The global crisis of antibiotic resistance has reached a point where, if action is not taken, human medicine will enter a postantibiotic world and simple injuries could once again be life threatening. New antibiotics are needed urgently, but better use of existing agents is just as important. More appropriate use of antibiotics in medicine is vital, but the extensive use of antibiotics outside medical settings is often overlooked. Antibiotics are commonly used in animal husbandry, bee-keeping, fish farming and other forms of aquaculture, ethanol production, horticulture, antifouling paints, food preservation, and domestically. This provides multiple opportunities for the selection and spread of antibiotic-resistant bacteria. Given the current crisis, it is vital that the nonmedical use of antibiotics is critically examined and that any nonessential use halted. Antibiotic resistance threatens the effective treatment of many bacterial infections. This Essay proposes that as any exposure to antibiotics promotes resistance, the extensive nonmedical use should be reduced to preserve this life-saving resource. The crisis of antibiotic resistance compromises the ability to treat infections and threatens many areas of medicine, including surgery. This problem has been exacerbated by the drastic decline in the development of new antibiotics. Even with greater public awareness of the scale of this problem, only two new antibiotics with activity against infections for which there is most need, those by gram-negative bacteria, have been launched in the 12 months preceding March 2015, and these have limited indications. These two new drugs are combination agents where an inhibitor of a resistance mechanism (β-lactamase) is provided with a currently licensed antibiotic (ceftolozane/tazobactam and ceftazidime/avibactam). Most new antibiotics only target gram-positive bacteria.The enormous threat posed by antimicrobial resistance has been recognised at national, regional, and global levels. In May 2014, WHO documented the global extent of antimicrobial resistance, reiterating the need for urgent action .Antibacterial drug  resistance is an illustration of natural selection. When bacteria are exposed to an antibiotic, those able to survive in its presence survive, proliferate, and spread. Resistance can arise de novo from spontaneous mutations in patients during antibiotic treatment , or in A crucial factor in the selection of antibiotic-resistant bacteria is the concentration of antibiotic that organisms are exposed to. The concentration of antibiotic that is required to inhibit the visible growth of bacteria is known as the “minimum inhibitory concentration” (MIC); the concentration required to kill bacteria is known as the “minimum bactericidal concentration” (MBC). The MIC and MBC values are higher for a resistant strain than for a susceptible strain. A concentration that is above the MIC for a susceptible strain but below that for a resistant strain allows resistant bacteria to multiply, while susceptible forms cannot . FurtherMuch attention has focused on overprescription and misuse of antibiotics in medical practice. Although the contribution that medical use has had to the antibiotic resistance burden cannot be overstated, this emphasis has overshadowed the widespread use of antibiotics outside human medicine. Resistance developed during nonmedical use of antibiotics can confer resistance to clinically useful antibiotics. Given the alarming depletion of the antibiotic arsenal, there is an urgent need to assess the extent and implications of antibiotic use outside medicine. Controversy has surrounded nonmedical uses of antibiotics due to a historical dearth of studies that directly correlate such use with the development of resistance and an impact upon human medicine. However, absence of evidence is not evidence of absence. This article outlines the available evidence that nonmedical use can select for antibiotic resistant bacteria.Use of antibiotics in feed or water is used in some countries, e.g., the United States, to accelerate the growth and increase the size of animals bred for food. Remarkably, more antibiotics are used in the US for animal growth promotion than in human medicine. Dosing of antibiotics in growth promotion is not controlled leading to some animals receiving subinhibitory doses. This creates conditions that can favour the selection of resistant bacteria, which subsequently contaminate the environment.Following the development of cross-resistance to antibiotics used in human medicine, several compounds were withdrawn from the European Union (EU) market. Use of growth-promoting antibiotics was further curtailed in 2006, with the implementation of an EU regulation banning the use of specific antibiotics of relevance to human health as animal growth promoters .Arguments by industry for the continued use of antibiotics as growth promoters have focused on the potential economic impact of a ban and a possible effect on animal welfare . FollowiIn 1999, the US National Academy of Sciences calculated that a ban of antibiotic growth promoters would have minimal impact on the US livestock industry and consumers (adding US$4.84–9.72 to bills each year) .Antibiotics are also widely used in veterinary medicine to treat sick animals, to control the spread of disease, and to prevent infections. Few antibiotic classes are specific to human or veterinary medicine, so the emergence of resistance in one area has the potential to select resistant bacteria that infect man (and vice versa). Herds and flocks of animals are generally treated collectively, with antibiotics provided in food or water. This makes it very hard to control the dose, raising the risk that animals experience suboptimal levels of antibiotic and increasing the likelihood that antibiotic-resistant bacteria will be selected and cause infections in people that are difficult to treat. Of concern is that coupled with use for growth promotion, the use of antibiotics in animals is predicted to rise .Aquaculture (farming of fish and other marine life) is a huge global enterprise, particularly in Asia. Industrial scale farming is generally stressful to fish, impairing their immune systems and leaving them more vulnerable to infections. This has been used to justify widespread prophylactic use of antibiotics in aquaculture. Antibiotics are generally given as part of feed but may not be wholly consumed by the fish on a farm. Moreover, poor sanitary conditions encourage the growth of pathogenic bacteria, while high fish densities promote the rapid spread of antibiotic-resistant bacteria on a farm. Resistant microbes can also be rapidly disseminated through water to other farms and to wild animals . As in aVarious other approaches could be used as alternatives to antibiotics in aquaculture, including vaccination or innovative methods such as phage therapy, probiotics, or disruption of signalling between bacteria to prevent biofilm formation. Vaccination is already widely used in countries such as Norway.fabI gene can confer triclosan