than that which could be safely achieved in the patient (maximum plasma concentration clinically achieved safely is ca. 0.5 mg/L). Nevertheless, interest in chlorpromazine as an anti-tubercular drug continued and when Crowle and his group [65] showed that clinically relevant concentrations of chlpromazine in the medium could promote the killing of intracellular Mycobacterium tuberculosis [65], interest in this agent was increased. Soon thereafter, the milder neuroleptic thioridazine was shown to have activity against all encountered antibiotic resistant strains of Mycobacterium tuberculosis (mono-resistant; multi-drug resistant and extensively drug resistant strains of Mycobacterium tuberculosis) [66]. Later studies demonstrated that thioridazine promoted the killing of intracellular multi-drug resistant [67] and extensively drug resistant strains [68] of Mycobacterium tuberculosis and could cure the mouse infected with antibiotic susceptible [69] and multi-drug resistant [70] strains of Mycobacterium tuberculosis.These studies laid down the foundation for the first demonstration that thioridazine in combination with antibiotics to which the Mycobacterium tuberculosis was initially resistant, could cure rather quickly, patients infected with extensively drug resistant Mycobacterium tuberculosis [71]. The mechanism by which these cures have been achieved involves the activation of lysosomal hydrolases resulting from the inhibition of potassium efflux from this organelle [72,73,74] and by inhibition of the source of energy needed for adequate function of the efflux pump system that afforded a multi-drug resistant phenotype of the infecting organism [72,73,74]. It should also be mentioned that the use of thioridazine as monotherapy of the extensively drug resistant tuberculosis patient results in rapid improvement in the quality of life in that the patients regain their appetite, put on weight, night sweats are reduced and even obviated, and because of the neuroleptic activity of thioridazine, stress that results from this infection is markedly reduced [75]. These latter studies have been expanded by Utwadia et al. [76], and as a result, thioridazine has been recommended for use as a “salvage drug” for therapy of the extensively drug resistant TB patient [75].Antipsychotics block D2 receptors in the dopamine pathway of brain such that dopamine released in this pathway has a lesser effect. The tricyclic compound phenothiazines are used as antidepressant and anxiolytic and antipsychotic agents. They accumulate in the brain provoking blockade of dopamine receptors inasmuch as excess release of dopamine in the mesolimbic pathway has been linked to psychotic experiences. High potency antipsychotic drug like haloperidol can be applied in doses of a few milligrams causing sleepiness and a calming effect in patients within minutes, while low potency antipsychotics like CPZ or TZ require doses of several hundred milligrams to produce the same action. These have a much greater anticholinergic and antihistaminic actions that can counteract dopamine related side effects. Most of the antimicrobial phenothiazines are of this order. Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources, energy providing enzymes, such as ATPase and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for treatment of these dreaded disease that is claiming more and more lives every year throughout the world. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered to patients suffering from specific bacterial infections. Trimeprazine is synergistic with trimethoprim [77]. Fp has been found to be synergistic with penicillin [78] and CPZ plus some antibiotics are also synergistic [16].Along with antibacterial action described in this review, many phenothiazines possess plasmid curing activities, which render the bacterial carrier of the plasmid sensitive to antibiotics [55,77,78,79,80,81]. Thus simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.

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+These authors contributed equally to this work.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Acinetobacter baumannii may exhibit phenotypic heterogeneous growth under exposure to antibiotics. We investigated the in vitro characteristics of A. baumannii isolates grown heterogeneously in the presence of meropenem and their virulence evaluated in experimental infections treated with meropenem. Five clinical A. baumannii isolates and the respective heterogeneously grown subpopulations were tested by agar dilution minimum inhibitory concentration (MIC) testing, pulsed field gel electrophoresis (PFGE), population analysis using meropenem and growth curves. The virulence of isolates and the therapeutic efficacy of three meropenem dosing schemes was evaluated in a neutropenic murine thigh infection model. The clinical isolates were meropenem-susceptible (MICs 1 to 4 mg/liter) and exhibited three distinct PFGE patterns. In all clinical isolates, population analysis yielded heterogeneously grown colonies. After seven subcultures in antibiotic-free media, resistant MIC levels were retained in two isolates (heteroresistant), while three isolates were reversed to susceptible MICs (persisters). Clinical isolates and heterogeneous subpopulations had similar growth rates. The heterogeneously grown A. baumannii subpopulations had reduced virulence, killing considerably fewer animals than the respective clinical isolates without treatment. The meropenem treatment outcome was similar in infections caused by the clinical and the heterogeneous isolates, irrespective to their MICs. In vitro meropenem exposure induces phenotypic heterogeneous growth in A. baumannii. Compared with the parental clinical isolates, the heterogeneously grown subpopulations exhibited lower virulence, killing fewer mice and responding equally to meropenem treatment, despite their higher MICs.Acinetobacter baumannii has been an important nosocomial pathogen for the past 30 years, frequently implicated in ventilator-associated pneumonia, bloodstream infections and urinary tract infections [1]. Ubiquity and propensity to develop antibiotic resistance make A. baumannii a common, yet difficult-to-treat, hospital pathogen [2], which often needs the use of carbapenems as a treatment of last resort. During the last few years, reduced susceptibility or resistance to carbapenems is increasingly observed among A. baumannii clinical isolates [3,4]. A further worrisome observation is heteroresistance of A. baumannii to antibiotics, such as colistin or carbapenems [5,6,7], which may have implications for the treatment of A. baumannii infections. We have previously demonstrated that meropenem pressure on A. baumannii can produce subpopulations with heterogeneous expression of resistance, reflected by colonies grown within the zone of inhibition around meropenem disks or Etest strips [7,8]. These colonies may represent truly resistant subpopulations with stable changes in their genome associated with antimicrobial resistance (heteroresistance), or they may be related to subpopulations able to persist in a latent state in the presence of antimicrobials (bacterial persistence) [9]. Inherent heterogeneity of bacterial populations may contribute to their adaptation to fluctuating environments and to the persistence of bacterial infections [10]. So far, there are scarce reports of experimental infections caused by A. baumannii and treated with carbapenems, such as meropenem [11]. Also, to the best of our knowledge, there is no data on the therapeutic activity of meropenem against infections caused by A. baumannii with phenotypic heterogeneous growth against carbapenems. We present herein the characteristics of A. baumannii clinical isolates with phenotypic heterogeneous growth against meropenem, as well as their infectivity and the meropenem therapeutic efficacy evaluated in neutropenic murine thigh infections. The characteristics of the study isolates are shown in Table 1. All clinical isolates were initially classified as meropenem-susceptible by the Vitek2 system and exhibited susceptible agar dilution MICs for meropenem that ranged from 1 to 4 mg/L. However, population analysis assays yielded colonies that grew in the presence of meropenem at 8 to 32 mg/L for all clinical isolates. All isolates were susceptible to colistin and exhibited various susceptibility profiles to other antimicrobials. Characteristics of the study isolates.a CIP, ciprofloxacin; COL, colistin; GEN, gentamicin; MEM, meropenem; MIC, minimum inhibitory concentration; PFGE, pulsed field gel electrophoresis; SAM, ampicillin/sulbactam; TOB, tobramycin; b These MICs were estimated after one week of daily subcultures in antibiotic-free medium.The growth rates of the clinical isolates did not differ considerably from those of the heterogeneously grown subpopulations (data not shown).After seven daily subcultures in antibiotic-free medium, the colonies of two clinical isolates (Ab1 and Ab5) that grew in the highest meropenem concentration, exhibited stable meropenem resistance (heteroresistance). In the remaining three clinical isolates (Ab2, Ab3 and Ab4), heterogeneously grown colonies were reversed to low meropenem MICs, which were similar to those of the respective clinical isolates (persistence). PFGE analysis discriminated three distinct genotypes among the A. baumannii clinical isolates, with the patterns of the heterogeneously grown subpopulations to be indistinguishable from those of the respective clinical isolates. PCR was positive only for the intrinsic blaOXA-51-like carbapenemase, while it was negative for ISAba1 elements upstream of the blaOXA-51-like carbapenemase gene and also for genes encoding acquired carbapenemases.The results of the experimental infections are presented in Table 2. Untreated mice. The experimental infections were initially performed without treatment, to estimate the virulence potential of the study isolates and the respective heterogeneous subpopulations. All 15 mice that were infected by the five clinical isolates and were not given treatment died within 24 h. The six untreated mice infected by two meropenem-persisting subpopulations (Ab3 and Ab4) also died within 24 h, while the nine mice infected by one persister and two heteroresistant subpopulations (Ab1h, Ab2h and Ab5h) survived 24 h. These results indicate a relatively higher virulence of the clinical isolates compared with the heterogeneously grown subpopulations.Results of the experimental infections.Mice treated with 20 mg/kg meropenem. Experimental data relative to carbapenem dosing schemes for the treatment of infections due to A. baumannii were very limited, and there was only one study that applied 20 mg/kg meropenem [11]. Under this meropenem regimen, 8/15 mice infected with clinical isolates died within 24 h, in contrast with only 1/15 mice dying when infected with heterogeneous subpopulations, also indicating a relatively higher virulence of the clinical isolates. No significant decrease in colonies grown was observed for all 30 mice that received 20 mg/kg treatment compared with untreated ones (p > 0.05), suggesting a poor efficacy of this dosing scheme.Mice treated with 100 mg/kg meropenem. Since the above treatment regimen of 20 mg/kg exhibited very poor outcomes in the initial experiments, we applied a higher dose of 100 mg/kg that was used in murine infections caused by another Gram-negative non-fermenting species, Pseudomonas aeruginosa [12]. The outcome of 100 mg/kg meropenem treatment was similar for the clinical isolates and the heterogeneously grown subpopulations (13/15 mice infected by clinical isolates and 14/15 mice infected by heterogeneous subpopulations survived 24 h), despite the higher MICs of the latter populations. No significant decrease in colonies grown was observed for all 30 mice given 100 mg/kg treatment compared with untreated ones (p > 0.05), also suggesting the poor efficacy of this treatment.Mice treated with 400 mg/kg meropenem. Another dosing scheme previously used also against pseudomonal murine infections [13] was further tested. All 30 mice infected by the clinical isolates and by the heterogeneously grown subpopulations survived 24 h under 400 mg/kg meropenem treatment. A significant decrease (p < 0.05) in colony counts was observed for 12/15 mice infected by four clinical isolates and for all 15 mice infected by heterogeneous subpopulations, compared with untreated mice, suggesting therapeutic efficacy of this dosing scheme. For the three mice infected by the clinical isolate Ab1, no significant decrease in colonies grown was observed, despite treatment, compared with untreated ones (p > 0.05), although Ab1 was meropenem-susceptible, indicating its higher infectivity compared with the respective heterogeneous subpopulation that responded more favorably even being meropenem-resistant.In contrast with the A. baumannii isolates, all 12 mice infected by the Escherichia coli ATCC 25922 strain survived 24h with 20, 100 and 400 mg/kg meropenem or without treatment. A significant decrease (p < 0.05) in colony counts was observed only for the highest used dosing scheme. During the last few years, A. baumannii isolates that exhibit carbapenem resistance are increasingly isolated and pose substantial therapeutic problems in many regions worldwide [14]. Furthermore, the ability of A. baumannii cells to survive under considerably higher carbapenem concentrations (phenotypic heterogeneous growth) may have implications for the treatment of multiresistant A. baumannii infections [15] and poses concerns. The observations of a previous study suggested that A. baumannii isolates that are apparently meropenem-susceptible by standard susceptibility testing may contain a certain amount of phenotypically meropenem-resistant subpopulations [7]. These subpopulations may have stably elevated meropenem MICs due to permanent genomic changes and are considered heteroresistant or may simply persist in a latent state in the presence of antimicrobials and exhibit susceptible MICs when they grow without meropenem exposure (persisters) [10]. Drug resistance in bacteria can be associated with a biological fitness cost [16]. It has been proposed that the magnitude of this cost is the primary factor that influences the rate of resistance development, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. However, mutants with no measurable cost have also been observed. It has been proposed that environmental conditions affect fitness cost and the cost of drug resistance can be reduced by regulation of the resistance mechanism or cost compensation [17].In the present study, heteroresistant phenotype was observed in two isolates, which retained meropenem MICs of 32 mg/L or higher, while three isolates exhibited a persister phenotype, with MICs to return to the susceptible range after seven daily subcultures without antibiotic presence. To investigate the hypothesis that the acquisition of resistance causes a fitness cost to the bacterial cells and to estimate the therapeutic response of meropenem treatment, we performed experimental neutropenic thigh infections. Three meropenem dosing schemes were used, as there is a paucity of data on A. baumannii infections treated with meropenem, and the only scheme previously applied on A. baumannii (20 mg/L, reference [11]) exhibited very poor therapeutic responses in our preliminary experiments. The results of the experimental infections suggested that heterogeneously grown A. baumannii subpopulations have lower virulence compared with the clinical isolates, as 9/15 untreated animals infected by persisters or heteroresistant subpopulations survived, while all untreated mice infected by clinical isolates died. These results probably reflect a fitness cost conferred by mutations related to the expression of the heterogeneous mode of growth against meropenem. The lower virulence of the phenotypic heterogeneous subpopulations is also reflected by the observation that the lowest meropenem dosage (20 mg/kg/8 h) conferred survival of 14/15 mice infected by persisters or heteroresistant subpopulations in contrast with only 7/15 mice infected by clinical isolates to survive. Finally, the infection outcome under meropenem treatment was overall not correlated with the meropenem MICs, even in stably resistant heterogeneous populations, further suggesting their impaired virulence. The study included five A. baumannii clinical isolates from our collection and the E. coli ATCC 25922 and P. aeruginosa ATCC 27853 strains as controls for the experimental infections and the phenotypic assays, respectively. Susceptibility status to β-lactams, aminoglycosides, quinolones and colistin was performed by the Vitek2 automated system (BioMerieux, Marcy l’ Etoile, France) and disk diffusion [18]. Agar dilution meropenem MIC testing [18] was performed for the clinical isolates and for subpopulations grown at the highest meropenem concentrations in population analysis assays. The genetic relationship of the isolates was tested by PFGE of ApaI-digested genomic DNA [7], and the banding patterns were compared visually using previously proposed criteria [19]. PCR for the intrinsic blaOXA-51-like allele and for genes encoding known class B and D carbapenemases were performed as described previously [20,21,22]. Population analyses using meropenem were performed in triplicate for all isolates. Phenotypically heterogeneous subpopulations were yielded by spreading approximately 108 bacterial CFU on Mueller-Hinton agar plates with meropenem concentrations ranging from 0.5 to 32 mg/liter and incubating the plates for 48 h. Colonies grown in the highest drug concentration (heterogeneous subpopulations) were tested for meropenem MIC directly from the population analysis plates and after one week of daily subcultures in antibiotic-free medium to test for the stability of the phenotype. One colony of the seventh subculture for each strain (Ab1h, Ab2h, Ab3h, Ab4h and Ab5h) was selected for use in the thigh-infection model, along with the respective clinical isolates (Ab1, Ab2, Ab3, Ab4 and Ab5).Growth curves were determined by diluting 0.1 mL of overnight Mueller-Hinton broth culture of the clinical isolates and heterogeneously resistant subpopulations in 15 mL of broth followed by incubation at 37 °C under constant shaking. The optical density of a 1 mL aliquot of each broth culture was determined at each hour for 16 h.Animal studies were approved by the Ethics Committee of Medical School, University of Thessaly, and conformed to the European Union guidelines. In each model, six-week-old, specific-pathogen-free, female Bagg inbred albino c-strain (BALB/c) mice (12-poster) weighing 23 to 27 g were used in each test group [23]. Mice were rendered neutropenic (neutrophils < 100/μL) by intraperitoneal cyclophosphamide on day 4 (150 mg/kg) and on day 1 (100 mg/kg) before thigh infection [24] and were anaesthetized with ketamine/xylazine, and thigh infections were produced by injecting 0.1 mL of bacterial suspension of 107 CFU/mL for each study and control isolate, 2 h before giving antibiotic therapy, as described previously [25]. The infections were done in triplicate for all isolates. After mice were infected, they were given meropenem treatment for 24 h. As therapeutic protocols of experimental infections caused by A. baumannii have been scarcely reported, we used three meropenem dosing schemes: (i) 20 mg/kg/8h that was applied previously in a study with A. baumannii isolates [11], (ii) 100 mg/kg/12 h [12] and (iii) 400 mg/kg/8 h [13], which were both applied on mice infections caused by P. aeruginosa. Equal numbers of animals remained untreated as controls. Mice either died from infection or were sacrificed at 24 h; thigh muscles were homogenized in 10 mL of saline, serially diluted and cultured quantitatively after serial dilutions, for CFU determination. The level of detection of this assay was 100 CFU/thigh. When no organisms were cultured from the thighs, the number of CFU was arbitrarily set at 100 for further calculations. The thigh CFU count was expressed as log10 CFU/thigh muscle. A t-test was used for statistical analysis. For all experiments, a p-value of ≤0.05 was considered indicative of statistical significance. All statistical analyses were performed using Minitab software (version 13.31). In conclusion, in vitro meropenem exposure of A. baumannii isolates may induce phenotypic expression of heterogeneous resistance. This phenomenon is reversible for some isolates (persisters), but could also be permanent when associated with stable changes in the genome of A. baumannii (heteroresistance) [9]. However, the clinical importance of this phenomenon remains unknown. Our results indicate that the effect of the meropenem heterogeneous growth to the infectious process seems to be equivocal, as the virulence of the heterogeneous isolates is lower than that of the clinical isolates, while the meropenem treatment outcome seems not to be affected due to the elevated meropenem MICs.This work was supported in part by the Research Committee of University of Thessaly, Greece.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Resistance mediated by efflux has been recognized in Staphylococcus aureus in the last few decades, although its clinical relevance has only been recognized recently. The existence of only a few studies on the individual and overall contribution of efflux to resistance phenotypes associated with the need of well-established methods to assess efflux activity in clinical isolates contributes greatly to the lack of solid knowledge of this mechanism in S. aureus. This study aims to provide information on approaches useful to the assessment and characterization of efflux activity, as well as contributing to our understanding of the role of efflux to phenotypes of antibiotic resistance and biocide tolerance in S. aureus clinical isolates. The results described show that efflux is an important contributor to fluoroquinolone resistance in S. aureus and suggest it as a major mechanism in the early stages of resistance development. We also show that efflux plays an important role on the reduced susceptibility to biocides in S. aureus, strengthening the importance of this long neglected resistance mechanism to the persistence and proliferation of antibiotic/biocide-resistant S. aureus in the hospital environment.Efflux pumps are membrane proteins that have the function of detoxifying cells by expelling noxious molecules [1]. The extrusion of antimicrobial compounds, such as antibiotics and biocides, is considered to be an “accidental function” of such efflux systems [2,3]. Nevertheless, efflux-mediated resistance towards antimicrobial compounds is increasingly recognized as an important resistance mechanism in bacteria [4]. Efflux pumps present different substrate specificities; some are specific to an antibiotic or a class of antibiotics, whereas multidrug efflux pumps, as the name implies, have the capacity to extrude more than one class of antibiotics and/or other antimicrobial compounds [5]. These latter efflux systems are of foremost relevance, since they can bestow the bacterial cell with a phenotype of resistance to multiple drugs in addition to promoting cross-resistance between antibiotics and other antimicrobial compounds usually used to prevent and control healthcare associated infections [4].In Staphylococcus aureus, several specific efflux pumps have been associated with resistance to antibiotics, such as tetracycline (Tet(K), Tet(L)) and macrolides (Mef(A), Msr(A)) [5]. Also, several multidrug efflux pumps have been described that are associated with resistance to antibiotics (e.g., fluoroquinolones) and to biocides, such as NorA, NorB, NorC, MepA and MdeA [5]. Other multidrug efflux pumps expel only biocides, as is the case of QacA/B and Smr [5]. In general, specific efflux pumps can be found either in the chromosome or in plasmids, while multidrug efflux pumps are mainly located in the chromosome, with the exception of QacA/B and Smr, which have only been described in plasmids [5].Despite the increasing number of S. aureus efflux pumps identified with the potential to contribute to the resistance towards clinically relevant antibiotics and other antimicrobial compounds, few studies have been undertaken to ascertain the collective and individual contribution of efflux systems to resistance phenotypes in clinical isolates [6,7,8], resulting in little information being available. One of the underlying reasons for this is the lack of established methods to assess efflux activity, mainly in the definition of threshold values for the attribution of “basal” versus “increased” levels for efflux activity, as well as the determination of the role of each pump on the overall efflux activity. Several approaches have been used to identify active efflux systems in bacteria, such as the use of radiolabelled substrates, fluorometric assays or the determination of the minimum inhibitory concentration (MIC) for different substrates in the presence of efflux inhibitors (EIs) [7,9,10]. In our group, we have developed methods based on ethidium bromide (EtBr), a substrate of the majority of the S. aureus multidrug efflux pumps, that has been proven reliable for the assessment of efflux activity in bacteria [11], namely the EtBr-agar cartwheel method [12], that allows the screening of large collections of clinical isolates to detect isolates with increased efflux activity. The monitoring of EtBr efflux in clinical isolates by real-time fluorometry [13] permits a more extensive characterization of that efflux activity and can be used to confirm, on a real-time basis, the results of the EtBr-agar cartwheel method. The use of EtBr has also been proven useful by other groups in detecting isolates with increased efflux activity [14]. The information obtained from these methods can then be complemented by the determination of MICs of effluxable substrates in the presence of efflux inhibitors. This work describes the use of these approaches to study the role played by efflux on the resistance to antimicrobial agents, including antibiotics and biocides, on a collection of S. aureus strains of clinical origin and how this efflux activity may contribute to the persistence of S. aureus cells over-expressing efflux pumps on the clinical environment.The EtBr-agar cartwheel (EtBrCW) method is a practical methodology to assess the presence of increased efflux activity in large collections of clinical isolates of different bacterial species [12]. This method allows the comparison of different isolates on the basis of their capacity to extrude EtBr. The isolates are streaked in solid media containing increasing concentrations of EtBr and the fluorescence emitted, which is inversely proportional to their capacity to extrude the compound, is compared to the fluorescence of control strains. Using this approach to test a collection of 52 ciprofloxacin-resistant S. aureus, we could discriminate these isolates in three distinct groups: a group of twelve isolates that showed fluorescence only at the highest EtBr concentration tested, presumably with increased efflux activity and designated EtBrCW-positive; a group of thirty-three isolates that showed fluorescence at the lowest EtBr concentrations tested and denominated EtBrCW-negative; and a third group of seven isolates showing fluorescence at intermediate concentrations of EtBr and denominated EtBrCW-intermediate isolates [7] (Figure 1).Further characterization of these isolates by a fluorometric assay that detects EtBr efflux by assessing in real-time the loss of EtBr fluorescence in bacterial cells previously loaded with this dye enabled us to corroborate the preliminary characterization of the isolates by the EtBrCW method (Figure 1). In particular, the increased efflux activity present in EtBrCW-positive isolates (Figure 1, red) is demonstrated by the lack of fluorescence in EtBr-agar plates together with a prompt EtBr efflux by real-time fluorometry, whereas EtBrCW-negative isolates (Figure 1, blue) emit a strong fluorescence in EtBr-agar plates and show only slight EtBr efflux. On the other hand, EtBrCW-intermediate isolates show intermediate fluorescence and EtBr efflux (Figure 1, orange). This analysis also showed that basal efflux activity is always present in S. aureus, as shown by the reference strain. ATCC25923 (Figure 1, green). Altogether, these two EtBr-based assays proved to be valuable tools to screen for increased efflux activity in clinical isolates of S. aureus, making it possible to differentiate strains with varying levels of efflux activity. As stated previously, EtBr is a common substrate of multidrug efflux pumps, which can also extrude other antimicrobial compounds, such as the antibiotics fluoroquinolones and biocides and, thus, used as a screening marker for efflux, leading to resistance towards fluoroquinolones and biocides. Characterization of reference and clinical isolates according to their efflux capacity. In green: reference strain S. aureus ATCC25923; in blue: EtBr-agar cartwheel (EtBrCW)-negative isolate SM5; in orange: EtBrCW-intermediate isolate SM44; in red: EtBrCW-positive isolate SM1. Central figure: screening of efflux activity by the EtBrCW method. Trypticase soy agar (TSA) plate supplemented with 2.5 mg/L EtBr streaked with representative isolates. (−) and (+): strains ATCC25923 and ATCC25923EtBr, used respectively as negative and positive controls for EtBr efflux. Graphics: evaluation of efflux activity by real-time fluorometry. Efflux assays for representative isolates are shown for cells resuspended in Phosphate buffered saline (PBS), in PBS plus glucose (0.4%) or PBS plus glucose and the efflux inhibitor, verapamil (at the sub-inhibitory concentration of 200 mg/L). The data presented was normalized against the data obtained in conditions of no efflux (absence of glucose and presence of 200 mg/L of verapamil). Fluoroquinolones are a class of antibiotics that possess a broad spectra of activity, including methicillin-resistant S. aureus (MRSA) [15]. However, the swift development of resistance to these antibiotics has impaired their clinical relevance [16]. At present, in Europe, around 25% of S. aureus isolates are resistant to fluoroquinolones, a percentage that increases to 90% among MRSA isolates [17].Resistance to fluoroquinolones in S. aureus is usually associated with the occurrence of mutations in the target genes, grlA/B and gyrA/B, that code for Topoisomerase IV (GrlA/B) and DNA gyrase (GyrA/B) proteins, respectively [16]. These mutations usually occur in a precise region denominated quinolone resistance-determining region (QRDR) and generate proteins with lower affinity for fluoroquinolones [16]. Several studies have shown that Topoisomerase IV is the primary target of fluoroquinolones in S. aureus. Accordingly, in vitro studies have demonstrated that emergence of fluoroquinolone resistance is associated with acquisition of mutations first in the grlA gene followed by mutations in the gyrA gene [18]. Also, fluoroquinolone resistant clinical isolates with mutations only in the gyrA gene are uncommon [19]. Moreover, quinolone resistant isolates with a single or double mutation in the grlA gene present high-level resistance when a gyrA mutation is acquired [19,20]. The occurrence of mutations in the grlB and gyrB genes has been shown to be infrequent [16]. Altogether, mutations in the QRDR of these genes are linked to high-level resistance to fluoroquinolones in S. aureus clinical isolates [16].Resistance to fluoroquinolones mediated by efflux has been described in S. aureus clinical isolates for the last two decades [9,18,21,22,23,24,25,26,27,28]. However, these studies related only to the role of the NorA efflux pump. Nowadays, it is known that at least three other multidrug efflux pumps, namely NorB, NorC and MepA, have been described as having fluoroquinolones as a substrate [29,30,31], although their actual contribution to clinical fluoroquinolone resistance remains uncertain. A few studies have been conducted with clinical isolates where an association between fluoroquinolone resistance and these efflux systems was explored [6,7,8]. The effect of the known efflux inhibitors, thioridazine (TZ) and verapamil (VER) [7], on the MIC levels of fluoroquinolones was evaluated, and mutations conferring fluoroquinolone resistance were screened. Reserpine is usually used to assess efflux activity in S. aureus, but it was not tested in this study, since previous data by our group has revealed that this compound has a mild inhibitory activity [7]. The isolates presented in Figure 2 are representative of each of the groups established previously, according to efflux capacity. Detailed data on MIC values is provided in Supplementary Table S1.The data presented in Figure 2 shows that independently of the mutations carried by each strain in both grlA and gyrA genes, which alter the target affinity of these antibiotics, efflux is an important component of the resistance level. This can be observed in the effect of the efflux inhibitor, TZ, on the MICs of the two fluoroquinolones tested for these isolates. The inhibitory effect of TZ was shown to be higher for strains with higher efflux activity, namely the ones classified as EtBrCW-positive or EtBrCW-intermediate, for which a two- to eight-fold reduction in the MICs of the two fluoroquinolones was observed, whereas this reduction was only two-fold for the EtBrCW-negative isolates. Verapamil showed a weaker effect, resulting in MIC reductions that ranged from none to four-fold for EtBrCW-positive and -intermediate isolates and none to two-fold for EtBrCW-negative isolates. Comparing the data gathered for the EtBrCW-intermediate isolates with the data of the remaining isolates, it could be observed that EtBrCW-intermediate isolates are more similar to EtBrCW-positive isolates. All these isolates carried mutations in both grlA and gyrA genes that have been described in the literature as being involved in high-level resistance to fluoroquinolones [22,32]. Accordingly, our isolates present ciprofloxacin MICs that range between 16 to 256 mg/L for EtBrCW-positive and -intermediate isolates and 8 to 32 mg/L for EtBrCW-negative isolates; whereas the norfloxacin MICs vary between 64 to 1,024 mg/L for EtBrCW-positive and -intermediate isolates, and 64 to 128 mg/L for EtBrCW-negative isolates. However, part of this resistance can be attributable to efflux, as seen in the degree of MIC reductions by the efflux inhibitors tested in Figure 2. We also observed that although isolates carrying a double mutation in grlA and a single mutation in gyrA presented higher MICs, as described in the literature, they also suffered higher MIC reductions with the efflux inhibitors, particularly with TZ (two- to eight-fold) in comparison with isolates carrying a single mutation in both genes (none to two-fold). Regardless of the type of combination of QRDR mutations presented by the isolates, the presence of TZ reduced the MICs of ciprofloxacin to 8–32 mg/L and the MICs of norfloxacin to 32–128 mg/L (Figure 2 and Supplementary Table S1). However, the fluoroquinolone resistance phenotype was not fully reverted by TZ, since all isolates remained resistant to either ciprofloxacin or norfloxacin in the presence of the inhibitor, as expected, due to the presence of mutations. Effect of the efflux inhibitors thioridazine (TZ) and verapamil (VER), at subinhibitory concentrations (12.5 mg/L and 200 mg/L, respectively), on the minimum inhibitory concentration (MIC) values of ciprofloxacin and norfloxacin for representative isolates of the EtBrCW-positive, EtBrCW-intermediate and EtBrCW-negative groups, each carrying different mutations conferring fluoroquinolone resistance (data for EtBrCW-positive and -negative strains from our previous study, [7]).These results reveal that efflux is an important contributor to fluoroquinolone resistance in S. aureus. While mutations in the QRDR of grlA and gyrA genes confer resistance up to a certain level, in particular 8 to 32 mg/L for ciprofloxacin and 32 to 128 mg/L for norfloxacin, the remaining resistance may be attributable to efflux, thus demonstrating that efflux is a relevant component of the level of fluoroquinolone resistance in these clinical isolates. This last observation is further supported by data on strain SM15, classified as EtBrCW-intermediate and showing low level resistance to fluoroquinolones and the single isolate among the EtBrCW-intermediate isolates that carry solely a single mutation in grlA QRDR and no mutation in gyrA (Figure 3). This strain presented MICs of 8 mg/L for ciprofloxacin and 16 mg/L for norfloxacin, which are near the breakpoint concentrations for an isolate to be considered resistant to these antibiotics (according to CLSI guidelines [33]). These values are in accordance to data in the literature for isolates carrying a single GrlA mutation [32]. However, the addition of efflux inhibitors lead to a reduction of these MIC levels, which, in the particular case of TZ, drop to the susceptibility levels for both ciprofloxacin and norfloxacin (1 mg/L and 4 mg/L, respectively) (Figure 3). Effect of the efflux inhibitors thioridazine (TZ) and verapamil (VER), at a subinhibitory concentration (12.5 mg/L and 200 mg/L, respectively), on the MIC values of ciprofloxacin and norfloxacin for the EtBrCW-intermediate strain SM15. WT: wild-type sequence (no mutations).These results further strengthen the importance of efflux on fluoroquinolone resistance in S. aureus, in particular in the early stage of the acquisition of mutations in the QRDR of grlA and gyrA genes. Strain SM15 may represent an intermediate stage of the emergence of fluoroquinolone resistance, with balanced contributions from both efflux and mutation to the resistance phenotype. In fact, recent studies from independent groups, working with different bacterial species, suggest that efflux systems may be a first response of the cell to cope with antimicrobial agents, enabling the cell to survive and acquire other, more stable resistance mechanisms, such as target gene mutations for fluoroquinolones, that will then provide a high-level resistance phenotype, as was recently demonstrated for Escherichia coli [34] and Mycobacterium tuberculosis [35]. In S. aureus, some evidence has also been found for this role of efflux pumps as a first-line defense mechanism towards noxious compounds [36,37] that are supported by data on clinical strains [6,7,8,38].Biocides differ greatly from antibiotics in their mechanism of action; whereas antibiotics have precise cell targets, biocides usually act upon several cellular targets [39]. They have an important role in infection control in healthcare settings, where they are currently used in a variety of products that are applied in the washing and disinfection of the environment and medical devices. They are also used as antiseptics for patients and healthcare professionals in hand hygiene, skin disinfection prior to invasive procedures and mucous disinfection [39]. Among these biocides, antiseptic formulations containing chlorhexidine or quaternary ammonium compounds, together with alcohol-based preparations, are the most commonly used for skin disinfection and hand hygiene, respectively [40]. Apart from healthcare settings, these compounds are widespread in industry, being also increasingly employed in the community setting [41]. Concern regarding the emergence of clinical strains showing reduced susceptibility or tolerance to biocides has been increasing in the last decade, with particular focus on the potential role of biocides as a selective force of antibiotic-resistant bacteria [42]. Reduced susceptibility to biocides in S. aureus is mainly associated with efflux pumps that are encoded in plasmids, including the efflux systems, QacA/B and Smr [5]. Nevertheless, the several chromosomally-encoded multidrug efflux pumps that have been described so far in S. aureus also have in their substrate profile a wide variety of biocides. Therefore, it is important to ascertain the contribution of these efflux systems to the biocide “susceptibility” profile in clinical isolates. The collection of 52 ciprofloxacin-resistant S. aureus isolates was also tested for susceptibility to biocides by determination of MICs of several biocides, namely the quaternary ammonium compounds, cetrimide, cetylpyridinium chloride and benzalkonium chloride, the bisbiguanidine chlorhexidine digluconate, pentamidine, tetraphenylphosphonium bromide and dequalinium chloride. MICs of EtBr were also determined, since this compound was used as a marker for efflux activity. Data for representative isolates can be found in Figure 4. Among the compounds tested, it could be observed that the MICs for EtBrCW-positive and -intermediate isolates were generally higher than the ones for EtBrCW-negative isolates. In particular, EtBrCW-positive and -intermediate isolates presented MICs in the following range: EtBr, 8 to 16 mg/L; the quaternary ammonium compounds cetrimide, 4 to 8 mg/L, cetylpyridinium chloride, 1 to 4 mg/L and benzalkonium chloride, 2 to 4 mg/L; tetraphenylphosphonium bromide, 16 to 64 mg/L; chlorhexidine digluconate, 0.00006% to 0.000125%, and dequalinium chloride, 4 to 16 mg/L. The range of MICs for the EtBrCW-negative isolates varied as follows: EtBr, 2 to 8 mg/L; cetrimide, 2 mg/L; cetylpyridinium chloride, 0.5 mg/L; benzalkonium chloride, 1 mg/L, tetraphenylphosphonium bromide, 16 to 32 mg/L, chlorhexidine digluconate, 0.00003% to 0.00006%; and dequalinium chloride, 2 to 4 mg/L (detailed MIC data is provided in Supplementary Table S2, Table S3). No significant difference was found between the groups of isolates for the MICs of pentamidine (data not shown). In sum, the EtBrCW-positive and -intermediate isolates presented MIC values that were two- to eight-fold higher than the ones presented by the EtBrCW-negative isolates (Figure 4). Although this difference is not extensive, it reveals that the efflux-positive and -intermediate isolates can withstand higher concentrations of these biocides. Moreover, it demonstrated a variation of the MICs of biocides according to the efflux capacity of the three groups of isolates (Figure 4). Effect of the efflux inhibitors thioridazine (TZ) and verapamil (VER), at subinhibitory concentrations (12.5 mg/L and 200 mg/L, respectively), on the MIC values of several biocides for representative isolates of the EtBrCW-positive, EtBrCW-intermediate and EtBrCW-negative groups.To assure that the difference observed in the MIC values of the biocides and EtBr was the result of the higher efflux capacity of the EtBrCW-positive and EtBrCW-intermediate isolates, the effect of TZ and VER on the MICs of these compounds was also evaluated (Figure 4 and Supplementary Table S2, Table S3).The effect of the efflux inhibitors upon the MIC values of the biocides selected showed that efflux activity has a strong involvement in the reduced susceptibility of S. aureus to biocides. For all isolates tested, independently of their efflux capacity, the MICs of all the biocides were reduced two- to 16-fold in the presence of TZ and none to eight-fold (mostly, about two-fold reduction) in the presence of VER, with the highest inhibitory effects observed for the quaternary ammonium compounds and for tetraphenylphosphonium bromide. The biocide for which this effect was lower was dequalinium chloride, with none to two-fold reduction of MICs in the presence of the efflux inhibitors. It could also be observed, in general, that for EtBrCW-positive and -intermediate isolates, the efflux inhibitors could reduce the MICs of the several compounds to levels similar or lower than the ones presented by the EtBrCW-negative isolates. Altogether, these results indicate that efflux activity contributes to reduced susceptibility to biocides in S. aureus. We have previously referred to the lower capacity of fluoroquinolone MIC reduction by VER, when compared to TZ [7]. Here, we provide experimental data on this difference and show that the same effect is observable for biocides, as well.Among the EtBrCW-positive isolates, SM52 is the only to carry a plasmid with the gene for the efflux pump Smr [7]. This pump is associated with low-level resistance to biocides and EtBr [43] and is found in a low prevalence (around 10%) in clinical S. aureus isolates [44,45,46]. The MIC values presented by the isolate SM52 are equal or lower than the ones of the remaining EtBrCW-positive and -intermediate isolates (that carry no Smr or QacA/B efflux pump), thus showing that the potentially active chromosomal multidrug efflux pumps can confer a resistance level to biocides similar or higher than the Smr plasmid-encoded efflux pump. The results described suggest that active efflux systems can be responsible for reduced susceptibility to biocides in S. aureus. The concentration in use in which these compounds are applied for washing and disinfection in healthcare settings is higher than the MICs values determined for these isolates. For example, chlorhexidine is used in concentrations that range between 0.2 to 4%, much higher than the highest MIC found in these isolates. This may suggest that this observed reduced susceptibility to biocides is not clinically relevant. Nevertheless, the misuse of these biocide formulations, especially in terms of the application time, together with contaminating residues left after use, may provide opportunities for more tolerant bacteria to be maintained and proliferate in these environments [47,48,49]. Furthermore, concern arises regarding biocide-resistant/tolerant strains and their role in the selection of antibiotic-resistant strains. Studies have related contradictory data concerning the relation between antibiotic and biocide resistance, but much evidence has been gathered that supports the potential co-selection of strains with reduced susceptibility to biocides by antibiotic-resistant bacteria, and vice versa. In the particular case of S. aureus, studies have shown that exposure to biocides can induce the overexpression of multidrug efflux pumps in both reference and clinical strains, leading to reduced susceptibility to the inducing biocide, as well as to other biocides and to antibiotics [38,50]. These findings strengthen the importance of increasing our knowledge of efflux as a resistance mechanism in S. aureus. A collection of 52 ciprofloxacin-resistant S. aureus isolates was studied [7]. The pan-susceptible reference strain, S. aureus ATCC25923, and its ethidium bromide-adapted counterpart, ATCC25923EtBr [50]. were used as controls. All strains were grown in tryptic soy broth (TSB) at 37 °C with shaking or in trypticase soy agar (TSA) (Oxoid Ltd., Basingstoke, UK). The strain ATCC25923EtBr was grown in TSB/TSA supplemented with 50 mg/L of EtBr. Antibiotics were purchased from different sources, as follows: ciprofloxacin (Fluka Chemie GmbH, Buchs, Switzerland) and norfloxacin (ICN Biomedicals Inc., Aurora, OH, USA). EtBr, biocides (benzalkonium chloride, tetraphenylphosphonium bromide, pentamidine isothionate salt, cetylpyridinium chloride, cetrimide, dequalinium chloride and chlorhexidine digluconate) and efflux inhibitors (thioridazine and verapamil) were acquired from Sigma-Aldrich (Madrid, Spain). All efflux inhibitors solutions were prepared in deionized water on the day of the experiment and kept protected from light. For the EtBr-agar cartwheel method [12], each culture was swabbed onto TSA plates containing EtBr concentrations ranging from 0.5 to 2.5 mg/L in increments of 0.5 mg/L EtBr. S. aureus ATCC25923 and ATCC25923EtBr were used as negative and positive controls for efflux activity, respectively [50]. The plates were incubated at 37 °C during 16 hours, after which the minimum concentration of EtBr associated with the bacterial mass that produced fluorescence under UV light was recorded in a Gel-Doc XR apparatus (Bio-Rad, Hercules, CA, USA). Isolates showing fluorescence at lower EtBr concentrations have potentially less active efflux systems than isolates for which fluorescence is only detected at higher concentrations of EtBr [12]. Thus, isolates were classified according to the emitted fluorescence registered, namely isolates showing emission of fluorescence at 0.5–1 mg/L EtBr were denominated EtBrCW-negative (with no potential active efflux systems); isolates showing emission of fluorescence at 1.5–2.0 mg/L EtBr were denominated EtBrCW-intermediate; and isolates emitting fluorescence only at the maximum concentration of EtBr tested (2.5 mg/L) were denominated EtBrCW-positive (with potential active efflux systems).Efflux assays by real-time fluorometry were performed in a Rotor-Gene 3000™ thermocycler, together with real-time analysis software (Corbett Research, Sydney, Australia) [13]. Cultures were grown in TSB medium at 37 °C with shaking until an optical density at 600 nm (OD600) of 0.6. The cells were collected by centrifugation at 13,000 rpm for 3 minutes and the pellet washed twice with a 1X phosphate buffered saline (PBS) solution. EtBr-loaded cells were prepared by incubating a cellular suspension with an OD600 of 0.3 with 0.25, 0.5 and 1 mg/L EtBr for EtBrCW-negative, -intermediate and -positive cultures, respectively, plus 200 mg/L of verapamil (a sub-inhibitory concentration) at 25 °C for 60 minutes. After EtBr accumulation, cells were collected by centrifugation and resuspended in 1X PBS to an OD600 of 0.6. Several parallel assays were then run in 0.1 mL final volume corresponding to 0.05 mL of the EtBr loaded cells (final OD600 of 0.3) incubated with 0.05 mL of (1) PBS only, (2) glucose 0.8% only (final concentration of 0.4%), (3) 400 mg/L of verapamil only (final concentration of 200 mg/L) and (4) glucose 0.8% plus 400 mg/L of verapamil (final concentrations of 0.4% and 200 mg/L, respectively). Efflux assays were conducted in the Rotor-Gene 3000™ at 37 °C, and the fluorescence of EtBr was measured (530/585 nm) at the end of every cycle of 10 seconds, for a total period of 10 minutes. The raw data obtained was then normalized against data obtained from non-effluxing cells (cells from the control tube with only 200 mg/L VER), at each point, considering that these correspond to the maximum fluorescence values that can be obtained during the assay. The relative fluorescence thus corresponds to the ratio of fluorescence that remains per unit of time, relatively to the EtBr-loaded cells.For determination of minimum inhibitory concentrations (MICs), cultures were grown in Mueller-Hinton broth (MH, Oxoid) at 37 °C. MICs for antibiotics were determined by the two-fold broth microdilution method and evaluated according to the CLSI breakpoints [33]. MICs for EtBr and biocides were also determined using the two-fold broth microdilution method. After an 18 hour incubation period at 37 °C, the MIC values were recorded, corresponding to the lowest concentration of antimicrobial compound that presented no visible growth. To evaluate the effect of efflux inhibitors on the MIC values, parallel cultures were tested in media containing varying concentrations of the antimicrobial compound in the absence and presence of the efflux inhibitors thioridazine and verapamil at the sub-inhibitory concentrations of 12.5 mg/L and 200 mg/L and equivalent bacterial inoculums. The cultures were incubated for 18 hours and growth evaluated visually. All assays were determined in triplicate.For the screening of mutations conferring fluoroquinolone resistance, internal fragments comprising the QRDR of grlA and gyrA genes were amplified using primers previously described [7]. The reaction mixture (50 μL) contained 2.5 U of Taq Polymerase (Fermentas Inc., Ontario, Canada), 1X Taq buffer (Fermentas), 25 pmol of each primer, 0.2 mM of dNTP and 1.75 mM of MgCl2. The PCR reactions were conducted in a thermocycler Mastercycler personal 5332 (Eppendorf AG, Hamburg, Germany). The amplification conditions were as follows: DNA was denatured at 94 °C for 4 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 50 °C for 30 seconds and extension at 72 °C for 1 minute, followed by a step of final extension at 72 °C for 5 minutes. Amplification products were purified and sequenced in both strands using the same set of primers. Sequences were analyzed and aligned using the freeware programs, BioEdit and ClustalW, respectively. The results described demonstrate that the two EtBr-based approaches used, namely the EtBrCW method and real-time fluorometry, are valuable techniques to screen and characterize efflux activity in clinical isolates of S. aureus. These isolates were classified according to their capacity to efflux EtBr and the MIC determination in the presence of efflux inhibitors allowed the correlation of this efflux activity with resistance to fluoroquinolones and some biocides, including quaternary ammonium compounds and chlorhexidine, antimicrobials widely used in healthcare settings. These results show that EtBr is indeed a good screening marker for efflux activity leading to resistance to fluoroquinolones and biocides.The data compiled in this study, together with results from previous studies from our group and other colleagues, indicate that efflux is a major mechanism in the first stage of development of resistance to antimicrobial compounds, in this case, fluoroquinolones. That is, activation of efflux systems by fluoroquinolones could promote S. aureus survival in “stress conditions”, allowing the bacteria to acquire and accumulate target gene mutations that are associated with high-level resistance. Furthermore, the demonstration that this same efflux activity can sustain higher tolerance of S. aureus cells to clinically relevant antiseptics and disinfectants, which could also, in turn, potentiate antibiotic resistance, strengthen the importance of this long neglected resistance mechanism to the persistence and proliferation of antibiotic/biocide-resistant S. aureus in the hospital environment.This work was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal), through grants PEst-OE/BIA/UI0457/2011 and PTDC/BIA-MIC/105509/2008. S. S. Costa and E. Junqueira were supported by grants SFRH/BD/44214/2008 and PTDC/BIA-MIC/105509/2008, respectively, from Fundação para a Ciência e a Tecnologia (FCT, Portugal).The authors declare no conflict of interest.

Med-MDPI/antibiotics/antibiotics-02-01-00100.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Nowadays, the emergence and spread of antibiotic resistance have become an utmost medical and economical problem. It has also become evident that subinhibitory concentrations of antibiotics, which pollute all kind of terrestrial and aquatic environments, have a non-negligible effect on the evolution of antibiotic resistance in bacterial populations. Subinhibitory concentrations of antibiotics have a strong effect on mutation rates, horizontal gene transfer and biofilm formation, which may all contribute to the emergence and spread of antibiotic resistance. Therefore, the molecular mechanisms and the evolutionary pressures shaping the bacterial responses to subinhibitory concentrations of antibiotics merit to be extensively studied. Such knowledge is valuable for the development of strategies to increase the efficacy of antibiotic treatments and to extend the lifetime of antibiotics used in therapy by slowing down the emergence of antibiotic resistance. With the early breakthroughs of Fleming and Waksman, the discovery of novel natural antibacterial compounds with important pharmaceutical applications revolutionized medicine and experienced an exponential phase, especially between 1950s and 1960s, the so called “Golden Age”. At that time, the potential problem of antibiotic resistance was unfortunately underestimated, in spite of the fact that the first multi-drug resistant strain appeared already in 1955 (reviewed by [1]). It soon became evident that bacterial resistance can be acquired through mutations or horizontal gene transfers [1]. Today, the worldwide spread of antibiotic resistance is a major healthcare and economic problem because it directly challenges our ability to treat infectious diseases. To extend the lifetime of current and future antibiotic-based therapies, it is increasingly urgent to enlarge our knowledge of how new antibiotic resistances emerge and spread in bacterial population. In this review, we will firstly focus on the bacterial responses to subinhibitory concentrations of antibiotics, with particular emphasis on the induction of the SOS and RpoS regulons. We will then describe how subinhibitory concentrations of antibiotics promote genetic variation by increasing the rates of horizontal gene transfer and mutations. Finally, we will discuss how these molecular mechanisms are directly responsible for the emergence and the spread of resistance determinants.Antibiotics are low molecular weight molecules (<3,000 Da) found in all kinds of terrestrial and marine environments. They are mainly produced by fungi (60%) and actinomycetes (30%), especially by the genus Streptomyces, but also by other bacteria (10%), such as Bacillus, Pseudomonas, Myxobacteria and Cyanobacteria [2]. Antibiotics are metabolites often produced by organisms that undergo morphological differentiation or by organisms that experience nutrient limiting conditions. This production is usually triggered by specific cellular signaling. Antibiotics can be grouped on the basis of their chemical structures, which are extremely varied and complex, or of their mechanism of action [3]. The targets of antibiotics might be either cellular structures or enzymes. The most common mechanisms of action involve the inhibition of the bacterial cell wall biosynthesis (e.g., β-lactam, glycopeptides); the inhibition of protein, RNA or DNA synthesis (macrolides, ansamycins, quinolones, respectively); and the damage of cell membranes (polymyxins). Antibiotics can also be divided in two major classes according to their biological effects: bactericidal compounds that kill bacteria (e.g., β-lactam, fluoroquinolones and aminoglycosides) and bacteriostatic compounds that inhibit bacterial growth (e.g., macrolides, tetracyclines). Recently, a common mechanism of cell death has been described, which is distinct from the classical described targets and shared by several classes of bactericidal antibiotics [4]. This mechanism involves the stimulation of endogenous reactive oxygen species (ROS) production, which damages lipids, proteins and DNA, thus leading to a sort of programmed cellular death that shares several characteristics with apoptosis [5,6]. Two hypotheses have been formulated on the possible natural role of antibiotics. The first postulates that antibiotics are biological weapons that protect the producer strain from bacterial competitors present in the same environment, in particular under stress conditions such as nutrient starvation [7]. For example, filamentous fungi and actinomycetes have a non-motile and saprophytic life cycle in complex habitats, such as the terrestrial soil, where competition with other inhabitants is high. Nevertheless, few examples in literature have demonstrated the role of these microbial products as antibacterials in the natural environments [8,9].The second hypothesis that suggests that antibiotics are involved in signaling mechanisms is supported by several lines of evidence. Many studies have demonstrated that exposure to subinhibitory concentrations of antibiotics induces changes in the expression profile of a wide range of genes in many different bacterial species, resulting in different phenotypes [10,11]. In addition, in situ, antibiotics are produced by bacteria and fungi at a very low concentration that would not have any lethal effects. Subinhibitory concentrations of antibiotics have also been found in aquatic and terrestrial environment due to their use in human and veterinary medicine and in agriculture [12]. Because antibiotics are not always well metabolized in human and animal bodies, they are excreted in active form. A similar fate is shared by the antibiotics used in agriculture. In the soil or in sewage, antibiotics are not fully biodegraded or removed by chemical treatments. For all these reasons, active antibiotics end up polluting soil, surface and ground water. Hence, we live surrounded by subinhibitory concentrations of antibiotics that can have important consequences on the resident microbial communities. The initial definition of antibiotic given by Waksman, i.e., “a natural chemical substance, derived from living microorganisms, which has the capability to inhibit growth or even destroy other microorganisms without harming the eukaryotic host” does not encompass chemically synthesized antibiotics, such as quinolones, sulphonamides or oxazolidinones. In this review we will indiscriminately consider the effects of natural and synthetic antibiotics. As discussed above, antibiotics are found in natural environments at subinhibitory concentrations. Transcriptome analyses, gene reporter fusions and classical genetic studies have shown that subinhibitory concentrations of antibiotics trigger wide changes in the transcriptional profiles and in the phenotypes of various bacterial species, such as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa or Staphylococcus aureus [10]. Most of the genes differentially expressed in the presence of subinhibitory concentrations of antibiotics encode functions not obviously linked with those targeted by the antibiotics. These observations support the idea that antibiotics may act as signaling molecules in nature.Because antibiotics target essential cellular functions, it is not unexpected that even at subinhibitory concentrations they can induce different stress responses, such as the SOS response or the RpoS regulon [13,14,15]. The SOS response is induced by the presence of unprotected single-strand DNA resulting from DNA damage and DNA replication arrest. The induction of the SOS response is mediated by the autocleavage of the LexA repressor, which can be stimulated by the co-protease activity of the RecA protein when it is associated with single-strand DNA. The inactivation of LexA results in the expression of about 40 genes encoding functions mostly involved in the DNA repair [16,17]. Antibiotics such as quinolones impair the function of enzymes interacting with DNA and thus promote the formation of DNA damage and replication arrest [18]. Therefore, the SOS induction observed after quinolone treatment results directly from their mechanism of action. In E. coli, lethal doses of β-lactams promote the induction of the SOS response, which is mediated by the two components system DpiAB [19]. This induction is therefore independent of DNA damage unlike the SOS induction by quinolone antibiotics. Other antibiotics that do not target functions directly linked with DNA, like aminoglycosides, may induce the SOS response through the stimulation of cellular ROS production, as suggested by the work of Kohanski et al. [4,20]. We recently showed that in E. coli, subinhibitory concentrations of different bactericidal antibiotics stimulate the induction of the general stress response, which is controlled by the RpoS sigma factor [15]. The induction of the RpoS regulon is controlled by many different factors acting at the level of the rpoS gene expression, rpoS mRNA stability or RpoS protein translation and stability [21]. The RpoS regulon was historically linked with stationary-phase gene expression. However, today we know that this regulon may be induced by a wide range of stress conditions like heat shock, starvation, low pH or osmotic shock [21]. The alternative sigma factor RpoS is conserved in many bacterial species and controls the expression of many genes involved in cell shape determination, stress response, biofilm formation, DNA repair, metabolism or genes coding for virulence factors [22]. Therefore, the functions of the RpoS-regulated genes may have an important impact on the emergence of antibiotic resistance and on the virulence potential of stressed bacterial populations. For instance, we showed that the induction of the RpoS regulon is required for increased mutagenesis in cells treated with subinhibitory concentrations of β-lactam antibiotics [15]. This is of high relevance because even slight modifications in the mutation rates can significantly influence the evolution of antibiotic resistance [23]. Numerous studies on the effect of subinhibitory concentrations of antibiotics report modulation in the expression of genes coding for virulence factors, such as exo and endotoxins or adhesins, in several human pathogen species [24,25,26,27,28]. In S. aureus treated with different β-lactam antibiotics, promoter-lux reporter constructions allowed observing and quantifying the induction of the spa, lukE and agr genes, known to code for virulence functions [28]. Induction of virulence genes by subinhibitory concentrations of antibiotics is of high importance from a clinical point of view because it may increase virulence of pathogens and hence contribute to increased morbidity and mortality. However, subinhibitory concentrations of antibiotics can also inhibit virulence gene expression, as demonstrated by Grimwood et al. [25], in the case of the exotoxins production of P. aeruginosa. The majority of the studies on gene expression or on phenotypic changes induced by subinhibitory concentrations of antibiotics have been conducted in homogenous liquid environments. To extend knowledge on the effects of subinhibitory concentrations of antibiotics, it is important to perform studies in structured environments, such as biofilms, because bacteria are predominantly found in such environments in nature. For instance, Zhang et al. [29] have already shown that the emergence of antibiotic resistance is accelerated in structured environments. The microfluidic device used in this study consists of an interconnected multi chamber chip that favors the formation of an antibiotic gradient. This structure mimics the environmental conditions, such as the human body, where cells encounter nutrient and chemical gradients. In such a structured environment, antibiotic resistant mutants appearing in areas of low antibiotic concentration can move and overtake sensitive bacteria in areas of higher antibiotic concentration. In addition, among the different phenotypes induced by subinhibitory concentrations of antibiotics, the stimulation of biofilm production was observed in numerous human pathogens, such as Staphylococcus or Pseudomonas species [11]. Biofilms can directly challenge the treatment of infectious diseases by greatly reducing the antibacterial efficacy of antibiotics [30,31]. Biofilm structures are heterogeneous environments in which bacteria face gradients of physical and chemical parameters, such as nutrients, oxygen, pH. Consequently, bacteria are in distinct physiological states, which endow them with variable capacity to tolerate antibiotics. The physical barrier created by the biofilm structure can also slow the diffusion of antibiotics [32,33] and therefore can promote the appearance of zones of subinhibitory concentrations of antibiotics. Because subinhibitory concentrations of antibiotics can induce stress responses, which in turn increase the capacity of bacteria to resist to higher doses of antibiotics, a vicious circle could be created. The formation of biofilms is induced by different environmental signals through molecular pathways often involved in quorum sensing or second-messenger signaling [34]. Biofilm formation, induced by subinhibitory concentrations of antibiotics targeting ribosomes, such as aminoglycosides, phenicols or tetracyclines, was shown to involve cyclic-di-GMP signaling both in P. aeruginosa and E. coli [35]. In P. aeruginosa, biofilm induction requires the presence of an inner membrane protein, coded by the arr gene, containing an EAL domain [35]. The EAL domain is commonly present in enzymes involved in the degradation of the cyclic-di-GMP, thus aminoglycosides may modulate the level of this second messenger by acting on the inner membrane protein. In E. coli, translational inhibitors promote the induction of poly-GlcNAc through the up-regulation of the activity of the pga genes products [36]. As for P. aeruginosa, cyclic-di-GMP is essential in this process and requires the di-guanilate-cyclase activity of the enzyme YdeH. The stimulation of biofilm production by subinhibitory concentrations of β-lactam antibiotics has been also demonstrated in E. coli, P. aeruginosa, S. aureus and Streptococcus pneumoniae [37,38,39,40]. For example, in E. coli, β-lactam treatments can increase the expression of cps genes, which code for colonic acid production pathway, while in P. aeruginosa, subinhibitory concentrations of imipenem increase the production of alginate, thus favoring the formation of thicker and more robust biofilms.Bacterial responses to subinhibitory concentrations of antibiotics raise the question of whether these responses result from a specific signal triggered by the antibiotics or whether they are only the consequences of a perturbation in the cellular homeostasis resulting from the antibiotic action. Even though the above described responses to antibiotics, like SOS, RpoS regulon or biofilm formation, are induced by a variety of different stresses, the presence of signaling pathways dedicated to respond specifically to antibiotics cannot be excluded. Subinhibitory concentrations of antibiotics strongly stimulate the transfer of mobile elements such as transposons, insertion sequences (ISs), integrons, integrating conjugative elements (ICEs) or pathogenicity islands (PIs), through transformation, conjugation or transduction [41]. These mobile elements can contain genes coding for different antibiotic resistance, heavy metal resistance or virulence factors, thus conferring a multi-resistant phenotype to the host cell. For example, it was shown that subinhibitory concentrations of β-lactam antibiotics enhanced the rate of conjugative plasmid transfer in S. aureus [42] and that pre-treatment of donor Bacteroides cells with tetracycline enhanced the conjugal transfer of different ICEs [43,44].The soil microcosm is one of the largest and diverse reservoirs for antibiotic resistant determinants in the form of mobile elements [41,45,46]. Subinhibitory concentrations of antibiotics found in the terrestrial environment through manure fertilization or sewage exposure may significantly contribute to the mobilization of these elements. The presence of antibiotics in the soil increases the horizontal gene transfer and generates diversity in the mobile elements [47]. In a soil treated with sulfadiazine, Heuer and colleagues found a novel low G+C content plasmid harbouring different antibiotic resistance genes, including tet(X) able to confer resistance to the third-generation of tetracyclines. The putative hosts for this new plasmid are Actinobacter spp, of which A. baumannii is one of the recent emerging multi-drug resistance strains in hospitals. The SOS response can promote the expression of genes involved in horizontal gene transfer. Subinhibitory concentrations of some antibiotics induce the SOS response, thus indirectly inducing horizontal transfer. The frequency of transfer of the SXT ICE of Vibrio cholerae is increased more than 300-fold when the E. coli donor cell is grown in the presence of subinhibitory concentrations of mitomycin C or ciprofloxacin, which both induce the SOS response and consequently genes necessary for the SXT transfer [48]. Similarly, subinhibitory concentrations of fluoroquinolones induce the SOS response in S. aureus and promote the replication and transfer of the pathogenicity island SaPIbov1, as well as the induction of the prophage encoding Shiga toxin [49]. In addition, the SOS response, induced by subinhibitory concentrations of antibiotics, was recently demonstrated to promote the expression and recombination of integrons [50]. However, the SOS induction by subinhibitory concentrations of antibiotics is not the only known mechanism that stimulates horizontal gene transfer. For example, subinhibitory concentrations of different antibiotics induce genetic transformation in the naturally competent S. pneumoniae, which lacks a SOS-like response [51].In the last decade, the intestine, an enormously dense and diverse microcosm, has come to the fore as another important reservoir of antibiotic resistance determinants [52]. The current metagenomic studies of the mammalian gut microbiome highlight the abundance and diversity of mobile elements carrying resistance determinants. In addition, previous works have demonstrated that intra and inter-species horizontal gene transfer occurs between commensal and pathogenic bacteria resident in or passing through the gut. In the human and animal intestine, bacteria can encounter a gradient of antibiotic concentrations because of therapeutic use/abuse with direct and important consequences [52]. Since the 90’s, several studies have demonstrated that subinhibitory concentrations of antibiotics induce the transfer of conjugative plasmids harboring antibiotic resistance in the digestive tracts of gnobiotic mice [53,54]. However, no evidence yet demonstrates that horizontal gene transfer is enhanced by the presence of low antibiotic concentrations in the human intestine, but this is likely in our view. Over the years it has become evident that antibiotics can directly affect the rate of emergence of antibacterial resistance determinants in a bacterial population [55,56,57]. Lethal doses of antibiotics select for pre-existing resistant strains. Sub-lethal doses can also select for pre-existing resistant strains [58], but in addition, they can favor the emergence of new resistant determinants by increasing the mutation rate and their spread through the stimulation of the horizontal gene transfer [59] (Figure 1). Emergence of resistance through mutations is especially relevant for resistance resulting from the modifications of the antibiotic targets, such as quinolone or rifampicin families, but also for the evolution of genes conferring resistance through enzymatic antibiotic modifications, such as β-lactam and cephalosporins. Molecular mechanisms by which subinhibitory concentrations of several unrelated classes of antibiotics induce mutagenesis have been characterized in different bacterial species, but most exhaustively in E. coli (Table 1).Impact of antibiotics on bacterial population.The most studied mutagenic antibiotics belong to the quinolone family, such as ciprofloxacin or nalidixic acid. The primary targets of quinolones are type II DNA topoisomerase, the DNA gyrase and topoisomerase IV coded by the gyrA, gyrB and parC, parE genes, respectively [18]. The inhibition of the “ligase activity” of these enzymes leads to the formation of double-strand breaks in DNA, potentially resulting in mutations and cell death [60]. In E. coli, the repair of double-strand breaks requires the homologous recombination pathways: RecBCD or RecFOR [61]. Processing double-strand breaks is also known to trigger the induction of the SOS response [62]. The SOS stress response controls the expression of the error-prone DNA polymerases PolII, PolIV and PolV coded respectively by the polB, dinB and umuCD genes [16]. Cirz and coworkers [63] found that subinhibitory concentrations of ciprofloxacin promote mutagenesis through the same pathways as UV, X or gamma rays, i.e., through induction of the error-prone DNA polymerases. These polymerases are required for the emergence of ciprofloxacin-induced resistant mutants in vitro and in vivo mouse model. As quinolones promote DNA damage, it is not surprising that they can promote mutagenesis by the induction of genes coding for the error-prone DNA polymerases, thus provoking the emergence of antibiotic resistant mutations. This also applies to folate inhibitor antibiotics, like sulfonamides, which by perturbing the nucleotide pool increase the error rate of the DNA polymerases and consequently increase the rate of emergence of antibiotic resistance [64]. Molecular mechanisms involved in antibiotic-induced mutagenesis in different bacterial species.Mutagenesis is not only induced by antibiotics that target functions related to DNA replication or DNA metabolism, but also by antibiotics targeting ribosomes (i.e., aminoglycosides or tetracyclines) or cell wall synthesis (i.e., β-lactam antibiotics) [13,14,15,55]. Ribosome-targeting antibiotics promote mutagenesis in different bacterial species [13,55]. However, the molecular factors involved have been investigated in detail only for streptomycin. Streptomycin is a bacteriostatic antibiotic that targets the 16S rRNA of the 30S subunit of the ribosome, promoting mistranslation. In E. coli, mistranslation induced by streptomycin is a key factor for the increase in mutagenesis [70]. One possible explanation is that mistranslation generates aberrant proteins, among which a mutator allele of the proofreading subunit of the replicative polymerase, hence increasing the error rate during DNA replication. This process is closely related to mistranslation-induced mutagenesis caused by the error-prone alleles coding for tRNA or 16S rRNA [67,71,72,73]. Subinhibitory concentrations of other aminoglycosides, like gentamicin or kanamycin, also increase mutagenesis in E. coli and in other species [15,55]. The molecular mechanisms involved in this process can be mistranslation as for streptomycin. However, their ability to increase ROS production could also be responsible for the increase in mutagenesis, as we will discuss below. β-lactam antibiotics exhibit a mutagenic activity in different unrelated bacterial species such as E. coli, V. cholerae and P. aeruginosa [15]. In all three species, mutagenesis induced by subinhibitory concentrations of β-lactam antibiotics almost exclusively depends on the PolIV activity [15,65]. Although an increase in the PolIV cellular amount was observed in cells treated with subinhibitory concentrations of ampicillin, the expression of the dinB gene was not induced and mutagenesis did not depend upon the SOS repressor LexA cleavage [15]. In vitro and in vivo studies showed that PolIV has a mutagenic activity by generating base substitutions and frameshifts or by incorporating oxidized nucleotides [74,75,76,77]. Our analysis of the mutational spectrum of cells treated with a subinhibitory concentration of ampicillin showed indeed that the PolIV promotes a wide variety of mutations including IS mobility [15]. We have recently demonstrated that the induction of the RpoS regulon is also a key factor in the increase of mutagenesis induced by subinhibitory concentrations of ampicillin. Among the RpoS-controlled genes induced by ampicillin, sdsR, which encodes a small RNA [78], has been proposed to negatively control the level of the MutS protein [15]. Hence, our study provides the first example of a molecular mechanism that directly controls the replication fidelity, thus the mutation rate, in response to an antibiotic-induced stress (Figure 2). The molecular factors required for the mutagenesis induced by β-lactams, i.e., RpoS, DNA PolIV induction and MutS depletion, were shown to be conserved among E. coli, V. cholerae and P. aeruginosa [15]. Schematic representation of how Escherichia coli cells modulate mutation rates in response to subinhibitory concentrations of bactericidal antibiotics. Bactericidal antibiotics, like ampicillin, induce ROS production by stimulating cellular respiratory activity. ROS damage all cellular macromolecules, thus promoting, for example, protein oxidation, DNA replication arrest and oxidation of dNTPs pool. In the presence of subinhibitory concentration of ampicillin, the amount of RpoS and PolIV proteins is increased, most likely because the ClpPX protease-chaperon complex, which degrades both RpoS and PolIV, becomes titrated by an increased amount of oxidized proteins. At the same time, the arrest of the DNA replication forks together with higher level of the PolIV error-prone DNA polymerase favors the incorporation of oxidized dNTPs into the DNA, which eventually results in generation of mutations. However, antibiotic-increased mutagenesis is possible only because the mismatch repair system is not able to repair all the PolIV-generated mutations in ampicillin treated cells. The reduction of mismatch repair activity in antibiotic-treated cells is mediated by SdsR, an RpoS-controlled small RNA, which interacts with the mutS mRNA [15]. Kohanski and co-workers [4] showed that different bactericidal antibiotics promote ROS formation at both lethal and subinhibitory concentrations [4]. In E. coli, the amount of ROS induced in the presence of subinhibitory concentrations of bactericidal antibiotics is correlated with the mutagenic effect of the antibiotics [55]. ROS are known to damage intracellular macromolecules, as lipids, proteins and DNA. However, we showed that the increase in ROS production is required but not sufficient for the increase in mutagenesis. In an rpoS deficient mutant treated with a subinhibitory concentration of ampicillin, the increase in ROS levels is similar as in the wild-type strain, without an accompanying increase in mutagenesis although both strains possess a functional dinB gene [15]. As described above, an RpoS-dependent genetic regulation appears to be the key factor in antibiotic induced mutagenesis in E. coli (Figure 2). Due to the extensive use and abuse, low concentrations of antibiotics are commonly polluting natural environments. It is becoming increasingly clear that exposure of bacterial populations to low concentrations of antibiotics has important ecological and evolutionary consequences. Low concentrations of antibiotics do not only contribute in selecting pre-existant resistant strains, but also have a non-negligible effect on the induction of phenotypic tolerance to antibiotics as well as on the emergence of new antibiotic resistances (Figure 1). Subinhibitory concentrations of antibiotics increase mutation rates, horizontal gene transfer, biofilm formation and the expression of virulence factors. Hence, the signals and the molecular mechanisms, involved in the responses to low concentrations of antibiotics, merit to be extensively studied. Targeting the key molecular actors involved in these responses or inhibiting the mutagenic pathways could indeed be a valuable strategy to increase the efficacy of antibiotic treatments and to extend the lifetime of antibiotics used in therapy, slowing down the emergence of antibiotic resistance. The authors want to thank Edwin Wintermute and Anthony Woo for carefully reading the manuscript. This work was supported by the FP7-HEALTH-F3-2010-241476, ANR-09-BLAN-0251, Idex ANR-11-IDEX-0005-01/ ANR-11-LABX-0071, AXA and Mérieux Research Grants. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).An environmental risk assessment (ERA) for the aquatic compartment in Europe from human use was developed for the old antibiotic Trimethoprim (TMP), comparing exposure and effects. The exposure assessment is based on European risk assessment default values on one hand and is refined with documented human use figures in Western Europe from IMS Health and measured removal in wastewater treatment on the other. The resulting predicted environmental concentrations (PECs) are compared with measured environmental concentrations (MECs) from Europe, based on a large dataset incorporating more than 1800 single MECs. On the effects side, available chronic ecotoxicity data from the literature were complemented by additional, new chronic results for fish and other organisms. Based on these data, chronic-based deterministic predicted no effect concentrations (PNECs) were derived as well as two different probabilistic PNEC ranges. The ERA compares surface water PECs and MECs with aquatic PNECs for TMP. Based on all the risk characterization ratios (PEC÷PNEC as well as MEC÷PNEC) and risk graphs, there is no significant risk to surface waters. The topic of pharmaceuticals in the environment (PIE) has gained a lot of attention in environmental discussions. Active pharmaceutical ingredients (APIs) are suspected of causing unintended adverse effects in environmental compartments, based on their intended property of high biological activity. For human APIs, which are excreted into wastewater, this primarily means concern for the sewage treatment plants (STPs) or surface waters. Such concerns have been fuelled by ubiquitous detections of APIs in STP effluents and surface waters since the 1970s, in concentrations in the ng/L to µg/L range. It is mostly older APIs that are regularly monitored and detected. While for the registration of new APIs an environmental risk assessment (ERA) has been requested in the European Union since the early 1990s [1], this was not the case beforehand, meaning that exactly for these older APIs there often is a lack of environmental fate and toxicity data.The old antibiotic trimethoprim (TMP) was first put on the market by F. Hoffmann-La Roche Ltd (Roche) in the 1960s in combination with sulfamethoxazole (SMX) under the brand name of Bactrim®. TMP has been regularly detected in the environment. Like all antibiotics, TMP has come under suspicion for the potential of selecting for, maintaining or increasing antibiotic resistance in environmental bacteria. The first in-depth aquatic ERA for TMP is presented here. It is based on both predicted and measured environmental concentrations (PECs and MECs, respectively) and on published and new chronic ecotoxicity data. Some of the latter were specifically commissioned in order to produce a solid effects assessment for TMP. Acute ecotoxicity data are integrated as well. In view of sufficient data available, this ERA was supplemented with a probabilistic comparison of percent-ranked MECs and chronic effects species sensitivity distributions in addition to the standard deterministic procedures.The diaminopyrimidine TMP (2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine; CAS Number 738-70-5) [2] is a bacteriostatic API that interferes with the bacterial dihydrofolate reductase enzyme, inhibiting the synthesis of tetrahydrofolic acid [2]. Bacteria are unable to take up folic acid from the environment, including their infection host in case of pathogenic species, and are dependent on their own de novo synthesis. Inhibition of dihydrofolate reductase starves the bacteria of nucleotides necessary for DNA replication. TMP is generally used in combination with sulfonamide antibiotics (mainly SMX), which interfere with another step of bacterial folate synthesis pathway; in combination, TMP and SMX act synergistically. TMP is rapidly absorbed after oral administration and widely distributed around the body to tissues and fluids. Serum therapeutic concentrations range from 1.5–2.5 mg/L up to 9 mg/L [3]. Metabolic reactions include oxidation of the methylene group to a hydroxymethyl group, N-oxidation, O-de-methylation and hydroxylation in phase-1 metabolism as well as conjugation with glucuronic acid or sulfate in phase 2. Around 10%–20% of a dose is metabolized. The metabolites are excreted in the urine as conjugates, but the greater part of the dose is excreted as unchanged drug. Urinary excretion is pH-dependent and is increased in acidic urine. About 40%–75% of a dose is excreted in 24 h, up to 60% being in the form of unchanged drug, with about 4% each as the 3'-hydroxymethyl and 4'-hydroxymethyl metabolites and 2% as the N1-oxide. Less than 4% is eliminated in the faeces. The plasma half-life ranges from 8 to 17 h with an average of 11 h [2,3]. The World Health Organization defined daily dose of TMP is 400 mg [4]. This value will later be used for the first PEC derivation.TMP is not particularly toxic to humans and mammals by oral administration in the short or longer term, however, it can be irritant and sensitizing [2]. It was mutagenic in a bacterial test system and at high doses it can be teratogenic and embryotoxic through its mode of action, folate antagonism [2], as folic acid is required for normal development. However, due to these mutagenic and reprotoxic properties, TMP is classified by default as T for toxic for a persistence, bioaccumulation and toxicity (PBT) assessment.The basic data for the environmental fate and effects of TMP are listed in tables in the Appendix of this publication, starting on Page 136, for better readability of the text. A discussion of the most important, selected values from these tables is presented in the following sections.Physico-chemical data for TMP are listed in the Appendix in Table A1, Page 136 ff [2,3,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. TMP is an organic base with a reasonably high water solubility of ~300 mg/L and a first base dissociation constant pKa around the neutral pH point [2]. There are no hydrolysable bonds. The melting point is around 200 °C [2], vapor pressure is low at ~1.32 × 10–6 Pa [5], hence the Henry’s Law Constant is low as well and the substance will not volatilize from water. In addition, with a first base pKa around 7 [2,3,9], TMP is at least partly dissociated in most environmental waters, i.e., it will be more hydrophilic and will volatilize even less. With an n-octanol/water partition coefficient logKow between 0.64 and 1.115 [2,12] TMP is not particularly lipophilic. Therefore, neither strong adsorption to organic substrates nor bioaccumulation would be expected. In confirmation, moderate to low adsorption constants to organic carbon (OC), activated sludge (AS) and soil have been published [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27], although Lin & Gan [16] noted strong adsorption in one soil beside moderate adsorption in others. Specifically, sorption to AS in sewage treatment plants (STPs) has been independently described as ‘negligible’ [25,26,27]. However, sorption should still be kept in mind as Trapp et al. [28] have shown using physicochemical activity-based environmental fate modeling that as a weak base, TMP is non-dissociated and thus more prone to sorption or bioaccumulation at a higher environmental pH of 9 than at pH 6 where TMP is mostly dissociated. In general, based on this low to moderate sorption, most TMP is expected to remain predominantly in the aqueous phase, meaning that little is removed to sludge in STPs, the exposure of soil by landspreading of digested surplus sludge is low, mobility in soils is high and little will partition from surface waters to sediment. The available literature data for TMP for biodegradation, removal in STPs, environmental fate and derived half-lives as well as bioaccumulation are collated in Table A2, Table A3, Table A4, Table A5 at the end (Page 138 ff).TMP is recalcitrant to biodegradation in standard ready and inherent tests [18,21,29,31] and also in a standard STP model test at low concentration [32]. This first impression may be misleading, however, as on one hand, significant cometabolic degradation was observed in a closed bottle test with sodium acetate in the toxicity control [29]. Moreover, good removal (>50%) was seen in those tests performed with aerobic AS with a long sludge retention time (SRT), i.e., a high sludge age [21,25,33,34,35]. Indeed, as consistently shown by Göbel et al. [19,26], Perez et al. [34] and Schröder et al. [35], who compared the removal in different steps of STPs, low removal was found in inocula with a short SRT, e.g., from primary sludge or young AS, but high removal was noted for inocula with a high SRT, i.e., nitrifying AS and sand filters. Similarly, rapid primary degradation of TMP was also shown by Löffler & Ternes [36] for natural sediments and by Schmidt et al. [37] during river bank filtration. In addition, Bundschuh et al. [30] determined that TMP is even rapidly degraded in a ready-type system, exposing fallen leaves in natural water to low concentrations of TMP, where they determined ~80% degradation in 7 days. A similar difference may also exist for anaerobic degradation as Gartiser et al. [21] recorded no significant methane production in a standard ISO 11734 anaerobic degradation test, while other investigations with surplus sludge from an anaerobic digestor [19], with manure and anaerobic bacteria [38] or in pig slurry [39] found high and rapid removal. In soil [13,40] and seawater [41], however, biodegradation seems to be slow with correspondingly long half-lives of around or more than 100 days. The above differences, mainly relating to SRT respectively nitrifying conditions, are probably responsible for the inordinately high range of removal noted for many different STPs in Europe, North America and the Far East. These include negative removal, which may signify cleavage of glucuronide or sulfate conjugates [75], and range up to nearly 100% [19,23,48,52,53,54,55,56,57,58,59,60,61,62,63]. Some of the negative removal rates, like the extreme value of −550% described by Lindberg et al. [45] for one STP in Sweden, are highly improbable, seeing as 60%–80% of ingested TMP is excreted as the parent and only 20%–40% as metabolites and conjugates [2,3]. Therefore, conjugate cleavage of 550% is quite impossible, but either sampling, synchronization or analytical problems are suspected. In conclusion, for TMP in STPs there is but minor removal during inadequate primary and secondary treatment [19,34], but nitrifying sludge is able to biodegrade TMP [25,34], suggesting an important role for both aerobic conditions [76] and in particular for long SRTs in secondary treatment [55,62]. Similarly, anaerobic degradation may range from low [21] to rather high [19,38,39]. For later PEC refinement, the recorded removal rates for full-scale working STPs were collated from 26 references [19,23,25,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64] listed in Table A3 (with the exception of the above extreme value from [45]). The 107 remaining recorded removal rates, representing at least 63 STPs, ranged from −128% [48] up to >99% [63]. The average removal is 25.0% and the median removal 30.0% (Figure 1). These removal values are in agreement with the data by Fick et al. [60] who determined TMP to fall into an average removal range between 10% and 49% in their Swedish investigation in the year 2010. It is to be noted that all these empirically determined degradation rates depend on several circumstances, from time-corrected sampling of influents and effluents, to types and functional quality of the sewage works to the analytics themselves. While for the latter in most publications the analytical methods and recovery rates and ranges are described in detail, exact measured values are presented all the same, mostly without explicitly pointing out the uncertainty contained. This was recently shown by a group from Cleveland, Ohio sewage treatment works [77] who used two different contract labs to evaluate both intralaboratory and interlaboratory variability. They found discrepancies in TMP quantification of 40% in the same influent and of 168%–180% for the same effluents. This finding calls for caution in regarding all the measured concentrations of TMP (and other substances) as representing a true value; they could in fact be lower or higher.Distribution of 107 published degradation/removal rates of Trimethoprim (TMP) in 63 sewage treatment plants (STPs) worldwide.Hydrolysis [65,66] and aquatic photodegradation in fresh and seawater [68,71] are not significant for TMP, except where both hydrogen peroxide and scavengers are present at the same time as UV irradiation [66,70,71,72]. Michael et al. [66] and Wu et al. [72] have recently confirmed that TMP degrades only slowly under natural solar illumination, approximately 10% in 500 min in demineralized water [66], respectively up to ~2% in 72 h in natural water [72]. However, it degrades much faster by hydrolysis in the aluminum-foil-wrapped dark control (up to ~15% in 72 h at pH 4 and 7), due to the temperature increase in the dark control [72]. While dissolved organic matter, which can act as a scavenger, is common in natural waters, peroxides may be less so; moreover, in most instances the superficial temperature will not rise massively, due to water movement. Hence, only slow photodegradation is predicted for TMP in temperate zones and it is not expected to play a major role in the environmental fate of TMP.Total environmental half-lives (t½) of TMP have been derived for some compartments. In an experimental microcosm, Lam et al. [65] analytically determined a t½ of 5.7 ± 0.1 days; this short time may reflect the earlier findings of Bundschuh et al. [30] in their miniature fallen-leaf/natural-water system. Extrapolated, i.e., estimated environmental half-lives for TMP are available for freshwater with >42 days [67] and 20–100 days [73]. Boxall et al. [67] also estimated a freshwater sediment t½ of >60–100 days, while Hektoen et al. [74] predicted a marine sediment t½ of 75–100 days. Once more there seems to be a wide range of half-lives for TMP, from the measured 5.7 ± 0.1 days [65] up to an estimated 100 days. This may again reflect nitrifying vs. non-nitrifying conditions, but mainly it does attest to a high uncertainty. However, the half-lives are important as the EU Technical Guidance Document for Risk Assessment (TGD) [79] classifies substances for persistence in function of their environmental half-life. Thereby, compounds are classified persistent (P) in freshwater if the aquatic half-life is >40 days and very persistent (vP) if it is >60 days; both P and vP in seawater if the marine half-life is >60 days; P if the freshwater sediment half-life is >120 days and vP if it is >180 days; P and vP if the freshwater or marine sediment half-life is >180 days [79]. Based on one experimental half-life of 5.7 days in a microcosm [65], TMP is not P, but it may well be P or even vP in freshwater if the extra­polated half-lives of >42 days [67] respectively 20–100 days [73] are correct.Data on bioaccumulation for TMP are scarce or indirect. The lipophilicity data for TMP range from a logKow of 0.64 to 1.15 [2,6,11,12], which argues against bioaccumulation. In an early experimental study, Bergsjø & Søgnen [9] exposed trout to a high TMP concentration of 75 mg/L in fresh and saltwater, but only for a short time of 84 h, which might not suffice for rigorous bioaccumulation assessment. They found a maximum bioconcentration factor (BCF; concentration in fish ÷ concentration in medium) of ~0.32 in marine fish liver and ~0.16 in freshwater fish liver, but from some of the graphs given the internal concentration seems to be still on the rise. However, Bergsjø et al. had dosed rainbow trout orally with radio-labeled TMP earlier [83] at a dose of roughly 0.02 mg TMP/g fish at 7 or 15 °C. Following the radio-label by autoradiography they noted a slow (maximum disintegrations per minute, DPM, around 48 h at 7 °C) to more rapid (max. DPM around 12–24 h at 15 °C) uptake, followed by a decrease that was rapid in muscle at 15 °C but slower in liver at 7 °C. Still, the maximum body concentration from a single dose reached at 48 h and declining thereafter does not speak for significant bioaccumulation. More recently, Fang et al. [81] dosed Japanese bass (Lateolabrax japonicus) once daily with 125 mg sulfamethazine and 25 mg TMP over five days. They derived the minimum holding period, unstated in the abstract but presumably until the analytes were below the limit of detection, from analysis in muscle, blood, liver and kidney as 26 days at 22 °C water temperature and 30 days at 16 °C. While no further information is given in the available abstract, a minimum 90% depuration time of 30 days at 16 °C does not seem inordinately long, suggesting reasonably rapid depuration and thereby relatively low bioaccumulation. Using multi-compartment, physico-chemical-activity-based modeling, Trapp et al. [28] showed that, contrary to expectation, TMP accumulates less in biota at pH 9 than at pH 6, due to increased relative partitioning to the sediment at pH 9 where TMP is mostly non-ionized. Conversely, according to Trapp and colleagues, at pH 6 TMP partitions more to biota than to sediment, but based on their data the worst-case water-biota BCF would still be <100 (approximate value from Figure 1 in Trapp et al.) [28]. This is indirectly supported by the reports of Ramirez et al. [82], who sampled common local fish from five wastewater-influenced streams in the eastern and southern USA as well as in one pristine control river, and Fick et al. [60], who did a comparable sampling in Swedish rivers and associated fish. Both groups never detected TMP in any of their fish samples. Fick and colleagues analyzed both surface water and biota samples at the same places; based on their range of surface water TMP concentrations, from 6.8 to 210 ng/L with no non-detects, and the fish concentration consistently below their LOQ of 0.1 µg/kg [60], the TMP BCF would be predicted to be <16 in the worst case. The regulatory limit for aquatic bioaccumulation is a BCF of 2000 for bioaccumulative (B) or of 5000 for very bioaccumulative (vB) according to the EU TGD [79]. Based on this mainly circumstantial evidence, TMP does not qualify as B. For uptake and bioaccumulation from spiked soil to plants over full growth duration for lettuce (103 days) and carrots (152 days), Boxall et al. [40] determined soil-based uptake factors of 0.06 for lettuce and 0.08 for carrots and soil-porewater-based uptake factors of 0.68 respectively 0.86 over the whole duration. Last, in a hydroponic exposure of two different sorts of cabbage plants, with 232.5 µg TMP/L in the nutrient solution over 51 days, Herklotz et al. [83] found a maximum wet-weight BCF of 0.3074. Even though for soil uptake in plants lower limits may apply for a B classification than for animals in water [85], with a BCF clearly <1 on chronic exposure there is no suspicion of bioaccumulation. Recently, Sabourin et al. [84] compared concentrations of pharmaceuticals and other substances in vegetables (sweet maize, carrot, tomato, potato) grown on soil fertilized with dried municipal sewage sludge or on non-amended control soil. Results for TMP are equivocal, as TMP was detected at comparable levels in tomatoes from one (0.432 ng TMP/g dry weight) of three amended soils and also from control soil (0.387 ng TMP/g dry weight), but was not detected in any other vegetable. The authors state that, by their own criterion that detections must be made in all three amended soils per vegetable, the results for all analytes including TMP are not significant. Hence, overall, there is no evidence for bioaccumulation of TMP. The EMA 2006 Guideline for ERA of human pharmaceuticals [86] derives the initial, crude surface water PEC for APIs with a simple formula, multiplying the maximum daily dose with a default penetration factor in the population of 0.01 and dividing by a default 200 L sewage per person and day and a default surface water dilution factor of 10, without factoring in any human metabolism or STP removal. For TMP, with a daily dose of 400 mg [4], this results in a crude surface water PEC of 2 µg/L. However, this initial PEC may be refined through incorporating actual use, either through published epidemiological data resulting in a lower penetration factor or through actual use figures. IMS Health is a company that collates sales figures for APIs, hence total TMP sales (i.e., pharmacies plus hospitals wherever available) were retrieved from the IMS Health database [87] for the years 1995–2003 for the following European countries: Austria, Belgium, France, Germany, Greece, Italy, The Netherlands, Portugal, Spain, Sweden, Switzerland and the United Kingdom, making up in 2003 a total of 370 million inhabitants [88]. Two results appear from this collation, first, the overall use declined from 55,578 kg in 1995 to 43,079 kg in 2003; such a decline was also noted by ter Laak et al. in a 2010 RIWA report on temporal and spatial trends of pharmaceuticals in the River Rhine [89] based on MECs in Dutch waters. Second, the average daily use per inhabitant for all these countries was 0.3955 mg TMP, with a range of 0.1937 mg for Greece to 0.5005 mg for the UK. For the last year in the series, the UK still has the highest per capita use per day of 0.5056 mg TMP.Inserting the highest of the above daily use figures for the UK in 2003 into the PEC equation results in a first refined surface water PEC for the UK of 0.253 µg TMP/L. For the European 1995–2003 average use figure the first refined surface water PEC is 0.198 µg/L. This PEC may be further refined by excretion rate of the parent API including glucuronide or sulfate conjugates, which will be hydrolyzed back to the API in STPs [75]. Based on a maximum 20% of ingested TMP being Phase-1-metabolized [3], 80% excretion as the parent or its conjugates will be assumed as a worst case; 60% excretion will be assumed as a best case. This results in second refined surface water PECs of 0.202 µg/L for the UK in 2003, respectively of 0.119 µg/L for all European countries for 1995–2003.A third PEC refinement may be made by incorporating STP removal of TMP. As derived above, based on a minimum of 107 measured removal rates, the average removal of TMP is 25.0% and the median (best case) removal is 30.0% (Figure 1). Using the lower, average removal for PEC refinement results in third refined surface water PEC of 0.152 µg/L for the UK in 2003, respectively of 0.089 µg/L for all European countries for 1995–2003. The serial PEC refinements are shown in Table 1.Surface Water predicted environmental concentrations (PECs) and their Refinement for TMP in Europe.Based on the available use, metabolism and STP removal data, a refined surface water PEC range of 0.089–0.152 µg/L seems realistic for Western Europe. This range can be compared with actual surface water MEC data.TMP has been measured in European surface waters at least since the mid-1990s and today very many MECs can be located. In total, data representing at least 1899 single MECs have been collated for this ERA; ‘at least’, because often the number of single analyses is not given and in such cases just one value was assumed. Most of the publicly available MECs (at least 754) are from Germany [90,91,92,93,94,95,96,97,98,99,100,101,102,103]. Other MECs are from France [47,104], The Netherlands [105,106], Spain [5,48,107,108,109,110], Sweden [44,60,111], Switzerland [112,113,114,115,116], Croatia [49] and the United Kingdom [50,117,118,119,120]. Special thanks to F Bonvin, T Kohn, M Lehmann and M Schärer (see Acknowledgements) for supplying single MEC data that have only been published as overviews.The values were collated into one single distribution as described by Straub [121,122] and Metcalfe et al. [123], detailed in the Experimental Section further below. Figure 2 is based on at least 1899 back-distributed single measurements that were percent-ranked. Datapoints (blue crosses) were inserted at those concentrations where at least one MEC is either explicitly reported or can be allocated with certainty. In view of many MECs being reported as below the limit of detection or quantitation, there are already a cumulative 8 percentiles of all MECs at 0.001 µg/L, corresponding to an estimated 150 MECs. The 50th and 95th percentiles (MEC50 respectively MEC95) are indicated in Figure 2 by drop lines; the MEC50 is ~0.012 µg/L, the MEC95 ~0.129 µg/L. For comparison, the highest single surface water MEC located in the literature, from the USA [124], is 0.710 µg/L (pink cross in Figure 2), very close to the highest European MEC of 0.690 µg/L [109].Compiled European surface water measured environmental concentrations (MECs) for TMP. It is recognized that this procedure does not deliver exact results but, on the other hand, it is the only possibility of compiling different MEC data into one single distribution and getting a consolidated overview comprising all data, instead of many smaller distributions presented in different formats. Moreover, the more data there are in this distribution, the better will it reflect the actual environmental distribution, in particular at the 95th percentile level, where indeed most references and their respective MEC values are fully integrated already.The MEC95 and MEC50 values lend themselves for comparison with the refined PEC (and alter also the PNEC) values. Recalling the refined PEC range of 0.089–0.152 µg/L, it would seem that the higher PEC is close to the MEC95 but that the best-case PEC is a factor of ~7.5 higher than the MEC50. Both PECs, however, could actually be too high, possibly for the following reasons. The PECs assume that the whole amount sold is also used and excreted. Patient noncompliance seems to be relatively common, however [125,126,127]. Particularly with antibiotics, some patients stop taking the medicines when they start to feel better, without finishing the whole treatment course. As long as these discarded APIs are not drained into the wastewater, this will reduce the surface water PEC. The PECs assume that the average and median removal rates in STPs derived here are representative for the whole of Europe. Possibly more STPs have well nitrifying AS that results in higher removal and thereby in a lower surface water PEC.The PECs assume a TGD [79] default surface water dilution factor of 10. If the average dilution factor in Europe is higher this would result in a lower PEC.The PECs do not factor in environmental degradation beyond the STPs. However, TMP can be degraded by both aerobic and anaerobic biological mechanisms [30,65] and to some degree by physico-chemical transformation, also in surface waters [70,72]. Both would reduce the PEC.For STPs to perform their intended function, the AS micro-organisms must not be affected by the micropollutants in the influent. Hence an appraisal of bacterial toxicity of TMP is necessary, the basic data are collated in Table A6 [15,18,21,29,30,64,128,129,130,131,132,133,134,135].TMP is not highly toxic to AS bacteria in standard aerobic and anaerobic tests [18,21,128], with EC50 values ranging from 17.8 to >100 mg/L. Also, in Lumistox tests with the light-emitting marine bacterium Vibrio fischeri TMP is not highly toxic, however, toxicity increases with prolonged exposure [129,130,131]. On even longer exposure of 14 days, TMP completely inhibited human nanobacteria at 3.9 mg/L [132]; this would be expected as the human therapeutical serum concentration is in the range of 1.5–9 mg/L [3]. In a closed bottle ready biodegradation test, no inhibition was noted in the standard toxicity control at 3.25 mg/L TMP-naphthoate, while a significant reduction of colony-forming was noted at 4.6 µg/L TMP-naphthoate [29]. This possible discrepancy is not discussed in the paper, however, the toxicity control measures overall inhibition while the colony-forming units relate to cultivable bacterial species. Hence the observations by Alexy et al. [29] may signify that TMP exerts adverse effects only on certain bacterial species, which may be masked or compensated by the remaining, non-affected species in AS. This interpretation may be supported by the findings of a statistical EC10 in AS of 0.435 mg/L (in contrast to the NOEC observed at 100 mg/L in the same GLP test) [129], by NOECs to soil bacteria of 0.02 mg/L [133] and to nitrifying bacteria at 0.05 mg/L in one test [57], while another nitrification inhibition test under GLP showed no effect at 96 mg/L [134]. Moreover, when tested in combination with four other antibiotics (sulfamethoxazole, erythromycin, roxithromycin, clarithromycin; all at the same concentration), TMP reduced the growth of fungi on fallen alder leaves in natural water at 40 µg/L, while at 0.4 µg/L no inhibition was noted [30]. Altogether, the above findings are rather difficult to interpret. It seems that TMP can inhibit certain microbial species at concentrations of 4.6 µg/L (LOEC) [29] while other bacteria are not adversely affected at concentrations over 100 mg/L [21]. In an STP, the latter may take over some of the ecological functions of the affected species, as suggested by the AS respiration inhibition and nitrification inhibitions tests (both relying on overall functional endpoints), and thereby compensate functionally for the inhibited micro-organisms. This is supported by the observation that biodegradation (not only of TMP itself but in general) and nitrification in working STPs is not significantly inhibited by the influent concentrations of TMP and many other substances, as shown by overall functional parameters. Therefore, TMP may cause inhibition of specific bacteria and potentially shifts in species compositions at concentrations between 4.6 and 0.4 µg/L, but at current uses there is no evidence of adverse effects on the functions of STPs. This may also be related to a certain tolerance (or resistance) of STP bacterial communities toward many different micropollutants including TMP. Similarly, Liu et al. [135] found in an experiment with spiked natural soil that TMP decreases the total soil respiration in comparison with a blank control during the first 4 days of exposure from 20 mg TMP/kg soil (dry weight), whereas from day 5 to the end of the assay at day 21 no inhibition was noted, but either no change or increased respiration at all concentrations up to the highest of 300 mg TMP/kg soil (dry weight). This was interpreted as an initial overall inhibition followed by an adaptation of the collective of aerobic micro-organisms. In this work, Liu et al. [135] note a soil dissipation half-time (DT50) of 2–5 days for TMP and in a later publication the same group [15] gives a DT50 in aerobic soil of 4 days, which suggests that after about half of the spiked TMP is removed (by biodegradation or bound residue formation) the bacterial community adapts to the substance. In view of the very general endpoint of total respiration a persistent inhibition of certain species is still conceivable, but the ongoing dissipation of TMP in the soil through mainly biodegradation [15] suggests that in such a case at least the biodegradation functionality can be compensated by the remaining bacteria.For the appraisal of surface water ecotoxicity there are two extensive datasets for TMP, one acute (Table A7) and one chronic (Table A8). The acute dataset fully rests on published and some older Roche-internal tests (which are already used for the Roche safety data sheets) while for the chronic dataset some new tests performed specifically for this ERA are reported for the first time.Acute data exist for cyanobacteria, algae, hydrozoans, rotifers, crustaceans, molluscs, flowering plants and fish. The acute EC50 or LC50 data range from 5.1 mg/L for a marine alga (where TMP would be mostly non-dissociated in view of the basic pH of seawater) [137] to 296 mg/L for daphnids [149] for those tests where a concise value is given (i.e., not a ‘>highest tested concentration’). For fish in particular, all highest tested concentrations did not result in an LC50, which would be expected in view of the fact that fish are not intended to be target organisms for antibiotics. The one apparent exception to this is an LC50 value of 3 mg/L cited by Kolpin et al. [124], which proves to be a miscitation: The original paper by Bergsjø et al. [80], which is actually referred to by Kolpin et al. [124], gives a single oral dose of approximately 0.02 mg radio-labelled TMP per gram of fish, but not a concentration. Moreover, none of the fish used is reported by Bergsjø et al. [80] to have died of TMP. Hence, this erroneous citation is not used for toxicity assessment.Antibiotics are used to inhibit bacterial infections, which is why Holten Lützhøft et al. [138] noted that ‘to perform a proper environmental risk assessment of antibacterial agents, it would be necessary to include a cyanobacteria as test organism in the test battery’; this request has been adopted in the EMA guideline for ERA of human APIs [86]. But at least in the case of TMP the cyanobacterian species tested are neither the most sensitive nor is the range of cyanobacterian EC50s limited to low concentrations; on the contrary, the EC50s range from 11 to >200 mg/L [136,138]. This may suggest that for some reason TMP is not as highly toxic to cyanobacteria than to human nanobacteria [132]; possibly, photosynthetic cyanobacteria are not as dependent on their own de novo folate biosynthesis as human pathogenic bacteria. By extension, the comparatively high threshold for ecotoxicological effects over the broad array of groups and species tested confirms the statement by Blaise et al. [129] that TMP is relatively nontoxic.The new chronic tests under GLP quality assurance commissioned with the aquatic flowering plant Lemna minor [143] and the zebrafish Danio rerio [153] bring the total number of systematic groups tested chronically to 8 (including the three standard groups algae, daphnids and fish) and the number of species to 17. Once more, the marine alga that was already the most sensitive on an acute scale has the lowest EC50 [137], which (again as expected) suggests that TMP would be more toxic while mostly non-dissociated in view of the basic pH of seawater. Also on a chronic level the cyanobacterians have a wide range of NOECs, from 3.1 to ≥200 mg/L [136]. A FETAX larval test with the toad Xenopus laevis is included among the chronic data despite the short duration, as this test takes place during a very sensitive phase of development and has therefore been accepted as chronic by the recent EU Technical Guidance Document for Deriving Environmental Quality Standards (EQS) in the scope of the EU Water Framework Directive [155]. Together with the new zebrafish early life stage NOEC at the highest tested concentration of 100 mg/L [153], the fish and amphibian data once more suggest that vertebrates are not particularly sensitive to TMP and that generally speaking, also on a chronic level TMP is relatively nontoxic [129] as well.This copious compilation of acute and chronic ecotoxicity data allows for both a solid deterministic and a well-founded probabilistic PNEC or HC5 (hazardous concentration for 5% of species tested) according to the requirements of the TGD [79]. Where more than one result was available for the same species, the geometrical average was calculated and this will be used for PNEC derivation, in line with the EU EQS derivation guidance [155]. According to the TGD [79], when at least three endpoints are available for a minimum dataset of algae, daphnids and fish, the acute deterministic PNEC is the lowest EC50 or LC50 divided by an assessment factor (AF) of 1000; the chronic deterministic PNEC is the lowest NOEC or EC10 divided by an AF of 10.The lowest chronic value retrieved, a NOEC of ≥1 mg/L (the highest tested concentration) for the duckweed Lemna gibba [142] will not be used for deterministic or probabilistic PNEC derivation, however, because (a) basing a PNEC on a lower bound of a NOEC generates a high uncertainty in general, in particular (b) because from a ‘≥’ value no unambiguous deterministic or probabilistic PNEC can be derived, but again only a ‘≥’ value, and (c) because the closely related Lemna minor showed a clear NOEC of 53.5 mg/L in a GLP test [143] with measured exposure concentrations, about 50 times higher than the disputed value. In addition, other, very low, highest tested concentrations published without any biological effects noted whatsoever, like the above value in Brain et al. 2004 [142], were not used. This concerns the daphnid NOEC of 10 µg/L (highest tested concentration) published by Flaherty & Dodson 2005 [152], which included the endpoints survival, adult and neonate morphology, ephippium production, fecundity and offspring sex ratio. However, it was based on a duration of only 6 days, whereas the OECD guideline stipulates 21 days, and was therefore not used for derivation of PNECs. Also, in a recent test with the marine rotifer Brachionus koreanus, Rhee et al. [145] tested nominal concentrations of 10 and 100 µg/L TMP for 10 days and noted ‘gradual’ or ‘slight growth retardation’ [145] (pp. 109 and 116, respectively) at 100 µg/L. However, while a slight retardation in growth may be seen from their graph on p 115, Rhee and colleagues do not comment on the fact that TMP-exposed Brachionus seem to fully compensate their delayed reproduction by the end of the test on day 10, when the error bars of controls and the two tested concentrations overlap. As the test runs over ten days, as there is no significant adverse effect at the end of the test and as the authors did not test sufficiently high concentrations to unambiguously demonstrate such an effect, this endpoint will not be used here. Last, the biomarker data for the zebra mussel Dreissena polymorpha published by Binelli et al. [156] are not used for PNEC derivation, either, as the acute-based NOEC is based on ambiguous inhibition or mortality endpoints. The same holds for the biomarker endpoints in the rotifer paper by Rhee and colleagues [145]. For the time being there is no regulatory guidance on extrapolation from biomarker responses to organism- or population-relevant endpoints that may be used within the scope of an ERA. Rejecting them for the PNEC derivation is in line with the EU EQS guidance document [155] which states that ‘data from studies describing endpoints that do not include direct measurements of survival, development or reproduction but, rather, describe e.g., behavioral effects, anatomical differences between control and treatment groups, effects at the tissue or sub-cellular level, such as changes in enzyme induction or gene expression … generally … are unsuitable as the basis for EQS derivation’. Based on these provisions the deterministic acute-based aquatic PNEC for TMP is 5.1 µg/L, derived from the marine algal EC50 of 5.1 mg/L (Phaeodactylum tricornutum) [137], applying an AF of 1000. Also the deterministic chronic aquatic PNEC of 240 µg/L relies on the same algal species with a NOEC of 2.4 mg/L [137] and an AF of 10. The chronic-based PNEC is considered more relevant in view of reflecting long-term, continuous exposure. However, the fact that the most sensitive organism for both the acute and chronic endpoints is a marine alga, suggests that the high pH of seawater renders TMP more toxic due to a higher non-dissociated fraction, beside the algae-typical phenomenon of ion trapping [157]. The first probabilistic PNEC was derived as described in the EU TGD [79] by calculating the HC5 or 5th percentile of the chronic NOECs distribution and dividing this figure by an additional AF between 1 and 5. While there is some information given on the choice of this additional AF, no unequivocal, hard criteria exist. Hence for this ERA, a range for the chronic probabilistic PNEC will be given, from HC5/5 to HC5. The HC5 calculated by Excel is 2.93 mg/L, therefore the probabilistic PNEC range is 586–2,930 µg/L, with an average of 1,758 µg/L. The derivation of the PNECs is shown graphically in Figure 3.Acute and chronic ecotoxicity data, deterministic and probabilistic predicted no effect concentrations (PNECs) for TMP. In Figure 3, the acute aquatic EC50/LC50 values (red dots) and chronic aquatic NOEC values (filled dark green triangles) for TMP are shown, both percent-ranked and plotted on a log-probabilistic scale, with deterministic PNECs (open symbols; AF 1000 for acute data, AF 10 for chronic NOECs) and the light green TGD-calculated probabilistic PNEC band ranging from HC5÷5 (586 µg/L) to the HC5 (2,930 µg/L).In addition to the TGD probabilistic PNEC, the chronic NOECs were entered into the Webfram application (http://www.webfram.com) [158], which calculates a probabilistic HC5 based on a Bayesian algorithm [159]. Moreover, Webfram also computes goodness-of-fit values according to Kolmogorov-Smirnov, Cramer-Von Mises and Anderson-Darling algorithms; for all three tests the goodness-of-fit of the chronic TMP NOECs is accepted at a p value of 0.01. The probabilistic HC5 as determined by Webfram is 1,778 µg/L, with a 95% confidence interval between 334 and 4,832 µg/L (Figure 4). This HC5 compares nicely with the average of the EU probabilistic PNEC range, 1,758 µg/L. Webfram: chronic aquatic NOEC values and HC5. Chronic aquatic NOEC values (black dots) for TMP, percent-ranked and plotted by Webfram on a log-probabilistic scale; the 95% confidence interval is given as dashed lines. The Webfram-calculated probabilistic HC5 is 1,778 µg/L (middle green arrow) and the 95% confidence interval for the HC5 lies between 334 and 4,832 µg/L (left and right green arrows). With sufficient exposure and effects information, both transformed into PECs or MECs and PNECs, the formal ERA for the surface waters in Europe can now be addressed. The various PECs and compiled MECs are compared with the PNECs in Table 2.All risk characterization ratios without exception are <1, which means no significant risk overall. In particular, all risk characterization ratios that use any chronic-based, deterministic or probabilistic PNEC, which is taken to better reflect the permanent exposure to APIs, range from <0.01 to <0.00001. This firmly corroborates the first conclusion of no significant risk from TMP in surface waters in Europe and beyond.TMP risk assessment for European surface waters: PECs, MECs, PNECs and PEC/PNEC and MEC/PNEC risk characterization ratios respectively margins of safety.The whole information for this ERA including the margins of safety determined here can also be illustrated in one single risk graph for TMP (Figure 5).In the risk graph (Figure 5) the whole exposure and effects information for TMP is brought together. Acute aquatic EC50/LC50 data are shown as red dots, with the derivation of the deterministic PNEC of 5.1 µg/L (hollow red circle) by application of an assessment factor (AF) of 1000. Chronic aquatic NOEC values are shown as filled dark green triangles, with the derivation of the deterministic chronic PNEC of 240 µg/L (hollow green triangle) by application of an AF of 10. Further, the bright green probabilistic TGD chronic PNEC band ranging from 586 to 2,930 µg/L is depicted as well as the Webfram-calculated HC5 of 1,778 µg/L (green star in the band). European MECs are shown by dark blue crosses, with the European MEC95 at 0.129 µg/L; in addition, the highest MEC from the USA of 0.710 µg/L is shown as a pink cross. For illustration, selected margins of safety (MOS) are shown by horizontal arrows from the MEC95 to the corresponding PNECs. TMP risk graph for European surface waters. Beyond the MOSs between the MEC95 for Europe and selected PNECs, the risk graph also shows very clearly that, at least within the confines of the 1st and 99.95th percentiles, the MEC regression lines and the chronic NOECs regression line do not overlap. This illustrates graphically that there is no perceivable risk. In view of the fact that TMP use has been declining in Europe over the past 10–15 years, this conclusion is further strengthened.Synergistic or cocktail effects arising from the exposure to many micropollutants, comprising not only APIs but quite a diverse group of substances, are not included in this ERA. However, the data collated and presented here can serve for developing the TMP ERA further to include at least some other APIs, mainly sulfamethoxazole or other sulfonamides, with which TMP is often combined. But while some aspects of mixtures ERA are reasonably well understood [160], it is not easy to do a combined ERA for a few substances and it becomes practically impossible to do it for a large number. Hence, the present TMP ERA does not address mixture toxicity.The PECs on which this ERA relies only refer to human use of TMP. But TMP is also used on a large scale for veterinary purposes, again mostly in combination with sulfonamides. While total European quantitative data are not readily available, there are both veterinary and human use data published for Denmark over the past 15 years (DANMAP) [161]. Denmark is a European country with intense agricultural production, both of farm animals like pigs, cows or poultry as well as of fish in freshwater and marine aquaculture. Hence, extrapolating from the Danish data to the European level is judged to add a worst-case exposure from animal use of TMP. DANMAP 2012 data show that the total veterinary usage of TMP plus sulfonamides has been rising in the decade from 2001 to 2010, with a maximum of 14,950 kg in 2009 and the 2010 figure at 13,900 kg. Assuming also a 5:1 ratio of veterinary sulfonamides to TMP (as with human sulfamethoxazole and TMP in Bactrim) would translate to an annual veterinay use of 2,333 kg TMP for Denmark. Specifically for aquaculture, 3,060 kg antimicrobials were used in 2010, of which 66% or 2,020 kg sulfonamides plus TMP, which again corresponds to 337 kg TMP for direct aquatic usage and 1,996 kg TMP (2,333 minus 337) for mammals and poultry. On the human-use side, in 2010, 417 kg TMP and derivatives were used beside 252 kg of sulfonamides plus TMP, which latter amount translates to 42 kg TMP, hence a total of 459 kg TMP from human use. Assuming that the farm animal use will not get directly into surface waters and therefore adding only the aquaculture TMP, which is used directly in water, to the total human use, results in a supplement of 337 kg to the 459 kg, or 73% more. Hence, as a very crude worst-case extrapolation, 173% of the human-use PECs will be used as an overall surface water PEC from human plus veterinary use for ERA. Multiplying the various PECs in Table 2 (above) with a factor of 1.73 will increase the PEC/PNEC ratios, but even for the rather unrealistic EMA crude PEC of 2.0 µg/L, now increased to 3.46 µg/L, the acute-based risk characterization ratio is still <1; it is still lower by dimensions for the MECs (which at least for Denmark include that part of veterinary TMP that ends up in surface waters) as well as for chronic PNECs. Hence, even including a reasonable worst-case contribution from veterinary use to aquatic TMP PECs will not lead to a significant surface water risk.Another topic that is far beyond the scope of this ERA is antibiotic resistance development or maintenance due to the presence of antibiotics like TMP in STPs, surface waters or other environmental compartments [162,163]. While multi-antibiotic resistance has been shown for certain environmental compartments, notably sewage treatment, surface waters and soils [164,165], it is difficult to causally relate solely the presence of antibiotics (as opposed to the input of resistant bacteria from human patients or livestock) to the development or maintenance of such resistance. Indeed, some researchers found no maintenance, but on the contrary loss, of resistance in a laboratory sewage treatment plant despite the continued presence of antibiotics [166]. Moreover, so far there is no accepted regulatory methodology to assess this question. Hence, the question of potential resistance must remain for other investigations.According to the TGD ERA methodology [79], substances may be transferred from wastewater to the soil by way of land-spreading of surplus sewage sludge and from surface water to sediment by partitioning or to groundwater by infiltration. For all of these pathways there are insufficient data for a serious assessment of TMP, both on the environmental fate, distribution, partitioning or MEC side and in particular on the effects side in the receiving compartments. In view of this situation, no attempt will be made to characterize risk for these compartments by discussing the meager data or by read-across. Environmentally relevant peer-reviewed and non-reviewed (‘grey’) literature for TMP was searched for using dedicated search engines on the internet (ACS SciFinder, Google Scholar, chemical data collections like OECD Chemicals Portal http://www.echemportal.org/ or the European Union Chemical Substances Information System http://esis.jrc.ec.europa.eu/ as well as safety data sheet search engines such as https://www.eusdb.de), beside company-internal substance documentation and archives. The information was sighted, ordered and collated. Reference lists in the retrieved documents often allowed to supplement the literature dataset with further, mostly older publications and also online sources for MECs. All retrieved published STP removal rates, viz. effluent concentration as a percentage of influent concentration, worldwide were entered into a spreadsheet with removal rates ranging from −550% (the highest negative removal reported, which eventually was not used in the analysis, see argument on page 118) to 100% removal, with a value of 1 per documented removal rate into one column per each reference. All rates were horizontally added to a total per removal rate in per cent. Then, these values were multiplied by 100 and divided by the known total number of removal rates plus 1, in a percent-ranking procedure. Then, a plot was drawn using SigmaPlot 12 software (Scistat, Inc., San Jose, CA, USA) with the percentiles on a probabilistic ordinate and the removal rates on a linear abscissa. The average and median removal rates were determined by excel spreadsheet functions.All reported, discrete, single European surface water MECs were entered into one column per reference into a spreadsheet with a 1-ng/L-gradation ranging from ≤1 ng/L up to 1,000 ng/L. Then, the remaining (non-specified) MEC data were back-distributed per publication into the same column, based on total number of analyses, number below LOQ, between LOQ and median, between median and 90th percentile and between 90th percentile and the maximum value, to an average expected fraction or number of detections per ng/L-gradation for these ranges. For instance, if the LOQ in a particular publication was 5 ng/L and there were 7 MECs <LOQ, 7 was divided by 5 and the resulting fraction of 1.4 was entered into all 5 gradations from ≤1 to ≤5. Similarly, the number of MECs given between LOQ and the median, or between the median and the 75th or 90th percentile if indicated, were back-distributed. Then, both the precisely known numbers and the expected, back-distributed fractions of detections were horizontally added per ng/L-gradation, multiplied by 100 and divided by the known total number of analyses plus 1, in a percent-ranking procedure. This procedure resulted in a theoretical 690 values computed, 0.690 µg/L being the highest published surface water MEC in Europe, from a series that sampled only 100 m downstream of sewage works effluents in Madrid Region [109]. Out of these 690 values, however, only those values were kept for plotting and graphical regression where at least one actual analytical detection was certain. The plot was drawn using SigmaPlot software with a probabilistic ordinate and a logarithmic abscissa. The associated regression line then allows the graphical estimation of the overall 50th and 95th percentile MEC values (MEC50 respectively MEC95) based on at least 1899 single European MECs (Figure 2).Based on the chronic aquatic ecotoxicity dataset retrieved and critically analyzed, it was found that chronic fish studies were totally lacking and that a chronic study with the angiosperm Lemna gibba [142] was not adequate for risk assessment as the NOEC found was the highest tested concentration. To fill these data gaps, two additional chronic ecotoxicity studies with the duckweed Lemna minor following OECD test guideline 221 [143] and the zebrafish Danio rerio following OECD TG 210 were commissioned at reliable contract labs. Moreover, an activated sludge respiration inhibition test according to OECD TG 209 [128] and a dedicated activated sludge nitrification inhibition test following ISO TG 9509 [134] were also made. All newly commissioned tests were performed under GLP quality assurance, in the cases of the duckweed growth inhibition and the fish early life stage tests also with full analytical determination of the exposure concentrations by HPLC and statistical determinations of EC10 and EC50s as applicable, beside the NOECs. All additional tests were financed by Roche.Deterministic and probabilistic ERA methods were applied, following the EU TGD [79] for both acute- and chronic-based deterministic PNEC derivation as well as for TGD probabilistic PNEC band calculation. Additionally, the Webfram online tool (http://www.webfram.com) [158] was used for deriving a second probabilistic PNEC or HC5 based on a Bayesian algorithm.An extended ERA was developed for the aquatic compartment in Europe for the old antibiotic TMP from human use. This ERA relies on both crude and refined surface water PECs for TMP, the latter integrating actual use figures, human metabolism and documented STP removal rates; these PECs range from the crude EMA PEC of 2 µg/L to the third refined PEC of 0.089 µg/L. The PECs are complemented by a veritable host of at least 1899 single MECs from European countries that were compiled into one distribution, allowing the approximation of median (0.012 µg/L) and 95th percentile (0.129 µg/L) values for surface water concentrations, with the European maximum at 0.690 µg/L. On the environmental effects side, existing and newly developed ecotoxicity data were used to derive deterministic acute and chronic PNECs of 5.1 respectively 240 µg/L. The 16 chronic data from 8 different systematic groups were also used to derive a probabilistic PNEC range of 586–2,930 µg/L (EU TGD) or a probabilistic HC5 (PNEC) value of 1,778 µg/L (95% CI: 334–4,832 µg/L; Webfram). All acute (EC50/LC50) and chronic (NOEC/EC10) ecotoxicity data for cyanobacteria, green algae, marine algae, angiosperms, hydrozoans, rotifers, crustaceans, fish and amphibians are above 1 mg/L, supporting low ecotoxicity for TMP. All PEC/PNEC or MEC/PNEC risk characterization ratios are <1, all of the chronic-based risk ratios are <0.01 to <<0.01, showing no indication of risk due to the presence of TMP in surface waters. Moreover, while the available data suggest that TMP is persistent in surface waters, there is no evidence that TMP bioaccumulates and there are no experimental ecotoxicity data that suggest inordinately high toxicity; hence TMP is not a PBT substance, either.Based on this extended ERA, no significant risk is seen for TMP from human use in the aquatic compartment in Europe.Insufficient environmental fate and effects data were available for a reasonably well founded ERA for the compartments sediment and soil, but evidence is given that these two compartments in all probability are not central for TMP from human use. Also, there is a plausibility presentation that the additional veterinary use of TMP does not lead to significantly increased surface water levels and thus not to significant increased risk. The issues of mixture toxicity and antibiotic resistance could not be addressed based on available data and risk assessment procedures.My sincere appreciation to Florence Bonvin and Tamar Kohn, both of EPFL Lausanne (CH), for making available their original MEC data for Vidy Bay in Lake Geneva, to Markus Lehmann, Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg (LUBW), Karlsruhe (DE), for German MECs from Baden-Württemberg and to Michael Schärer, BAFU, Berne (CH) for the Swiss BAFU-Oberflächengewässer-Messdaten. These values placed the compiled MECs distribution on a much more solid footing and thereby strongly improved the MEC50 and MEC95 for Europe. Best thanks to Daniela Oggier of BMG Engineering, Schlieren (CH) and to Daniel Gilberg of ECT Oekotoxikologie, Flörsheim (DE) as well as their collaborators for excellent chronic ecotoxicity tests. Many thanks to Mathieu Guillaume (F.Hoffmann-La Roche, Basle, CH) for help with the IMS Health data and to Stefan Trapp (Technical University of Denmark, Kongens Lyngby, DK) for help with veterinary use data of TMP. F. Hoffmann-La Roche Group SHE (Basle, CH) is acknowledged for financing the new chronic ecotoxicity tests. Thanks to two anonymous peer reviewers for suggestions to improve this article.IMS Health is acknowledged for the use of API sales data; however, analysis of IMS Health data was arrived at independently by the author of the present paper on the basis of the data and other information and IMS Health is not responsible for any reliance by recipients of the data or any analysis thereof.The author is a full-time employee of the pharmaceuticals and diagnostics company F. Hoffmann-La Roche Ltd in Basle, Switzerland, where he works as the Environmental Risk Assessor for Roche. Roche first put on the market the antibiotic combination of trimethoprim and sulfamethoxazole under the trade name of Bactrim® in the late 1960s. Physico-Chemical Data for TMP. Biodegradability and elimination of TMP. Removal of TMP during sewage treatment.Environmental Fate of TMP.Bioaccumulation data for TMP.HRT = Hydraulic retention time; LOD = limit of detection; ND = not detected; NS = not significant; SRT = sludge retention time.Micro-organism and activated-sludge toxicity data.Acute ecotoxicity data for TMP.Note: In case of several values for the same species, the geometrical average was calculated [155]. Values in bold italics are the values used for PNEC derivation while the single value in brackets was not used for the PNEC, see text. HTC = Highest tested concentration.Chronic Ecotoxicity Data for TMP.Note: In case of several values for the same species, the geometrical average was calculated. Values in bold italics are the values used for PNEC derivation. HTC = Highest tested concentration. Endpoints/ values in brackets were not used. * = See text.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Resistance-Nodulation-Division (RND) efflux pumps are one of the most important determinants of multidrug resistance (MDR) in Gram-negative bacteria. With an ever increasing number of Gram-negative clinical isolates exhibiting MDR phenotypes as a result of the activity of RND pumps, it is clear that the design of novel effective clinical strategies against such pathogens must be grounded in a better understanding of these pumps, including their physiological roles. To this end, recent evidence suggests that RND pumps play an important role in the virulence of Gram-negative pathogens. In this review, we discuss the important role RND efflux pumps play in different facets of virulence including colonization, evasion of host defense mechanisms, and biofilm formation. These studies provide key insights that may ultimately be applied towards strategies used in the design of effective therapeutics against MDR Gram negative bacterial pathogens. Discovery of antibiotics in the early part of the 20th century is considered to be one of the major advances of modern medicine. Our ability to successfully treat bacterial infections has in part led to an unprecedented increase in human life expectancy [1]. However, even around the time antibiotics were first introduced, a number of studies reported resistance to these antibiotics [2]. Nonetheless, since the incidents of resistance were not very widespread and also because newer antibiotics were being discovered quite regularly, antibiotic resistance was not considered a significant problem at the time. This led to the notion that the era of infectious diseases was more or less over [3]. Unfortunately, as resistance has continued to spread around the world at a rapid pace, our search for newer antibiotics has become increasingly difficult and has almost come to a standstill in the last few decades. Bacteria utilize various strategies to gain resistance against antibiotics and these mechanisms can be acquired or are intrinsic in nature. Among intrinsic mechanisms of resistance, energy-dependent efflux of antibiotics is considered one of the most important mechanisms of resistance in Gram-negative bacterial pathogens [4,5]. Energy-dependent efflux of antibiotics by bacterial cells was first described by Stuart Levy’s group at Tuft’s University, when they showed that tetracycline-resistant strains of E. coli were able to pump out the tetracycline molecules thus reducing their effective concentration inside the cell [6]. Efflux of tetracycline may very well have been the underlying mechanism in one of the first reports of tetracycline resistance in the late 1940s [7]. In this classical paper, Demerec showed that tetracycline resistance in E. coli was a two-step process, wherein the first step was responsible for low level resistance while the second step conferred high-level resistance. This is consistent with recent findings where efflux has been shown to confer the first but low-level of resistance allowing for the subsequent accumulation of other resistance mechanisms that lead to a higher degree of resistance [8]. Efflux proteins identified to date have been classified into five different families: (1) the major facilitator superfamily (MFS); (2) the ATP-binding cassette (ABC) superfamily; (3) the small multidrug resistance (SMR) family; (4) the resistance-nodulation-division (RND) superfamily; and (5) the multidrug and toxic compound extrusion (MATE) family [9]. Of these five families, proteins belonging to the RND family play an important role in the intrinsic resistance of Gram-negative bacteria. Even though these pumps play a critical role in the development of multidrug resistant phenotypes, it is clear that the efflux of antibiotics is not their primary physiological function. An understanding of their natural functions is important in tackling the problem of antibiotic resistance mediated by these pumps, as it will aid in identification of environmental conditions that promote their expression. This knowledge can be used in clinical settings to foresee the emergence of multidrug resistant phenotypes which in turn can help in application of more effective therapeutic interventions. The objective of this review is to underscore the role of RND pumps in the virulence of Gram-negative bacteria with an emphasis on the likely interplay between resistance and virulence.RND pumps were first described by two different groups in E. coli [10] and Pseudomonas aeruginosa [11] independently in the early 1990s. These pumps function as a tripartite complex composed of the RND protein (the inner membrane component), membrane fusion protein (MFP: the periplasmic component), and the outer membrane protein (OMP: the outer membrane protein). These three proteins form a continuous channel across the Gram-negative cell envelope ensuring that the substrate molecule, captured from the outer leaflet of the inner membrane bilayer, is effluxed directly across the periplasm and the outer membrane (which is characterized by low permeability itself) into the external medium with the aid of the proton-gradient as an energy source. In the recent past, crystal structures of the representatives of all three components of the RND complex have been solved [12,13,14,15], providing invaluable insights regarding the mechanism of assembly and action of these pumps. For more information on the mechanism of action of these proteins and their role in multidrug resistance, readers are advised to refer to several excellent reviews published recently [5,16,17]. During the initial years after the discovery of RND pumps, it was widely believed that they evolved as a result of antibiotic selection pressure in the bacterial environment. However, it was later shown that RND pumps are part of an ancient family of proteins, homologs of which are found in all three domains of life [18]. This finding led to the belief that since these pumps are omnipresent they must be involved in some important physiological roles. In addition, a single bacterial species can contain multiple RND pumps with overlapping specificities for different antibiotics. For example P. aeruginosa contains 12 different RND pumps that share a number of antibiotic substrates. If the primary function of these pumps is to efflux antibiotics, then the redundancy in substrate specificity among different pumps within the same species cannot be easily explained. In addition, antibiotics are not the only known substrates of RND pumps and they are known to efflux a wide range of non-antibiotic compounds including dyes, detergents, disinfectants, fatty acids, etc., further indicating that efflux of antibiotics is not necessarily their primary function. The regulation of expression of RND pumps is often mediated by an intricate balance between local regulators (generally repressors encoded upstream of the operon) and global regulators. It is therefore not surprising that the expression of these pumps is very tightly regulated. Overexpression of RND pumps may result from a variety of mechanisms. One mechanism is interaction of selected molecules with the local repressor, thus resulting in derepression of the operon. For example, overexpression of the CmeABC pump of Campylobacter jejuni occurs as a result of interaction of its repressor CmeR with salicylate resulting in its inactivation [19]. A second possible mechanism leading to overexpression is mutations in the repressor encoding genes as seen for the overexpression of the MexAB-OprM pump of P. aeruginosa or the AcrAB pump of E. coli resulting from mutations in their repressor encoding genes mexR [20] or acrR [21], respectively. Thirdly, activation of expression by means of a global regulator can also lead to pump overexpression. This has been observed with the E. coli AcrAB pump which is induced by bile salts and fatty acids as a result of their interaction with the global regulator Rob [22], which is one of the several global activators controlling the expression of this pump.As a result of their broad substrate specificity, exposure of cells to one substrate that causes overexpression of these pumps can result in cross-resistance to multiple antibiotics. For example, exposure of P. aeruginosa cells lacking MexAB-OprM and MexCD-OprJ to triclosan results in isolation of mutants that overexpress the MexJK pump which is also capable of effluxing antibiotics like ciprofloxacin and tetracycline [23]. Various environmental stress conditions, mediated by global regulatory mechanisms, are also known to cause the overexpression of RND pumps and thus contribute to the MDR phenotype. For example ethanol has been shown to cause overexpression of the AdeABC pump of Acinetobacter baumannii [24] and the AcrAB pump of E. coli [25], incubation temperature has been shown to impact the expression of the EmhABC pump of Pseudomonas fluorescens [26] and the AcrAB pump of Salmonella Typhimurium [27], and osmolarity has been shown to impact the expression of the AcrAB pump of E. coli [28]. The extremely broad substrate specificity of RND efflux pumps is also a major hindrance in the discovery and design of new antibiotics, as studies have shown that a majority of promising lead compounds with antibacterial activity can easily be missed in the initial screening process due to their activity [29,30]. In spite of significant advances over the last few years towards a better understanding of the structure and mechanism of the action of RND pumps, the answer to one question remains elusive: what role do they play in bacterial physiology? Studying natural functions of RND pumps poses multiple challenges. For example, broad substrate specificity of these pumps means that not all molecules pumped out are necessarily their ‘natural’ substrate. In addition, although much attention has been paid to their overexpression in response to antibiotics, studies demonstrating their overexpression in the absence of antibiotic selective pressure remain few and far between. For a bacterial pathogen to be able to successfully establish infection in the host, it has to be capable of surviving the host defense mechanisms. Various strategies employed by these pathogens to do so contribute to their virulence. These strategies include overcoming the physical barrier, surviving host antimicrobial components, production of toxins to damage host cells, etc. There is now accumulating data that shows that overexpression of efflux pumps in Gram-negative pathogens can occur concomitantly with the process of infection, leading to the question: Is there a correlation between the overexpression of RND pumps and the virulence of a pathogen? In the following sections, we discuss the contribution of RND efflux pumps to various processes contributing to the virulence of bacterial pathogens including; colonization, protection from host defense mechanisms, toxin production, and biofilm production (summarized in Table 1). Several studies have demonstrated the role of RND pumps in host colonization possibly due to their ability to export host-derived antimicrobials as well as bacterial toxins. One of the first such studies was with P. aeruginosa which showed that mutants overexpressing the MexAB-OprM, MexCD-OprJ, or MexEF-OprN pumps could be rapidly isolated in the absence of any antibiotic treatment from an acute pneumonia model in rats [31]. Isolation of such mutants in the absence of any antibiotic treatment suggested that overexpression of the RND pumps provided some sort of selective advantage to the bacteria, most likely defense from the host factors. Yet another study showed that the MexAB-OprM pump of P. aeruginosa was required for the invasion of Madin-Darby canine kidney (MDCK) cell lines [32]. A third study that supports these findings demonstrated that inhibition of the MexAB-OprM pump resulted in reduced invasion of MDCK cells lines by P. aeruginosa [33]. Moreover, MuxABC-OpmB, a recently characterized RND pump in P. aeruginosa [34], was shown to contribute to the twitching motility of bacteria [35]. Twitching motility is likely to contribute to the virulence of P. aeruginosa as gene deletion mutants of this pump were found to display attenuated virulence in Brassica pekinensis and Drosophila melanogaster infection models [35]. Examples of Resistance-Nodulation-Division (RND) pumps from Gram-negative species with their antibiotic substrates and proposed physiological role.In contrast to evidence supporting a role of RND pumps in enhancing virulence of P. aeruginosa, there is also evidence to the contrary. For example, one study has shown that overexpression of RND pumps (MexCD-OprJ or MexEF-OprN) can lead to downregulation of type III secretion proteins in P. aeruginosa [64]. This in turn is likely to cause a reduction in the virulence of this organism as exhibited by reduced cytotoxicity of pump overexpression mutants on a macrophage cell line. It was also shown in the same study that the overexpression of MexCD-OprJ or MexEF-OprN in clinical isolates of P. aeruginosa resulted in a downregulation of exsA gene expression [64]. The gene product of exsA is a regulator protein that activates the expression of various genes in the type III secretion system regulon [65]. The mechanism by which the overexpression of MexCD-OprJ or MexEF-OprN results in the downregulation of exsA gene remains unclear. However, the ExsA protein is known to autoactivate the expression of the exsA gene in response to various stimuli [64]. It is therefore possible that overexpression of either of the two RND pumps causes an increased efflux of unknown effector molecule(s) that activate exsA expression through the ExsA protein, and this could conceivably cause a reduction in exsA expression. Taken together, these studies suggest that different RND pumps may have a different impact on the virulence of P. aeruginosa. The impact of RND efflux pumps in the pathogenicity of bacteria has also been shown in Salmonella Typhimurium. For example, it was found that AcrAB-TolC mutants were attenuated in their ability to colonize mice [59] and chicks [57]. However, the attenuation of colonization was more profound in a TolC deletion mutant than the AcrAB pump, the major RND pump identified in this organism [66]. This is because, while the AcrAB mutant failed to invade macrophages, TolC mutants failed to both adhere to and invade the macrophages [67]. Since TolC is known to function as the outer membrane component of various efflux pumps, it appears that the AcrAB pump may not be the only pump involved in the colonization of Salmonella Typhimurium, and that the attenuation of colonization in TolC deletion mutants is a cumulative effect of inhibition of multiple pumps. Studies from Vibrio cholerae have also provided some interesting insights into the cumulative role of several RND pumps in virulence. It was first shown that four of the six RND pumps present in this organism namely, VexAB, VexCD, VexIJK, and VexGH contributed to the colonization of V. cholerae in an infant mouse model [62,63]. Using gene deletions created for the RND protein-encoding genes, the authors of this study showed that the colonization of the small intestines of infant mice was compromised about 50-fold in a strain of V. cholerae that lacked all four of the above pumps, while no significant attenuation was observed for various combinations of double knockouts, ΔvexB ΔvexD [62], ΔvexB ΔvexK [62], and ΔvexB ΔvexH [63]. Interestingly, the attenuation of colonization in the strain lacking all three of vexB, vexD, and vexK genes could be reversed by complementing the strain with the vexB gene on a plasmid alone. This presumably results from plasmid-derived overexpression of vexB which is likely to be several folds higher than chromosome-based overexpression of the gene. It is clear from these studies that the presence of multiple RND pumps is required for the colonization of the infant mouse intestine by V. cholerae. Bile salts are one of the common substrates of all of these pumps and thus it is conceivable that they aid in successful colonization by offering resistance to bile and possibly other antimicrobials present in the host gut. However the purpose of this apparent functional redundancy of RND pumps in the processes of host colonization by V. cholerae remains unclear.In addition to colonization, the same group showed that RND efflux pumps are required for the optimal production of cholera toxin (CT) and the toxin regulated pili (TCP) in V. cholerae [62]. It is hypothesized that the VexGH pump in V. cholerae is involved in the efflux of effector molecules that suppress the transcription of toxin genes and that their export by RND pumps removes this suppression leading to an increase in expression of CT and TCP [63]. This suggests that RND pumps in V. cholerae not only aid in colonization but also in optimum toxin production. In addition to the preceding examples of P. aeruginosa, Salmonella Typhimurium, and V. cholerae, RND pumps have also been shown to be important for the colonization of hosts by several other pathogens. For example, the MtrCDE pump of Neisseria gonorrhoeae [43] was shown to be critical for the survival of this bacterium in the genito-urinary tract of female mice [44,46]. This environment is rich in fatty acids, bile salt, and steroids, all of which have been shown to be substrates of the MtrCDE pump [44,45]. In addition, this pump has also been shown to impart protection against antimicrobial peptides [68], underscoring the important role the MtrCDE pump plays in resisting the host defense mechanisms and thus aiding in the colonization process. The role of RND pumps in colonization has been illustrated in plant pathogens as well. For example, in Pseudomonas syringae, a pump similar to MexAB-OprM of P. aeruginosa not only effluxes a variety of antimicrobials including dyes, β-lactams, fluoroquinolones, and tetracycline among others, but was also found to play a role in the survival of the organism in bean plants, presumably by protecting cells from plant secondary metabolites [56]. Similarly, the AcrAB pump of Erwinia amylovora effluxes secondary plant metabolites with antimicrobial activity, thus aiding in improved colonization of the host [40]. During the process of infection, a pathogen first encounters the elements of innate or non-specific immunity. An important part of the innate immune response is the process of phagocytosis. Here we discuss the correlation between RND pumps and the oxidative and nitrosative stress generated during the process of phagocytosis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important defense mechanisms employed by phagocytic cells against microbial pathogens [69]. As such, pathogenic bacteria must be able to withstand the effects of ROS and RNS in order to survive phagocytosis and cause disease. A number of studies have shown that both oxidative and nitrosative stress can induce the expression of RND pumps suggesting that these pumps are a part of the bacterial defense system against ROS and RNS compounds. One of the first suggestions about the link between oxidative stress and RND efflux pumps came from studies in E. coli showing that the overexpression of the soxS gene can result in upregulation of the AcrAB pump [28,42]. SoxRS is a two-component regulatory system that responds to oxidative stress and therefore plays a role in protection against oxidative damage by the host immune system. SoxR is the sensor protein that activates the expression of soxS in response to superoxide and nitric oxide ions. SoxS is subsequently responsible for the activation of genes whose products impart protection against oxidative damage [70]. In Salmonella Typhimurium, the SoxRS system has been shown to be involved in the regulation of the AcrAB pump [58]. Indeed clinical isolates of both E. coli [70] and Salmonella Typhimurium [71] have been isolated that harbor mutations in soxR and display the MDR phenotype. The SoxRS system has also been shown to regulate the expression of the AcrAB pump in Klebsiella pneumonia [72] and Enterobacter cloacae [73] suggesting that the role of oxidative stress in the expression of RND pumps is widespread. In our laboratory, we have also observed upregulation of the AdeABC RND pump in A. baumannii in response to oxidative stress (Andrei Bazyleu and Ayush Kumar, unpublished data). Although we are not sure yet if this is a result of the activity of a SoxRS-like system in A. baumannii, this does provide further evidence that oxidative stress modulates the expression of RND pumps in bacteria. An association between oxidative stress and RND pump expression has also been observed in P. aeruginosa. The MexXY pump of P. aeruginosa has been shown to be highly expressed in isolates of P. aeruginosa from cystic fibrosis (CF) lungs [55] and the expression of this pump has also been shown to be induced by oxidative stress [53]. The MexXY pump is responsible for resistance to aminoglycoside antibiotics [54]. These antibiotics target the ribosome and it is hypothesized that compounds that act on the ribosome are substrates of this pump [74]. Remarkably, the CF lung has been shown to be rich in oxygen radicals [75] and it is therefore likely that an environment rich in oxygen radicals such as CF lungs induces the expression of the MexXY pump which provides a fitness advantage to P. aeruginosa in the presence of aminoglycosides [76], the group of antibiotics widely used against this bacterium in CF cases [77]. The interplay between oxidative stress and antibiotic resistance is quite interesting as recent studies from the James Collins laboratory at Boston University show that the bactericidal action of a number of unrelated antibiotics results from their ability to generate hydroxyl radicals which ultimately contribute to cell death [41]. The study also revealed that sublethal concentrations of antibiotics induce mutagenesis in E. coli by stimulating the production of ROS. One of these mutations was shown to be present in the promoter region of the acrA gene that encodes the MFP of the AcrAB efflux pump, thus revealing an intriguing relationship between oxidative stress and RND efflux pumps. If antibiotics elicit their antimicrobial action via generation of ROS, then overexpression of these pumps serves as a useful means to alleviate the bactericidal effects of antibiotics by effluxing these molecules out of the cell.A recent study in Campylobacter jejuni suggests that RND pumps could be part of a complex protection mechanism employed by bacteria against oxidative damage [38]. This study showed that CosR, an OmpR-type response regulator, modulates the expression of almost 500 genes in this organism [78]. Among these are the cmeABC operon that encodes an RND efflux pump and the katA gene that encodes the sole catalase enzyme in C. jejuni. CosR was shown to be a negative regulator of the cmeABC operon and a positive regulator of katA expression. The CmeABC pump not only effluxes antimicrobials [39] but also plays a critical role in the colonization of chicken intestines, most likely as a result of its ability to efflux bile salts [79]. On the other hand, KatA plays a vital role in resistance to oxidative stress by detoxification of ROS to less toxic compounds. Since efflux pumps have been speculated to play a role in minimizing the effects of oxidative stress [80], possibly by exporting the toxic products of oxidative damage, it can therefore be speculated, based on the counter regulation of cmeABC and katA by CosR, that the CmeABC pump may be involved in protection against oxidative damage (possibly by effluxing toxic products of oxidative damage) particularly when expression of katA is downregulated. In addition to ROS, RNS are also an important part of the innate immune system. Just as in oxidative stress, nitrosative stress was also recently shown to induce the expression of the MexEF-OprN pump of P. aeruginosa [50], a pump that effluxes a number of antibiotics including chloramphenicol, trimethoprim, and ciprofloxacin [51]. Chloramphenicol however, is the only known antibiotic substrate that is capable of inducing the expression of the mexEF-oprN operon [50] whereas a chloramphenicol derivative that lacks the nitro moiety is unable to induce its expression. It is therefore suggested that nitrosated products generated as a result of nitrosative stress are probably natural substrates of the MexEF-OprN pump and that chloramphenicol can induce this pump’s expression as it shares structural similarity with these yet unknown nitrosative products. Homologs of the MexEF-OprN pump have been described in other organisms including the CeoAB-OpcM pump in Burkholderia cenocepacia [81], the BpeEF-OprC pump in Burkholderia pseudomallei [82], and the AdeFGH pump in A. baumannii [83,84]. All of these homologs have been shown to efflux chloramphenicol and the expression of the BpeEF-OprC pump has also been found to be induced by chloramphenicol [85]. However it remains to be seen if chloramphenicol has a similar effect on expression of other homologs of the MexEF-OprN pump. Biofilms are microbial communities that grow attached to a variety of surfaces. Bacterial biofilms are clinically very important and are implicated in various persistent or recurrent infections that are very difficult to treat with antibiotics. Resistance to various antibiotics in a biofilm results from a multitude of factors that include their impermeability to antibiotics [86], upregulation of various resistance genes [87], and the phenotypic heterogeneity of the cells forming the biofilm [88]. Quorum sensing (QS) is an important mechanism that controls biofilm formation in a cell density-dependent manner [89]. Sensing of the cell density is achieved by means of extracellular signaling molecules called autoinducers (AIs). As the concentration of AIs reaches a certain threshold level, they trigger a signal to bacterial cells which respond by altering the expression of various genes in a coordinated manner. The role of RND efflux pumps in biofilm formation has been studied in various organisms. For example, the MexAB-OprM and MexEF-OprN pumps of P. aeruginosa have been shown to efflux AI molecules [47,52] effectively reducing their intracellular concentrations. As a result, MexAB-OprM or MexEF-OprN overexpressing strains of P. aeruginosa exhibit reduced expression of AI-dependent genes including those involved in AI synthesis itself along with other virulence genes. For the same reason, such strains also exhibit compromised biofilm formation ability. Work from our laboratory also supports this. When comparing two different strains of P. aeruginosa, PAO1 (a strain that constitutively expresses the mexAB-oprM pump) and PAO200 (a PAO1 derivative lacking mexAB-oprM), we observed that even though the secretion of AI, homoserine lactone, was higher in PAO1, PAO200 formed more biofilm than PAO1 (Sarah Warren and Ayush Kumar, unpublished data). Similar observations have been made in B. cenocepacia where deletion mutants of two different RND pump-encoding genes BCAL1675 and BCAL2821 show approximately 30% less accumulation of AI in the growth medium compared to the wild-type strain [36], further suggesting that RND efflux pumps are involved in the export of quorum sensing signals. In addition to a relationship between efflux pump activity and QS, it has also been shown that RND pump expression can impact flagellar motility. Flagellar motility also plays an important role in biofilm formation as it has been shown to impact the entry of macromolecules in the biofilms and also the dissolution of biofilms [90]. Interesting data related to this has come from studies on B. cenocepacia. Deletion of an RND pump-encoding gene BCAL2821 in B. cenocepacia was shown to impact the expression of over 200 genes and that of another RND pump encoding gene BCAM1946 resulted in the altered expression of over 150 genes. In addition, a double deletion mutant of both genes showed differential expression of 550 different genes [37]. A large proportion of genes whose expression was altered in these knock-out strains were those that are involved in flagellar motility. Interestingly, some of the flagellum-associated genes whose expression was upregulated in the BCAL2821 deletion mutant were found to be downregulated in the BCAM1946 deletion mutant, leading to speculation that these pumps play a balancing role in flagellum-associated functions. In the same study, the authors showed an increase in biofilm formation in both BCAL2821 and BCAM1946 deletion mutants. Since flagellar motility plays a key role in biofilm formation and since the BCAL2821 pump (but not BCAM1946) also effluxes acylhomoserine lactone [36], it is possible that these pumps in B. cenocepacia play a role in biofilm formation at multiple levels. For example, they may be involved both in transporting the acylhomoserine lactone and modulating the expression of flagellum-associated genes. However, another study in P. aeruginosa failed to show any role of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps in biofilm formation [91]. A lack of correlation between the expression of RND efflux pumps and autoinducer synthetase genes lasI and rhlI has also been reported [92]. Nevertheless, in the presence of azithromycin, a macrolide, MexAB-OprM or MexCD-OprJ pumps were found to be required for azithromycin-resistant biofilm formation [93]. These studies show that the role of the MexAB-OprM and MexCD-OprJ pumps in biofilm is complex and dependent on various factors such as the presence of antibiotics. Furthermore, azithromycin has been shown to improve respiratory function in cystic fibrosis patients [94] and the study by Gillis et al. [93] shows that the MexAB-OprM and MexCD-OprJ pumps may play a critical role in the formation of biofilms in cystic fibrosis lungs during azithromycin therapy. Yet another, albeit indirect, evidence of overexpression of RND efflux pumps in biofilms in P. aeruginosa came from the analysis of its multidrug resistance under hypoxic conditions [95]. It was observed that under low oxygen concentrations (1%), P. aeruginosa exhibited an increased resistance to various antibiotics. This increased resistance was reversible in the presence of the efflux pump inhibitor (EPI), Phenyl alanine arginine β-naphthylamide (PaβN), suggesting that activity of RND efflux pumps was required for the formation of biofilms. Under hypoxic conditions the expression of MexEF-OprN pumps was found to predominate expression of other RND pumps. Biofilms are structurally very heterogeneous and the center of the microcolonies can have almost no oxygen [87]. It is therefore possible that expression of RND pumps, in this case the MexEF-OprN pump, varies within different layers of biofilms with the highest expression in the deepest layers where the oxygen concentration is the lowest. Because biofilms are characterized by the presence of distinct microenvironments, bacterial cells within them can exist in different metabolic states and the outer layers of the biofilm tend to consist of cells that are the most metabolically active. This observation is also relevant with respect to efflux pump activity. For example, the expression of MexAB-OprM has been shown to be dependent on the metabolic state of P. aeruginosa with the expression being highest in metabolically active cells [96]. Therefore, cells present in the outer layers of biofilms are more likely to overexpress the MexAB-OprM pump compared to cells present in the inner layers. Though it is not clear whether overexpression of efflux pumps in biofilms serves any other purpose than antibiotic resistance, it is evident that the expression of efflux pumps can vary considerably within different layers of biofilms. The role of RND efflux pumps in biofilm formation was also examined in a study where EPIs were shown to significantly reduce biofilm formation in E. coli and K. pneumonia [97]. The authors of this study used thioridazine (an inhibitor of the NorA major facilitator superfamily efflux pump in Staphylococcus aureus [98]) along with two different inhibitors of RND pumps, PAβN and 1-(1-naphthylmethyl) piperazine (NMP). It was observed that the combination of thioridazine with either PAβN or NMP was most effective in inhibiting the formation of biofilms, suggesting that inhibition of multiple efflux pumps belonging to different families may be required for the complete inhibition of biofilm formation. However, since the authors did not explore the effect of EPI combinations on planktonic cells, it is difficult to say if the effect seen was simply the result of deleterious effects on cells from the accumulation of various metabolic byproducts and toxins or if it was specific to biofilm formation. Although RND pump encoding genes have been shown to be present in various bacterial species including pathogenic and non-pathogenic bacteria, the role they play in the multidrug resistance of Gram-negative pathogens makes them extremely clinically relevant. Even though they are arguably the most important contributor to the multidrug resistance phenotype in these pathogens, it is widely accepted that efflux of antibiotics is not the natural function of RND pumps. Study of their natural functions will lead to a better understanding of conditions that promote their expression, information that will be critical in designing improved and more effective therapy options for multidrug resistant organisms. To this end, there is increasing evidence that RND efflux pumps are involved in the virulence of Gram-negative bacteria, thus revealing interplay between resistance and virulence and further highlighting the clinical relevance of these pumps. One key implication of these data is that RND efflux pumps are a lucrative target for successful treatment of Gram-negative infections. For example, inhibitors of these pumps can conceivably not only neutralize resistance but also impact their ability to cause successful infection. However, studies on the role of RND pumps in virulence are met with their own challenges. For instance, because of their extremely broad substrate specificity, the mere appearance of a phenotype upon overexpression of these pumps is not enough to ascertain their role in virulence. In addition, functional redundancy of these pumps in colonization or efflux of various ligands (for example, non-antibiotic substrates like quorum sensing molecules and host defense molecules) further complicates efforts to study their contribution to virulence. One approach that promises to provide invaluable information on correlation of antibiotic resistance and virulence mediated by RND pumps is the study of common regulatory mechanisms. For example, identification of common regulatory pathways that control the expression of RND pumps and virulence genes will provide more concrete evidence of their role in virulence, information that can be used to design novel therapeutic options for MDR Gram-negative infections. The authors declare no conflict of interest.The work in AK’s laboratory is supported by grants from the Natural Science and Engineering Research Council (NSERC), Canada Foundation for Innovation (CFI), Ontario Research Funds (ORF), and JP Bickell Foundation. The authors are grateful to Kari Kumar for the critical reading of the manuscript. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The use of light-activated bactericidal agents—photobactericides—is suggested in local infection in order to conserve conventional antibacterials for more systemic disease. Local administration of a photobactericide such as methylene blue coupled with locally-targeted red light illumination ensures the production of non-specific reactive oxygen species and thus a rapid and localised antibacterial response, regardless of the conventional resistance status. To this end, the response of photobactericides to conventional resistance mechanisms, and their potential use in infection, is discussed.Given the bewildering numbers of drug-resistant strains of bacteria in modern healthcare, it is clear that conventional antibacterial approaches are no longer widely effective. This is truly a terrible situation when considered from the viewpoint of those involved in producing the “Golden Age” of antibiotics when all of mankind’s infectious diseases were thought to be susceptible to Fleming’s legacy. However, in the early 21st Century, with the benefit of considerable hindsight, there is a sound understanding of the failure of conventional antibacterial chemotherapy, based with greater emphasis on bacterial evolution than on the initial good fortune and subsequent—if predictable—egotism of Homo sapiens. This understanding counts the rapidity of bacterial genetic turnover and adaptability as key, thus explaining the apparent ease—on a human timescale, at least—with which our bacterial colonists become immune to the chemical battery deployed against them.It is not in the least surprising that the early, successful antibacterial types such as the sulphonamides and penicillins were used with such alacrity. In the period 1935–1945 patient mortality from bacterial disease was high, and obviously there was a massive requirement for wound and infection therapy during World War II. However, the phenomenon of drug resistance was appreciated at least by those involved in the field of chemotherapy—Fleming himself alluded to it in his Nobel Prize Lecture in 1945 [1]. Despite this, antibacterial drugs have been used, even in well-organised healthcare systems, with breathtaking profligacy until relatively recently. Again, with hindsight, bacterial evolutionary kinetics have always meant that specific antibacterial drugs, or drug classes, would have a finite period of usefulness.Despite this deterministic outlook, our clinical use of antibacterial agents has been flawed. Investigation of the modes of action of these valuable commodities has, in most cases, shown that each drug type has a single site and mode of action—for example, the mimicking of the D-alanine-D-alanine terminus of the incipient peptide crosslink in bacterial cell wall peptidoglycan by the bicyclic penicillin nucleus, which leads to the inhibition of action of bacterial transpeptidase. Resistance to the initially-introduced penicillin drugs was seen in the immediate post-war period due to endogenous hydrolytic enzymes, later known as β-lactamases, the overexpression of which furnished mutant organisms with an evolutionary advantage, and the adaptation of which allowed the clinical survival of their progeny. While early penicillin resistance was particularly problematic in Gram-positive cocci [2], one of the most significant clinical threats in recent years can be thought of as an elaboration in Gram-negative pathogens, i.e., the extended spectrum β-lactamases (ESBLs) which inactivate the carbapenems, important modern drugs expensively developed for use against serious Gram-negative infection [3]. These again obey the single site/single mode of action paradigm, and act in the same way as the original penicillins developed by Florey and the Oxford group in the 1940s.A further problem in drug resistance lies in the ability of bacteria to remove or to exclude toxic substances. Generally cells are able to expel xenobiotic substances from the interior using protein pumps, and in drug resistant forms, this facility can be over expressed. However, given the relatively non-specific nature of the substrates involved this may be considered to be a far greater problem in terms of drug therapy since the potential structural range of candidate therapeutics is wide, i.e., efflux capability does not pertain to a single chemical class of agent. Similarly, the exclusion—i.e., non-admittance-of antibacterial agents from the cell is normally achieved via the lowered expression of small porin channels in the cell exterior. Again, this can have no relevance to a particular chemical class of therapeutic.Thus, 21st Century bacterial infection often represents a considerable—and increasingly insurmountable—problem to those administering the conventional armamentarium. In addition, new drugs are very slow in arriving, and most which have been accepted for clinical use still obey the single site/single mode of action paradigm. Consequently, not only will these drugs have a limited period of utility, they will also add to the selective evolutionary pressure already driving bacterial resistance development.The photoantimicrobial approach to infectious disease offers multifactorial attack—i.e., multiple and variable sites of action coupled with a non-selective, oxidative mode of action. As the term suggests, photoantimicrobials require light activation so local application and activation are required, but this has important ramifications regarding treatment, particularly from the point of view of effects on local flora in comparison to those seen with conventional systemics, and in the lack of resistance development.The interaction of light with photosensitising molecules, of which the photoantimicrobials are a sub-class, is slightly different to that of the larger group of dyes and pigments. Light absorption by both classes of compounds entails the removal of a certain wavelength range of the incident light energy, depending on the chemical make-up of the absorber, leading to electronic excitation. In dyes and pigments, the excitational energy is lost rapidly, whereas in photosensitiser molecules the excited state is sufficiently long-lived for electronic rearrangement to occur, promoting electron transfer reactions and the transfer of excitational energy to molecular oxygen. Both of these routes result in the formation of reactive oxygen species which are highly damaging in the cellular milieu. However, they are short-lived species, so oxidative damage is highly localised (Figure 1).Photosensitisation pathways leading to bacterial cell death. Dashed arrows indicate possible deactivational (non-destructive) routes.The history of photoantimicrobials is based on the observed antimicrobial activity of a handful of dyes which were associated with the burgeoning science of biological staining at the end of the 19th Century. The lead compound resulting from this was the phenothiazinium derivative methylene blue (MB, Figure 2), which was shown to kill bacteria, viruses, fungi and protozoa under illumination during the same momentous period (1928–1935) which covered both Fleming’s and Domagk’s major antibacterial discoveries. There remains a strong rationale for the use of methylene blue as a chemical lead by researchers involved in photoantimicrobial discovery, as it also represents the first-in-clinic example, being currently licensed for oral disinfection [4].Photobactericides from the main classes of photosensitiser. Methylene blue (MB) and toluidine blue (TBO), phenothiazinium class; TMPPP [tri(N-methylpyrid-4-yl)phenylporphyrin] and Ce6, porphyrin; RLP068 and ZnTSPc, phthalocyanine. Cationic examples are broad-spectrum, anionic examples are active mainly against Gram-positive bacteria.The reason for the truly broad antibacterial spectrum of methylene blue and its congeners lies in the possession of a permanent positive charge. This guarantees the activity against Gram-negative bacteria not seen in anionic (negatively charged) or neutral photosensitisers, such as the haematoporphyrin derivatives used far more successfully in the photodynamic therapy of cancer (PDT) [5].However, broad-spectrum photoantibacterial activity is not limited to the phenothiazinium class. Indeed it should not be limited by chemical class at all. The relevant criterion here is that the photosensitiser in question has a positive charge. Consequently, there are examples of broad-spectrum photoantibacterials in the synthetic porphyrin and phthalocyanine classes also (Figure 2) [6]. Another important aspect in the development of a clinically-useful photobactericide is the associated absorption spectrum. The reason for this is the presence of other absorbing species at the infection site. Such species are the natural materials present—e.g., blood in a wound contains haem pigments which absorb both ultraviolet and visible wavelengths, while soluble aminoacids and vitamins absorb only in the ultraviolet region. A considerable portion of photobactericidal research is thus entailed in the production of molecules which may be excited at longer wavelengths. Practically, since the longest wavelength of absorption of haem is a weak band at 630 nm, intense absorbers beyond this wavelength may be easily discovered among both the phenothiazinium and phthalocyanine series, e.g., methylene blue (660 nm) and RLP068 (668 nm) respectively (Figure 2). Clearly efficient excitation is also necessary, but this is relatively straightforward given access to diode lasers and light-emitting diode-based equipment.Logically, a structure-specific mechanism of resistance such as β-lactamase activity requires that structural motif for activity. Conventional agents not containing this motif should be immune to attack. Similarly drug activity may be nullified by the alteration of the active site, for example in tetracycline resistance via ribosomal protection. In either of these scenarios, substitution of the original therapeutic with one which is chemically distinct (i.e., from a different class), should regain efficacy. However, where there is more than one resistance mechanism at work—as is increasingly the case—this approach becomes less successful. The utility of photoantimicrobial agents lies in efficacy regardless of the conventional drug resistance mechanism. This is considered below.As noted above, target alteration as a drug resistance mechanism usually pertains to changes to minor morphological or chemical changes which result in lowered affinity of the drug for its target, for example ribosomal protection in tetracycline resistance [7], or the change from D-alanine-D-alanine to D-alanine-D-lactate at the glycopeptide-active site in the developing bacterial cell wall in vancomycin-resistant enterococci [8]. Such changes are specific for the incoming drug molecule and may often thus confer class resistance.The effects of a photobactericide in such a case should be consistent between drug-sensitive and drug-resistant cells. This is due to structural dissimilarity between the photobactericidal molecule and conventional drugs, and to the non-specific nature of the oxidising species produced on illumination. Such activity has been reported for methylene blue derivatives against vancomycin-susceptible and vancomycin-resistant enterococci [9].As with target alteration, drug inactivation as a resistance mechanism may rely on target discrimination. The β-lactamases provide an excellent example of this, being inactive against other chemical classes of antibacterial agent. Clearly the same argument pertains for photobactericidal activity in such cases, given the difference in chemical structures employed and also in the reactive oxygen species produced, and effective photobactericidal activity has been reported for methylene blue against extended-spectrum β-lactamase (ESBL)-producing strains of Escherichia coli [10]. Such activity may also be expected against New Delhi metallo-β-lactamase-1 producing bacteria, underlining the utility of the light-activated approach.It might be expected that antioxidant enzymes would present a resistance route against photobactericidal agents, acting to nullify the reactive oxygen species produced on illumination. However, it has been demonstrated that enzymes such as catalases, peroxidases and superoxide dismutase are themselves inactivated on exposure to singlet oxygen (SOD, Route 3, Figure 3) [11]. It is therefore not surprising that upregulation of SOD as a consequence of Staphylococcus aureus exposure to protoporphyrin-initiated photodynamic treatment reportedly does not affect the cell-killing outcome [12].Photobactericide action and resistance mechanisms.In comparison to the two previous routes, decreased cell permeability is much less structure specific, being based on significantly-reduced numbers of porins in the outer membrane of Gram-negative bacteria. Consequently, most conventional antibacterial agents are effectively barred from their targets. However, it has been demonstrated for cationic photobactericides, such as the pyridinium phthalocyanines, that they gain entry to the Gram-negative cell via self-promoted uptake [13]. This process involves the disruption of the outer membrane by the positive charge, or charges, on the photosensitiser via displacement of the divalent metal ions required for the stabilisation of the membrane’s anionic head groups. Subsequently, the oxidising species produced by photobactericides may then produce non-specific oxidation, causing damage to the outer membrane sufficient to cause catastrophic breakdown to a similar extent to that caused by peptide antibiotics such as the polymyxins. Significant disturbance to the outer membrane will also allow ingress to the interior of the cell and photodamage to underlying structures/organelles [14]. While anionic photosensitisers are generally ineffective against Gram-negative bacteria, this may be reversed by attaching a polycationic residue to the photosensitiser. This has been reported for chlorin e6 (Ce6, Figure 2) and polyethyleneimines [15].In addition, it has been established for a considerable time that the exclusion of a photobactericide from the target cell does not inhibit cell damage, since polymer-bound examples have been shown to cause cell death on illumination, allowing the development of photoantimicrobial plastics and textiles [16,17].A similar argument can be made for the assisted exclusion of photobacterides from the target cell, although Hamblin has shown that both the phenothiazinium derivatives methylene blue and toluidine blue (MB and TBO, respectively, Figure 2) can act as substrates for efflux pumps in bacteria [18], and that this behaviour may be reversed via the addition of inhibitors such as verapamil [19]. Other cationic photosensitisers, from different chemical classes of significantly greater moleclular weight, such as the porphyrins and phthalocyanines are reported to avoid transport by such efflux pumps. In addition, it is theoretically possible to inactivate the efflux pump via illumination during transport of the photosensitiser, as indicated in Figure 3 (Route 2). This is an approach currently under investigation.It is emphasised that, unlike conventional antibacterial agents, photobactericides are intended for local/topical application—i.e., where there is a focus of infection rather than dissemination. Clearly local application allows for concentration of the active agent at the site of infection, rather than relying on transport through the bloodstream following systemic administration. Light is also applied in a focused manner. In terms of the major resistance mechanisms discussed above, only that involving exclusion via efflux appears to offer any difficulty for the photobactericidal approach, and this has not been encountered in the clinic. In addition, local administration to an infection site should produce a higher concentration of photosensitiser external to the target cell than would be required for cell death—it should be recalled that photobactericides can produce reactive oxygen species on illumination outside the cell.The multiple site of action/mode of action paradigm associated with photoantimicrobial agents is underlined in reported passaging experiments. For example, twenty daily passages using the cationic phthalocyanine RLP068 (Figure 2) against strains of Staphylococcus aureus and Pseudomonas aeruginosa, with a range of conventional drug resistance profiles, demonstrated no development of resistance to the photodynamic approach [20].Given the demonstrable efficacies of various photobactericides against a broad-spectrum of bacterial pathogens, regardless of conventional resistance status, the clinical potential of this class of agents is considerable. For clinicians it is, however, apparently tempered by the fact that light activation is required—this being seen as a deviation from the standard antibacterial paradigm. It should be pointed out both that the standard paradigm is no longer successful and that light activation is routinely used in other areas of medicine, such as the treatment of psoriasis with PUVA. Clearly, if mankind is to keep up with its bacterial colonists, changes will need to be made to the clinical approach to microbial—not just bacterial—disease, and photobactericides should be part of that change.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Among the class of pollutants considered as ‘emerging contaminants’, antibiotic compounds including drugs used in medical therapy, biocides and disinfectants merit special consideration because their bioactivity in the environment is the result of their functional design. Antibiotics can alter the structure and function of microbial communities in the receiving environment and facilitate the development and spread of resistance in critical species of bacteria including pathogens. Methanogenesis, nitrogen transformation and sulphate reduction are among the key ecosystem processes performed by bacteria in nature that can also be affected by the impacts of environmental contamination by antibiotics. Together, the effects of the development of resistance in bacteria involved in maintaining overall ecosystem health and the development of resistance in human, animal and fish pathogens, make serious contributions to the risks associated with environmental pollution by antibiotics. In this brief review, we discuss the multiple impacts on human and ecosystem health of environmental contamination by antibiotic compounds.For millennia, long before humans made footprints on the earth’s surface, bacteria have been performing crucial biological services in diverse ecological niches. Humankind learned of the advantageous use of many natural processes in which bacteria play a pivotal role and forged a relationship that has served human enterprise ever since. However, the consequences of this relationship are not exclusively positive. A comparatively small proportion of bacterial species are pathogens but some of these are responsible for infectious diseases that can have devastating impacts on human and animal health. Humans have exploited the activities of antibiotic compounds, many of which are derived from nature, to fight bacterial infections for centuries. Despite human efforts to manipulate nature, our unintended consequences on ecological processes in the natural environment can influence human health outcomes. Development of antibiotic resistance in pathogens is a shining example of health issues in which understanding the central role of the environment is crucial to managing this potential risk. Here we discuss recent contributions to our understanding of relationships between health, antibiotic resistance and the environment.Although widespread use of antibiotic drugs to treat human infections grew tremendously since penicillin was introduced into medical therapy [1], the beneficial antibiotic properties of many bacteria and plant species have been known for a very long time. Recent advances in analytical chemistry have provided evidence that tetracycline derived from Streptomyces bacteria in beer fermentation was used for medical treatment of infection in ancient (350–550 AD) Nubian patients [2]. The ancient Greeks and Romans valued wild indigo root as both a blue dye and for its ability to treat respiratory tract infections and some soft tissue infections [3]. Interestingly, woad obtained from the plant Isatis tinctoria produces blue dye similar to indigo and has antibiotic properties [4] for which historical documents confirm its use in traditional Chinese medicine and in European medicine to treat certain infections since the 14th century. Chromobacterium violaceum is a Gram-negative, heterotrophic bacterium common in soil or aquatic ecosystems in many tropical regions and frequently found in black water in the Amazon River [5]. This particular bacterial species is well-known for its purple pigment and has demonstrated antimicrobial properties that have activity against Leishmania species, Mycobacterium tuberculosis and some viruses [6]. Although many plant species are known to produce compounds with antibiotic activity, few show promise for applications in human medicine as potent broad-spectrum antimicrobials [7]. Processes that lead to the development of antibiotic resistance have likely occurred throughout all of microbial evolutionary history. Microbial analyses of rock surfaces of the Lechuguilla cave system in New Mexico, dating back some 4 million years, has revealed the presence of bacteria that are resistant to many structurally and chemically diverse antibiotics currently used in human medicine [8]. Phylogenetic analyses showed that the OXA genes which encode a class of β-lactamases that confers resistance to a broad range of β-lactam antibiotics have existed on plasmids for millions of years [9]. This evidence counters the popular argument that mobilization of antibiotic resistance genes is completely due to modern use of antibiotics in medical therapy. However, despite evidence that the evolutionary processes responsible for development of antibiotic resistance in environmental bacteria probably occurred for billions of years, it is clear that selection pressure has become more intense with increasing use and disposal of antibiotics and that this selection is geared more towards ensuring survival in hostile environments rather than improving fitness in slowly evolving populations [10,11,12]. Development of antibiotic resistance in bacteria is yet another example for which the metaphor of the ‘ecological footprint’ [13] aptly describes the increase in impacts on natural processes that occur as a result of human activities—in this case via underuse, overuse and misuse of antibiotics in medical therapy.Ever-changing environmental conditions influence the biological, chemical, physical and ecological processes that govern the health of all ecosystem species. Just as humans must adapt to environmental stressors, the survival of healthy populations of bacteria depends on their ability to respond quickly to overcome environmental threats. Gene exchange is a common property of all bacteria. When human activities amplify the effects of exposure to stressors, the development of resistance to these represents one of the most striking illustrations of Darwinian selection and survival. Scale is an important factor in considering the response of organisms to environmental change. The effects of processes that occur at the cellular level often translate to effects at different environmental scales. For example, the mechanisms of development of antibiotic resistance in bacteria resident in biofilms lining the catheter tubes of a human patient can be very similar to those in the biofilms lining a wastewater treatment reactor. Microbes interact with a myriad of small molecules in their lifetime and it is likely that resistance develops as a self-defense mechanism, an altered response to chemical signals or as a way to metabolize molecules as a food source.It is now accepted that resistance is a natural property of all bacteria [14,15,16] and the term ‘resistome’ is used to describe the framework that encompasses all forms of resistance and precursor elements [17]. More and more evidence is being collected to support the idea that the environment acts both as a reservoir for antibiotic resistance and a means by which this resistance can be broadly disseminated. Genes can move quickly through a bacterial population via vertical or horizontal transfer mechanisms and combined with the grand magnitude of the resistome, it is no wonder that bacteria can quickly adapt to resist new drugs soon after they are introduced for medical or agricultural use. Resistant bacteria, antibiotic resistance genes, degradative enzymes that work to inactivate antibiotics, and antibiotic molecules are present in the environment at all times and thus distinguishing naturally occurring resistance in organisms from resistance as a result of environmental pollution is a complicated task. Each of these contributors adds a layer of complexity to our understanding of the environmental footprint of antibiotics. Superbugs exist everywhere in nature. Several well-known pathogens have demonstrated disturbing trends for developing resistance to antimicrobials. Since Snow’s first epidemiological study of cholera in 1854 [18], water contamination with toxigenic Vibrio cholerae has been a well-understood risk to public health of ongoing relevance in many developing countries. Vibrio cholerae exists in the environment and, during its quiescent stage, distribution of the pathogen through bodies of water spreads the threat to human health, particularly when strains are resistant to multiple classes of antibiotics. Outbreaks of cholera are wide-spread in the developing world (particularly after natural disasters) and among the recent examples of emergence of multi-drug resistant strains of the pathogen are reports from India [19], China [20], and Haiti [21]. Although comparatively few of the species of bacteria in the environment are pathogens, those that have earned the reputation as ‘superbugs’ make substantial contributions to the environmental footprint of antibiotics by increasing the amount and diversity of drugs needed to combat infection, increasing the cost of treating illness and altering the ecological conditions of healthy functioning ecosystems.Emergence of multi-drug resistance in community acquired pathogens such as Mycobacterium tuberculosis and Streptococcus pneumoniae illustrates a worrisome scenario in which the effectiveness of the antibiotics that previously successfully treated the infections has deteriorated over time. Nocosomial pathogens associated with hospital acquired infections, such as methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa and Vancomycin-resistant Enterococcus faecium occur with high frequency of drug-resistance traits and have been detected in wastewater treatment plants that receive hospital effluent discharge [22,23,24,25]. The discharge of effluents from wastewater treatment plants provides the pathway for introduction of contaminants into the receiving environment. Given that secondary treatment of municipal wastewater depends on maintaining conditions for healthy populations of useful bacteria in reactors to degrade sewage, it is highly likely that some resistant pathogens could also be among the organisms released after waste processing.Environment has multiple meanings. Ecosystems describe communities of living organisms and their relationship to the non-living environmental condition in which they reside. Hospitals and each of their sub-units represent unique ecosystems that, in some cases, have environmental conditions that facilitate rapid development of antibiotic resistance in pathogens. Among the hospital acquired pathogens, the emergence and spread of resistant strains of Clostridium difficile is demonstrating a disturbing upward trend. Increased multi-drug resistance and virulence in Clostridium difficile is well-documented [26,27,28] and this is of concern since outbreaks of C. difficile infections occur in long-term care facilities with increased frequency [29,30]. In addition, zoonotic transfer of pathogens via companion animals may be an under-recognized mode of transmission of organisms including Clostridium difficile. Although a common pathogen among several animal species, within the past decade, C. difficile has become accepted as an enteric pathogen in horses [31,32,33]. Humans and pets often share the same environment in close proximity making transmission of bacteria likely but particularly problematic if the same species of enteric pathogen can thrive in human and animal hosts. When infections that were previously associated with the hospital environment occur in the community, the implications connected with development of resistance in the pathogens responsible for these outbreaks amplify the public health concern. Another important pathogen that has a long history of association with hospital acquired infections is Acinetobacter baumaii. This species of gram negative bacilli are common soil dwelling organisms that are widely distributed in nature. Because they can survive under a broad range of environmental conditions and can exist for long periods of time on dry surfaces, the risk of transmission between individuals is high. Multi-drug resistant Acinetobacter baumaii is a rapidly emerging pathogen in health care settings where antimicrobial resistance has seriously limited options for treatment [34]. As a last resort for multi-drug infections, carbapenems are used to treat Acinetobacter baumaii associated infections however, carbapenem-resistant strains of the pathogen are being reported at an alarmingly high rate [35,36,37,38]. Outbreaks of severe multi-drug resistant A. baumannii were observed in military personnel active in the conflicts in Iraq and Afghanistan [39,40,41,42,43]. Originally, environmental exposures to soil-dwelling strains of the pathogen were thought to be responsible for the outbreaks. However, subsequent culture-based studies suggested that exposure in health care facilities is the more likely pathway of transmission between patients [44,45,46,47,48]. Environmental contamination plays a critical role as an important reservoir in outbreaks of A. baumannii and in extending the need for more frequent use and higher potency antibiotic drugs.Microbes travel. Environmental transport of antibiotic resistant bacteria, especially human and animal pathogens, extends opportunities for exposure of non-target organisms in air, water, soil and sediment. People travel. Increased global mobility of humans and animals has influenced the rates of exposure to pathogens in unprecedented ways. Environmental contamination by antibiotic compounds is inextricably linked to development of antimicrobial resistance in non-target species of bacteria. Whether the bacteria perform critical ecosystem services, pose a health threat as pathogens or have incompletely understood functions in nature, development of antimicrobial resistance as a result of human activities is problematic. Susceptibility characteristics of microbes can be altered by incorporation of genetic information encoding for resistance or by mutation in their DNA. Antibiotic resistance genes are recognized as important environmental contaminants [49,50,51]. Bacteria strains containing genes that code for resistance to fluoroquinolones [52,53,54,55,56], macrolides [57,58,59], sulfonamides [60,61,62] and trimethoprim [63,64] have been isolated from environmental samples. Resistance can spread as a result of distribution of resistant bacterial strains or genetic elements of resistance throughout the receiving environment, evolution and selection of new resistant strains or the amplification of pre-existing resistant strains of bacteria. Introduction of antibiotics as environmental contaminants have important influences on these processes that alter abundance of resistance genes in the environment in multiple ways. Genes of some strains of bacteria encode for the production of enzymes that are responsible for resistance to antibiotics which have the four-carbon beta lactam ring in their chemical structure. These enzymes, known as beta lactamases, are capable of breaking the ring structure that is common to antibiotics, such as penicillins, cephamycins and carbapenems, thereby inactivating the antimicrobial properties of the molecule. Extended-spectrum beta lactamases (ESBLs) are a sub-group of this class of enzymes that are often plasmid encoded and also carry genetic traits for resistance to cephalosporins and other antibiotic drugs used for treatment of serious pathogenic infections of human patients. These classes of antibiotics represent the last resort in treatment for some infections. Incidence of infection by ESBL-producing organism is being reported with increasing frequency [65,66,67]. This is an extremely important concern in the medical community since the range of antibiotic treatment options is progressively shrinking. One of the latest examples of this disturbing trend is the emergence of New Delhi metallo-beta-lactamase (NDM-1) in some strains of Gram negative bacteria that have demonstrated resistance to virtually all antibiotic drugs in common use. The role of the environment in the transport and dissemination of bacteria producing deactivating enzymes cannot be overlooked given that ESBL-producing Escherichia coli has already been isolated in samples collected from wastewater treatment effluents [68] and in isolates collected from household pets [69].It is accepted knowledge that antibiotics are present as contaminants in a variety of environmental systems [70,71,72,73,74,75,76,77]. Among the class of pollutants referred to as ‘emerging contaminants’, antibiotic compounds have a suspicious reputation because their biological activity is an intrinsic characteristic of their functional design. Antibiotics are introduced into the environment via multiple pathways that include effluents from disposal of human waste, waste from agricultural food animal production and aquaculture of finfish, direct application to some plants, industrial effluents from pharmaceutical production, agricultural run-off and disposal of ethanol production waste products. Acute and chronic bioassays have provided evidence that environmental exposure to low concentrations of some antibiotic drugs have toxic effects in species such as Daphnia magna [78], Selenastrum capricornutum [79] and Artemia [80]. Toxicity evaluations of enrofloxacin and ciprofloxacin using representatives of four photoautotrophic aquatic species found that enrofloxacin did not inhibit growth although toxic responses in some of the macrophytes were observed for exposure to ciprofloxacin at the same environmentally relevant concentrations [81]. Failure to detect antibiotics in environmental samples (especially those with high organic content) does not mean antibiotic residues are not present. The diverse chemical and structural properties of antibiotic compounds in environmental sample matrices make chemical determination problematic in many circumstances. Biodegradation [82,83,84], photodegradation [85,86,87,88,89], chemical complexation or chelation [90,91,92,93,94] and adsorption to particulate matter [95,96,97,98,99,100,101,102,103,104,105] alter the concentrations of antibiotic residues that can be reliably measured in samples of environmental origin. The current analytical method of choice is liquid chromatography combined with tandem mass spectrometry using a variety of extraction methods, frequently solid phase extraction techniques. Despite excellent improvements in analytical determination of antibiotic residues in environmental samples, analyte recoveries, detection limits and reproducibility can be highly variable.The discharge of effluents from wastewater treatment plants represents important point sources of contaminants in the environment. Wastewater treatment plants have been described as ‘hotspots’ for antibiotics [106] and for antimicrobial resistance [107] although some treatment options appear promising for reducing the load of antibiotic residues that could be delivered to the receiving environment. Processes for the removal of oxytetracycline [108], sulfamethoxazole [109,110], trimethoprim [111], and some fluoroquinolones [112,113] have been demonstrated. Biological waste treatment processes rely on complex ecological interactions among the microbial species present in the system reactors. Our understanding of these ecological factors is growing and processes to treat wastewater have been shown to influence the contribution of antimicrobial resistance elements and resistant strains of bacteria released into the environment [114,115,116].Direct application of antibiotic compounds can contribute to the contaminant load on the receiving environment. Oxytetracycline and streptomycin are frequently used in orchards to prevent Erwinia infection in apples and pears. Alternatives to the application of antibiotics on trees to control conditions such as fire blight include spraying orchards with a copper sulphate solution known as ‘Bordeaux mixture’. Although the practice is common and effective in the treatment of fire blight, recent studies have demonstrated that long-term application of copper to agricultural soil altered microbial community structure and altered gene expression in some soil macrofauna [117]. As in the case of human and veterinary medicine, treatment of infections in plants requires judicious use of antimicrobials such that the beneficial ecosystem services provided by a plethora of bacteria in the environment are not compromised.The original concept behind the ecological footprint model was to compare human demands on nature with nature’s ability to regenerate the resources needed to accommodate human consumption for lifestyle needs. Under the rubric of mitigating anthropogenic impacts on climate change and reducing human dependence on limited resources of fossil fuels, production of ethanol is one case where effects to reduce human ecological footprint may contribute to the increase in the microbial ‘resistance footprint’. Conversion of corn starch to ethanol is highly susceptible to contamination by bacteria that will compete with yeast during the fermentation process [118]. In order to control excessive growth of bacterial populations in the production process, antibiotics such as penicillin, erythromycin, virginiamycin and tylosin are frequently added to the tanks where corn mash is mixed with warm water to ferment ethanol. The waste by-product of the ethanol fermentation is nutrient-rich corn mash known as ‘distillers grains’ and is a common ingredient in livestock or poultry feed. Evidence supports the hypothesis that macrolide antibiotics in dried distillers’ grains can persist through the fermentation process and remain active after incorporation in livestock feed [119,120,121]. Environmental contamination by antibiotics is but one factor in the equation that defines the health consequences of antibiotic resistance in bacteria. The combined impact of resistant strains of bacteria, antibiotic resistance genes, degradative enzymes that inactivate antibiotics, and antibiotic compounds can have profound influence on human and ecosystem health. It will take a concerted effort involving antibiotic stewardship, judicious use of antibiotics in human and animal medicine, surveillance of drug use and the incidence of antimicrobial resistance, public awareness campaigns, and government commitment to leading coordinated initiatives for society to be protected from the deleterious consequences of excessive development of antibiotic resistance in pathogens. The environmental footprint of antibiotics must be minimized to ensure that the drugs that we use in human and veterinary medicine remain effective. This is our path of “least resistance”. 

Med-MDPI/antibiotics/antibiotics-02-02-00206.txt

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Seventy years after the introduction of antibiotic chemotherapy to treat tuberculosis, problems caused by drug-resistance in Mycobacterium tuberculosis have become greater than ever. The discovery and development of novel drugs and drug combination therapies will be critical to managing these problematic infections. However, to maintain effective therapy in the long-term and to avoid repeating the mistakes of the past, it is essential that we understand how resistance to antibiotics evolves in M. tuberculosis. Recent studies in genomics and genetics, employing both clinical isolates and model organisms, have revealed that resistance to the frontline anti-tuberculosis drug, rifampicin, is very strongly associated with the selection of fitness compensatory mutations in the different subunits of RNA polymerase. This mode of resistance evolution may also apply to other drugs, and knowledge of the rates and mechanisms could be used to design improved diagnostics and by tracking the evolution of infectious strains, to inform the optimization of therapies.Tuberculosis, caused by infection of the airways and lungs by Mycobacterium tuberculosis, is a major cause of infectious disease-associated morbidity and mortality worldwide [1,2]. Treatment of tuberculosis is difficult and requires relatively long courses of antibiotic treatment. This leads to problems of resistance development during therapy, and as a result, effective therapy is only achieved through the use of combinations of antibiotics [3]. Over the years, new anti-tuberculosis drugs have been discovered, developed and tested, and this discovery process continues today [4], with several anti-tuberculosis drugs in clinical trials [5]. Ongoing drug development has also lead to changes and refinements in the recommended combinations of antibiotics and in therapeutic duration, to improve the effectiveness of therapy against drug-susceptible M. tuberculosis [3]. The increasing prevalence of multidrug-resistant tuberculosis in recent years, in part associated with HIV infections, is driving continuing efforts in drug discovery and development and testing of novel drug combinations, in an attempt to achieve more effective therapy against resistant infections [6]. While the development of new anti-tuberculosis drugs is critical for dealing with the immediate therapeutic problems, in the longer-term, it will also be very important to gain a better understanding of the evolutionary processes that drive drug resistance development, so that we have the possibility to develop rational approaches to reducing the problem of resistance [7].Rifampicin is an oral rifamycin that was shown, in the 1960s, to be effective for treatment of tuberculosis [8,9]. Despite an increasing incidence of resistance, rifampicin remains an important antibiotic and is, together with isoniazid/ethambutol and pyrazinamide, an essential part of short-course anti-tuberculosis treatment [3,10]. The target of rifampicin is the β-subunit of the RNA polymerase, where it binds and inhibits the elongation of RNA transcription shortly after initiation [11,12]. Resistance to rifampicin arises due to single amino acid substitutions in the β-subunit. Classic studies in E. coli [13] supported by data from clinical isolates of M. tuberculosis [14] and from other organisms [15,16] show that rifampicin resistance is nearly always caused by any one of many different point mutations affecting a relatively small part of the β-subunit of RNA polymerase close to the catalytic center of the enzyme [12,17,18]. In M. tuberculosis, the clinical breakpoint for resistance to rifampicin has recently been revised downward, from a minimal inhibitory concentration (MIC) of 1.0 mg/L to 0.0625 mg/L, based on population pharmacokinetic studies [19,20]. In E. coli and Salmonella, where genetic studies are frequently carried out, rifampicin MIC for wild-type strains is approximately 12 mg/L, while a resistant mutant can have an MIC ≥3,000 mg/L [21].The development of resistance to antibiotics in M. tuberculosis is associated with chromosomal mutations rather than with horizontal genetic transfer events, as is common in many other infectious bacteria [22]. Genetic and physiological studies in model organisms, like Salmonella typhimurium (S. enterica serovar Typhimurium) and Escherichia coli, have shown that most chromosomal mutations causing resistance to antibiotics are associated with significant fitness costs, both in vitro and in vivo [23,24,25]. The close association of significant fitness costs with antibiotic-resistance mutations is not surprising when one considers that the processes targeted by antibiotics are usually of central importance to bacterial growth and include the machinery of protein synthesis, DNA replication and transcription and the processes of cell wall synthesis [23]. The concept of a biological fitness cost usually refers to a measured reduction in relative bacterial growth rate in a particular environment, but the concept is broader and embraces for example reductions in transmission efficiency or changes in relative virulence in bacterial pathogens, including for M. tuberculosis [26]. The clinical significance of having fitness costs associated with resistance mutations is that it implies that in mixed bacterial populations and in the absence of sufficient antibiotic selective pressure, that antibiotic-resistant strains would be out-competed and progressively replaced by higher-fitness antibiotic-susceptible strains [25]. If that were the case, then it might be possible, in principle, to restrict the proportion of resistant strains in a population by restricting antibiotic use in general or by periodically cycling different antibiotics in clinical practice. However, there are several serious caveats to that hypothesis. Thus, experiments in model organisms, testing many different antibiotics and resistance mutations, have shown that secondary fitness-compensatory mutations arise frequently in resistant strains (Figure 1) and that these can significantly ameliorate the fitness costs of resistance [23,25]. Compensatory evolution can even make a bad situation worse. Thus, for fluoroquinolones, it has been shown in different species that some compensatory mutations that reduce the fitness costs of resistance mutations simultaneously increase the level of resistance to the antibiotic [27,28]. Accordingly, increased resistance can evolve by Darwinian selection for increased fitness even in the absence of continued antibiotic selection pressure. Also, experimental studies in Pseudomonas aeruginosa have shown that different deleterious rifampicin-resistance mutations can exhibit antagonistic epistasis, partially or completely compensating for each other’s fitness costs [29]. A second caveat concerns resistance mutations that are low-cost or cost-free (Figure 1). At sub-MIC antibiotic concentrations, a condition likely to be relevant for TB, where there is long-term treatment with multiple antibiotics, low-cost resistance is preferentially selected, and such mutants can outcompete the susceptible wild-type, even at very low antibiotic concentrations [30]. The prediction is that low-cost resistance mutations should be selectively enriched among resistant clinical strains, and there is some evidence that such enrichment occurs in M. tuberculosis [31,32,33]. A third complication, that may be particularly relevant for resistance arising in tuberculosis patients treated with combination therapy, is the possibility of epistatic interactions between mutations causing resistance to different antibiotics. It was shown, using P. aeruginosa as a model organism, that the fitness costs of individual mutations causing streptomycin-resistance varied significantly, depending on the presence of particular mutations causing resistance to rifampicin, and also on whether the antibiotic rifampicin was present or absent from the environment [34]. This conclusion, that epistatic interactions may play a role, is supported by a recent analysis of the relationships between resistance mutations and genetic background in multidrug-resistant isolates of M. tuberculosis [35]. Thus, epistasis and genotype-by-environment interactions may each have a significant influence the evolution of multidrug-resistance in M. tuberculosis. Ideally, the fitness costs of antibiotic resistance should be measured in defined groups of infected patients. However, for practical and ethical reasons, such an approach is, at least with current technologies, ruled out. Instead, fitness costs are usually measured in controlled laboratory experiments, using model environments and model organisms, as surrogates for the clinically relevant situations [23]. In measuring fitness, there are several aspects and parameters to consider. (i) It is critically important that isogenic strains be used for the comparison. This makes it practically difficult to use most clinical isolates, unless they can be genetically manipulated or selected, to generate an isogenic pair. In most cases, this problem is solved by selecting or generating mutations in laboratory strains that mimic the mutations found in clinical isolates. Thus, the direct clinical relevance of a particular genetic background usually has to be sacrificed in favor of making a genetically controlled experiment; (ii) It is important that the species being studied is amenable to genetic manipulation, so that individual mutations or combinations of mutations can be evaluated for their effects on resistance and fitness in standard or ‘wild-type’ genetic backgrounds, without the confounding effects of an uncontrolled number of additional genetic variations typically present in different clinical isolates. Such genetic manipulations have been made to determine the evolutionary path to aminoglycoside resistance in M. smegmatis [36], but they are so difficult to achieve in M. tuberculosis, that the use of more genetically amenable species is strongly favored; (iii) It is important that the model system used can be evaluated with a variety of different fitness assays, such that an assay with sufficient discriminatory power to differentiate between relevant isogenic variants can be chosen as appropriate [7,23]. Taking each of these factors into consideration, S. typhimurium represents a practical, genetically amenable, model system to measure the fitness costs associated with mutations to rifampicin resistance [21].Evolution of resistance is usually a two-step process. Most frequently, resistance is initially associated with a reduction of relative fitness that can subsequently be ameliorated by acquisition and selection of additional fitness compensatory mutations.Rifampicin resistance is strongly correlated with mutations in a small stretch of DNA in the gene rpoB, which encodes the β-subunit of RNA polymerase. The so-called rifampicin-resistance-determining-region (RRDR) covers 81 base pairs encoding for the amino acids 507 to 533 in the β-subunit. The great majority (96%) of unrelated clinical rifampicin-resistant M. tuberculosis isolates were found to carry mutations or small deletions in this region that are absent in rifampicin-sensitive isolates [18]. That these mutations in the RNA polymerase are responsible for the rifampicin resistance was finally proven by the construction of mycobacterial shuttle plasmids containing different versions of the rpoB gene. Recombinant Mycobacterium isolates harboring a plasmid containing the wild-type rpoB gene showed no rifampicin resistance, while isolates that had a plasmid carrying a rpoB gene with a proposed RifR mutation were shown to have a rifampicin-resistance phenotype [37].For the remaining 4% of isolates, no rpoB mutations were identified, neither within the RRDR of rpoB nor outside of it, which left open the possibility of mutations outside rpoB being associated with rifampicin resistance. Hypothesizing that efflux pumps might be responsible for the rifampicin-resistance phenotype in this 4% of isolates, transcriptional analyses of 20 efflux pump genes were performed on M. tuberculosis isolates that were phenotypically resistant, but carried no mutation in rpoB. Three different efflux pumps (Rv2936, Rv0783 and Rv0933) were overexpressed in the resistant isolates, suggesting these pumps might be involved in reducing the intracellular rifampicin concentration of the cell. To further analyze whether overexpression of these efflux pumps conferred rifampicin resistance, the relevant pump genes were cloned onto expression vectors and moved into E. coli strains. Overexpression of Rv0783 caused a two-fold increase in rifampicin MIC, while overexpressing Rv2936 increased the rifampicin MIC four-fold. In contrast, overexpression of Rv0933 had no effect on rifampicin resistance. These data proved that overexpression of two different efflux pumps could generate a rifampicin-resistance phenotype in M. tuberculosis [38].In addition to the mutations identified within the RRDR of rpoB that confer rifampicin resistance, two other regions of rpoB have been associated with low-level rifampicin-resistance in the E. coli rpoB gene. These are amino acids 148–153 and 1244–1260 [39]. The amino acids 148–153 lie within a 97 amino acid long insert in the E. coli rpoB gene that does not exist in the M. tuberculosis gene, and so far, no clinical isolates have been reported to carry any mutations in either of these regions, suggesting that these mutations are not of clinical importance.As with many other resistance mutations, RifR mutations do not come without a cost. Competing laboratory-derived rifampicin-resistant isolates against the rifampicin-sensitive parental strain shows a competitive disadvantage of the RifR mutation in the absence of the drug. Depending on the particular mutation, the strain genetic background and the type of competition assay, the fitness of RifR mutants has been found to vary from being indistinguishable from the susceptible wild-type strains down to a relative fitness of approximately 0.2 [33,40,41]. In general, resistance mutations with a lower measured fitness cost in in vitro assays are also the resistance mutations that are more frequently identified in clinical isolates. However, it is notable that resistant clinical isolates have been found to have lower or even no fitness cost, compared with the costs associated with the same resistance mutations measured in vitro-derived isolates. Competition of clinically-derived rifampicin-resistant isolates against their drug-susceptible ancestors indicates that the fitness cost of RifR mutations is generally lower the longer the resistant strain has infected the patient, suggesting that fitness-compensatory evolution may have occurred in vivo [40]. Genetic evidence for compensatory evolution to reduce the fitness costs associated with rifampicin-resistant RNA polymerase was first shown in E. coli. Four strains with different RifR mutations were evolved in vitro by serial passage for increased competiveness fitness. After 200 generations of evolution, isolates of three of the four strains showed a significant increase in growth rate, proving that compensatory evolution had taken place. Partial sequencing of the rpoB gene of the evolved isolates showed secondary mutations within the gene in about half the isolates, suggesting that secondary mutations within the rpoB gene can compensate the fitness cost of the initial RifR mutation [42]. Since clinical M. tuberculosis isolates sometimes contain multiple mutations within the RRDR of rpoB and the majority of the putative compensatory mutations in E. coli were found within this region, it is reasonable to conclude that the presence of multiple mutations in clinical isolates also indicates a mixture of primary RifR mutations and secondary compensatory mutations. Even so, in about half of the evolved E. coli strains, no compensatory mutation was identified within the rpoB gene, suggesting that the fitness cost caused by the primary RifR mutation could also be compensated by mutations outside this gene. In a more recent study, a S. typhimurium strain containing the high-cost RifR mutation rpoB R529C was evolved for 60 generations by serial passage to select increased fitness. Faster growing isolates were identified, and the genes, rpoA, rpoB and rpoC, encoding for the RNAP α, β and β' subunits, respectively, were sequenced. In agreement with the previous E. coli data, about half of the strains were found to contain a secondary mutation within rpoB. In the other strains, secondary mutations were found in rpoA or rpoC (Figure 2). With the exception of one secondary mutation in rpoB that by itself causes a RifR phenotype, the secondary mutations selected in E. coli and Salmonella did not significantly increase or decrease the MIC [21,42]. Genetic reconstructions were made and showed that each of these secondary mutations was necessary and sufficient to compensate for the fitness cost caused by the primary RifR mutation, proving that mutations within three different subunits of RNA polymerase can compensate for the fitness cost caused by a primary RifR mutation [21]. The reasons why fitness costs are frequently associated with RifR mutations are not fully understood. However, at least some mutations in rpoB destabilize the interaction between RNAP and ribosomal RNA promoters in E. coli, a phenotype that could explain reduced fitness and also could plausibly be compensated by secondary mutations affecting other subunits of RNAP [43,44]. Fitness compensatory mutations that ameliorate the fitness costs of the rifampicin-resistance mutation, rpoB R529C, occur in rpoA, rpoB and rpoC genes, coding for different subunits of RNA polymerase.Recent studies on rifampicin-resistant M. tuberculosis have underlined the clinical importance of these compensatory mutations. Whole genome sequencing of ten paired clinical rifampicin-resistant isolates and their susceptible ancestors showed that four of the ten isolates had a putative compensatory mutation in the rpoA or rpoC gene. Local sequencing of the rpoA, rpoB and a part of the rpoC genes from 117 MDR strains representing the five main global MTBC lineages and 212 MDR strains from high-burden countries (Abkhazia/Georgia, Kazakhstan and Uzbekistan) showed that about 20% of the global and 32% of the high-burden MTBC isolates, contained a secondary mutation in one of the three RNAP genes [45]. Another study analyzing whole genome sequences of M. tuberculosis strains of the Beijing family isolated in Russia further confirmed these results. Twenty-six rifampicin-resistant and seven susceptible strains were analyzed. While none of the susceptible strains carried a mutation in any of the RNAP genes, except one mutation in rpoC that arose before the evolution of rifampicin-resistance, each one of the 26 RifR strains had at least one known RifR mutation in rpoB and at least one additional mutation in rpoA, rpoB or rpoC [46]. These studies show that secondary mutations in the RNA polymerase genes occur frequently, and based on the conclusions from the genetic reconstructions made in Salmonella [21], they presumably compensate for the fitness costs of primary RifR mutations in clinical M. tuberculosis. Another study of compensatory mutations in the rpoC gene of rifampicin-resistant M. tuberculosis suggests that these secondary mutations may increase not only the fitness of the RifR strains, but may also enhance the spread of the resistant strain. A set of 286 drug-resistant and 54 drug-sensitive clinical isolates from Cape Town, South Africa, were analyzed for the presence of mutations in a small part of the rpoC gene, which had earlier been found to be a hot spot for putative compensatory mutations [45] and classified according to their IS6110 RFLP patterns. Interestingly, putative compensatory mutations in rpoC were found in 31% of the isolates belonging to recognized RFLP clusters, while only 9% of the isolates with non-clustered RFLP patterns harbored secondary mutations in rpoC. Since clustered RFLP patterns are associated with ongoing transmission, the data suggest that rifampicin-resistant strains with a secondary mutation in rpoC are more likely to spread within the population than strains without a secondary mutation [47].The data from genomics and genetics analyses support the conclusion that the evolution of rifampicin-resistance in M. tuberculosis is strongly associated with selection for fitness-compensatory mutations, occurring in different subunits of the RNA polymerase. This knowledge could be applied to study the evolution of resistance to other drugs and in the design of improved diagnostics. By tracking the evolutionary trajectories of infectious strains, it may be possible to use genomics information as an aid in the optimization of tuberculosis therapies.Work in the authors’ laboratory is supported by grants from the Swedish Research Council (Vetenskapsrådet), EU Project Predicting Antibiotic Resistance (PAR, Grant No. 241476), Swedish Strategic Research Foundation (SSF), Swedish Innovation Agency (Vinnova) and the Knut and Alice Wallenberg Foundation (RiboCORE Project) to D.H.The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Bacterial infections caused by antibiotic-resistant isolates have become a major health problem in recent years, since they are very difficult to treat, leading to an increase in morbidity and mortality. Fosfomycin is a broad-spectrum bactericidal antibiotic that inhibits cell wall biosynthesis in both Gram-negative and Gram-positive bacteria. This antibiotic has a unique mechanism of action and inhibits the initial step in peptidoglycan biosynthesis by blocking the enzyme, MurA. Fosfomycin has been used successfully for the treatment of urinary tract infections for a long time, but the increased emergence of antibiotic resistance has made fosfomycin a suitable candidate for the treatment of infections caused by multidrug-resistant pathogens, especially in combination with other therapeutic partners. The acquisition of fosfomycin resistance could threaten the reintroduction of this antibiotic for the treatment of bacterial infection. Here, we analyse the mechanism of action and molecular mechanisms for the development of fosfomycin resistance, including the modification of the antibiotic target, reduced antibiotic uptake and antibiotic inactivation. In addition, we describe the role of each pathway in clinical isolates.Infectious diseases are the second-major cause of death worldwide and the third-leading cause of death in economically advanced countries [1]. Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent and represent a priority health problem [2]; hence, the problem of antibiotic resistance needs an urgent response. Developing a new antibiotic can take years and millions of dollars. Therefore, in the meantime, the rational use or retrieval of old antibiotics, like polymyxins, fusidic acid, co-trimoxazole, aminoglycosides, chloramphenicol and fosfomycin, may be a short-term solution [3].Here, we focus our analysis on fosfomycin (also termed phosphomycin and phosphonomycin), a natural antibiotic compound produced by several Streptomyces and Pseudomonas species, exerting a powerful bactericidal activity against a wide range of Gram-negative and Gram-positive bacteria [4]. Fosfomycin is a phosphonic acid derivative containing an epoxide and a propyl group [(2R,3S-3-methyloxiran-2-yl) phosphonic acid] with a unique chemical structure (Figure 1). This molecule, with a very low molecular weight, is within a class of its own and is unrelated to any other antibiotic family, in addition to having an exclusive target, the initial step in peptidoglycan biosynthesis [5]. Chemical structure of fosfomycin [(2R,3S-3-methyloxiran-2-yl) phosphonic acid].Fosfomycin is mainly used for the treatment of uncomplicated urinary tract infections (UTIs) [6], and various formulations are available. The form of the medication for intravenous use is fosfomycin disodium salt. For oral use, the antibiotic is combined in calcium salt or formulated with tromethamine. Fosfomycin tromethamine is primarily administered in a single dose, reaching a very high antibiotic concentration that is able to successfully kill the most common urinary pathogens [7]. It has also been used in combination with other antibiotics in the treatment of patients suffering serious infections, systemic infections with sepsis or nosocomial infections [8]. Fosfomycin shows a powerful bactericidal activity against enteric Gram-negative bacteria, such as Escherichia coli, although some of them have decreased susceptibility, as Klebsiella pneumoniae and Enterobacter cloacae. Fosfomycin is also very effective against Gram-positive cocci, like Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis and Enterococcus faecium [9].The analysis of its activity against nine commonly encountered bacteria associated with urinary tract infection has revealed a high susceptibility in E. coli isolates and most K. pneumoniae, E. cloacae and S. aureus strains [10]. In addition, E. faecalis and E. faecium isolates were also quite susceptible to fosfomycin, yet with higher MIC values. However, Acinetobacter baumannii isolates were resistant to fosfomycin, while P. aeruginosa and Stenotrophomonas maltophilia showed moderate susceptibility [10]. Fosfomycin has been successfully evaluated as a treatment option for infections caused by multiple drug resistant (MDR) Gram-negative and Gram-positive bacteria [11,12]. For example, a survey of clinical MDR Enterobacteriaceae isolates, including producers of extended-spectrum β-lactamases (ESBL), showed that >90% of E. coli and >80% of K. pneumoniae isolates were susceptible to fosfomycin [13]. Fosfomycin is a bactericidal antibiotic that inhibits the initial step in the biosynthesis of peptidoglycan in prokaryotes [5]. Peptidoglycan is assembled from a building block composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid with an attached pentapeptide. Fosfomycin acts as a phosphoenolpyruvate (PEP) analogue and binds MurA (UDP-GlcNAc enolpyruvyl transferase), an essential enzyme for peptidoglycan biosynthesis [14], preventing the formation of UDP-GlcNac-3-O-enolpyruvate from UDP-GlcNAc and PEP during the first step in peptidoglycan biosynthesis, leading to bacterial cell lysis and death [5] (Figure 2). The antibiotic can enter into the active site of MurA and inhibits this enzyme by covalently binding via a thioether bond formation with a key residue in the active site, Cys115 [15,16]. The crystal structure of E. coli MurA complexed with UDP-GlcNAc and fosfomycin has revealed that the Cys115-bound molecule is tightly packed between the enzyme and the substrate, forming strong electrostatic interactions between three conserved positively charged residues of MurA (Lys22, Arg120 and Arg397) and the phosphonate group of the antibiotic [16]. There are different mechanisms leading to fosfomycin resistance:(i) Reduced permeability to fosfomycin. Since the discovery of fosfomycin, it was established that the main mechanism for the acquisition of antibiotic resistance was an impaired fosfomycin transport, due to mutation of any of the target genes encoding the antibiotic permeases. In E. coli, two main nutrient transport systems are responsible for fosfomycin uptake, the glycerol-3-phosphate transporter (GlpT) and a hexose phosphate transporter, the glucose-6-phosphate transporter (UhpT) [5]. The expression of the GlpT and UhpT transporters is induced by their substrates, glycerol-3-P and glucose-6-P, respectively, and requires the presence of cAMP-CRP (Figure 3). Mutations in any of the structural genes of those pathways produce a decrease in antibiotic uptake, conferring different levels of fosfomycin resistance [5,17,18]. Strains defective in fosfomycin uptake are not able to grow using some substrates as the sole carbon source, glycerol-3-P in GlpT-deficient strains or glucose-6-P (and other hexose phosphates) in Uhp-deficient strains. Mutants affected in both systems are often unable to grow using multiple carbohydrates. In fact, it has been observed that the addition of glucose-6-P induces fosfomycin sensitivity in resistant GlpT-deficient strains, due to the induction of UhpT synthesis [5]. Therefore, the measurement of MIC to fosfomycin in E. coli is performed using media with and without glucose-6-P [19]. However, the addition of glucose-6-P recommended by the CLSI manual provides a more reliable MIC result, due to its activity as inducer of fosfomycin transport.Although transporters are usually very selective, the chemical structure of fosfomycin mimics both glycerol-3-P (G3P) and glucose-6-P (G6P), which are transported under normal conditions. MurA catalyses the formation of UDP-GlcNac-3-O-enolpyruvate, a peptidoglycan precursor, from UDP-GlcNAc and PEP during the first step of peptidoglycan biosynthesis, allowing cell growth (A). In contrast, when fosfomycin (F) is present, it is transported inside the cell by GlpT and UhpT, blocking the UDP-GlcNac-3-O-enolpyruvate synthesis by mimicking the original substrate of MurA, PEP, avoiding cell wall synthesis and leading to cell death (B). For simplicity, only peptidoglycan and the inner membrane are shown.While fosfomycin uptake depends on GlpT and UhpT in E. coli, it has been reported that fosfomycin can only enter into the cells via GlpT in P. aeruginosa, due to the absence of UhpT permease. As a result, glpT is the only target gene whose inactivation confers antibiotic resistance in P. aeruginosa [20]. All the fosfomycin-resistant mutants generated in P. aeruginosa by spontaneous mutations are affected in glpT [20]. However, these mutations appear to be cost-free, probably because this species cannot use glycerol-3-P as a sole carbon source, even in glpT+ wild-type strains [21].Fosfomycin uptake is essential for antibiotic activity, and intrinsic resistance to the antibiotic in some pathogenic species is caused by the lack of transport. A paradigmatic example is Listeria monocytogenes. This species is unable to uptake the antibiotic in vitro and, consequently, is resistant to fosfomycin. Nevertheless, a central virulence regulator, PrfA, induces in vivo the virulence factor Hpt, a glucose-6-P permease that also mediates the uptake of fosfomycin, conferring antibiotic susceptibility during infection [22,23].Regulation of GlpT and UhpT. In E. coli and several Enterobacteria, the expression of glpT and uhpT requires the presence of the cAMP, which together with the receptor protein complex (CRP) forms the cAMP receptor protein complex (cAMP-CRP). This complex binds to the specific promoter sites of both genes, glpT and uhpT, and promotes their expression. Both transporters experience additional regulation. On the one hand, glpT gene expression is also controlled by the repressor, GlpR, which becomes inactive when it is bound to glycerol-3-P (G3P), and on the other hand, of uhpT; high-level expression also requires the regulatory genes, uhpA, uhpB and uhpC, which sense and transduce signals by phosphorylation when hexose phosphates are detected, thereby positively regulating the transcription of the gene.GlpT system. GlpT is a glycerol-3-phospate permease, a protein belonging to the organophosphate phosphate antiporter (OPA) family of the major facilitator superfamily (MFS). GlpT transporters are present in various bacterial species with a high degree of sequence conservation, and homologues are widely distributed in all phyla [24]. The structure of E. coli GlpT reveals two domains connected by a long central loop, with a substrate translocation pore located between the two domains opened to the cytoplasm [25,26]. As an integral inner membrane component, the GlpT protein contains 12 highly conserved transmembrane α-helices typical of all glycerol-3-P permeases [25,26]. GlpT catalyses an exchange of external glycerol-3-P for internal Pi, acting as a secondary active transporter for glycerol-3-P uptake [27].This permease also provides an entrance mechanism for fosfomycin, owing to this antibiotic mimicking glycerol-3-P. The acquisition of mutations affecting GlpT confers fosfomycin resistance, decreasing the antibiotic uptake into the bacterial cells [5,28,29]. The interaction between GlpT and fosfomycin has recently been characterized in proteoliposomes, showing that fosfomycin competes for the substrate-binding site of the permease and is transported by the protein in vitro [30]. The expression of GlpT in E. coli is induced by the presence of glycerol-3-P. The repressor, GlpR, blocks glpT transcription by binding to the operators near the glpT promoter. The interaction of GlpR with glycerol-3-P reduces its affinity for the glpT operator and activates GlpT synthesis [31,32,33] (Figure 3). Inactivation of GlpR leads to a constitutive expression of glpT. UhpT system. UhpT is a hexose phosphate transporter responsible for the accumulation of glucose-6-P and the uptake of fosfomycin into the bacterial cells. Fosfomycin-resistant strains defective in growth with hexose phosphates as a carbon source, such as glucose-6-P, carry mutations in the uhp genes [34].UhpT is also a member of the Major Facilitator Superfamily that exchanges a cytoplasmic phosphate Pi for a hexose phosphate [35]. UhpT transporters show an extensive amino acid sequence homology with glycerol-3-P transporters, GlpT [36]. In a similar way as GlpT, UhpT is a monomer, with twelve transmembrane alpha-helical segments [37,38]. In addition, the UhpT transport system is controlled by several regulatory components, including uhpA, uhpB and uhpC, whose products are necessary for high-level expression of the UhpT transporter (Figure 3). Inactivation of any of these regulatory genes also leads to fosfomycin resistance, due to an inhibited or reduced expression of the UhpT transporter, thereby preventing fosfomycin uptake into the cells [34].The integral membrane component UhpC detects the extracellular signal, glucose-6-P and activates UhpB [39]. UhpB is a sensor histidine kinase in a two-component regulatory system with UhpA based on His-to-Asp phosphoryl transfer. UhpA, a DNA-binding response regulator, binds to two adjacent regions, an upstream strong binding site and a downstream weak binding site, in the uhpT promoter. Phosphorylation of UhpA stimulates DNA binding, hence promoting uhpT transcription [40,41,42] (Figure 3).Regulation: levels of cyclic adenosine monophosphate (cAMP). High levels of cAMP are required for the full expression of the fosfomycin transporters, GlpT and UhpT, in Enterobacteria. cAMP synthesis depends on the activity of adenyl cyclase CyaA. cAMP levels are also regulated by the phosphotransferase enzyme, PtsI, a component of the PEP sugar phosphotransferase transport system. Mutations in cyaA or ptsI cause a decrease in the intracellular cAMP levels and, consequently, a reduced synthesis of both fosfomycin transporters, leading to a diminished antibiotic uptake [43,44,45,46].Inactivation of the cAMP receptor protein CRP impairs the expression of both transporter systems, reducing the susceptibility to fosfomycin [43]. cAMP-CRP recognizes several binding sites upstream of the glpTQ operon in a DNA stretch, controlled negatively by GlpR and positively by cAMP-CRP [47]. This global regulator also binds to the E. coli uhpT promoter at a single site upstream of the UhpA-binding sites (Figure 3). Transcription of the uhpT gene requires the response regulator UhpA and is stimulated by the global regulator protein, cAMP-CRP [48]. cAMP-CRP stabilizes the open promoter complexes for uhpT transcription and also enhances the rate of their formation [49].(ii) Modification of the antibiotic target MurA. Modification of the antibiotic target is one of the most common mechanisms to acquire antibiotic resistance in bacteria. MurA, an essential enzyme, is the target of the antibiotic fosfomycin, which, as mentioned, inactivates the enzyme by irreversibly binding to the protein. In E. coli mutation of the fosfomycin-binding site in MurA, Cys115, results in resistance to this antibiotic [50]. After a protein mutagenesis analysis, it has been proposed that the catalytic residue, Cys115, acts as a general acid-base catalyst, promoting the enzymatic reaction. When the enzyme mediates the enolpyruvyl transfer from PEP to the 3'–OH of UDP-GlcNAc, MurA-Cys115 reacts with PEP (or fosfomycin) to form a covalent phospholactoyl-enzyme adduct [15,51,52,53]. In addition, MurA-Cys115 seems to be essential for product release, i.e., inorganic phosphate and UDP-GlcNAc-3-O-enolpyruvate [54].MurA shows an enzymatic activity susceptible to be blocked by fosfomycin in a dose-dependent manner. However, the Cys115 to Asp mutation in the E. coli MurA generates a fully active enzyme, yet completely insensitive to inhibition by fosfomycin, while the Cys115 to Glu mutant shows no enzymatic activity [50]. The impact of these types of mutations in the acquisition of fosfomycin resistance is reflected by the presence of an Asp residue in the catalytic site of MurA proteins encoded by pathogenic bacteria with intrinsic resistance to fosfomycin, such as Mycobacterium tuberculosis, Chlamydia trachomatis and Borrelia burgdorferi [55,56,57]. In addition, antibiotic resistance to fosfomycin was acquired in E. coli by the expression of this naturally resistant enzyme when endogenous E. coli murA was conditionally inactivated [57]. Conversely, mutation of the wild-type aspartate residue in the MurA active site to a cysteine renders an enzyme sensitive to fosfomycin in M. tuberculosis and B. burgdorferi [55,56]. Modification of the fosfomycin target to acquire antibiotic resistance seems to be rare in clinical isolates. A fosfomycin-resistant E. coli mutant affected in MurA affinity to the antibiotic was characterized [58]. However, very few reports of clinical isolates show mutations in the murA gene, and none in the catalytic site of MurA. Recently, mutations in the MurA sequence of clinical isolates of E. coli, Asp369 to Asn and Leu370 to Ile, have been suggested to contribute to the development of fosfomycin resistance in vivo [59]. These mutant proteins are less susceptible to the inhibitory activity of this antibiotic. Both highly conserved residues could be important for PEP substrate binding and, thus, may affect interaction between the enzyme and fosfomycin [59]. Since fosfomycin produces covalent modification of MurA, increased synthesis of the enzyme confers a resistant phenotype [60]. Indeed, the analysis of a complete E. coli library of gene amplifications (the ASKA collection) has revealed that murA is the only gene in the entire E. coli genome capable of conferring clinical levels of antibiotic resistance when overexpressed [61]. Increased MurA levels in E. coli correlate with higher levels of fosfomycin resistance, reaching clinical resistance levels (32 µg/mL) at a low fitness cost. In this sense, it has been shown that the enhanced expression of the murA gene contributes to the acquisition of fosfomycin resistance in several E. coli clinical isolates [62].(iii) Antibiotic modification. Several enzymes are able to modify fosfomycin, producing chemical changes that inactivate it. Microbial resistance to fosfomycin by antibiotic modification in pathogenic strains involves one of three different fosfomycin resistance proteins (FosA, FosB or FosX). All of them catalyse the opening of the oxirane ring of the antibiotic, rendering it ineffective. Nevertheless, they differ in terms of chemical mechanism, using different substrates to add chemical groups to the antibiotic [63]. Structure-based sequence alignments of Fos proteins show remarkable sequence homology among them, with a limited set of residues that differ among Fos enzymes and confer different catalytic properties to each class (Figure 4). This allows different fos genes to recombine via homologous recombination, leading to recombinant enzymes that confer fosfomycin resistance, as shown between Mesorhizobium loti FosX and P. aeruginosa FosA [64].Amino acid sequence alignment generated by ClustalX (under Bioedict) of three representative sequences of fosfomycin resistance proteins (Fos) present in different bacterial species. Represented sequences are FosA [P. aeruginosa 18A], FosA3 [E. coli], FosB [S. aureus subsp. aureus USA300_TCH1516], FosC2 [E. coli] and FosX [Clostridium botulinum Ba4 str. 657]. Fos enzymes belong to the same divalent metal-ion dependent metalloenzymes, the vicinal oxygen chelate superfamily (VOC), sharing a high number of core-conserved or identical residues in their sequences.Fosfomycin resistance proteins (FosA, FosB and FosX) are members of the same metalloenzyme superfamily, the divalent metal-ion dependent enzymes [63]. They are evolutionarily related and form a group of enzymes related to glyoxalase I, methylmalonyl-CoA epimerase and extradiol dioxygenases, all members of the same metalloenzyme superfamily. The members of the metalloenzyme superfamily, the vicinal oxygen chelate superfamily (VOC), share a common structural fold that provides a very versatile metal coordination environment, mediating the catalysis of a very diverse set of reactions [65,66].FosA. FosA was first identified as a fosfomycin resistance determinant able to modify and inactivate the antibiotic in conjugative multiresistance plasmids from Enterobacteriaceae clinical isolates [67,68,69]. The gene fosA was found to reside in a transposon, Tn2921, in some plasmids [70]. The nucleotide sequence of this transposon has revealed that fosA is flanked by two identical insertion sequences (ISs) and associated with genes showing striking similarity to a genomic segment from Enterobacter cancerogenus [71]. In fact, close relatives of FosA, with catalytic properties very similar to those of the plasmid-encoded enzyme, also appear in microbial genomes, such as that of the pathogen P. aeruginosa [72]. The fosfomycin resistance protein, FosA, is an Mn2+-dependent glutathione S-transferase that inactivates fosfomycin by the addition of glutathione to the oxirane ring of fosfomycin, rendering it inactive [73,74] (Figure 5). Overexpression of FosA from a plasmid in E. coli confers fosfomycin resistance in a wild-type strain, but not in cells deficient in glutathione biosynthesis [75]. FosA acts as a homodimeric metalloenzyme with an Mn2+ molecule bound to each subunit in a metal binding site that interacts strongly with the substrate fosfomycin [72,75,76]. In addition, FosA also requires K+ for optimal activity, due to the 100-fold activation effect of the monovalent cation when it interacts with the catalytic site [77]. Functional analysis of the FosA sequence has revealed several residues involved in substrate binding and ligands to the Mn2+ and the K+ ions that are essential for enzymatic activity [78].Reactions catalysed by Fos metalloenzymes (FosA, FosB and FosX) and fosfomycin kinases (FosC, FomA and FomB). Fosfomycin-inactivating enzymes modify the antibiotic, rendering it inactive by opening the oxirane ring (metalloenzymes) or by phosphorylation (fosfomycin kinases). Substrates and the metal requirement for each enzyme are also shown. FosB. FosB is a thiol-S-transferase related to FosA that was first detected in a plasmid conferring resistance to fosfomycin in Staphylococcus epidermidis [79,80]. Fosb has been widely detected in the chromosomes and plasmids of many low G+C Gram-positive bacteria, including Bacillus subtilis, Bacillus anthracis, Bacillus cereus, S. aureus, S. epidermidis and E. faecium. In B. subtilis, intrinsic fosfomycin resistance depends on the presence of the fosB (yndN) gene in the bacterial chromosome. Expression of fosB requires the extracytoplasmic sigma factor, SigW [81], a regulator with a prominent role in providing inducible resistance to antimicrobial compounds [82]. Therefore, fosB or sigW mutants are fosfomycin-sensitive in B. subtilis [81].FosB was initially characterized as an Mg2+-dependent l-cysteine thiol transferase that catalyses the addition of a thiol group using l-cysteine as a donor substrate [81]. B. subtilis FosB is a dimer whose activity is almost 10-fold greater with Mg2+ or Ni2+ than with Mn2+ as a cofactor. In contrast to FosA, FosB shows no monovalent metal dependence [81].Recently, it has been suggested that bacillithiol, the α-anomeric glycoside of l-cysteinyl-d-glucosamine with l-malic acid could be the thiol donor in vivo for FosB [83] (Figure 5). Cells lacking bacillithiol show a dramatic increase in fosfomycin sensitivity in B. subtilis, B. anthracis and S. aureus [83,84,85]. The increase in fosfomycin sensitivity, due to the lack of bacillithiol in B. subtilis, was similar to a fosB null mutant and to a fosB and bacillithiol biosynthesis double mutant [83]. Kinetic analysis of FosB from S. aureus has confirmed that the enzyme is a divalent metal-dependent (Mg2+ and Mn2+) thiol S-transferase, and bacillithiol is its preferred thiol substrate under physiological conditions [85].FosX. FosX hydrolases are a subfamily of enzymes related to FosA and FosB, sharing 30%–35% sequence identity with both groups of enzymes and detected in the survey of the microbial genome sequences [86]. fosX homologues have been identified in the chromosome of several microorganisms, including Mesorhizobium loti, and the pathogens, L. monocytogenes, Clostrium botulinum and Brucella melitensis.FosX is a Mn2+-dependent epoxide hydrolase that catalyses the hydration of fosfomycin [86,87] (Figure 5). The enzyme mediates the addition of water to the C1 position of the antibiotic, breaking the oxirane ring, producing a diol product. The reaction involves an essential glutamic acid residue in the FosX active site acting as a general base catalyst for the reaction [86,87].Other fosfomycin-inactivating enzymes. Microorganisms able to synthesize fosfomycin, such as some strains of Streptomyces and Pseudomonas syringae, are resistant to high concentrations of the antibiotic. Antibiotic-producing organisms usually associate biosynthetic genes to resistance genes in gene clusters in order to protect cells from the harmful effect of the antibiotic [88,89]. Fosfomycin producers have antibiotic kinases unrelated to Fos metalloenzymes that modify and detoxify the antibiotic inside the cells. In Streptomyces spp., two fosfomycin kinases sequentially modify the antibiotic in the presence of ATP and Mg2+. FomA converts fosfomycin to fosfomycin monophosphate, while FomB produces fosfomycin diphosphate using the monophosphate form as a substrate [88,89] (Figure 5). FomA shares homology with the amino acid kinase family and also with the resistance protein, FosC, from P. syringae and differs from FomB. The structure of the FomA enzyme bound to fosfomycin shows a typical fold of the amino acid kinase family of enzymes, and important structural similarities [90]. Intrinsic resistance to fosfomycin in P. syringae relies on a fosfomycin kinase named FosC, which is able to phosphorylate the antibiotic to produce fosfomycin monophosphate using ATP in the reaction [91] (Figure 5). However, a recent article demonstrated that FosC from P. syringae is actually an ortholog of FomA [92].In clinical isolates, the main mechanism for the development of fosfomycin resistance is a reduced permeability of the cell membrane. The contribution of plasmid-encoded inactivating enzymes to the overall resistance to fosfomycin in clinical strains seems to be low [93,94]. Modification of the antibiotic target, MurA, also seems to be a very rare mechanism in fosfomycin-resistant clinical isolates, although it is clearly very important for intrinsic resistance in some pathogens [55,56,57]. Only a few reports have described mutations in the murA sequence or murA expression that could be associated with fosfomycin resistance [59,62]. This suggests that changes in the MurA sequence reducing its affinity for fosfomycin can also affect the essential process of peptidoglycan biosynthesis. This could be explained by the fact that MurA is an essential enzyme and, apparently, can be modified by point mutations in a few specific residues without rendering it inactive. In our experience, the probability of finding a murA mutant resistant to fosfomycin in a bacterial population is around 10−9 to 10−10 in vitro, while gene inactivation (glpT, for instance) is three orders of magnitude higher.An analysis of plasmid-encoded fosfomycin resistance in pathogenic bacteria has revealed a relatively low percentage of fosA and fosB genes among fosfomycin-resistant strains. Only five strains out of 219 fosfomycin-resistant isolates carried plasmids harbouring fosA (three Enterobacteria) and fosB (two staphylococci). The mechanism of antibiotic resistance in the other isolates was caused by the alteration of the chromosomally encoded antibiotic uptake systems [93]. No transferable plasmid-encoded fosfomycin resistance was found among a collection of P. aeruginosa antibiotic resistance strains [95].Although the incidence of plasmid-encoded fosfomycin-modifying enzymes is still low, plasmids that encode Fos enzymes are very often associated with other antibiotic resistance genes, leading to the emergence of multidrug resistance strains. In a survey of CTX-M β-lactamase-producing E. coli clinical isolates, three out of ten fosfomycin-resistant strains contained two different FosA-like enzymes with glutathione S-transferase activity, FosA3 and FosC2, encoded in transferable multiresistance plasmids simultaneously conferring a cefotaxime resistance phenotype [96]. Multiresistance plasmids encoding fosA3 and, to a lesser extent, fosC2 are emerging among CTX-M β-lactamase-producing E. coli and K. pneumonia isolates in Asia (China, Japan and South Korea) [96,97,98,99]. For instance, the multidrug resistance plasmid, pHN7A8, carries the bla(TEM-1b), bla(CTX-M-65), fosA3 and rmtB genes conferring resistance to penicillins, cephalosporins, fosfomycin and aminoglycosides, respectively [100]. Plasmid pKP96 carries nine genes (fosA among them), conferring resistance to several antibiotics, including penicillins, cephalosporins, fosfomycin, aminoglycosides, tetracycline, quinolones and sulfamethoxazole [101]. Therefore, the acquisition of fosfomycin resistance mediated by antibiotic-modifying enzymes shows a higher incidence in multidrug resistance strains. From a collection of 21 isolates with extended-spectrum β-lactamase, seven strains (five E. coli and two K. pneumoniae) harboured both fosA3 and blaCTX connected via insertion sequences in different multiresistance plasmids [102]. Fos enzymes encoded in the chromosome contribute to intrinsic resistance, but they could also be important in pathogenic bacteria for the development of antibiotic resistance, as seen in P. aeruginosa overexpressing chromosomally encoded fosA [103].A recent study of six fosfomycin-resistant E. coli clinical isolates showed that all of them contained glpT mutations with an impaired GlpT transport system, and five of them were unable to grow using glycerol-3-P. Two of these strains with a high level of fosfomycin resistance were also defective in UhpT by gene loss, but all the other resistant isolates were functional in UhpT transport growing with glucose-6-P [59]. A previous report by Nilsson et al [104] about the molecular mechanism of fosfomycin resistance in 13 E. coli clinical isolates revealed that the highest level of fosfomycin resistance required simultaneous inactivation of both transport pathways, GlpT and UhpT (a glpT stop codon + uhpA deletion in this strain). By contrast, most of the resistant strains analysed were only defective in the UhpT transport system, growing with glycerol-3-P, but not with glucose-6-P as a sole carbon source. Nevertheless, among those strains, the uhpT and/or uhpA genes, inactivating mutations were only detected in four of them, although it is possible missense mutations were not evaluated [104]. In a survey of fosfomycin-resistant isolates in the urinary isolates of E. coli producing extended-spectrum beta-lactamases, a cluster of five isolates carried an uhpA deletion [105], showing that mutations targeted to chromosome genes could be important for the development of antibiotic multiresistance. Although mutations in uhpA are often detected in clinical isolates, this is not the case for uhpB or uhpC. Experiments of in vitro mutagenesis by insertions in uhpA led to the loss of uhpT expression; however, a high proportion of uphB or uhpC mutations retained uhpT expression [39]. A high level of fosfomycin resistance has also been described by the concurrent effects of increased murA expression/murA point mutations and alteration of the GlpT or/and UhpT transport systems in two Shiga-Like Toxin-producing E. coli strains [59,62].Finally, it is known that mutations in cyaA or ptsI, which in turn decrease the level of cAMP, provoke a profound disturbance in carbohydrate metabolism of bacteria and may have a high biological cost [104]. Therefore, it is expected that these mutations per se lack clinical relevance unless bacteria find compensatory mutations. The need for this compensation certainly diminishes the probability of finding these mutants in clinical settings.The sequence changes in fosfomycin-resistant strains isolated both in vitro and in clinical settings show that large and small deletions are the main source of gene-inactivating mutations, followed by insertions/duplications. In addition, a considerable number of point mutations have been detected, including truncations by nonsense and missense mutations [20,104,106]. As an example, the sequence of glpT in 20 fosfomycin-resistant isolates of P. aeruginosa, where all fosfomycin resistance mutations are targeted to glpT, has been explored detecting 14 deletions (nine frameshifts), five-point mutations and one insertion/duplication [20,106].Fosfomycin has been used for a long time, but the emergence of antibiotic resistance and the decline in newly developed antibiotics has increased interest in the treatment of bacterial infections with this antibiotic. The mechanisms of acquisition of fosfomycin resistance should be considered in detail so as to optimize therapy and avoid the further development of antibiotic resistance. Evaluation of fosfomycin susceptibility in clinical strains is widely performed, but the molecular bases are frequently unexplored. In addition, several clinical trials have recently been performed to evaluate the potential application of fosfomycin in the treatment of bacterial infections, including those caused by multidrug resistant isolates, most of them with promising results. Therefore, a more-in-depth knowledge of the molecular mechanisms leading to fosfomycin resistance in clinical strains could improve the successful use of fosfomycin for the treatment of bacterial infections.This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, co-financed by the European Development Regional Fund “A way to achieve Europe” ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015 and FIS PI10/00105) and the PAR Project (Ref 241476) of the EU 7th Framework Programme. ACG was supported by a postdoctoral grant in life sciences from the Fundacion Ramon Areces. ARR was supported by The Collaborative Research Centre (CRC) 973 “Priming and Memory of Organismic Responses to Stress”. Some of the figures were generated from the online pathways chart of SABioscience.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The development of antibiotic resistance is usually associated with genetic changes, either to the acquisition of resistance genes, or to mutations in elements relevant for the activity of the antibiotic. However, in some situations resistance can be achieved without any genetic alteration; this is called phenotypic resistance. Non-inherited resistance is associated to specific processes such as growth in biofilms, a stationary growth phase or persistence. These situations might occur during infection but they are not usually considered in classical susceptibility tests at the clinical microbiology laboratories. Recent work has also shown that the susceptibility to antibiotics is highly dependent on the bacterial metabolism and that global metabolic regulators can modulate this phenotype. This modulation includes situations in which bacteria can be more resistant or more susceptible to antibiotics. Understanding these processes will thus help in establishing novel therapeutic approaches based on the actual susceptibility shown by bacteria during infection, which might differ from that determined in the laboratory. In this review, we discuss different examples of phenotypic resistance and the mechanisms that regulate the crosstalk between bacterial metabolism and the susceptibility to antibiotics. Finally, information on strategies currently under development for diminishing the phenotypic resistance to antibiotics of bacterial pathogens is presented.Most studies on the development of antibiotic resistance deal with inheritable resistance. Under this view, the acquisition of a resistance phenotype requires a genetic change; either mutations (including point mutations, deletions and insertions), or the acquisition by horizontal gene transfer of antibiotic resistance genes [1,2,3,4,5]. However, other situations in which bacteria become transiently resistant to antibiotics, in the absence of a genetic change, have been also described [6]. Among them, the most studied are drug indifference, the growth in biofilms, and the phenomenon of persistence. However, there are other situations wherein bacteria present changes in their susceptibility to antibiotics depending on their metabolic state. Whilst this phenotype of resistance to antibiotics has been in occasions attributed to a situation of growth arrest that precludes the activity of some bactericidal antibiotics as beta-lactams, a growing number of evidences indicates that the link between the metabolic state of bacterial populations and their phenotype of susceptibility to antibiotics is far more complex. Although increased resistance can also be achieved because of the induction of specific resistance mechanisms (for instance induction of chromosomally-encoded β-lactamases by β-lactams [7]), this topic is not usually considered as phenotypic resistance and will not be discussed in the article.Some recent works have analyzed the determinants that contribute to the intrinsic resistance (intrinsic resistome) of bacterial pathogens [8,9,10,11,12,13,14,15,16]. Notably, in all cases in which a comprehensive study has been performed the number of genes involved in the phenotype of resistance is larger than could be predicted if they had evolved as specific elements for counteracting the action of the drugs. Furthermore, several of such genes encode key elements of the bacterial metabolism. Altogether, these results indicate that the specific phenotype of susceptibility to antibiotics is under metabolic control and hence that changes in the bacterial metabolism can consequently alter the susceptibility to antibiotics [17,18]. The fact that the susceptibility to antibiotics can change depending on the bacterial metabolic state is a two-way road. Bacteria can be transiently more resistant to antibiotics, a situation that may compromise therapy. Conversely, there can be specific metabolic conditions that increase the susceptibility to antibiotics. This situation has been recently exploited for developing nutritionally-based strategies for fighting persistent cells [19]. Similarly, the comparison of the metabolism of Mycobacterium tuberculosisin vitro and in vivo has served to understand the reasons why some lead compounds with very good activity in vitro and good pharmacological properties were not useful in a tuberculosis model of infection. The understanding of the causes of this phenotypic resistance, which is due to differences in the carbon metabolism of M. tuberculosisin vivo and in vitro, is important for the development of novel anti-tuberculosis antibiotics [20].Several articles have reviewed the acquisition of antibiotic resistance determinants by human pathogens. However, information on phenotypic resistance is lower in comparison. In the current article, some specific examples of situations that produce a transient, non-inherited, state of antibiotic resistance are reviewed. Soon after the introduction of antibiotics for treating infections, it was described that resting cells are less susceptible to penicillin [21], a situation (Figure 1) that was named ‘drug indifference’ [22]. Some recent works have shown that this effect is specific for the type of antibiotic considered. Non-dividing cells are fully resistant to ampicillin and tetracycline, whereas ciprofloxacin and streptomycin are active against stationary cells although their level of activity is lower than when cells are actively growing [6].Some situations of transient changes of bacterial susceptibility to antibiotics are described. (A) Drug indifference occurs when the antibiotic is effective only in a specific bacterial physiological condition. This situation was first described for β-lactam antibiotics that do not kill non-dividing growing cells; (B) In a bacterial population, there is a subpopulation of cells called persistents that are not killed by antibiotics in conditions that kill the bulk of the population. Once growth resumes, these persistent bacteria become susceptible to antibiotics, indicating that the phenotype of resistance is transient, not being the consequence of a genetic change; (C) Bacteria usually grow forming biofilms when attached to surfaces and under these conditions they are more resistant to antibiotics. Gradients of nutrients and of oxygen cause different metabolic states in the bacteria depending on their depth inside the biofilm, which can affect their susceptibility to antibiotics. Moreover, compounds of the matrix can impair the diffusion of the antibiotic and eventually bind the drug thus reducing its free concentration. Quorum sensing, which can be triggered at dense regions of the biofilm, may also alter bacterial susceptibility to antibiotics. (D) Bacteria change their permeability to antibiotics in response to several environmental and internal factors such as temperature, the presence of specific inducers, reactive oxygen species (ROS) or specific metabolic situations. Changes can affect antibiotic susceptibility at several levels. First, reducing the binding of the antibiotic by modification of the lipopolysaccharide (LPS), or generating outer membrane vesicles that give rise to more surface and minimizes the effective amount of the antibiotic per cell. Second, modifying the number or type of bacterial porins, the aqueous channels used by the antibiotic to penetrate inside bacteria, might help keeping the antibiotics out of the cell. Third, expressing multiple efflux pumps that extrude the antibiotic once it has reached the cytoplasm decreases the toxic activity of the antibiotic.The reduced susceptibility to antimicrobials that depends on the growth rate can be relevant for bacteria growing at host locations where growth is restricted or in situations where the bacteria have made use of the nutritional resources provided by the host and hence growth rate is reduced. This might be particularly relevant for long-lasting infections. In fact, it has been described that the antibiotic concentrations required for curing an infection are directly related to the duration of the infection [23]. Further works using a model in which mice were infected with a mixture of susceptible and resistant bacteria showed that during infection bacteria become increasingly refractory to treatment [24]. This reduced susceptibility is not inheritable and is the consequence of changes in the bacterial physiology. More recently, it has been shown that eight hours after infection bacteria are in a resting situation [6], which can cause the observed drug indifference, at least in this experimental model of infection.These finding are of relevance for the treatment of human infections. It has been suggested that the existence of drug indifferent, slow growing (or resting) cells can be the cause of relapse after antibiotic treatment that can be observed for some bacterial infections [25,26]. It has been stated as well that this low susceptibility is the cause for the need of using very long treatments for slow-growing bacteria as Mycobacterium leprae [27] or Mycobacterium tuberculosis [28]. Finding drugs that can act on these ‘drug refractory’ cells is thus of interest for the treatment of long lasting infections or infections due to slow-growing pathogens. An example of this view is the potential use of antibiotics that specifically kill anaerobes for the treatment of infections by M. tuberculosis, since it has been described that dormant, hypoxic M. tuberculosis cells are resistant to isoniazid, but susceptible to metronidazol [29,30,31]. Although most studies on bacterial susceptibility to antibiotics are performed using bacteria growing planktonically, microorganisms can grow forming biofilms when they are attached to a surface [32,33,34,35]. For human pathogens, this situation can be particularly relevant in the case of chronic infections of prosthetic devices. Different works have shown that bacteria forming biofilms are less susceptible to antibiotics than those growing planktonically [36,37,38,39,40,41,42,43]. Furthermore, it has been described that subinhibitory concentrations of antibiotics can trigger the formation of biofilms by bacterial populations [44,45], suggesting that antibiotics can induce transient, non-specific resistance to themselves. There are several potential reasons why biofilms are more resistant to antibiotics (Figure 1). The most obvious one is that biofilms are complex structures in which the free diffusion of compounds as antibiotics [46,47] is more difficult that in liquid cultures (planktonic cells). The capability to diffuse into the biofilm will differ depending on the structure of the antibiotic [48]. Increasing the diffusion of the antibiotics inside biofilms might thus help in enhancing their activity [49]. However, this would be just a part of the problem [50]; in several occasions the speed in the penetration of the antibiotics into the biofilm does not seem to be the key element for explaining the phenotype of resistance [51]. The biofilm structure itself may contain elements that bind antibiotics, sequestering them and hence reducing the freely available concentration of these compounds within the biofilm structure. More recent articles have focused on the metabolic state of bacteria when growing forming biofilms. It has been suggested that the biofilm-forming microorganisms can display different metabolic situations, including actively growing cells that should be killed by antibiotics and resting cells that would be transiently resistant to these compounds. These resting cells might be a subpopulation of persisters (see below) present in any biofilm [52]. The different metabolic situation of each subpopulation is highly dependent on the oxygen and nutrients availability, which is higher at the upper layers of the biofilm and lower at the deeper ones [53,54]. Surprisingly, the effect of these metabolic situations is different depending on the antibiotic family. It has been found that the aerobic regions of Pseudomonas aeruginosa biofilms are susceptible to quinolones and resistant to cationic peptides whereas the opposite occurs at the hypoxic regions [55]. One of the potential causes of the resistance to cationic peptides of biofilms relies on their structure itself. One of the compounds forming the biofilm extracellular matrix is DNA. This macromolecule chelates cations, and reduced concentrations of divalent cations trigger expression of the master sensor regulator of resistance to cationic peptides PhoP-PhoQ [56]. It has been then suggested that the use of DNase, which can break the biofilm matrix, may increase biofilm susceptibility to antibiotics [57]. In addition to the effect of metabolic changes on the susceptibility to antibiotics to biofilm-growing bacteria, classical resistance determinants may also be involved in the phenotype of resistance. For instance, the efflux pump MexAB-OprM contributes to the lack of susceptibility of P. aeruginosa biofilms [58], whilst it is not relevant in the case of planktonic cells. Altogether, these results indicate that there are several, simultaneous mechanisms that alter the susceptibility to antibiotics of bacteria growing in biofilms. Understanding the signals and mechanisms involved in the formation of biofilms can thus help in eliminating them. In the case of P. aeruginosa, the quorum sensing (QS) response triggers biofilm formation. Inhibition of QS thus reduces the formation of biofilm and consequently increases susceptibility to antibiotics. One of the compounds that inhibits biofilm formation is the macrolide azithromycin [59]. Although Gram-negative bacteria as P. aeruginosa are intrinsically resistant to macrolides, azithromycin has been proposed as a drug of choice for the treatment of chronic P. aeruginosa infections given its strong anti-QS and anti-biofilm activity [60,61].One of the main factors in the effectiveness of the antibiotics is their penetration inside the bacterial cell. This means that the first barrier against antibiotics consists on the bacterial envelopes. Consequently, changes in bacterial permeability may affect the susceptibility to antibiotics [62,63]. The permeability to antibiotics of microorganisms can be modulated by means of three different mechanisms (Figure 1). First, bacteria may alter their surface by modifying the lipopolysaccharide, reducing the molecular interactions with the antibiotic, and consequently, its penetration. Production of membrane vesicles increases the available membrane surface, which can also reduce the effective concentration of antibiotic available to enter the cell. Second, alterations in the number or type of porins or antibiotic transporters may change the susceptibility to antibiotics. Third, once the antibiotic reaches the cytosol, it can be pumped actively outside the cell through efflux pumps. In Gram-negative bacteria, the first barrier to antibiotic penetration is the outer membrane, mainly composed in its outer face by the lipopolysaccharide (LPS). The LPS presents anionic groups where the cationic antibiotics bind, representing the first step of antibiotic uptake [64]. In P. aeruginosa, it has been described that the alteration of the negative charge of the lipid A of the LPS by adding 4-aminoarabinose, increases the resistance to polymyxin, aminoglycosides and cationic antimicrobial peptides. This response can be induced by limiting concentrations of the divalent cations Mg2+ and Ca2+, or of polymyxin and cationic antimicrobial peptides. In P. aeruginosa, there are at least three two-component systems (PhoP-PhoQ [65,66], PmrA-PmrB [67], and ParS-ParR [68]) that upregulate the arnBCADTEF operon, whose products are responsible for the changes in the LPS. The induction of resistance to cationic antibiotics by environments with low amounts of Mg2+ has been also described in Salmonella typhimurium [69]. Especially relevant in the clinic is the induction of antibiotic resistance by host-defense peptides belonging to innate human defense [70], and for the new peptides that are being developed to be used in the frame of novel anti-infective therapeutic strategies [71].A similar phenomenon of LPS modification leading to transient resistance to aminoglycosides has been described in Stenotrophomonas maltophilia [72]. The authors found a correlation between the LPS pattern and an increase in the resistance to aminoglycosides when the bacteria were grown at 30 °C. Although the molecular basis of transient induction of resistance in such conditions has not been clarified, these results strongly suggest that the growth temperature affects the LPS composition challenging the binding or the uptake of aminoglycosides.Another mechanism that might transiently induce resistance in Gram-negative microorganisms is the formation of Outer Membrane Vesicles (OMVs). It has been proposed that OMVs can trigger an initial defense mechanism against different environmental stresses including antibiotics [73]. OMVs are made with fragments of the bacterial outer membrane enclosing periplasm and active proteins. Among the roles that OMVs might play, it has been suggested that they are involved in secretion, intercellular signal trafficking and in bacterial survival-related functions [74]. Like LPS, OMVs can bind cationic peptides and antibiotics, which suggests that they can provide transient resistance to these compounds just by a trapping mechanism that reduces the free concentration of the drug [73]. It is known that some factors like temperature and nutrient limitation can increase the production of OMVs. Furthermore, the composition and number of OMVs are different in biofilms and in planktonic cultures [73]. Whether or not induction of OMVs formation under such environmental conditions can induce transient antibiotic resistance is still an open question that merits to be explored.To enter into bacterial cells, antibiotics can make use of regular transporters (porins and inner membrane transporters) used for the microorganisms to allow the entrance of different substrates, including nutrients [63,75]. Escherichia coli harbors two main porins, OmpC and OmpF. These two porins are involved in the transport of some antibiotics such as quinolones, tetracycline, chloramphenicol or β-lactams. Low-level expression or inactivation of porins impairs the intracellular accumulation of the antibiotic and consequently renders resistance [75]. This implies that changes in the expression of porins due to environmental signals may alter bacterial susceptibility to antibiotics [62]. It has been described that OmpC and OmpF are inversely regulated. OmpF is highly produced in environments with low osmolarity and temperature, whereas OmpC is the prevailing porin when bacteria are growing under conditions of high osmolarity and high temperature, like for example when they are colonizing a nutrient-rich animal gut [76]. Since each porin has different substrates profile, the consequences of this regulation for antibiotic resistance will have some degree of specificity [75]. The expression of porins is finely tuned in response to changes in the environment; because of this, they are under control of complex regulatory networks. The phosphorelay system EnvZ-OmpR senses osmolarity and controls at the transcriptional level the OmpF/OmpC ratio [77,78]. On top of this regulation, there are two small antisense RNAs (micF [79] and micC [80]) which participate in the post-transcriptional regulation of OmpF and OmpC, respectively. micF is known to respond to changes in osmolarity [81], temperature [82] and nutrient availability [83], and is the link to other regulatory networks such as the Mar-Sox-Rob regulon, which responds to salicylate [84]. In this way, fast changes in the environment can be rapidly faced up by the microorganisms and this has consequences in bacterial susceptibility to antibiotics.As above stated, multidrug efflux pumps can extrude a broad range of substrates chemically different, including antibiotics [85,86,87,88,89,90]. Genomic studies have shown that multidrug efflux pumps can account for 10% of all transporters in some bacterial species [91,92,93]. Together with their high conservation, this suggests that efflux pumps have important roles for the bacterial physiology in addition to being antibiotic resistance determinants [89,90]. Expression of efflux pumps is usually tightly down-regulated, which means that transient expression is achieved just in the presence of the right effectors [94,95]. Among those effectors that trigger expression of MDR efflux pumps, some might be relevant during infections. These include biocides such as triclosan, which is found in several products from soaps to toothpastes, and which has been shown to induce transiently the expression of the SmeDEF efflux pump from S. maltophilia [96]. More relevant from the therapeutic point of view is the induction of efflux pumps (and consequently resistance) in the presence of bile, cationic peptides or fatty acids [97,98,99,100,101], because bacteria can encounter these compounds and therefore might display a phenotype of transient resistance in the course of an infection. In addition to their induction by specific effectors, expression of efflux pumps can be altered by more general changes to their habitat. For example, it has been described that oxidative and nitrosidative stress [102] might alter the expression of multidrug efflux pumps. In E. coli, the exposure to oxidative agents like paraquat, which produces anion superoxide O2−, induces the SoxRS operon. As a consequence, the expression of the AcrAB-TolC multidrug efflux system is induced [103] and bacteria become transiently antibiotic resistant. In P. aeruginosa, another regulator, MexR, which is the repressor of mexAB-oprM MDR efflux pump, is directly oxidized by oxidative agents. As a consequence, MexAB-OprM is overexpressed leading to transient antibiotic resistance [104]. A similar response to oxidative and nitrosidative stress has been described for other P. aeruginosa efflux pumps, like MexXY-OprM [105], and MexEF-OprN [106]. Since bacteria can face oxidative and nitrosative stress during infection, it is likely that these efflux pumps are overexpressed and bacteria present a phenotype of reduced susceptibility to antibiotics in this situation. However, this topic has not yet been addressed in detail.We have presented examples in which changes in membrane permeability trigger transient antibiotic resistance. However, the opposite might also happen. In some cases, in vivo growing conditions can lead to an increase in antibiotic susceptibility allowing a better response to anti-infective therapy. This is the case of susceptibility to fosfomycin of Listeria monocytogenes [107]. Listeria is an intracellular pathogen. In vitro, Listeria is resistant to fosfomycin. However, it has been shown that when growing inside the host cells L. monocytogenes becomes susceptible to this antibiotic. Fosfomycin enters in bacteria using Hpt, the same transporter as hexoses-phosphate and glycerol-phosphate [108]. The Hpt transporter is controlled by the global regulator of virulence, PfrA, which is highly expressed during macrophage infection [107,109]. One of the nutrients that L. monocytogenes uses at the intracellular milieu is glucose-phosphate. For an efficient uptake of this nutrient, the expression of its transporter Hpt is induced in intracellular bacteria, hence causing the observed increased susceptibility to fosfomycin. In this way, an antibiotic whose use has been discarded based on classic susceptibility tests, would have a potential use in clinics, to treat Listeria infections.From these results, it is clear that the metabolic status of the microorganisms is of relevance for their susceptibility to antibiotics [17]. It is thus possible that global metabolic regulators may also modulate the antibiotic resistance. This is the case of Crc, a global post-transcriptional regulator of carbon metabolism in P. aeruginosa [110,111,112,113]. It has been described that in addition to regulating the utilization of alternative carbon sources in nutrient-rich environments, Crc modulates the virulence and antibiotic susceptibility of P. aeruginosa [18]. Bacterial transporters are targets for Crc. For example, the expression of the OprD2 protein, involved in the uptake of basic amino acids and of the antibiotic imipenem, and of GlpT, involved in the uptake of glycerol phosphate and of the antibiotic fosfomycin, are downregulated by Crc. As a consequence, a Crc defective P. aeruginosa mutant is more susceptible to these antibiotics. This makes Crc a good target in the search of drugs, to be used in combination, for enhancing the activity of antibiotics currently in use. Several reports have shown that the size of the bacterial inoculum is important for the activity of antibiotics [114,115,116,117]. It has been suggested that this effect can be the cause of the failure for treating infections with a high bacterial load, because the actual minimal inhibitory concentrations of such bacterial populations are higher than those determined using classical laboratory tests [115,118]. In the case of bacteria producing antibiotic-inactivating enzymes, this effect is due to the activity of the enzyme that degrades the antibiotic at higher rates when more cells are present [116,117,119]. However, the situation is less clear in the case of bacteria that do not harbor a mechanism of resistance that involves the degradation of the antimicrobial. For these microorganisms and antibiotics, two situations can be envisaged. One is a reduction in the antibiotic concentration at high cell densities due to its binding to cell envelopes and debris of alive and killed cells. Another is the existence of less molecules of antibiotic per cell at high cellular density. Using experimental approaches and mathematical modeling, it has been shown that most likely both situations are relevant for inoculum-dependent antibiotic resistance [120], and that other processes can be important for this phenotype. Taken into consideration that high cell densities trigger the QS response and the fact that QS might affect the susceptibility to antibiotics it is worth considering that the QS response might have a role in the inoculum-dependent antibiotics susceptibility of bacterial populations. Nevertheless, to the best of our knowledge, this possibility has not yet been explored.Persistence to antibiotic treatment has been described for several different bacterial species, and consists (Figure 1) on a situation in which a subpopulation of the bulk of bacteria under treatment presents a refractory state to the action of antibiotics [121,122,123]. The relative fraction of cells in this situation can range from 10−6 for exponentially growing bacteria to 10−2 for bacteria in the stationary growth phase. Resistance is transient; persistent cells resume growth once the antibiotic is removed, but are killed at a similar rate as the original susceptible population if the antibiotic is added again [124]. For most infections, phenotypic resistance to antibiotics of a very small number of cells is not problematic, since these cells are removed by the action of the immune system. However, there are situations in which persistence can be a relevant problem for the success of the treatment of the infection.One possible explanation for persistence is that, in any bacterial population, a fraction of the cells is in a non-dividing (dormant) situation that makes them refractory to the action of the antibiotics. However, the situation is more complex [125], with a number of global regulators and metabolic enzymes involved in the process [126,127]. Whilst bacterial cultures can contain a subpopulation of pre-existing dormant cells (Type I persisters), there is another subpopulation (Type II persisters) that emerges as the consequence of the inherent bistability of growing cells, from normal to persisters [121]. The main factor triggering Type I persistence is starvation. Nevertheless, the phenomenon is not a direct response to a non-growing situation of bacterial cells. On the contrary, the fact that Type I persisters are fully susceptible to antibiotics in a short time window after the beginning of growth of the whole population indicates that persistence consists on a metabolic shift that occurs after the stationary phase is over [128]. One of the key elements for the establishment of Type I persistence is the stringent response as shown by the relevant role that SpoT and RelA have for persistence in E. coli [129]. However, some different works have shown that there are different mechanisms triggering persistence, among them the inherent bimodal distribution of toxin/antitoxin proteins that can render non-uniform bacterial populations in which those cells containing high levels of toxin arrest their growth for long periods of time [39,130,131]. It is important to notice that infection-linked situations can trigger persistence. For instance, quinolones can induce persistence in E. coli through the SOS-mediated induction of the toxin TisB [132] and intracellular pathogens can present non-dividing, persistent, subpopulations when growing inside their host cells [133].The increased knowledge on the mechanisms of persistence has led to propose different approaches for eradicating these bacterial subpopulations that are refractory to antibiotic treatment. In the case of P. aeruginosa, it has been described that the QS response is relevant for persistence [134].Inhibition of QS has been suggested as an anti-virulence approach for fighting P. aeruginosa infections [135,136]. Reduction of the fraction of persistent cells or enhancing their susceptibility to antibiotics by this kind of inhibitors will also be of benefit for the treatment of P. aeruginosa infections. Recent work has shown that the QS inhibitor (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one restores the antibiotic susceptibility of P. aeruginosa PAO1 persisters. However, the effect is not due to the inhibition of QS and the mechanisms for this activity remain to be fully determined [137].Some works have shown that the knockout of genes coding for metabolic enzymes, such as ygfA that encodes an enzyme involved in folate biosynthesis or yigB, encoding a flavin mononucleotide phosphatase decreases persistence [126], whereas their overexpression, increases tolerance. Given that persistence can be under metabolic control, another approach for eliminating persisters is by shifting the bacterial metabolism. The feasibility of this approach has been demonstrated in a set of recently published articles. In one of them, the authors were able to kill persister cells with aminoglycosides just by adding specific metabolic substrates that allow the recovery of bacterial proton motive force without resuming growth [19]. Following the same approach, it has been found that the addition of 3-[4-(4-methoxyphenyl)piperazin-1-yl]piperidin-4-yl biphenyl-4-carboxylate to bacterial cultures causes the reversion of persisters to antibiotic-sensitive cells [138].A recent article explored another possibility for eradicating persisters. It was observed that M. tuberculosis persisters require a small reduction in oxygen availability for their survival to antibiotics. At high concentrations of oxygen, persisters were killed at the same rate as normal cells. Since his situation could be a consequence of a reduced capability of persisters to generate reactive oxygen species (ROS) in the presence of bactericidal antibiotics, priming ROS generation will eliminate persisters. In line with this reasoning, the authors demonstrated that antibiotic clofazimine, which increases ROS, successfully eradicates the persister population [139].Most studies on the mechanisms of antibiotic resistance address the development of resistance as the consequence of a genetic, inheritable change, which can be a mutation or the acquisition of a resistance gene. These are off/on directional situations; bacteria are either susceptible or resistant and reversion from resistance to susceptibility is not a frequent event. Several works have shown that this is a part of the resistance landscape and that there are situations in which resistance is not driven by a genetic change. Transient, reversible resistance can be achieved by different mechanisms, which are linked to the physiological state of bacteria and to the inputs that microorganisms receive when confronted to different habitats and stressors. The study of the mechanisms of phenotypic resistance is of relevance to understand the reasons of therapeutic failures for bacteria that are classified as susceptible using classical testing methods, but also to increase the susceptibility to antibiotics of bacterial pathogens. Classical methods for improving the efficacy of antibiotics are mostly based on the development of permeabilizers and of inhibitors of mechanisms of resistance, mainly of antibiotic inactivating enzymes and of multidrug efflux pumps [9,140,141,142]. The study of the mechanisms leading to phenotypic resistance may also serve for this purpose. As stated in the review, this knowledge may serve, among other issues, to eliminate persister cells by priming their metabolism, to implement novel strategies for treating infections by intracellular pathogens, which are more susceptible to some specific antibiotics when growing inside the cell, to develop antimicrobials with anti-biofilm activity, or to define novel targets, which inactivation would increase the susceptibility to antibiotics.Thanks are due to Fernando Rojo and Mary Higgins for proof-reading the manuscript. Work in our laboratory is supported by Grants BIO2011-25255 from the Spanish Ministry of Science and Innovation, PROMPT from CAM, REIPI from the Instituto de Salud Carlos III, and HEALTH-F3-2011-282004 (EVOTAR) and HEALTH-F3-2010-241476 (PAR) from the European Union. FC is the recipient of a JAE predoctoral fellowship.

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+Formerly associated with Regions Hospital, St. Paul, MN 55101, USA.This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Regions Hospital started a multidisciplinary antibiotic stewardship program (ASP) in 1998. The program effectively shut down from 2002–2004 as key personnel departed and was then restarted but without the dedicated pharmacist and infectious diseases physician. Purchasing data (in dollars or dollars/patient/day) unadjusted for inflation served as a surrogate marker of antibiotic consumption. These data were reviewed monthly, quarterly, and yearly along with antibiotic susceptibility patterns on a semi-annual basis. Segmented regression analysis was use to compare restricted antibiotic purchases for performance periods of 1998–2001 (construction), 2002–2004 (de-construction), and 2005–2011 (reconstruction). After 4 years (1998–2001) of operation, a number of key participants of the ASP departed. For the following three years (2002–2004) the intensity and focus of the program floundered. This trend was averted when the program was revitalized in early 2005. The construction, deconstruction, and reconstruction of our ASP provided a unique opportunity to statistically examine the financial impact of our ASP or lack thereof in the same institution. We demonstrate a significant economic impact during ASP deconstruction and reconstruction.Antibiotic stewardship is gaining universal acceptance as a valuable tool to limit bacterial antibiotic resistance, maximize clinical outcome with antibiotic therapy, minimize antibiotic induced adverse events, and to control cost. A recent policy statement by the Society for Healthcare Epidemiology of America (SHEA), the Infectious Diseases Society of America (IDSA), and the Pediatric Infectious Diseases Society (PIDS) recommends (1) antibiotic stewardship programs (ASP) be mandated through regulatory mechanisms; (2) antibiotic stewardship be extended into the ambulatory healthcare setting; (3) there should be formal education regarding antibiotic resistance and stewardship; (4) antibiotic usage data be collected and shared for inpatients and outpatients, and (5) antibiotic stewardship research is needed [1,2]. Regions Hospital (formerly Ramsey Medical Center) is a 455 bed, level-one trauma center, with ~120,000 patient days of care per year. Among the hospital units are multiple intensive care units, including a burn unit. In 1998, Regions Hospital started an ASP with many of the key elements identified later in the IDSA ASP position paper published in 2007 [2]. (Table 1) The program was initially staffed with 0.25 FTE infectious diseases (ID) physician and 1.0 FTE ID trained pharmacist. The program was interdisciplinary involving the pharmacy director, hospital administrator, microbiologist, information systems (IS) personnel, infection control, and the physician chair of the pharmacy and therapeutics committee. Initial ASP goals included optimizing clinical outcomes with antibiotic therapy, limitation of collateral damage due to overuse or inappropriate use of antibiotics, and cost savings.Hospitals that have committed to an ASP experience a significant reduction in antibiotic cost following implementation [1,2,3,4,5]. We report here a somewhat unique situation where our ASP was constructed, de-constructed, and reconstructed over a period of 14 years and the resultant economic impact.Monthly antibiotic purchasing for 1998 through 2010 were considered and analyzed in terms of total restricted and unrestricted antibiotics purchases, restricted antibiotics per patient/day, total antibiotics, total antibiotics per patient/day, and percent restricted to total antibiotics, with total purchase costs serving as a surrogate marker for utilization. Total patient days used census figures for adult inpatients including ICUs. Segmented regression analysis for interrupted time series [6] was used to determine significance for the difference in levels and slopes over time due to the two interruptions (changes in the nature/status of the stewardship program): (1) deconstruction (stop) of team in June 2001, and (2) a subsequent reconstruction (restart) of the core stewardship team in March 2005, Figure 2) The Durbin-Watson statistic was used to test for autocorrelation. If autocorrelation was detected, the parameter was corrected with Yule-Walker estimation. Significance was determined at the 0.05 level. Recommendations by Infectious Diseases Society of America (IDSA) for Antimicrobial Stewardship Program [2].a As per the Infectious Diseases Society of America-United States Public Health Service grading system for ranking recommendations in clinical guidelines; b Includes pharmacy director, patient care committee, and medical executive committee; c Restriction of antibiotics was primarily utilized. Restricted antibiotics did not need pre-authorization, but directed the attention of antibiotic surveillance for patient evaluation. A handful of antibiotics were selected for the requirement of pre-authorization; d Antibiotic forms were utilized during some point over the 11 years, but it was hard to gain acceptance of the use of the forms. For this reason, they are no longer utilized; e The health care information technologies were recently implemented in the past two years of the antimicrobial stewardship program.Data were missing or questionable for 4/156 monthly time periods. Thus we used the mean total restricted antibiotics for all other time periods to replace these four values of total restricted antibiotics. Other variables for restricted antibiotics per day and percent-restricted antibiotics were adjusted accordingly based on this modification.The antibiotic formulary was divided into two groups of agents, restricted and non-restricted; restricted classification was driven primarily by cost, dosing difficulty and risk of toxicity (e.g., aminoglycosides), and/or agent novelty (Table 2). A patient receiving a restricted antibiotic initially did not require prior-authorization but these patients would be identified on the IS daily report and would be evaluated by the ASP team. Over time and with the introduction of new antibiotics, the “restricted” list expanded with some products requiring prior authorization by an infectious diseases physician.Example of antibiotic formulary classifications.a Must be approved by ID prior to use; b During the time period, the formulary carbapenem was switched from imipenem to meropenem.A list of patients receiving restricted antibiotics was generated daily by the hospital’s information systems department, and the ID physician, ID pharmacist, pharmacy, and chair of the P&T committee received copies to identify for possible interventions. In most cases, the pharmacy personnel and ID consultation personnel worked in tandem; however, if the pharmacist encountered a patient not also being seen via an ID consultant, the pharmacist contacted the prescribing physician and discussed possible treatment options. Unresolved cases between the pharmacist and prescriber were then referred to the ID consultant for further review and action.In 1998, the only readily available and widely used metric to measure antibiotic consumption was antibiotic purchase data. As external comparisons of our performance were not possible, a decision was made to collect data over time and compare the internal performance of our ASP yearly. Over time, to maintain consistency in our internal comparisons, we have continued to use this metric. As a result, parameters of performance included total antibiotic purchases for the calendar year, total antibiotic cost per patient per day, restricted antibiotic cost per patient per day, percent of antibiotic dollars spent on restricted antibiotics, and the percent of antibiotic purchases as compared to total in patient drug purchases for the calendar year. The obvious drawback in using purchase data is that increases or decreases in annual antibiotic purchasing patterns are subject to the magnitude of inflation, better purchasing strategies, contract price changes, rebates, generic drug status, introduction of expensive new agents that are widely used, and now the purchasing and hording of antibiotics in short supply. However, this variability remained in play over the duration of the program. The clinical pharmacist and pharmacy director reviewed antibiotic use in the form of purchasing data monthly. These data were summarized and reviewed quarterly by the antibiotic subcommittee and passed on through the hospital committee structure. Annually and semiannually, the antibiotic subcommittee reviewed bacterial antibiotic susceptibility for Gram positive and Gram-negative pathogens as an alert for changing patterns of antibiotic susceptibility. The clinical microbiologist stratified these data by intensive care units and as comprehensive (hospital-wide) data. Based on antibiotic subcommittee decisions, any changes of 5% of more in susceptibility from year to year were considered to be significant and warranted further action as necessary. Antibiotic susceptibility data were reviewed for the first six months of the year and at the end of the 12 months. The clinical microbiologist also alerted the team to any unusual organisms or unusual resistance patterns between the 6 and 12 month periods.A comprehensive yearly presentation of antibiotic purchase data and bacterial susceptibility data was prepared and shared with the infectious diseases physicians group, our hospital vice president, key physicians, the Pharmacy and Therapeutics Committee, the antibiotic subcommittee, and the pharmacy director. Minutes of the report are passed along to our Patient Care and Executive Committees.After initially establishing a baseline in 1998–2001, antibiotic costs (total restricted and unrestricted, total and restricted cost/patient/day) initially rose with the patient census 2002–2004 during ASP deconstruction and cost fell after reconstruction 2005–2010 despite continued increases in patient census (Figure 1). Restricted antibiotics (Table 2) consumed 55%–75% of the antibiotic dollar. Four or five antibiotics (levofloxacin, ceftriaxone, imipenem/meropenem, ertapenem, and vancomycin) depending on time period made up ~50% of the antibiotic budget. Over the fourteen years, antibiotic purchases represented <15% of the inpatient drug budget.Total antibiotic purchases, Total antibiotic cost per patient per day, and annual census by year. Beginning in 2001, the antibiotic stewardship program experienced the loss of several key initial personnel on the ASP team, including the primary ID physician, the clinical microbiologist, and two clinical pharmacists who worked primarily with the stewardship program. With the departure of these individuals, the intensity and operation of the ASP floundered. Instead of a focused daily responsibility, these tasks were reassigned to the decentralized clinical pharmacists and made part of other daily clinical responsibilities. The program was revitalized in 2005 with a re-focusing of efforts on antimicrobial stewardship.Using cost data for 2002, 2003 and 2004 and projecting future costs using the slope of that data into 2005 and beyond versus what actually happened after reconstruction clearly demonstrate the successful impact of restoring the program (Figure 2). Figure 2 reveals a significant positive change (increasing use) in both total antibiotics (p = 0.0021) and restricted antibiotic cost/patient/day (p = 0.0037) due to the deconstruction of the stewardship team (interruption #1–1st solid vertical line). However, once reconstruction of the team occurred (interruption #2–2nd solid vertical line), a significant negative change (p ≤ 0.001, respectively for both) was observed (R2 = 0.51 and 0.45, respectively).A significant increase in cost for both overall total antibiotics (p = 0.0039) and total antibiotics per day (p = 0.01) was also observed related to the deconstruction of the team. However, when reconstruction of the team occurred, a significant decrease in cost (p < 0.0001 for both) was observed (R2 = 0.34, 0.30, respectively). Further, a significant positive change in percent restricted to total antibiotics (p = 0.0135) was observed related to deconstruction. Once the reconstruction occurred, a significant negative change (p = 0.0033) was observed (R2 = 0.58).Segmented regression series analysis of total restricted antibiotic purchases by year during antibiotic stewardship program (ASP) construction (start), deconstruction (stop), and reconstruction (restart) See text for statistic description.In 1998, we initiated our hospital wide effort at antibiotic stewardship. Elements of our initial program aligned well with the position adopted by the IDSA with regard to antibiotic stewardship goals and daily practice elements. On a monthly, quarterly, and yearly basis, we monitored overall antibiotic purchases (Figure 1), the percent of the inpatient pharmaceutical budget made up by antibiotics, overall antibiotic cost per patient per day (Figure 1), restricted antibiotic cost per patient per day, and the percent of our antibiotic dollar represented by restricted antibiotics. As we could not benchmark our performance/results against other similar institutions in 1998, we made a decision to compare ourselves to ourselves over time. While the sensitivity of antibiotic purchase dollars is not an ideal metric today, the comparison is tempered by the longitudinal period of time involved in our internal comparison. In retrospect, use of defined daily doses or antibiotic days of therapy may have provided a better reflection of antibiotic consumption. However, in 1998, the readily available and retrievable parameter for our purpose was dollars expended. Another expected lesson over the 14-year experience is that a program of antibiotic stewardship is likely to change over time. Pharmacy and medical personnel will change and the various tools that can be used to affect prescriber behavior may need to be rotated into and out of the stewardship program. Further, some elements, such as our trial of the antibiotic order form, did not work for us and failed to have the desired impact in our system. Stewardship programs over time are also likely to experience a fatigue factor especially in a teaching institution as prescribers rotate in and out throughout the year. Likely each institution will have to find the right stewardship niche that works for them and that may change somewhat from year to year. Lastly, while these programs are attractive, barriers often exist to procuring enlisting the specified personnel identified in the IDSA position paper and the provision of financial support for dedicated time on task [2].The IDSA primary goal for antibiotic stewardship is to optimize antibiotic clinical outcomes while minimizing unintentional consequences of antibiotic therapy [2]. We have no definitive data that suggesting that that goal was realized in our program. However, in regard to the secondary goal of reducing healthcare costs, this study demonstrates a statistically significant alteration in antibiotic costs when the antibiotic stewardship effort is interrupted and then restarted. (Figure 2) Most ASPs can demonstrate an initial reduction in antibiotic costs. In this report we demonstrate the initial financial effect of our ASP, the increasing costs associated with the interruption of the program, and the return of financial stability when the program was restarted. These unique circumstances lent themselves to a segmented time sequence series analysis, which provided statistical evidence of the ASP effect on antibiotic costs.We demonstrate the need for the development and continued support of an ASP. Antibiotic costs were significantly influenced by the presence or absence of a functioning ASP during 14 years of operation/observation at Regions Hospital. The successes reported here should encourage hospitals and health-care providers to initiate and continue ASPs.We would like to thank Liann Walker and Julia Moody for their efforts in the development of the stewardship program and Elizabeth Hermsen for reviewing the manuscript as well as Patrick Mauldin for his assistance with interrupted time-series analysis.John Rotschafer received honoraria as a consultant from Cubist, Optimer, and Pfizer Noe Mateo received honoraria from Gilead as a consultant. Remaining authors have no conflicts.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).We investigated the effects of two novel copper-based inorganic formulations for their activity against 60 isolates of Helicobacter pylori (Hp). The two copper-based formulations were tested against three NCTC Helicobacter pylori isolates and 57 clinical strains isolated from the UK and Italy in time-kill assays. Both copper-based formulations were bio-cidal against all Helicobacter pylori strains tested reducing the viable count by 4–5 log within 2 h. These two copper-based anti-microbial agents deserve further study in relation to the treatment of H. pylori-related gastric disease.Helicobacter pylori is a gram negative, microaerophilic bacterium found principally in the stomach [1]. Infection with this organism is one of the most widespread human infections that affects up to 50% of the world’s population. Epidemiological studies have clearly demonstrated a major aetiological role for Helicobacter pylori in peptic ulcer disease, gastric MALT [mucosal associated lymphoid tissues] lymphoma, and distal gastric cancer [2,3,4,5]. Clarithromycin or metronidazole combined with amoxicillin and a proton pump inhibitor is the most frequently used regimen for first-line Helicobacter pylori therapy (standard triple therapy) in clinical practice in Europe as well as US [6]. This regimen is also advocated in the European Helicobacter Study Group (EHSG) Maastricht 2006 Guidelines. However, there are a number of problems with this approach. Currently, standard triple therapy fails to eradicate Helicobacter pylori infection in more than a third of patients [7]. Increasing primary resistance to clarithromycin or metronidazole, reported by different studies [8,9,10], is widely claimed as the most important factor reducing the efficacy of therapy. The prevalence of clarithromycin resistance varies between 6.1% to 14.5% in the USA and may be as high as 24% in some European countries, while the prevalence of metronidazole resistance ranges from 25% to 38% in Western countries, and up to 77% in some Asian countries. Amoxycillin resistance is rare. In a recent study, increasing rates of clarithromycin resistance (8.7%, 23.5% 26.7%, 34.5% were paralleled by falling eradication rates (90.6%, 80.2% 76% 74.8%) respectively [11]. A recently suggested first line treatment is sequential therapy, where the main aim is to overcome clarithromycin resistance [12]. Amoxycillin is given for five days followed by the addition of clarithromycin and a nitroimidazole for five days with a proton pump inhibitor given for the whole 10 days. Reported eradication rates are over 93.4% and are thus much better than current triple therapy results [13]. However, even this is only a Grade B success: a Grade A success is defined as >95% eradication [14,15,16]. It has also been suggested that sequential therapy should be a second-line therapy not a first line therapy. In areas where clarithromycin resistance is low, various modifications of sequential therapy have been used but none achieving Grade A eradication. The management of Helicobacter pylori infection after a failed course of initial treatment is considered more difficult, mainly due to selection of bacterial strains resistant to the antibiotics used in the primary eradication attempt. Thus, optimal therapy after first-line treatment failure remains controversial due to the lack of large randomized cohort studies and the lack of homogeneity of patients studied. In second or third line therapy, only a few antibiotics against Helicobacter pylori infection are suitable, such as bismuth compounds, levofloxacin, rifabutin or furazolidone, which are used in triple or quadruple regimes [6]. In summary, the treatment of Helicobacter associated disease is beset by the following problems: (1) Increasing antibiotic resistance; (2) Decreasing eradication rates from >90% to ~70%; (3) The need to take lots of tablets and therefore compliance problems leading to antibiotic resistance and failure of eradication; (4) The association of proton pump inhibitor use with Clostridium difficile colitis [17]. For these reason potential alternative treatments have been investigated such as inhibition of binding [18], photodynamic therapy [19], biocidal action of spices [20], anti-malarial drugs [21], essential oils [22] and other botanical extracts [23]. In this study, we have investigated the efficacy of novel highly reductive copper compounds for their bactericidal activity against Helicobacter pylori. Both CuAL42 and CuPC33 had significant antimicrobial activity against Helicobacter pylori. A kill curve of ATCC strain J99 is illustrated in Figure 1. The kill curve of two NCTC isolates that were either Type II (cagA negative) or Type I (cagA positive) and 2 clinical isolates that were separately metronidazole resistant or clarithromycin resistant is illustrated in Figure 2 (a–d respectively) and demonstrated both compounds had similar activity and killed or reduced the number of bacteria by log >6 at 12 ppm, irrespective of virulence marker or antibiotic sensitivity. Time-kill curves with CuAL42 and CuPC33 on the antibiotic-sensitive H. pylori strain, J99.Star, control; closed square, CuAL42 0.5 mg/L; closed triangle, CuAL42 1 mg/L; closed upside-down triangle, CuAL42 5 mg/L; closed diamond, CuAL42 12 mg/L; open square, CuPC33 0.5 mg/L; open triangle, CuPC33 1 mg/L; open upside-down triangle, CuPC33 5 mg/L; open diamond, CuPC33 12 mg/L. The results shown are the means of triplicate observations. Error bars representing standard deviations (less than 10%) are not shown for reasons of clarity.Time-kill curves with CuAL42 and CuPC33 on (a) CagA−; (b) CagA+; (c) metronidazole-resistant and (d) clarithromycin-resistant H. pylori strains. Star, control; closed square, CuAL42 5 mg/L; closed triangle, CuAL42 12 mg/L; open square, CuPC33 5 mg/L; open triangle, CuPC33 12 mg/L. The results shown are the means of triplicate observations. Error bars representing standard deviations (less than 10%) are not shown for reasons of clarity.The average reduction of viability for the 57 isolates is shown in Figure 3. The reduction in viability for both CuPC33 and CuAL42 was significant (p = 0.0001). The number of strains that were killed at 1 and 2 h is shown in Figure 4 where CuAL42 was slightly more active than CuPC33. The resistance rates of the clinical isolates were clarithromycin, (41%) metronidazole (58%) and levofloxacin (20%). Antibiotic resistance bore no relationship to the efficacy of the copper compound. Mean Activity of CuAL42&CuPC33 against 53 clinical isolates of H. pylori. (AL42 = CuAL42; PC33 = CuPC33). Number of H. pylori isolates killed at one hour (T1) and two hours (T2) by CuAL42 and CuPC33 (PC33 = CuPC33; AL42 = CuPC42). Previous studies have demonstrated these copper complexes killed Acinetobacter baumanii (ACCB) and methicillin resistant Staphylococcus aureus (MRSA) at a concentration of 150 ppm within 2 h, compared to equivalent concentrations of copper sulfate and the inorganic binders. At a concentration of 20 ppm the copper-based compounds also completely inhibited colony formation of ACCB and MRSA, but this required 16 hr of exposure [24]. More recently organo-metallic complexes including copper-fluoroquinolone have shown good antimicrobial activity against a small number of Helicobacter pylori strains [25]. In this study, we have analyzed the activity of CuPC33 and CuAL42 copper complexes against 60 stains of Helicobacter pylori and shown bactericidal activity against all isolates at a low concentration (12 ppm) within a period of 2 h. With increasing time, increasing numbers were killed. The specific advantage of the CuPC33 and CuAL42-complexes in this study, over the antibiotic-metal complexes [25], is the absence of any antibiotic. This suggests a lack of generating antibiotic resistant strains and possible not predisposing the patient to Clostridium difficile associated disease, although these effects would have to be confirmed by future work. Several mechanisms and target sites of action have been proposed for this very broad spectrum of biocidal activity of copper. Firstly, copper is a highly redox-active metallo-ion that promotes membrane lipid peroxidation, which in turn damages bacterial plasma membrane integrity [26]. Secondly, copper binds and disorders helical DNA by engaging this structure at two different binding sites, explaining its activity against a wide range of viruses [27]. Thirdly, copper facilitates free radical–mediated degradation of proteins, which includes on the one hand oxidation of specific amino acid residues, and on the other degradation of sulfydryl moieties [28]. The mechanism by which the inorganic binders so effectively enhance copper’s biocidal activity is currently under investigation. Preliminary experiments using scanning electron microscopy indicate that exposure of ACCB to CuAL42 (150 ppm) for 20 min causes extensive membrane “blebbing” [24]. Previous work has indicated a lack of cytotoxicity for eukaryotic cells indicating a selective toxicity for bacterial cells at the concentrations used [24,29]. Eukaryotic cells have a recognized ability to withstand copper toxicity by well characterized, often highly complex mechanisms of intracellular protein-dependent coordination, chelation, and transport, and to well characterized extracellular binding pathways in multicellular higher organisms [30]. Finally, the use of copper complex for Helicobacter pylori-associated peptic ulcer disease has one further possible advantage: that of enhancing ulcer healing. One of the earliest events in ulcer healing is rapid epithelial cell migration leading to reconstitution of the epithelium [31]. This is followed by cell proliferation, which restores the mucosal epithelium to its normal thickness. Angiogenesis is essential for providing blood flow to the re-establishing epithelium and for the creation of new glands [32]. In animal models, it has been shown that the ulcer repair process can be accelerated by the administration of epidermal growth factor (EGF) via an effect on both angiogenesis and re-epithelialization [33]. Copper is recognized to have angiogenic properties by stimulating VEGF and hypoxia-inducible factor-1 alpha [34,35] and it is not unreasonable to suppose that these copper complexes may have a similar action, although experimental data is currently lacking. Bacteria: 3 NCTC cultures—NCTC11637, NCTC 12908, ACTC J99 and 57 clinical isolates, 43 from Italy and 14 from the UK were used. Copper compounds: The two copper-based formulations—CuPC33 and CuAL42—were provided by Remedy Research Ltd. These formulations are composed of copper sulfate and two inorganic compounds that form the metallo-ion binders. The binders comprise an ammonium salt and an inorganic acid—ammonium phosphate and phosphoric acid in PC33 and ammonium sulfate and sulfuric acid in AL42. The concentration of elemental copper in each stock solution was 30.43 g/L.Clarithromycin, levofloxacin and metronidazole sensitivity was determined on all isolates using European Guidelines [36]. Briefly, testing was performed on Muller Hinton agar containing 10% horse blood with an inoculum of 0.5 × 109. Plates were incubated at 37 °C in a micro-aerobic atmosphere (CampyGen Oxoid, Basingstoke, UK) for 3 days. Clarithromycin and levofloxacin resistance was determined by E test (AB Biodisc Solna Sweden) with resistance recorded as > or equal to 1.0 g/mL for both and resistance to metronidazole determined by breakpoint agar streak with growth at 8.0 g/mL denoting resistance. Cag A positive (NCTC11637); CagA negative (NCTC 12908 and ACTC J99) and 57 clinical isolates were tested. For 5 of the isolates (3 Type strains and 2 clinical isolates) a time-kill curve was performed with an inoculum of 107−8 cfu/mL in sterile water at differing concentrations (0.5, 1.0, 5.0, and 12 ppm) of the two copper compounds CuAL42 and CuPC33 against a control containing either sterile water, copper sulfate or binders, for 30, 60 and 120 minutes. At each time point, samples were withdrawn, decimal diluted into ¼ strength Ringers lactate, plated onto blood agar (Oxoid Ltd.) and incubated. Subsequently all the isolates were tested by agar incorporation using Muller Hinton agar with 10% horse blood at the same inoculum against 12 ppm for 60 and 120 mins. The plates were incubated for 5 days at 37 °C in an atmosphere generated by CampyGen (Oxoid Ltd.). A student’s t-test was performed comparing the mean viable count of the control against the test for both agents at T1 and T2. Our conclusions are that these reductive copper compounds deserve further studies in relation to the mechanism of killing, resistance rate and effects on ulcer healing separate from the bactericidal activity reported here. The work was carried out in the Department of Clinical Microbiology, University College London Hospital Trust and financial support was from Departmental and private grant funds. The copper (CuPC33 and CuAL42) compounds were invented, patented, developed and provided by Stephen Hickok. We would like to specially thank Tony J. Hall for his important contribution. These copper biocides are available for research purposes upon request to Hickok.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).This study presents the evaluation of seven pharmaceutical compounds belonging to different commonly used therapeutic classes in seawater samples from coastal areas of Gran Canaria Island. The target compounds include atenolol (antihypertensive), acetaminophen (analgesic), norfloxacin and ciprofloxacin (antibiotics), carbamazepine (antiepileptic) and ketoprofen and diclofenac (anti-inflammatory). Solid phase extraction (SPE) was used for the extraction and preconcentration of the samples, and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for the determination of the compounds. Under optimal conditions, the recoveries obtained were in the range of 78.3% to 98.2%, and the relative standard deviations were less than 11.8%. The detection and quantification limits of the method were in the ranges of 0.1–2.8 and 0.3–9.3 ng·L−1, respectively. The developed method was applied to evaluate the presence of these pharmaceutical compounds in seawater from four outfalls in Gran Canaria Island (Spain) during one year. Ciprofloxacin and norfloxacin were found in a large number of samples in a concentration range of 9.0–3551.7 ng·L−1. Low levels of diclofenac, acetaminophen and ketoprofen were found sporadically.The rapid development of human civilisation has led to numerous environmental problems that affect water resources and has resulted in growing water shortages in various regions [1]. Exposure to hazardous chemicals including biocides, pesticides, and endocrine disrupters represents a threat that should be subject to assessment measures and the reduction and control of irrigation as specified by legislation [2]. Among the various laws relating to water is the EU Water Framework Directive 2000/60/EC (WFD) [3]. The WFD is a norm that provides a framework for community action in the area of water policy for the protection and management of inland waters, transitional waters, coastal waters and groundwater.Traditionally, scientists have studied the presence of chemical pollutants that are regulated by law in the environment. However, more sensitive analytical methods have been developed and have made possible the detection of other hazardous pollutants, referred to as emerging contaminants [4,5], including pharmaceutical compounds [6,7]. Emerging pollutants are anthropogenic in origin; therefore, there are no natural background levels in the environment. The presence of pharmaceuticals in water due to agricultural, industrial and urban activities has been detected, with major sites of pollution being in wastewater treatment plants (WWTPs), sediments of rivers and coastal waters [8,9]. The presence of pharmaceutical compounds has been reported by several authors [10,11,12].Currently, the preferred method for the determination of pharmaceuticals is high-resolution liquid chromatography with mass spectrometry detection (LC-MS) [13,14]. Despite the high sensitivity that can be obtained with this system, it is necessary to apply an extraction technique for sample preparation for analysis due to the low concentrations (ng·L−1) of these compounds in water samples. Solid phase extraction (SPE) is a commonly used sample pre-treatment technique. This methodology represents an attractive alternative to conventional liquid-liquid extraction techniques because it reduces the volume of organic solvent consumed, reduces the total analysis time and allows for automation [15,16].In this work, we developed an analytical method based on solid-phase extraction (SPE) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of pharmaceutical compounds from different therapeutic classes in seawater samples. Table 1 shows the compounds studied and their physicochemical characteristics, which influence their behavior in the environment. The method was applied to evaluate the presence of these pharmaceutical compounds in seawater near outfalls of wastewater in Gran Canaria Island (Spain) between January 2011 and January 2012.The pharmaceutical compounds selected in this study (atenolol, acetaminophen, norfloxacin, ciprofloxacin, carbamazepine, ketoprofen and diclofenac) were purchased from Sigma-Aldrich (Madrid, Spain). Their stock solutions (1 g·L−1) were prepared by dissolving appropriate amounts of pharmaceutical standards in methanol (HPLC gradient-grade PAI-ACS) from Panreac Química (Barcelona, Spain) and the solutions were then stored in glass stoppered bottles at 4 °C prior to use.List of pharmaceutical compounds, chemical structure and pKa values.* Extracted from Hazardous Substances Data Bank.LC-MS quality methanol and water were used to prepare the mobile phase for LC-MS/MS. All solvents and the ammonium formate and formic acid that were used to adjust the pH of the mobile phase were obtained from Panreac Química (Barcelona, Spain).Ultra high purity water was obtained from a Milli-Q (Millipore, Bedford, MA, USA) water purification system and was used for conditioning the process of solid-phase extraction and for preparing aqueous standard solutions.The seawater samples for the determination of pharmaceutical compounds were collected in coastal areas near four outfalls from different urban wastewater treatment plants located on the island of Gran Canaria (Spain) during one year: Las Palmas de Gran Canaria (15.39351° W, 28.09292° N), Jinámar (15.38990° W, 28.03802° N), Punta Salina (15.40751° O, 27.84004° N), Los Cochinos (15.54993° W, 27.76447° N). The samples were collected at a depth of 1 m at the coordinates described and were stored in 1 L amber glass bottles that were pre-rinsed with methanol, deionised water and a real seawater sample for each sampling point. The samples were filtered through 0.65 µm membrane filters (Millipore, Cork, Ireland) and stored in the dark in a refrigerator. All samples were analysed within 48 h of collection.The chromatographic system from Varian (Varian Inc., Madrid, Spain) used for analysis of the selected pharmaceuticals compounds consisted of a 212-LC Binary Gradient LC-MS Chromatography Pump fitted with a Prostar 410 HPLC Autosampler and a 320-MS LC-MS/MS system (triple quadrupole) equipped with an electrospray ionisation (ESI) interface. The system and the data management were controlled and treated by MS Varian LC-MS Workstation Version 6.9 SP1 software.The multiple reaction monitoring (MRM) parameters were optimised for subsequent quantitative analysis. This procedure was conducted using a 1 mL syringe (Hamilton Company, Reno, NV, USA) and a continuous flow rate of 20 µL·min−1. Each standard or mixture was prepared at a concentration of 10 mg·L−1 in methanol. Each solution was taken up with a Hamilton syringe at a volume of 0.1 mL and the remaining 0.9 mL of syringe volume was filled with mobile phase.Ionisation in the ESI source was achieved using nitrogen as the nebuliser and drying gas. The housing and desolvation temperatures were set to 60 °C and 250 °C, respectively, for the optimisation of the syringe pump injections for MS/MS. The drying and nebulising gas pressures were fixed at 30 psi and 65 psi, respectively. The capillary voltage was set to 4.5 kV in positive mode (ESI+) and −3 kV in negative mode (ESI−). The shield voltage was maintained at −600/600 V (ESI+/ESI−) and the cone voltage was optimised for each individual compound. Collision-induced dissociation (CID) was conducted with argon as the collision gas at a fixed pressure of 1.94 psi.The analytical column used for the separation was a 2.0 mm × 50 mm, 2.4 µm particle size Pursuit UPS column (Agilent Technologies). The mobile phase consisted of water (containing 0.2% formic acid and 5 mM ammonium formate, pH 2.6) and methanol. The program began at (90:10) (v/v) for 8 min, then 80:20 (v/v) (water/methanol) for 2 min, followed by an increase to 50% (v/v) of methanol over 5 min, a further increase to 100% (v/v) of methanol over 10 min, and lastly, a return to the initial conditions over 3 min. The system was then allowed to equilibrate for 2 min. The injection volume was 10 µL, and the flow rate was 200 µL·min−1. Calibration of the pharmaceutical compounds was performed in the range of 0.5–600 µg·L−1.The optimisation of the mass spectrometer parameters, such as cone voltage and collision gas energy, was carried out by directly injecting standard solutions of each individual compound into the mass spectrometer. The obtained fragment ions and collision potentials are displayed in Table 2. Figure 1 presents the total ion current (TIC) for target analytes (a) and the fragment ions that were achieved in the multiple reaction monitoring (MRM) mode (b). Two transitions were acquired for the confirmation of all analytes.Mass spectrometer parameter for the determination of target analytes.* Fragment ion used for cuantitation (MRM).(a) Total ion current of pharmaceutical compounds in a standard mixture with LC-MS/MS detection: 1-Atenolol; 2-Acetaminophen; 3-Norfloxacin; 4-Ciprofloxacin; 5-Carbamazepine; 6-Ketoprofen; 7-Diclofenac; (b) The fragment ions in the multiple reaction monitoring (MRM) mode.The SPE cartridges used were Oasis HLB (6 mL, 200 mg) from Waters. The cartridges were conditioned with 5 mL of methanol followed by 5 mL of Milli-Q water a flow rate of 10 mL·min−1 for each run. Water samples were then loaded onto the cartridges at a flow rate of 5 mL·min−1 and thereafter, the cartridges were washed with 5 mL of Milli-Q water at a flow rate of 10 mL·min−1 to remove possible interferences. Finally, the cartridges were dried under vacuum for approximately 5 min and further eluted with 2 mL of methanol at 1 mL·min−1. Blanks were run to evaluate any carryover during SPE. To perform LC-MS/MS analysis, the eluates were evaporated under a gentle nitrogen stream and reconstituted with 1 mL of LC-MS quality water.The extraction process was optimised prior to evaluating the presence of pharmaceutical compounds in seawater. The SPE optimisation included the evaluation of the following experimental variables: cartridge type, pH, ionic strength, sample volume and desorption volume. Different cartridges were tested (Oasis HLB, Sep-Pak C18, Bond Elut SCX and Lichrolut EN) and it was found that the Oasis HLB cartridge was the best for the majority of compounds. Furthermore, we evaluated different pHs (3, 7 and 8) and ionic strengths (0%, 15% and 30% NaCl) of the sample solution. pH values of 7 and 0% (w/v) NaCl were found to be the optimum values for the extraction. We also tested different sample volumes (100, 250, 500 and 1,000 mL) and found the optimum to be 500 mL. To evaluate the required volume for desorption step, we tested the use of 1 mL and 2 mL of desorption solvent applied in one portion and 2 mL of desorption solvent applied in two 1 mL portions. The best desorption volume for the extraction of the analytes was 2 mL of methanol applied in one portion. In summary, the optimum conditions for the solid phase extraction were to pass 500 mL of sample at pH 7 and 0% (w/v) NaCl through an Oasis HLB cartridge, followed by elution of the analytes with 2 mL of methanol. The eluates were evaporated under a gentle nitrogen stream and reconstituted with 1 mL of LC-MS quality water. These conditions resulted in 500 times more concentrated samples.The analytical parameters of the method are shown in Table 3. Calibration curves were established for each compound in the range of 0.5–600 µg·L−1, and the correlation coefficients were greater than or equal to 0.9906 in all cases. The recoveries of analytes using the optimised method (SPE extraction and LC-MS/MS detection) were evaluated at a concentration of 0.1 µg·L−1 for each compound in triplicate. The obtained recoveries were between 78.3% and 98.2%.Analytical parameters for SPE-LC-MS/MS method.a Limit of Detection; b Limit of Quantification; c Linear Dynamic Range.Six standard mixtures of pharmaceuticals (0.1 µg·L−1 of each compound) were extracted and then injected into the LC-MS/MS to calculate the reproducibility (RSD, %) for each compound. Satisfactory results were achieved for all compounds with RSDs lower than 11.8%. The limit of detection (LOD) was defined as the lowest concentration that gave a signal-to-noise ratio (S/N) equal to 3, and the limit of quantification (LOQ) was defined as the lowest concentration that gave a S/N equal to 10 [19]. LODs and LOQs were in the range of 0.1–2.8 ng·L−1 and of 0.3–9.3 ng·L−1, respectively. When compared with the results of other authors, we observed that the detection limit of our proposed method was appropriate for the detection of these pharmaceutical compounds [20].SPE extraction combined with LC-MS/MS was applied to monitor seawater near four different outfalls from urban wastewater located on the island of Gran Canaria (Spain). Figure 2 shows the localisation of sample collection points. Samples were collected during the period between January 2011 and January 2012. Sample collection was not performed during two months (February and April 2011) for the outfalls located in the south of the island due to weather problems.Localisation sampling points on the island of Gran Canaria. 1- Las Palmas de Gran Canaria; 2- Jinámar; 3- Punta Salina; 4- Los Cochinos. The results of these measurements are shown in Table 4. Forty-eight samples were analysed for the different selected points of the outfalls, and it was observed that two of the analysed pharmaceutical compounds (atenolol and carbamazepine) were not detected in any sample collected. The rest of the compounds under study were found in different concentrations ranging from 4.4 to 3,551.7 ng·L−1. Norfloxacin and ciprofloxacin were found in 56.2% and 60.4% of samples, respectively. Low levels of diclofenac, acetaminophen and ketoprofen were found at random. Figure 3 shows a chromatogram corresponding to a sample taken at the Las Palmas de Gran Canaria outfall in March 2011. The presence of acetaminophen, norfloxacin and ciprofloxacin can be observed in this chromatogram.By analysing the results of each outfall, it can be seen that for the outfalls to the south of the island (Punta Salina and Los Cochinos), higher levels were detected in the second half of the year during the summer and the beginning of winter. This may indicate the existence of higher dump of sewage from different wastewater treatment plants in the south of the island due to an increase in the population because of increased tourism during this time. However, for the outfalls located farther north (Las Palmas de Gran Canaria and Jinámar), the levels were more widely dispersed throughout the year, representing a more stable population in this area. The concentrations of pharmaceutical compounds in seawater obtained in this work are similar to those found in other studies for some of the monitored compounds (diclofenac and ketoprofen) [11,21] in seawater. However, we did measure higher levels for some of the monitored compounds, for example, the level of ketoprofen in Jinamar in February 2011.Concentrations in ng·L−1 found in seawater from four different outfalls in Gran Canaria island. aa n = 3; b nd = no detected.Chromatogram of seawater sample from Las Palmas de Gran Canaria outfall with LC-MS/MS detection: 2-Acetaminophen; 3-Norfloxacin; 4-Ciprofloxacin.Furthermore, the fluoroquinolone levels (norfloxacin and ciprofloxacin) we found were quite high. This fact can be seen by comparing our results with data obtained for the levels of these compounds in the effluents of wastewater treatment plants on the island of Gran Canaria, which did not exceed 13.62 µg·L−1 [22]. Upon reaching the seawater, there should be a dilution effect such that the concentrations in seawater are lower than those found in effluent water samples, as is the case in the sampling of norfloxacin in February 2011 at Las Palmas de Gran Canaria (3.6 µg·L−1). Fluoroquinolone levels that other authors have found in municipal wastewater effluents and in the receiving surface water [23,24] are lower (309.2 ng·L−1 maximum) than levels we found in this study, but this comparison is between different matrices. If we do the comparison with a similar matrix (seawater), the levels found in our study would be therein values obtained in others studies (7.5–103 ng·L−1) [25], (3.2–6,800 ng·L−1) [26]. Although these compounds are expected to be preferentially adsorbed on solid environmental matrices [27], the concentrations found in some cases in seawater are quite high. So, this may be due to the type of antibiotics (ciprofloxacin and norfloxacin) are used in aquaculture, Zou et al. suggest that fluoroquinolones (norfloxacin and ciprofloxacin) are extensively used in aquaculture and it is the cause of the high concentrations found for these compounds [26]. The aquaculture is an economic activity of production in Gran Canaria Island that has grown in the last years and it is possible that the unusual concentrations may result from the use of these compounds in fish ponds, which exist close to the sample collection points.In the present work, a study of the presence of seven pharmaceutical compounds (atenolol, acetaminophen, norfloxacin, ciprofloxacin, carbamazepine, ketoprofen and diclofenac) in seawater samples near four outfalls from urban wastewater on the island of Gran Canaria in Spain was executed. SPE using an Oasis HLB cartridge was used for the extraction step. Subsequently, detection was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Seawater samples were collected monthly in the period between January 2011 and January 2012. During the monitoring time, two of the analysed pharmaceutical compounds (atenolol and carbamazepine) were not detected in any of the collected samples. All compounds under study were found in variable concentrations ranging from 4.4–3,551.7 ng·L−1. The fluoroquinolone (norfloxacin and ciprofloxacin) compounds were detected more often and at the highest concentrations in the analysed samples.This work was supported by founds provided by European Union through the Programa de Cooperación Transnacional MAC 2007–2013 within the project Mejora de la Calidad de Aguas Recreativas y Costeras de la Macaronesia, CARMAC, (MAC/2/C011), leaded by Instituto Tecnológico de Canarias S.A. (ITC).
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+	  The authors declare no conflict of interest. 

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The emergence of multi-resistant bacterial strains is a major source of concern and has been correlated with the widespread use of antibiotics. The origins of resistance are intensively studied and many mechanisms involved in resistance have been identified, such as exogenous gene acquisition by horizontal gene transfer (HGT), mutations in the targeted functions, and more recently, antibiotic tolerance through persistence. In this review, we focus on factors leading to integron rearrangements and gene capture facilitating antibiotic resistance acquisition, maintenance and spread. The role of stress responses, such as the SOS response, is discussed.Since the discovery of penicillin by Alexander Fleming in 1928, antibiotics have been the major line of attack for combating infectious diseases. Extensive use of antibiotics, whether natural (isolated from bacteria or fungi) or synthetic, gives rise to development of antibiotic resistance, making it difficult to treat various infections, especially in hospitals where multi-resistant bacteria are involved in nosocomial infections.The mechanisms underlying bacterial resistance to antimicrobial agents reside in the ability of bacteria to quickly modify their genomes. This plasticity is a consequence not only of spontaneous mutations or genome rearrangements that can occur during the bacterial life cycle, but also of exogenous gene acquisition through genetic exchange between bacteria and gene capture in integrons.An integron is characterized by (i) an intI gene encoding a site-specific recombinase of the tyrosine recombinase family, (ii) an adjacent primary recombination site attI [1] and (iii) an array of gene cassettes, downstream of a constitutive promoter (Pc). A gene cassette is a promoterless open reading frame (ORF), flanked by site-specific recombination sequences (attC). The integrase IntI recognizes attC and attI recombination sites and respectively catalyzes excision (attC × attC recombination) and integration (attI × attC recombination) of the cassettes, generating combinations that allow the expression of genes that have been positioned close to Pc [2]. Other than integron rearrangements, IntI also allows the integration of exogenous circular promoterless gene cassettes carrying the attC recombination site [3]. The integrated gene cassettes can be expressed from the Pc promoter located upstream of the attI site in the integron platform [4].In this review we focus on the factors that lead to integron rearrangements and gene capture and that enable antibiotic resistance acquisition, maintenance and spread.Early on, the effective treatment of infectious diseases has been impeded by the development of antimicrobial resistance. Single drug resistant phenotypes were not entirely unforeseen, as demonstrated in early laboratory studies. In contrast, however, multi-drug resistance was not anticipated since the co-appearance of multiple mutations conferring such a phenotype was considered to be beyond the evolutionary potential of a given bacterial population. However, bacteria sometimes possess adaptive genetic resources—termed integrons—that can be used as a reservoir of silent genes mobilizable when needed. Integrons are natural gene expression systems that allow the integration of a gene cassette by site-specific recombination, transforming it into a functional gene [5].Multi-resistance integrons (RI) have been isolated on mobile elements responsible for the assembly and rapid propagation of multiple antibiotic resistances in Gram-negative bacteria and are all embedded inside transposons [6,7]. Integrons were only formally identified as agents of multiple antibiotic resistance gene recruitment in the late 1980s [8]. However, they have contributed to the initial multidrug resistance outbreaks in the 1950s, as indicated by the involvement of Tn21—an integron-containing transposon—in the resistances disseminated by plasmid NR1 (R100) [9]. Several genera including Enterobacteriaceae, Pseudomonas, Vibrio and Acinetobacter (see for example [10,11]) have been shown to harbor RIs. To date, more than 130 different resistance cassettes have been identified in these elements, allowing their hosts to resist to all classes of antibiotics except tetracycline [12,13,14].Several integron classes have been described since 1998, and are in many cases sedentary components of the genome of a number of environmental bacteria, especially members of the proteobacteria group [15,16,17,18,19]. Among these chromosomal integrons, the ones found in all Vibrio species, in many Pseudomonas, Treponema [20] and in Xanthomonas harbor large arrays of gene cassettes (up to 200), in structures called superintegrons (SI) [5]. The SIs are defined by the fact that they show a large array of cassettes (>20 and up to 200) that all share a high degree of identity of their associated attC recombination sites (>80%).Evidence suggests that the SIs and the other chromosomal integrons are the source of the RIs and their cassettes. Phylogenetic relationships point to freshwater b-proteobacteria as the recent ancestors of the most common RIs (Class 1) [21]. Several SIs have been found to carry proto-resistance genes that can provide a resistance phenotype once recruited in a RI [13] (for a review, [14]). Among these are the dfr trimethoprim resistance cassettes [22], the catB9 chloramphenicol resistance cassette [13], the ampicillin resistance cassettes blacarb7 and blacarb9 [23,24] and a more recently discovered qnrVC cassette conferring resistance to quinolones [25]. The discovery of integrons, and among these the SIs, in the chromosome of environmental strains has led to the extension of their role from the “simple” acquisition of resistance genes to a wider role in the adaptation of bacteria to different environments. Notably, all strains of V. cholerae carry a SI in their chromosome (for a review [5]).Studies on the dynamics of cassette recombination and on the regulation of integrase expression are ongoing. Integrons carrying multiple resistance cassettes are stable in the laboratory, even in absence of selection, suggesting the integrase is expressed at low levels in these conditions. The cassettes are nevertheless transferred at a high rate in the environment [26], pointing to a high level of integrase expression in some conditions. Elucidating the mechanisms that regulate the expression of the integrase in various environments would shed light onto the conditions that govern the acquisition of new genes. In the case of resistance and adaptation genes ensuring better fitness in a given environment, this could facilitate the development of new strategies preventing it.The SOS response is a bacterial stress response induced when an abnormal amount of single-stranded DNA (ssDNA) is present in the cell [27]. ssDNA is the substrate for RecA polymerization and the formation of a ssDNA-RecA nucleofilament. This activated state of RecA catalyzes the auto-proteolysis of a repressor, LexA, which normally represses the SOS regulon. Sequence analysis of the promoter regions of intI genes from various RIs and SIs has led to the identification of a conserved region of 16 nucleotides localized 20 to 40 nucleotides upstream of the intI gene. This sequence is similar to the Escherichia coli lexA boxes, which are the target for binding and repression by LexA [28]. It was further demonstrated that intI is indeed regulated by the bacterial SOS response: the activity of IntI from RIs and the V. cholerae SI is induced after treatment with mitomycin C, a DNA damaging agent known for inducing SOS. Moreover, the regulation of the intI promoter by the SOS response was confirmed in strains carrying different lexA alleles (constitutive or defective SOS mutants). In vitro tests have shown that LexA is capable of binding to and repressing the intI promoter [29,30]. Determining the conditions that lead to SOS induction is thus crucial to understand when and where cassette recombination takes place and how the integrase is activated. Figure 1 summarizes the current knowledge on integrase activation in various conditions.Model of intIA regulation and its implications for genome plasticity. Grey boxes represent mechanisms involved in IntIA regulation. Horizontal gene transfer (conjugation, transformation) induces SOS through ssDNA uptake by recipient cells, which in turn triggers intIA transcription. Carbon sources present in the environment also regulate IntIA expression through carbon catabolite control (adapted from [31], Copyright© American Society for Microbiology). The accumulation of ssDNA that triggers induction of the SOS response normally happens when cells try to replicate their damaged DNA. ssDNA is also produced by exogenous DNA uptake machineries that are involved in horizontal gene transfer (HGT), such as conjugation, transformation and occasionally transduction. Whether through conjugation or transformation, it is now clear that the integron plays a major role in bacterial adaptation to the local environment, as shown for V. cholerae, where integron-mediated interspecies gene capture allows for efficient adaptation to local growth conditions in a given environment [32].Conjugative transfer of several broad and narrow host range plasmids induces the SOS response in recipient E. coli and V. cholerae cells [33]. It had been previously shown that during inter-species Hfr conjugation, SOS is induced in the host cell [34,35], suggesting that the low level of homology impairs recombination of the incoming plasmid DNA with the chromosome and thus dramatically enhances the induction of the SOS response. This also suggests that the levels of SOS induction may reflect the ability of RecA to find homologous DNA molecules and initiate strand exchange [36]. In the case of plasmid conjugation, there is little or no similarity with the bacterial chromosome, which might explain the very high SOS induction levels observed. Interestingly, the strongest SOS-inducing plasmids are the ones that cannot replicate in the recipient cell, suggesting that even when the acquired exogenous DNA cannot be maintained, alterations in gene expression in the recipient cell can still occur and possibly lead to antibiotic resistance development. This is particularly relevant in environments where bacterial communities are concentrated. It was also proposed that narrow host range plasmids could constitute a pool of mobilizable suicide vectors that facilitate dissemination of resistance cassettes among evolutionarily distinct bacterial species without the fitness cost associated with plasmid maintenance [37].Triggering integron cassette recombination is one of the consequences of the induction of the SOS response by conjugative DNA transfer [33]. The expression of the RI integrase IntI1, and subsequent cassette recombination, was studied in E. coli during HGT [33]. Conjugative transfer of strong SOS-inducing plasmids increased expression of IntI1, leading to an enhanced SOS-dependent cassette excision rate, linking conjugation, site-specific recombination and genome remodelling. An important consequence of this is the emergence of antibiotic resistant clones, as demonstrated by the acquisition and expression of a chloramphenicol resistance cassette (catB9) in the SI of an originally sensitive V. cholerae, through conjugation-mediated SOS induction [33].V. cholerae alternates between two life styles: growth in biofilms on crustacean shells and colonization of the intestinal tract during infection [38,39]. In both cases, it cohabits with a variety of other bacteria. Induction of the SOS response can thus result not only in chromosomal cassette rearrangements, but also in the capture resistance genes by RIs located on conjugative plasmids that might later transfer to new hosts [5,40,41]. Moreover, conjugation commonly takes place in environments where bacterial populations are concentrated, such as wastewater treatment plants, and where they harbor a large diversity of integrons on conjugative plasmids [42,43]. A remarkable example of gene exchange between bacteria was shown for the Enterobacteria in the mammalian gut when, upon infection with pathogenic Salmonella, HGT was boosted between the invading bacteria and the resident E. coli [44]. Under these conditions the frequency of transconjugant formation approached 100% and lead to the spread of plasmid-encoded fitness, virulence and antibiotic resistance factors. If conjugation induces SOS in the recipient cell, and if the conjugative plasmid (or the host cell) carries an integrase, it is thus easy to imagine how rearrangements and gene capture can lead to the development and dissemination of resistance determinants, as was shown for V. cholerae [13]. This also underlines the role of stress in genome plasticity.Transformation is a second mechanism of HGT that relies on ssDNA uptake and processing [45]. Transformation occurs when a bacterial cell reaches a competent state rendering it capable of taking up DNA present in its environment and, in some cases, of integrating the acquired DNA in its genome by recombination [46]. It was observed in Bacillus subtilis that when lysogenic strains were rendered competent, the induction of a prophage led to the reduction in the frequency of transformation [47]. Prophage induction is in many cases SOS-dependent. It is likely that the reduced transformation efficiency of lysogenic cells compared to non-lysogenic cells is the result of induction of the SOS response by the ssDNA acquired during transformation. Several mechanisms, such as special growth conditions or stress, lead to the induction of competence for natural transformation [48]. Competence has been suggested to be a stress response that could substitute for the SOS response in some bacterial species that lack an SOS regulon but in which the DNA repair genes are part of the competence regulon (for a review see reference [49]).Transformation has been widely studied in several γ-proteobacteria (for a review see reference [48]). V. cholerae is one of the naturally competent gram-negative bacteria [50,51]. It is well established that natural competence for transformation in V. cholerae is regulated by TfoX (also called Sxy in Haemophilus influenzae). Competence is induced in V. cholerae by the presence of chitin [50], a component of the crustacean shells that are one of its natural growth substrates. In addition to genes required for its metabolization, the presence of chitin triggers the expression of TfoX [52,53] and of the entire competence regulon [54,55,56,57].Previously performed microarray studies following growth on chitin did not reveal an increase in the expression of the SI integrase or of the SOS regulon [58]. However, these studies were performed with the non-transformable V. cholerae laboratory strain, which naturally carries an inactivated hapR allele and is as such deficient in competence induction [59]. HapR is the major regulator of quorum sensing and it was shown that the expression of HapR is required for the V. cholerae natural transformation [59]. Moreover, no exogenous DNA was added to the medium in these studies. Redfield and collaborators also performed microarray analysis on H. influenzae and did not see any up-regulation of SOS regulated genes by TfoX/Sxy [57]. Again, no DNA was added to the reaction mixtures. However, when a V. cholerae HapR+ strain was used induction of the SOS response, as well as expression from the wild type SI integrase promoter, was detected upon addition of linear DNA to competent cells [31]. Thus, like conjugation [33], transformation is a mechanism of HGT that induces the bacterial SOS response and therefore also induces expression of the V. cholerae integron integrase.In another study, gene capture by Acinetobacter baylyi integrons during natural transformation with DNA from various integron-harboring bacterial species was assessed [60]. The absence of an SOS regulon in A. baylyi, and thus absence of the LexA repressor, leads to a constitutively active integrase promoter. Remarkably, the transient presence of foreign DNA in the cytoplasm of the recipient strain was shown to be sufficient for efficient interspecies cassette exchange. These results are reminiscent of the ones obtained with the abortive conjugation assays mentioned above [33]. Moreover, even when the integron integrase of the recipient strain was inactivated, an integrase gene encoded by the incoming donor DNA could efficiently replace the inactive copy in the host cell and allow full functionality of the chromosomal integron.The acquisition of resistance genes under these conditions did not appear to have a fitness cost, highlighting the fact that gene capture is not only efficient but also that captured gene cassettes can be stably maintained in the chromosome without a negative effect on cell fitness. Interestingly, the SOS response also regulates some toxin-antitoxin (TA) systems [61]. The presence of 13 cassettes encoding TA systems in the V. cholerae SI might play a significant role in integron maintenance [62].RIs, like other mobile elements such as transposons and ISCR2 elements are sometimes found associated with integrating conjugative elements (ICEs) of the SXT/R391 family [63,64,65]. ICEs are self-transmissible bacterial mobile elements that play a major role in the dissemination of antibiotic resistance genes in bacterial populations. They transfer by conjugation in a process similar to that of many conjugative plasmids, and their transfer was shown to induce the SOS response in recipients to the same extent to what was observed for conjugative plasmids [66]. However, their stable maintenance in their host cell usually depends on site-specific integration into the chromosome. A consequence of this feature is that they are also transmitted vertically with the host chromosome during cell division [67].SXT/R931 ICEs constitute the largest family of ICEs studied to date and are predominant in Vibrio cholerae, in which integrons also play a major role in gene exchange and genome plasticity. ICEs of the SXT/R391 family are now known to be widespread in clinically and environmentally relevant species of Vibrio and related γ-proteobacteria and are commonly associated with the resistance to multiple antibiotics such as sulfamethoxazole (sul2), trimethoprim (dfrA1, dfrA18), aminoglycosides (aphA, strBA), chloramphenicol (floR) and in one case to cephalosporins (blaCMY-2) ([68] and references therein). The common set of conserved genes shared by all members of this family encodes the functions necessary for their integration/excision, conjugative transfer and regulation. In discrete regions of this conserved scaffold each ICE contains additional variable DNA sequences which, besides resistance to multiple antibiotics, can encode heavy metal resistance, toxin/antitoxin systems [69,70], c-di-GMP turnover proteins [71] and multiples genes of yet unknown function [64]. C-di-GMP is a bacterial second messenger molecule involved in the transition between the motile and biofilm lifestyles of V. cholerae and thus directly impacts its epidemic potential by contributing to its survival in the aquatic environment (biofilm formation [72]) and its ability to colonize the human intestine (motility [73]).In manner reminiscent of the induction of the bacteriophage λ lytic cycle, the excision/integration and dissemination of SXT is triggered by exposure to DNA damaging agents. However, the expression of IntSXT—the ICE-encoded tyrosine recombinase required for both integration and excision of SXT from the chromosome—is not under the control of LexA, but rather under the control of two ICE-encoded transcriptional activators, SetC and SetD [74,75]. These transcriptional activators are themselves repressed by a master repressor, SetR, which bears similarities with the λ cI repressor. Repression by SetR is alleviated in conditions which induce the SOS response [74] and by analogy with λ cI it is thought that the intracellular pool of SetR is depleted by the action of RecA bound to ssDNA. Thus, exposure to antibiotics that induce the SOS response triggers the excision and transfer of SXT/R391 ICEs. Recently, it has been shown that most antibiotics induce the SOS response in V. cholerae [66], a phenomenon which would lead not only to the expression of a co-resident integron integrase, but also to increased conjugative transfer of SXT/R391 ICEs. Conversely, conjugative transfer of SXT from an E. coli strain to a V. cholerae strain has been shown to induce the SOS response and cause a 12-fold increase in the activity of the SI integrase (intIA) promoter [33]. Thus, transfer of SXT/R391 ICEs in Vibrio strains is expected to lead to cassette rearrangements in the co-resident SI.SXT/R391 ICEs are even further linked with integrons since a number of these elements contain a class 4 RI conferring resistance to trimethoprim (dfrA1) embedded in their sequence [30,64,76]. In all instances, this integron contains the same array of cassettes, to the exception of the one found in ICEVchMoz10, which lacks the dfrA1 cassette [63,64]. The absence of this cassette is the only marked difference between ICEVchMoz10, isolated in Mozambique in 2004 and ICEVchBan9, isolated in Bangladesh in 1994 [64]. It is tempting to speculate that this RI, which contains four other cassettes of unknown function, might confer an advantage beyond the resistance to trimethoprim that allowed its conservation across time and geographical distance. In contrast to the integrases of other RI classes, the integrase of the RI found in class 4 RIs, IntI9, is not predicted to be induced by LexA as its promoter region lacks a LexA binding site, and the conditions which induce its expression remain to be determined [30].Besides leading to increased integron cassette rearrangements, induction of the SOS response has been recently shown to participate in ICE plasticity through the induction of an ICE-encoded homologous recombination system capable of promoting the formation of hybrid ICEs [77,78]. This recombination system (bet/exo) is related to the Red recombination system encoded by bacteriophage λ. It is under the control of the ICE-encoded transcriptional activators SetCD and thus induced by exposure to DNA damaging agents [78]. SXT/R391 ICEs all share the same chromosomal attachment site (prfC) and their ability to co-exist in the same cell allows for the integration in tandem of two similar but non-identical ICEs [79]. Inter-ICE recombination events occur between two tandemly arranged SXT and R391 ICEs and lead to the formation of novel ICEs composed of sequences derived from both parental elements present in the donor cell [77,79,80], thus potentially participating in the diversity of the antibiotic resistance patterns carried by these elements. The discovery that this homologous recombination system is induced during the SOS response sheds new light on the adaptation of these mobile genetic elements to their host’s stress response and to the extent with which they profit of this for their own plasticity. Exposure to DNA damaging agents such as antibiotics increase hybrid ICE formation and ICE conjugative transfer which, in V. cholerae, also contributes to inducing the SOS response. Ultimately this leads to increased integron integrase expression, ICE diversity and to the spread of antibiotic resistance determinants.Interestingly, homologues of SXT bet/exo have been recently identified for the first time in a wide variety of bacterial species and on a number of conjugative elements, such as plasmids belonging to the A/C incompatibility group [64,78]. IncA/C plasmids are the closest parents of SXT/R391 ICEs and are largely recognized for their major role in multidrug resistance in Salmonella, Yersinia pestis and in aquatic γ-proteobacteria such as V. cholerae [81,82,83,84,85]. These broad host range plasmids also often carry class 1 RIs and encode, amongst others, resistance to β-lactams (blaSHV-1, blaCMY-2-1, blaCMY-2-2), tetracycline (tetRA), sulfonamides (sul1 in the 3'-CS of the RI, sul2), aminoglycosides (aadA integron cassette, aphA, strBA), chloramphenicol (cmlA7 integron cassette), and mercury (merRTPCADE) [64,85,86,87].Bacteria have the ability to adapt to various environments and efficiently switch between widely different growth conditions. For instance, V. cholerae alternates between two niches: the host’s intestines, which are full of nutrients, and the aquatic environment (crustacean shells) where nutrients are scarce and carbon sources vary.When the level of the favorite carbon source (such as glucose) is low in the medium, bacterial cells start to use “slow” carbon sources (such as chitin in the case of V. cholerae), which activates the carbon catabolite control regulon, also called CRP (cAMP receptor protein) regulon. Carbon catabolite repression, defined as the inhibition of gene expression by the presence of the favorite (i.e., rapidly metabolizable) carbon source, has been widely studied [88,89,90]. In γ-proteobacteria, growth in media lacking the “favorite” carbon sources leads to increased expression of the CRP-regulated genes, through the activation of the adenylate cyclase [91]. Under such conditions, the rising levels of intracellular cAMP control the CRP regulon through the action of the CRP-cAMP complex (referred to as CRP in the following lines). CRP binds to regulatory DNA sequences termed crp boxes [92] and modulates transcription. CRP not only controls specific catabolic pathways involved in carbon metabolism, but also many other genes involved in important aspects of cell physiology and interaction with the environment [93,94], such as quorum sensing, virulence and competence [95,96,97]. Many genes involved in the adaptation and survival of E. coli, such as the ccd toxin, or the plasmid F tra genes, are also known to be regulated by CRP [98]. In V. cholerae, CRP represses the cholera toxin and the toxin co-regulated pilus [99], favoring growth in low-nutrient environments (outside the host) and virulence in nutrient-rich environments such as the intestine.Interestingly, regulation of natural competence for transformation also depends on the CRP-regulon, mainly because an increase in cAMP levels triggers the expression of the competence regulon activator TfoX/Sxy [57,91]. Additionally, Δcrp mutants produce lower levels of HapR [100], essential for competence [50]. Finally, TfoX/Sxy, in complex with CRP, is involved in the induction of some competence-specific crp boxes [91,101,102]. H. influenzae and V. cholerae Δcrp mutants are thus unable to become competent [57,103]. Hence, carbon source-dependent regulation plays a role in genetic exchange through the regulation of natural competence for transformation.In V. cholerae CRP activation also acts by directly impacting the SI intIA integrase promoter activity in an SOS-independent manner [31]. The cAMP-CRP complex was in fact shown to directly bind the intIA promoter and activate its transcription, favoring cassette rearrangements in the SI [31]. CRP also enhances transcription from the primary cassette promoter Pc in the attI site (Krin and Mazel, unpublished results). Like the SOS response, CRP regulation can thus be considered a stress response because it modulates the expression of the catabolite control regulon when rapidly metabolizable carbon sources are scarce [88,89,90,94,104]. The regulation of the SI integrase by two different stress responses highlights the influence of the environment on bacterial genetic adaptability. One may speculate that outside the infected host, integron-harboring bacteria shuffle gene cassettes, creating genetic diversity in order to increase their odds of surviving in hostile conditions or when exposed to antibiotics.A large proportion of the antibiotics ingested are released intact in the environment [105,106] and found at trace levels or as gradients in various environments [107]. This is particularly relevant in the aquatic environment [108,109] and in the mammalian hosts of pathogenic and commensal bacteria, where antibiotics can play a very important role in the selection of resistant bacteria [110]. These low concentrations do not affect bacterial growth and are referred to as sub-MICs, for sub-minimal inhibitory concentrations.Unlike above-MICs, the biological effects of sub-MICs of antibiotics have not been studied in detail. Transcriptome and proteome analyses have shown that many antibiotics exhibit contrasting properties when tested at low and high concentrations [111]: sub-MICs modulate metabolism through altered transcription whereas higher concentrations of antibiotics inhibit growth (for a recent review, [112]). It has been noted for example that sub-MICs induce several changes in the expression profile of a wide range of genes unrelated to the target function, such as resistance to oxidative stress, motility, virulence and biofilm formation [113]. For instance, in Salmonella enterica, a sub-MIC of rifampicin was shown to modulate transcription of several promoters through the interaction with RNA polymerase [114], whereas sub-MICs of fluoroquinolones (FQs) modulate gene expression by inducing the SOS response through DNA damage [115]. Sub-MICs of macrolides also have an effect on gene expression in this bacterium [116]. It was thus proposed that antibiotic sub-MICs act as agents for bacterial communication and signaling. This would explain the natural occurrence of such concentrations in bacterial communities [113,117]. Biofilm formation (modified flagellar formation and motility) is another consequence of the exposure to sub-MICs of FQs, macrolides and vancomycin (Staphylococcus sp.) [118,119,120] and to sub-MICs of aminoglycosides (AGs) (P. aeruginosa and E. coli) [121]. Biofilm communities are known to be more resistant to antimicrobial agents [122]. Although the aim of this review is not to expose links between biofilms and resistance it is worth mentioning that biofilm-specific tolerance to FQs has recently been studied in detail [123] and that many reports describe SOS induction [124,125] and high horizontal gene transfer levels in biofilms [126,127].We depicted earlier in this review how SOS induction triggers integron rearrangements. Several classes of antibiotics (fluoroquinolones [128], β-lactams and trimethoprim [129,130,131]) are known to induce the SOS response and increase mutation frequencies in E. coli. By directly targeting DNA related functions (such as replication and repair) or the DNA molecule itself (through crosslinks or lesions) they lead to the accumulation of DNA damages. The resulting induction of the SOS response increases the frequency of mutations (shown for FQs and trimethoprim in Staphylococcus aureus [132,133]) and higher levels of homologous recombination (shown for FQ in E. coli [134]). Point mutations can result in antibiotic resistance acquisition. For instance, resistance to ciprofloxacin (FQ) and to rifampicin is due to mutations caused during the induction of the SOS response, through the action of the error-prone polymerases II/IV/V [135]. Sub-MICs of FQs were clearly shown to cause resistance development in S. aureus [136] and S. enterica [110]. The increased frequency of mutation in presence of FQs was proposed to lead to the overload of MutS-dependent mismatch repair, causing the accumulation of unrepaired DNA and the appearance of point mutations that lead to antibiotic resistance. On the other hand, antibiotics that do not target DNA—such as aminoglycosides (AGs), chloramphenicol, rifampicin and tetracycline—were initially discounted as SOS-inducers after studies in E. coli [66,131] and S. aureus [133]. Conversely, tetracycline was also shown to induce the appearance of mutations, which require SOS-regulated DNA polymerases [137], suggesting a link between tetracycline and the SOS response.Since many bacteria carry integrons that are under the control of the SOS response and that sub-MICs of antibiotics affect gene expression, it was essential to shed light onto the possible effects of sub-MICs on these genetic elements. Strikingly, and unlike for E. coli, sub-MICs of AGs, chloramphenicol, rifampicin and tetracycline induce the SOS response in V. cholerae [66], Photorhabdus luminescens and Klebsiella pneumoniae [138]. This unexpected induction of the SOS response suggests a role for intermediate factors that cause stress and lead to DNA damage in V. cholerae. Moreover, expression from the V. cholerae SI integrase promoter was shown to be induced in the presence of sub-MICs of antibiotics belonging to these classes [66]. A parallel can be made here with the effects of metronidazole, another antibiotic that does not cause direct DNA damage, on Pseudomonas aeruginosa. P. aeruginosa is known to be exposed to sub-MICs of antibiotics in the lungs of cystic fibrosis patients, where it causes chronic lung infections and where gradients of antibiotics exist. Strikingly, SOS-mediated integron rearrangements in P. aeruginosa led to β-lactam and ceftazidime resistance during metronidazole treatment of a patient [139]. It is interesting to note here that AGs and metronidazole are both capable of inducing the SOS response although neither causes direct DNA damage.Induction of the SOS response has other implications related to genome stability and antibiotic resistance [27,140]. On one hand, it facilitates acquisition of resistance. Induction of the SOS response increases the frequency of point mutations, as shown previously for FQs and ampicillin (mentioned above, [133]) and more recently for sub-MICs of AGs, rifampicin, tetracycline and chloramphenicol in V. cholerae [66]. Ampicillin at sub-MICs was found to down-regulate mismatch repair in E. coli, P. aeruginosa and V. cholerae hence increasing mutation frequencies [141]. The same phenomenon was observed in V. cholerae after treatment with AGs (Baharoglu and Mazel, unpublished results). Such modest increase in mutation frequency (from 10−9 to around 10−8) is of high importance since it was shown to influence the evolution of multidrug resistance in bacteria [142]. Indeed, strains characterized by low or high mutation rates actually have a lower resistance to antibiotics than strains that have an intermediate rate of mutation (around 10−8), and this, independently of the antibiotic tested [142]. Indeed, high mutation frequencies probably more often lead to deleterious mutations. Another way for FQ resistance acquisition through SOS induction is described in [143], where the qnrB gene conferring low resistance to quinolones was shown to be regulated by LexA. Resistance to quinolones is thus induced by the presence of quinolones themselves, at sub-inhibitory concentrations, or by other antibiotics that induce the SOS response. Another study has also suggested that increased resistance to one antibiotic (after sub-MIC FQ-dependent increase in mutation frequency) can lead to the development of resistance to other classes of antibiotics [133].Moreover, induction of the SOS response can favor the spread of these resistances. As mentioned earlier, SOS induction leads to spread of antibiotic resistance genes by inducing the dissemination of integrating conjugative elements (ICEs) [74]. SOS induction can also facilitate HGT and dissemination of virulence factors carried by mobile genetic elements [144,145]. Interestingly, AGs, FQs and mitomycin C (MMC) induce the competence (com) regulon in Streptococcus [146]. Streptococcus does not have a homologue of the SOS repressor LexA; however, its com regulon is considered a parallel of the SOS regulon since it contains most of the DNA repair genes, including recA. This means that upon becoming competent for natural transformation Streptococcus also becomes highly recombinogenic, which favors the acquisition and expression of new genes.Apart from the evolution of bacterial resistance, sub-MICs are also involved in the conservation of multiple resistances by the bacteria carrying them, through reduced fitness cost. Selection and enrichment of resistant bacteria has been observed for E. coli and Salmonella using three different antibiotics (tetracycline, FQs, AGs) at a hundred-fold below the MIC [147]. The authors show in this study that in the presence of sub-MICs of these antibiotics, the fitness cost of antibiotic resistance is overcome and resistant bacteria are maintained. Similarly, bacteria that show slightly increased mutation frequencies and harbor antibiotic resistances are found in greater proportions in the commensal flora of cystic fibrosis patients subjected to prolonged antibiotic treatment [148]. Another study even concludes that resistant strains have a selective advantage over others in presence of sub-MICs of FQs and tetracycline [149].Finally, induction of the SOS response by antibiotics also leads to the formation of persister cells [150,151,152]. Persisters are antibiotic tolerant cells that are not killed during treatment and resume growth when antibiotics are removed (for a review [152]). Dorr et al. showed that persisters are not pre-existing dormant cells, but rather that their formation is induced by the SOS response [150]. Interestingly, the appearance of persister cells was shown to be much higher during treatment with a sub-MIC of FQ than when higher antibiotic concentrations were used (stronger SOS induction). Persister cell formation can occur through the induction of toxins from the toxin-antitoxin family, such as TisB from the SOS regulon, which decrease the growth rate (drop of ATP, no active peptidoglycan synthesis, no ribosome, no replication), causing tolerance to multiple antibiotics [151]. Interestingly, 15 toxin-antitoxin modules are present in the V. cholerae SI [76,153], and TisB may not be the only toxin that leads to persistence. Therefore, the sub-MIC use of SOS-inducing antibiotics in V. cholerae may lead to persistence and eventually contribute to the development of multidrug resistance.The induction of the SOS response by sub-MICs of antibiotics that are not known to cause DNA damage formation is intriguing. A recent study demonstrated that β-lactams, FQs and AGs lead to cell death through the production of reactive oxygen species (ROS) in bacteria [154]. ROS damage DNA and damaged DNA is a potent inducer of the SOS response. This study suggests that all bactericidal antibiotics, regardless of their cellular target, have the potential to induce the bacterial stress response. Since ROS can damage DNA and proteins, and thus induce mutagenesis, [155,156] it could be the missing link between sub-MIC antibiotic treatment and the induction of the SOS response.Our group very recently showed that sub-MICs of tobramycin leads to an increase of intracellular ROS formation in V. cholerae, causing oxidative stress even at concentrations 100 times below the MIC [138]. At sub-MICs, tobramycin mediates induction of the SOS response mostly through the formation of ROS and the subsequent 8-oxo-G incorporation in DNA. The effect on the SOS-dependent intIA promoter was also assessed and we demonstrated an increase in integron recombination in presence of of sub-MICs of antibiotics. This means that not only numerous rearrangements may take place within the V. cholerae SI, but that other SOS-regulated integrases [29,76] (such as those from plasmid-borne RIs) are likely to be induced if present in a V. cholerae cell. AGs are commonly used against Gram-negative bacteria and the fact that they induce integrase activation and gene capture is of particular concern. Induction of the SOS response by AGs is a conserved trait among distantly related Gram-negative pathogens such as Klebsiella pneumoniae and Photorhabdus luminescens [138]. E. coli on the other hand, has a stronger resistance to the stress triggered by AGs [138]. This might indicate that some species counter their poorly efficient protection system against oxidative stress by being more easily capable of modifying their gene expression patterns [157]. Interestingly, numerous cassettes encoding resistance to all AGs were characterized in RIs (to date, a total of 43 cassettes [14]). The capture and selection of these cassettes can now be explained by the fact that exposure to AGs directly induces the SOS response and thus, the integrase. This implies that the use of these antibiotics may promote cassette rearrangements and expression of integron-borne resistances to all families of antibiotics, including to ones that do not induce the SOS response in E. coli.Induction of ROS formation by sub-MICs of antibiotics was shown to increase resistance development in various bacteria. In Proteus mirabilis for instance, for which sub-MICs of FQs were also shown to stimulate ROS formation [158], repeated cultures in the presence of a sub-MIC of FQs induces the formation of FQ resistant variants. These resistant phenotypes were not due to the typical mutations in the primary targets of FQs (like the gyrase or topoisomerase IV) but rather to enhanced resistance to oxidative stress [158]. Additional data on sub-MICs of antibiotics underline their important consequence of allowing bacteria to survive to antibiotic concentrations that would normally be lethal. FQ treatment at high concentrations leads to lipid and protein oxidation by ROS. Strikingly, less oxidation was observed at high FQ concentrations when bacteria had first been exposed to sub-MICs of FQs [158]. In a sense, one can think of sub-MICs of antibiotics as “homeopathic doses” that protect bacteria when later exposed to a normally effective antibiotic treatment.Interestingly, susceptibility to antibiotics increases in absence of ROS detoxification pathways [159,160]. In parallel, stabilization of a single oxidative stress-sensitive protein is sufficient to enhance oxidative stress resistance of V. cholerae [161], a fact that confirms the weight of protein oxidation on V. cholerae’s ability to cope with stress. Several very different modes of antioxidant molecule production have been discovered in bacteria and can have, for instance, a role in the stringent response [162] or in increasing superoxide dismutase and catalase levels through H2S production and therefore help counteract ROS formation during antibiotic stress [163]. By elevating the production of antioxidant enzymes, these mechanisms allow bacteria to grow in the presence of a wide range of antibiotics (ofloxacin, meropenem, colistin, gentamicin) and can be regarded as antibiotic tolerance mechanisms [164].Oxidative stress is known to induce the RpoS regulon [165]. RpoS, the stationary phase sigma factor, is induced in response to various stresses during the exponential growth phase [166,167,168] and increases resistance to stress [169]. Genes expressed following the induction of the RpoS regulon, namely catalases (KatE, KatG) and iron chelators, protect cells from ROS-related DNA damage [170] such as double-strand DNA breaks [171,172,173]. RpoS was also shown to play a role in antibiotic tolerance: when screening for mutants with altered antibiotic tolerance and decreased persistence genes dnaK, rssB, dksA and ygfA were identified, among others [174]. The proteins encoded by these genes regulate the stability of RpoS and the expression of the RpoS regulon, which points out RpoS as one of the determinants of persistence development.Furthermore, several lines of evidence suggest a role for RpoS in horizontal gene transfer. Recently, the RpoS pathway was shown to be linked with the expression of the integron integrase in E. coli in presence of sub-MICs of AGs [138]. Observations in V. cholerae also show that the integron integrase promoter is more active during the stationary growth phase, suggesting that there could be an effect of RpoS on the integrase promoter (Krin and Mazel, unpublished results). Furthermore, in Pseudomonas knackmussii RpoS controls the activation of the integrating conjugative element ICEclc [175]. In absence of RpoS, the ICEclc integrase promoter, which catalyzes ICE excision, is significantly less active. This considerably impairs horizontal transfer of ICEclc, as ICE excision is a prerequisite for its conjugative transfer.RpoS is conserved within α-, β- and γ-proteobacteria, but the composition of the RpoS regulon varies from one species to the other [176]. It has been suggested that these variations sometimes arise from the integration of horizontally transferred genes into the RpoS regulon [176,177]. Environmental pressure can also cause rapid loss or change in the RpoS regulon [178,179]. An additional contribution to coupling stress and cassette array expression could reside in the integration of exogenous open reading frames, captured by the V. cholerae integron, into the RpoS regulon. Nonetheless, further work is still needed in order to characterize the possible effect of RpoS on RI and SI cassette array expression. RpoS has also been proposed to be involved in double-stranded plasmid transfer in E. coli in laboratory conditions [180]; however, the existence of such a mechanism in nature remains to be proven. Finally, quorum sensing enhances oxidative stress response and survival by up-regulating RpoS [181]. If one considers sub-MICs of antibiotics as signaling molecules that activate oxidative stress response, then RpoS can be named as one of the key players that trigger gene exchange and genome plasticity.Sub-MICs of antibiotics appear to be potent agents of stress for bacteria. The SOS DNA damage response and the RpoS general stress response synergistically protect cells from this kind of aggressions. Horizontal gene transfer during conjugation and natural transformation also influences genome plasticity through acquisition of exogenous genes and through the induction of the SOS response. Moreover, several environmental factors such as carbon source or the presence of oxygen play a role in the activation of the integron integrase, as described throughout the manuscript. The possible effects of other factors such as pH, salinity and temperature are not excluded.In the light of the studies mentioned in this review, we believe that it is important to better understand how different ecological niches and different lifestyles modulate the evolution of bacterial stress responses, since they have a major impact on the evolution of genome plasticity and antibiotic resistance. Although no data on the induction of the SOS response in the mammalian gut is currently available, several lines of evidence suggests this is a possibility: (i) the presence of sub-MICs of antibiotics in the gut, (ii) increased levels of HGT [44] and (iii) other factors like oxidative stress can plausibly be expected to induce the SOS response and, ultimately, integron rearrangements.In the search for compounds that can potentiate the effect of antibiotics on bacteria, the ones that amplify ROS production are currently of high interest. Studies showed that bacterial metabolites can render E. coli persister cells more susceptible to AGs in the presence of certain carbon sources [182] and it was further proposed that the amplification of ROS production could have the same adjuvant effect on antibiotics [183,184]. These authors have identified 133 reactions that could be potential sources of ROS and demonstrated that the modification of these pathways can lead to increased antibiotic susceptibility through increased ROS formation.An engineered bacteriophage that suppresses the SOS response (by over-expressing the LexA repressor) has also been reported to enhance the lethal effect of quinolones, AGs and β-lactams on E. coli, to reduce the number of resistant bacteria that arise from the antibiotic treatment, and to increase survival of infected mice [185]. According to the authors, these observations would be the result of disabling DNA damage repair. Another way of preventing treatment failure is by combating the SOS-induced mutagenic DNA polymerase-dependent mutations that lead to FQ resistance [135].Unraveling the factors that control the expression of integron integrases is essential to determine the pertinence of the development of integrase inhibitors in the battle against the dissemination of multi-resistant strains. Understanding the molecular mechanisms that drive the emergence of drug resistance can facilitate the design of more effective treatments.Studies cited from the Mazel laboratory were funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525), by the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage) “Evolution and Transfer of Antibiotic Resistance” (EvoTAR) and the French Government’s Investissement d'Avenir program, Laboratoire d'Excellence “Integrative Biology of Emerging Infectious Diseases” (grant n ANR-10-LABX-62-IBEID). ZB was supported by a DIM Malinf postdoctoral fellowship (Conseil régional d’Île-de-France). GG is the recipient of post-doctoral research fellowships from the Fonds de Recherche Québecois—Nature et Technologies and from the Roux, Howard and Cantarini Foundation (Institut Pasteur). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare no conflict of interest.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Inappropriate antibiotic use in normally self-limiting acute respiratory tract infections (RTIs), such as sore throat and the common cold, is a global problem and an important factor for increasing levels of antibiotic resistance. A new group of international experts—the Global Respiratory Infection Partnership (GRIP)—is committed to addressing this issue, with the interface between primary care practitioners and their patients as their core focus. To combat the overuse of antibiotics in the community, and facilitate a change from prescribing empiric antibiotic treatment towards cautious deferment combined with symptomatic relief, there is a need to introduce and enhance evidence-based dialogue between primary care practitioners and their patients. Communication with patients should focus on the de-medicalisation of self-limiting viral infections, which can be achieved via a coherent globally endorsed framework outlining the rationale for appropriate antibiotic use in acute RTIs in the context of antibiotic stewardship and conservancy. The planned framework is intended to be adaptable at a country level to reflect local behaviours, cultures and healthcare systems, and has the potential to serve as a model for change in other therapeutic areas.Antibiotic resistance exerts a significant and pressing global healthcare challenge, with 25,000 people in Europe dying each year as a direct result of resistant infection [1]. Using 2007 data, the costs of related healthcare expenditure and productivity loss for the European Union member states, Iceland and Norway, were estimated to be as high as 1.5 billion Euros per year [1]. In America, hospital-acquired infections, most of which are caused by antibacterial-resistant pathogens, result in an estimated 99,000 deaths per year [2]. Gram-negative bacteria, in particular, become resistant to antibiotics, resulting in, for example, carbapenem-resistant Enterobacteriaceae spp. and extended-spectrum beta-lactamase-producing Enterobacteriaceae [3,4,5,6]. Certain gram-negative pathogens are now panresistant, where none of the currently available antibiotics are effective [6,7,8]. In South Asia, antimicrobial resistance is estimated to cause 300,000 infant deaths annually from neonatal sepsis-causing organisms, such as Staphylococcus aureus, Escherichia coli, Enterobacter and Acinetobacter species and Pseudomonas [9]. The problem is thought to be equally widespread, yet under-investigated, and consequently under-estimated and under-reported, in other developing countries.While antibiotic resistance has predominantly been a clinical problem in hospital settings, recent data show resistant organisms have also been detected in patients outside of this setting [10], with higher resistance rates in countries with high levels of antibiotic use [11]. Diseases associated with antimicrobial resistance outside the hospital setting include tuberculosis, gonorrhoea (specifically Neisseria gonorrhoeae), typhoid fever and Group B streptococcus [12]. Community-acquired antimicrobial resistance is of particular concern as these infections can be common and easily transmitted [10]. Infection with antibiotic-resistant bacteria may cause severe illness, increased mortality rates, an increased risk of complications and admission to hospital, often resulting in higher treatment costs [13,14,15]. Some recent emerging trends that are of growing importance should also be considered. One is the rise in prescribing for urinary tract infections. Even in countries such as the Netherlands, which has a low and stable antibiotic prescribing rate, a 32% increase in prescribing for these infections was noted between 2007–2011 [16]. In other European countries increased resistance to the standard urinary tract infection treatment, nitrofurantoin, is also developing [17]. This is being driven by increasing antimicrobial resistance of Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae [18]. This poses a significant public health problem, with therapeutic options to treat life-threatening K. pneumoniae infections diminishing [18]. In the context of few innovative or new antibiotics in the drug development pipeline, the World Health Organization (WHO) describes a future of a post-antibiotic world and warns that not only will this eliminate the advances in healthcare made over the past 100 years, that have ensured longer life in most parts of the developed and developing worlds, but it may also result in simple infections becoming unmanageable and potentially fatal [19,20]. The limited use of antibiotics in the future does not appear to be sufficient enough to change behaviour in antibiotic prescribing and self-initiated antibiotic use. The challenge facing societies around the globe is to encourage significant changes now to save lives in the future.GRIP is a new multi-disciplinary group of international experts, comprising of primary care and hospital physicians, microbiologists, researchers and pharmacists. GRIP members were selected due to their existing interest and work in the field of antibiotic use and resistance. In particular, all members are focused on implementing change in antibiotic use and the treatment of non-serious infections, such as respiratory tract infections (RTIs), in primary care. The need for a multidisciplinary group of experts with broad reach was also considered when identifying candidates, to maximise the impact GRIP is intending to have as a campaigning force for change. GRIP is committed to address antibiotic overuse-related problems using evidence-based studies to support suitable rationale for antibiotic use and promote antibiotic stewardship among healthcare professionals. A framework is being developed for non-antibiotic treatment options for symptoms of acute RTIs.In this first GRIP opinion paper, the group aims to stimulate debate between healthcare professionals and highlight the need for an integrated approach to facilitating behavioural change among antibiotic prescribers and users.A pilot for the development of such an integrated approach should be a single infection, the treatment of which is currently characterised by the overuse of antibiotics in the primary care sector—an ineffective and unnecessary treatment option for the majority of patients—and where alternative symptomatic relief options are available. GRIP identified that the majority of patients who develop acute RTIs, such as sore throat and common colds (with the exception of the more severe infections such as pneumonia), can adopt this approach. Once a coherent international approach has been developed to promote a change in the prescribing and use of antibiotics for the treatment of RTIs, this could serve as a model for change in other infections.Data show a direct correlation between the use of antibiotics and resistance. Countries where there is higher consumption of antibiotics show higher resistance rates [11,21]. The converse has also been shown, with Finnish data finding macrolide resistance of S. pyogenes reducing in parallel with a decrease in antibiotic consumption. Antibiotic resistance dropped from 9.2% in 1997 to 7.4% in 2000, with a statistically significant association between regional macrolide resistance and consumption rates [22].Taking Europe as an example, antibiotic usage varies widely, as do resistance rates. The most recent data from the European Antibiotic Surveillance Reports found that antibiotic resistance rates of E. coli and/or K. pneumoniae varied markedly between countries. Resistance of both were among the highest in Greece and Slovakia and the lowest in Sweden and Norway in 2011 [18]. Rates of resistant E. coli varied by a factor of 18 between Sweden (1.0%) and Greece (18.2%); for K. pneumoniae the differences were even more pronounced, ranging from 0.7% in Sweden to 64.1% in Greece [18]. This correlates to antibiotic consumption levels. On average, the European consumption rate for antibiotics is 18.3 defined daily doses (DDD)/1000 inhabitants/day in 2010, with a low rate seen in Sweden (~14 DDD/inhabitants/day) and the highest in Greece (~39 DDD/inhabitants/day), France and Luxembourg (both ~28 DDD/inhabitants/day) [23].The impact of high antibiotic use on antibiotic resistance is significant [24]. Global data from 2010/2011 for the percentage of extended spectrum beta-lactamase [ESBL]-producing bacteria present, indicating antibiotic resistance of E. coli and Klebsiella spp., are highest in India and China at ≥80% and ≥60%, respectively [13]. Rates were ≤30% in Latin America, East/Southeast Asia and Southern Europe, and 5%–10% in Northern Europe, Australasia and North America [13]. European data from 2011 demonstrate an alarming increase in resistance of these organisms, with around a third of European countries showing a rise in combined resistance to third-generation cephalosporins, fluoroquinolones and aminoglycosides over the previous four years [18].This will exert a major adverse effect on treatment options. A recent review describing patients with urinary tract and respiratory tract bacterial infections treated with antibiotics reported that individual resistance may persist for up to 12-months post-treatment, thereby creating situations for the need of second-line antibiotics [25]. When antibiotics are used the individual’s entire bacterial gene pool is sensitive to change. As resistance can be transferred between bacteria, new pathogens can evolve. Therefore, antibiotic use in an individual could have a direct impact on the level of resistance in the population [25].In developing countries, the impact of rapidly increasing resistance rates is of particular importance and concern. Respiratory disease has overtaken diarrhoea as the most frequent cause of child death in these countries, [26] with community-acquired S. pneumoniae among the main pathogenic species. This organism also demonstrates increasing resistance to a variety of antibiotic agents [27]. Misconceptions and uncertainties regarding the role of antibiotics exist among patients and primary care physicians [28]. For example, a survey in Australia revealed that only 50% of patients are conscious of the development of antibiotic resistance, with even fewer (40%) aware that antibiotics are ineffective against viruses [29]. This echoes European research reporting that around half of patients believed antibiotics were effective in treating viruses, cold and flu [30].RTIs are the most commonly treated acute problem in primary care [31], with the majority of infections of viral origin. In patients with RTIs at high risk of developing complications the use of antibiotics needs to be considered. However, complication rates have markedly reduced [31,32] and the need for antibiotics is no longer required in the majority of people with RTIs. Furthermore, in developed countries with comparatively low antibiotic use, complications are no more frequent than in developed countries with high antibiotic use.Despite this, antibiotic use for RTIs in primary care remains high. In Europe, upper RTIs accounted for 57% of antibiotics used, with a further 30% for lower RTIs; in contrast the next most common condition was urinary tract infections at 7% [30]. In the UK, 60% of all antibiotics prescribed are for patients with RTIs [33]. In Bangkok, Thailand, the prevalence of antibiotic prescription for some RTIs was as high as 78%–94% [34]. A lower prescription rate of 33.5% was observed in Norway for patients with RTIs seen in primary care [35]. In Italy, antibiotics were prescribed for 44% of croup, influenza and common cold cases presenting in primary care [36]. There also appears to be a dissonance between physician and patient expectations during a RTI consultation. Primary care physicians say they often feel under pressure by the patient to prescribe antibiotics [37]. Some studies report that patients strongly influence the antibiotic prescribing of physicians by using a number of different behaviours, such as “in the next two days I must leave for my holidays” or “I got antibiotics for this before and it worked” [38]. Other studies report that only a small proportion of patients explicitly expect a prescription [39], and even if they do, their primary concern is to find symptomatic relief [37,40]. In a Dutch survey, patients who, prior to the consultation indicated a desire for an antibiotic, were equally satisfied with the consultation afterwards, irrespective of whether they received an antibiotic or not [41]. Furthermore, a clear explanation about the expected course and duration of disease, and a proper physical examination correlated more to patient satisfaction than a prescription.Improved communication in primary care can help bridge this gap between physician and patient expectations. This can be achieved using various approaches. In Germany, training physicians in patient-centered communication resulted in a 40% reduction in antibiotic prescribing, with results lasting for over one year [28]. In comparison, the control group of doctors with no training in communication skills increased their antibiotic prescribing during the study period. In the UK, the paediatric antibiotic prescribing rate decreased by 50% as a result of physicians using an interactive information leaflet on the management of upper RTIs in children during the consultation with parents [31]. The limited use of diagnostic techniques in general practice to identify the aetiological agent (viral or bacterial) might also explain the overuse of antibiotic therapy. In fact, where diagnostic rapid tests are used there is a lower risk of antibiotic use [36]. However, as the majority of infections, bacterial or viral, are self-limiting and symptomatic relief is adequate, GRIP raises the issue of the need for testing. There are various national and global initiatives to try to counteract the threat of antibiotic resistance. In 2010, the Infectious Diseases Society of America (IDSA) started a new campaign, the “10×'20” initiative, which is a global commitment to support the development of ten novel, effective antibiotics by 2020 [42]. An update in 2013 on the incentive highlighted the slow progression in the clinical development of new antibacterial agents for resistant gram-negative bacteria [43]. In addition to the development of new antimicrobial agents, IDSA called for more focus on antibiotic stewardship and infection prevention in order to preserve the efficacy of current antibiotics [43]. The UK government published a report in March 2013 identifying antimicrobial resistance as an area requiring profound action due to the impact on global health [43]. Emphasis was placed on the preservation of antibiotics and tips were provided on effective antibiotic prescribing. The report stated: “vital to improving antimicrobial stewardship will be the education, training and workforce priorities of healthcare and public health professionals” and a detailed UK 2013–2018 antimicrobial resistance strategy and action plan will be published in 2013 [44].In many countries there are education campaigns that aim to change healthcare professional and patient behaviour on antibiotic consumption. These include initiatives from the US Center for Disease Control and Prevention, first launched in 1995, and the European Centre for Disease Prevention and Control that has been running a European-wide antibiotic awareness day since 2008. In addition, many countries have run their own national awareness measures, such as Australia (‘Resistance fighters’), France (‘Antibiotics, not automatic’, underway since 2001), Germany (the DART—Deutsche Antibiotika-Resistenzstrategie) and Thailand (‘Antibiotics Smart Use’, running since 2007).The impact of these campaigns was diverse. An analysis of 22 national or regional level campaigns in high-income countries did find a reduction in antibiotic use. As all but one campaign targeted the patient and healthcare professional simultaneously [45], it cannot be concluded whether patient education and awareness alone is an effective intervention to decrease antibiotic use. Yet, despite an 18-year awareness campaign in the US, antibiotic overprescribing for paediatric patients remains high at 60%, and research on a UK awareness campaign also found little impact. Polish data suggest that a third of the population changes behaviour due to the European Antibiotic Awareness day; prescribing rates however remain high [46].It is unclear from current study data whether these effects are sustainable. In France, the awareness initiative reduced antibiotic prescribing by 26.5% in the first five years [47]. A more recent survey, however, showed that 42% of patients had taken antibiotics in the previous year, of which a third were for upper RTIs [30]. It is also difficult to determine what is more effective: changing patient knowledge and perception of antibiotics or physician prescribing behaviour. European research showed that advice from a doctor and television advertising are the most effective means of educating patients on appropriate antibiotic use [30]. As the latter is often cost-prohibitive in many countries, the focus for change needs to be on changing doctor and supplementary healthcare professional behaviour. In a research context, this has been shown to work. An analysis of interventions to improve antibiotic prescribing for RTIs among primary care physicians resulted in, on average, an 11.6% reduction in antibiotic prescribing [48].Despite the range of campaigns and initiatives, educating healthcare professionals, and also training them how to educate patients on antibiotic use is essential. More could be done to optimise the interaction between patient and healthcare professionals during the consultation for an acute RTI. Numerous regional, national and international guidelines are in place outlining appropriate prescribing and many educational initiatives exist that focus on raising awareness of antibiotic resistance globally. GRIP believes that this is where the focus in countering antibiotic resistance should lie and calls for the development of an international framework for the non-antibiotic management of RTIs that specifically addresses the physician-patient interaction.In addition to setting out the rationale for appropriate antibiotic use in RTIs and outlining non-antibiotic management strategies, the framework should cover several key topics (Table 1). Framework outline for the non-antibiotic treatment of acute respiratory tract infections.Policy to advance antibiotic stewardship and conservancy imperativesPrevention of inappropriate antibiotic use by providing guidance on the indications, signs and symptoms of RTIs, and subgroups of patients where antibiotic use is appropriatePatient participation to encourage patient empowerment, combined with appropriate evidence-based symptomatic management of RTIsPrescriber guidance on strategies for an effective dialogue between primary care healthcare professionals and patients during and after consultation for a RTI, resulting in clear take-home messages, supplemented with appropriate materials or referral to available resources, including, but not limited to:
+			
+            acknowledging the reasons for patient consultation for an RTI
+			providing reassurance and counselling on non-antibiotic treatment, resulting in patient satisfaction with the consultation
+			offering evidence-based, symptom-focused advice
+			educating the patient on antibiotic conservancy
+			outlining what follow-up is required and what symptoms would necessitate further intervention
+            
+			acknowledging the reasons for patient consultation for an RTIproviding reassurance and counselling on non-antibiotic treatment, resulting in patient satisfaction with the consultationoffering evidence-based, symptom-focused adviceeducating the patient on antibiotic conservancyoutlining what follow-up is required and what symptoms would necessitate further interventionRTI: Respiratory tract infection.The development of this framework should be cognisant of effective behavioural change initiatives from various countries and involve multiple stakeholders. The framework has to be robust enough to set a benchmark for care, while allowing adaptation at country level to reflect local realities. For example, in some countries the enforcement of legal or fiscal measures would be of great value to restrict inappropriate antibiotic sales through pharmacies and to incentivise primary care physicians to reduce prescribing.GRIP is working to further develop this framework, which will be supplemented by practical materials to educate primary care healthcare professionals, pharmacists and patients in changing behaviour and perceptions around antibiotic use in RTI treatment, and to encourage symptomatic relief. A key component of this will be guidance for healthcare professionals on having a constructive dialogue with patients that allows patients’ needs for symptom relief to be met without the need for an antibiotic prescription. Materials will be available through a GRIP website.A summit meeting is to be held in the summer of 2013 to convene antibiotic resistance expertise from around the globe in order to finalise the framework into one that facilitates prudent antibiotic use via targeted, nationwide actions. Representatives will span both the developing world (Brazil, China, India, Malaysia, Russia, South Africa, Singapore and Thailand) and developed countries (Australia, Austria, Czech Republic, Germany, Ireland, Israel, Italy, Middle East, Netherlands, Switzerland, the United Kingdom and the United States of America). The core GRIP group also aims to include members from developing countries where antibiotic resistance poses particular problems, such as India and Thailand. Inappropriate antibiotic use in normally self-limiting RTIs is common in many countries and is contributing to the increase in antibiotic resistance. To reverse this tendency, a multi-faceted international, collaborative framework needs to be developed that facilitates behavioural change towards a non-antibiotic, patient-centered symptomatic management strategy in primary care. This framework should not only set out the rationale for why appropriate antibiotic use in RTIs is essential, but it should particularly outline how to enforce its implementation to change practice through improved dialogue between the healthcare provider and patient, as articulated in the framework outline. The framework should be adaptable at country level to reflect cultural sensitivities, differing healthcare provision systems and national guidelines, and could serve as a model for change in other therapeutic sectors where overuse of antibiotics in the primary care is of concern. This framework will be supplemented with practical materials that facilitate conversations between healthcare professionals and patients to promote appropriate antibiotic use.The authors would like to thank Mash Health Ltd, who were funded by Reckitt Benckiser Ltd for editorial support during the preparation of this manuscript. The Global Respiratory Infection Partnership is funded by an unrestricted educational grant from Reckitt Benckiser Ltd. Authors have served as advisory board members for Reckitt Benckiser Ltd.

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+This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The use of antimicrobials over the past six decades has been associated with the emergence and dissemination of antimicrobial-resistant bacteria. To explore local geographical patterns in the occurrence of acquired antimicrobial resistance (AMR), AMR of E. coli causing urinary tract infections (UTI) in the community in the West of Ireland was mapped. All adult patients consulting with a suspected UTI in 22 general practices in the West of Ireland over a nine-month study period were requested to supply a urine sample. Those with a laboratory confirmed E. coli infection were included (n = 752) in the study. Antimicrobial susceptibility testing was performed by standardized disc diffusion. Patient addresses were geocoded. The diameters of the zone of inhibition of growth for trimethoprim (5 μg) and ciprofloxacin (5 μg) for the relevant isolate was mapped against the patient address using ArcGIS software. A series of maps illustrating spatial distribution of AMR in the West of Ireland were generated. The spatial data demonstrated a higher proportion of isolates with AMR from urban areas. Some rural areas also showed high levels of resistant E. coli. Our study is the first to demonstrate the feasibility of using a geographical information system (GIS) platform for routine visual geographical analysis of AMR data in Ireland. Routine presentation of AMR data in this format may be valuable in understanding AMR trends at a local level. The spread of acquired antimicrobial resistance (AMR) in bacteria represents a significant public health threat, with growing limitations on the availability of effective drug treatment options for many common infections [1,2]. Studies of AMR in bacterial pathogens have emphasized changes in institutional settings, for example hospitals, or differences between institutions, regions or countries. Spatial differences in AMR at the local community level are less well described. Previous research has indicated that in addition to individual consumption of antimicrobials, geographical area-level factors also affect the risk of acquiring resistant bacteria [3,4,5]. This perspective is important, as individual use of an antimicrobial drug can impact on the risk of acquiring a resistant organism for a household and even a community [6,7,8].The use of maps for analyzing and illustrating the occurrence of AMR in bacterial pathogens at a national level has been employed on a European-wide basis since 1999 [9]. The European Antimicrobial Resistance Surveillance Network (EARS-NET) maps show levels of resistance for key antimicrobial agents for selected species of bacteria. However EARS-NET is limited to invasive human isolates and is reported on a national, countrywide level. A similar European platform reports the variation in antimicrobial consumption in thirty countries [10]. This can help to assess relationships at a national level between the consumption of antimicrobials and the occurrence of AMR [8]. Differences in the levels of AMR in bacteria at the regional level have been reported in numerous countries, and practitioners are advised in many countries to consult local resistance data when prescribing antimicrobials for bacterial infections [4,5,11,12]. There are currently no platforms available for the routine analysis or reporting of AMR bacteria at the local or regional level in Ireland. Reasons for spatial variation in the occurrence of resistance at a local level can be complex, but may be attributed to differences in antimicrobial consumption [13], environmental contamination with resistant bacteria [6], localized spread through direct person-to-person transmission [14] and socioeconomic factors [15]. Geographical information systems (GIS) have been used as a means of conveying health care related data to a wide audience, as they facilitate the visualization of disease patterns in a specified geographic region [16]. GIS are useful in epidemiological studies as they can combine several overlaid maps to view possible associations between different factors. They are being used more frequently in primary care research [16,17]. A previous study in the West of Ireland used a GIS to investigate the spatial and temporal distribution of human cryptosporidiosis over a three-year period and to highlight areas and seasons of high risk [18]. A major outbreak of cryptosporidiosis occurred during this period and the resulting GIS information was helpful in analyzing and presenting data on the outbreak. GIS have also been implemented for the study of other infectious diseases in Ireland, including a longitudinal study of bovine tuberculosis [19].Investigations into the spread of resistant pathogens in the wider community represent a major challenge for public health. Community based investigations generally involve broad geographical regions, and there are currently no established regional AMR reporting systems in place in Ireland. The appropriate medium would need to convey relevant health information, such as resistance data, to health care professionals at all levels, in particular general practitioners, on a routine basis. The ideal platform for this type of data would give an easy to understand, clear and concise overview of bacterial AMR on local, regional and national levels. This platform should also support analysis of spatial variation in the occurrence of AMR in the context of spatial data on antimicrobial consumption and other health indicators (e.g., deprivation indices, land use patterns). This information would promote health care professionals’ and communities’ awareness of the local occurrence of resistance and ultimately to understand to what extent factors such as local antimicrobial consumption impact on local resistance levels. The routine provision of AMR data in the primary care setting in this format could support a positive change in antimicrobial prescribing practices, as prescribing would be guided by local resistance patterns. GIS are currently underutilized for studying AMR in the community and have not been used previously for this purpose in Ireland. Previous research elsewhere applied GIS to study meticillin-resistant Staphylococcus aureus (MRSA) [20], Streptococcus pneumoniae [21] and E. coli [22]. The present study aims to use GIS to present AMR to two important therapeutic agents in E. coli causing UTI from a prospective study in a region in the West of Ireland, details of which have been previously reported [23]. The agents studied were trimethoprim, which is currently recommended as first line treatment for UTI in Ireland, and ciprofloxacin, a fluoroquinolone, recommended as a reserve second line agent [11]. Antimicrobial susceptibility data from all E. coli isolates associated with UTI in the community during the study period were mapped against the geo-coordinates of the patients. Since Ireland does not have postal codes, geocoding was done by hand, based on the townland address. This process aided a feasibility assessment for the routine use of GIS in analyzing and reporting AMR information. The current study included 682 females (91%). The overall patient mean age was 55 (±21) years (Table 1). Overall, female patients were younger compared to males (55 ± 21 compared to 64 ± 18 years).Summary of patient descriptive.The distribution of E. coli UTI cases, study practices and nursing homes in the study region are outlined in Figure 1. A high number (n ≥ 10) of E. coli cases per electoral divisions (ED) are indicated with a red or orange color. A low number (n ≤ 5) of cases are indicated with blue or green. Practices and nursing home locations are identified in each ED. High numbers of UTI cases are observed in more urban areas with a higher population density. Distribution and frequency of E. coli urinary tract infection (UTI) cases, practices and nursing homes in the study region at electoral division level.Heat maps showing the mean antimicrobial diameter of the zone of inhibition results for E. coli to ciprofloxacin and trimethoprim are presented in Figure 2a,b. These maps display results without the differentiation of the individual ED from where the isolates were obtained. The results are not interpreted according to any breakpoint criteria. Darker regions correspond to localities where isolates with lower zone of inhibition diameters, indicating higher levels of resistance to the antimicrobial agent, were obtained. The heat maps indicate that for both trimethoprim and ciprofloxacin, higher levels of antimicrobial resistance (lower zone diameters) seem to occur in urban areas. Higher levels of resistance were also noted in areas where isolates were obtained from patients in nursing homes, however this result is tentative given the low number of isolates included in the study. While resistance to trimethoprim is more widely disseminated in the region, similar hotspots of resistance for both ciprofloxacin and trimethoprim can be observed in both maps, in particular towards the extreme western and eastern regions of the maps. Average antimicrobial susceptibility results * for E. coli isolates for (a) ciprofloxacin (left) and (b) trimethoprim (right).A choropleth map (Figure 3a,b) displays the proportions of trimethoprim and ciprofloxacin resistance in each individual ED according to the current European Committee for Antimicrobial Susceptibility Testing (EUCAST). This map also shows similar hotspots of AMR as noted in the scalar format in Figure 2. Percentage antimicrobial resistance* for E. coli isolates for (a) ciprofloxacin (left) and (b) trimethoprim (right) based on European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines.The present study demonstrates the feasibility of the application of GIS for studying the dissemination of acquired AMR in a common bacterial pathogen in the wider community at a local level. GIS software allows for the analysis and visual representation of data in the form of a geographical map, which can be easily interpreted by health care professionals, members of the public and policy makers [16]. Results should be considered in the context that the limited number of E. coli UTI isolates occurring in many individual EDs contributes to potential differences in AMR that occur by chance. Nevertheless, the results of the current study suggest spatial clustering of resistance and potential areas of high-risk for antimicrobial-resistant E. coli.The study uses both clinically interpreted (EUCAST) and non-interpreted (zone diameter) results to present the data, which also allows for the visualization of a summative quantitative measure of resistance (mean zone diameter) in addition to the more commonly used percentage of isolates categorized as resistant by defined interpretive breakpoint criteria. The results suggest spatial clustering of resistance to trimethoprim and ciprofloxacin and therefore the possibility of local high-risk areas of resistant bacteria. The use of GIS allows one to examine spatial variations at the level of resistance in the context of particular geographical information, for example urban compared with rural areas or the occurrence at a nursing home in the locality. There is a subjective impression, for example, of a link between higher resistance among E. coli in a locality and the presence of a nursing home in the area, although this is both unconfirmed and difficult to interpret. The inclusion of isolates from nursing homes in the current study introduced bias. UTIs are very common in nursing home residents and urine samples are regularly taken to check for infection. A higher proportion of UTIs is therefore to be expected in areas with a nursing home. The proportion of resistant E. coli UTIs is also higher due to a combination of high antimicrobial consumption by the residents and interpersonal spread. The inclusion of isolates from nursing homes will inflate the overall AMR level of a particular area compared to townlands where no nursing home is present, in particular with the low numbers included in this study. Previous research has identified household and community risk factors for the acquisition of resistant bacterial pathogens. The role of the wider environment in the spread of AMR is now better understood [6]. A study investigating the geographic risk associated with community acquired MRSA in the US identified a significant cluster of MRSA isolates (n = 27) from patients within a specific area of a city [20]. The authors suggest that these maps be used to advise antimicrobial therapy for individuals within the region and that the patients address should be taken into consideration when appropriate empirical treatment options are being considered. The use of GIS to visually present the association between antimicrobial consumption and antimicrobial prescribing was previously carried out in Sao Paulo, Brazil [22]. This previous study focused on ciprofloxacin resistance in E. coli as a urinary tract pathogen and correlated high-level resistance with antimicrobial consumption expressed as defined daily doses (DDD). Similar to the current study, clustered hot-spots of resistance were observed along with significant spatial variation. Resistance hot spots correlated to higher usage (5–9 DDDs per 1,000 inhabitants) of ciprofloxacin in the community. General practitioners (GPs) could benefit from the routine use of GIS for displaying AMR data by aiding in the prescription choice of antimicrobial drugs for common infections. The maps could indicate if the GP is in an area where high or low resistance to certain drugs is prevalent in the bacterial population. This would result in less use of newer, reserve antimicrobials (particularly in areas of low resistance), hence more appropriate and better treatment outcomes for patients (particularly in areas of high resistance). Conversely, GIS could be used to show antimicrobial prescribing patterns as a direct measure of antimicrobial resistance in a specified region; however this was not carried out in the present study. While there are numerous advantages of using GIS to display health care information, appropriate precautions must be adhered to. When inferring a relationship based on the results of spatial analysis, caution should be used in applying the appropriate areal units (scale of the map) and analysis should be carried out at numerous scales to evaluate the effect [16]. The use of the Kernel function to generate heatmaps is sensitive to any bias that may be present in the original data used. The patient data used in the current study was limited to the patient address at the townland level to ensure that specific patients could not be identified and for feasibility of use. However there is a potential risk of bias due to the aggregation of E. coli information at the townland level. This is particularly apparent with the inclusion of isolates from nursing home residents, where a greater number of E. coli isolates with increased AMR can inflate the observed levels of resistance in that area, as previously discussed.The use of GIS for mapping health care data based on patient addresses in Ireland on a routine basis has several limitations. Geocoding of patient addresses can be a time-consuming task, in particular where postal codes are not in use, as is the case in Ireland. Numerous discrepancies exist between the spellings of various townlands, in particular where Irish is the first language (Gaelic-speaking areas). The introduction of postal codes in Ireland, or the automatic linking of patient addresses with x, y coordinates, would alleviate this problem and allow for automated patient data retrieval and subsequent analysis. This is the first study to report the use of GIS for the investigation of patterns of antimicrobial resistance in pathogenic bacteria in Ireland. The study highlights the potential of GIS to present antimicrobial resistance data for routine communication of current epidemiological trends in bacterial pathogens. As piloted in this study, this can allow the investigation into the impact of possible risk factors associated with antimicrobial resistance, such as the presence of a health care center such as a nursing home in the region. While not evaluated in the current study, the software can also be used to investigate the impact of other potential risk factors such as social deprivation or agricultural use in the region [18], as well as correlating antimicrobial prescribing data with resistance patterns, which is currently being analyzed in a separate study. The study area covered two counties in the West of Ireland, namely Galway and Roscommon (total area = 8,695 Km2). Patient data from a previous prospective case-control study analyzing AMR (trimethoprim and ciprofloxacin) in E. coli causing UTI in the community was used [3]. Details of ethical approval, practice selection and patient inclusion criteria are described in previous papers [3,24]. The previous prospective study was carried out over a nine-month period in 22 participating practices. All adult patients with a suspected UTI were requested to supply a urine sample for analysis at the regional diagnostic laboratory. A total of 778 patient records from 22 practices in the West of Ireland were selected. Of these patients, 23 were excluded because the patients lived outside the geographical area being studied. A further three were excluded on the basis of having incomplete data, giving a final sample size of 752 patients (Table 1). To obtain patient addresses at the townland level, the results from the patients’ analyzed urine samples were accessed at the University Hospital Galway (UHG). Antimicrobial susceptibility zone diameters (mm) to two antimicrobials (trimethoprim and ciprofloxacin) and patient townland addresses were geocoded with assignment of specific latitude and longitude coordinates, for use with ArcGIS software as described below.Patient townland data was converted to a vector point Shapefile by extracting the centroid (x,y location) of each townland polygon. Following this, a spatial join was carried out in ArcGIS to link the patient data with the geographic townland dataset. Electoral divisions (ED) (n = 342) were used as base scale features (polygons). EDs are the smallest legally defined administrative areas in Ireland, of which there are 3,440 [25]. The resultant dataset provided a geolocated townland and electoral division address for each patient. Each of the practices (n = 22) was manually geolocated on a base map in ArcGIS. Antimicrobial susceptibility testing was carried out using the disk diffusion method of the European Committee on Antimicrobial Susceptibility (EUCAST) [26]. The diameter of the zone of inhibition (mm) of growth was mapped against patient addresses and zone diameters were interpreted (resistant or susceptible) according to the EUCAST clinical breakpoints and were also mapped [26]. The minimum zone diameter that can be obtained is 6 mm (diameter of antimicrobial disk used in the test). Color themed maps were created to visually display the number of UTI cases per ED. Where an isolate originated from a nursing home patient, the nursing home was indicated in an ED. Heatmaps were generated by extracting the average zone diameter for each ED, while choropleth maps were generated using proportions of resistance for each ED. Heatmaps use varying degrees of color (yellow, orange, red) to represent a scale. In this study, a lighter yellow color indicates a larger zone diameter result and less resistance to the specific antimicrobial studied. A deeper orange color indicates a smaller zone diameter result indicating increased resistance to an antimicrobial, while a red color indicates complete resistance (i.e., a minimum zone diameter of 6 mm) to the antimicrobial. The spatial extent of heatmaps (Figure 2a,b) does not extend to the boundary of the two study area counties. This is a factor of the distribution of the sample locations. In an effort to convey accurate information pertaining to AMR within the study areas, regions that do not have corresponding AMR data have been left blank (no data).This research demonstrates that it is possible to spatially analyze community antimicrobial resistance in common bacterial pathogens. This suggests a potential application of geo-mapping to optimize antimicrobial prescribing practices across large geographical regions. This work was supported by a grant from the Health Research Board, Ireland and the Millennium Research Fund, National University of Ireland Galway. This work includes Ordnance Survey Ireland data reproduced under OSi Licence number NUIG220212. Unauthorized reproduction infringes Ordnance Survey Ireland and Government of Ireland copyright. ©Ordnance Survey Ireland, 2013.The authors declare no conflict of interest. Ethical approval for the original prospective study was obtained from the Irish College of General Practitioners. Ethical approval to access patient address information for the current study was obtained from Galway University Hospital ethical committee.