resistance [In the home, numerous products are available to kill potentially harmful bacteria. Many such as soaps and shampoos contain triclosan, a synthetic compound with antibacterial, antifungal, and antiviral properties. As well as cleaning products, it has also been incorporated into solid products such as fabrics and children’s toys. Triclosan is active against many bacteria, but resistance can develop through a range of mechanisms. Notably, triclosan targets the bacterial FabI protein, and mutations in the corresponding sistance . This alsistance . DevelopConcerns have also been raised about the use of quaternary ammonium compounds (QACs), detergents, and antimicrobial chemicals used in a range of products, including facial cleansers, sun creams, mouthwashes, and sterilising agents for healthcare surfaces. QAC resistance genes are carried on mobile genetic elements that also harbour antibiotic resistance genes, so widespread use of QACs leads to the spread of both QAC and antibiotic resistance .Antibiotics may not be fully metabolised by the human body, and a large amount may be excreted in faeces or urine and enter wastewater treatment facilities. The slow rate of degradation of some antibiotics and use of treated and untreated sewage as fertiliser can also lead to the accumulation of antibiotics, and antibiotic-resistant bacteria, to levels that create selective pressures facilitating the spread of resistance.Water systems are prone to the development of biofilms (slimy layers of communities of bacteria), which tend to be less susceptible to antibiotics . BiofilmPharmaceutical production facilities are a further potential source of environmental contamination with antibiotics. In India, significant levels of antibiotics have been detected in wastewater treatment plants handling water from 90 pharmaceutical facilities—suggesting that there is a minimum of 45 kg of antibiotics being discharged every day . FurtherUse of human or animal waste (manure) as a fertiliser on plants, which contains antibiotic residues or antibiotic-resistant bacteria is another possible route of dissemination of antibiotic-resistant bacteria or genes. Recently, culinary herbs exported from Southeast Asia have been found to harbour multidrug-resistant bacteria that are resistant to many antibiotics .Some sectors continue to dispute that the use of antibiotics outside of human medicine has any impact on medical practice. This has been because direct evidence of the complete sequence of events has been hard to demonstrate. This is for several reasons, not least the erroneous assumption that the identical strain of bacteria must be found, for instance, in both animals and humans . There iCampylobacter spp. have been isolated from poultry [Campylobacter spp. can take longer to resolve. Therefore, until there is evidence to show that the large numbers of fluoroquinolone-resistant Campylobacter (from 15%–80% isolates depending on the country) isolated to date have not arisen during exposure of animals to these drugs, it is prudent to assume that the majority are of foodborne origin [Transfer of antibiotic-resistant bacteria to humans via animals is accumulating. Two examples are (1) antibiotic-resistant zoonoses selected after exposure to fluoroquinolone antibiotics in animals. For instance, fluoroquinolone-resistant  poultry , poultry poultry . The chre origin . Pig fare origin . Recent e origin . Novel re origin . Infectie origin .Kluyvera spp. [Kluyvera spp. gene was mobilised and transferred to human commensal bacteria, which then caused an infection and/or transferred the resistance gene and element to human pathogenic gram-negative bacteria such as Escherichia coli. ESBL-producing gram-negative bacteria are a huge global healthcare burden and have caused the large increase in use of carbapenem antibiotics in human medicine. (2) There is evidence that antibiotic resistance genes arising in aquaculture have been transferred to bacteria that infect people. The human pathogen Salmonella Typhimurium DT104, for example, has been found to carry resistance genes that appear to have arisen in fish pathogens [Aeromonas plasmid may also have been transferred (with associated antibiotic resistance genes) to a range of human pathogenic bacteria, including Salmonella enterica, Yersinia pestis (the cause of plague), and Vibrio cholerae [Transfer of antibiotic-resistance genes to strains of bacteria that colonise and infect humans, through pets  or animals reared for food production, is also occurring. Two examples are (1) a high incidence of a particularly problematic antibiotic resistance gene encoding an extended-spectrum beta-lactamase  has been found in bacteria isolated from humans and retail chicken meat . The CTXera spp. , an orgaathogens . An Aerocholerae .Whilst some would still consider the evidence above as speculative, it is unlikely that each event in the process will be categorically identified, because it would require the agreement of many different individuals to participate, including farmers (sometimes large corporations as in the US), veterinarians, abattoirs, food-preparation and packaging plants, healthy volunteers, and patients. Obtaining ethical approval for such studies in people would be difficult and the cost for such a large study would be enormous and so prohibitive.The discovery of antibiotics was one of the most influential moments in human history. Antibiotics have transformed human medicine, and their bacteriostatic and bactericidal powers have been exploited in multiple applications. Unfortunately, society now takes antibiotics for granted. Their widespread use has undermined the very features that have made them such powerful contributors to human health and well-being, and resistant bacteria can be isolated from numerous environments including bees, plants, and ethanol production .We now need to value antibiotics more highly and recognise that for the foreseeable future, these drugs are a finite and diminishing resource whose use must be carefully managed. While improved “antibiotic stewardship” in medical practice is essential, use of antibiotics outside human medicine needs to be rigorously examined and reduced to the barest minimum. We have produced a series of recommendations to address unnecessary use outside human medicine . ImplemeA global ban on the use of all antibiotic growth promoters in animals.Increased monitoring of aquaculture and animal husbandry welfare practices to minimise the need for therapeutic drugs.Increased investment in research into minimising stress and disease in aquaculture and farm animals to limit the need for therapeutic drug use.Withdrawal of certain antibiotics from veterinary medicine, with specific classes of antibiotics reserved for human use.No use of any new classes of antibiotic in both animals and humans.Aquaculture facilities to be positioned distant from wild fish populations, to prevent dissemination of resistance genes.Increased research into diagnostics in animals and fish, to promote targeted treatment and prevent overuse of antibiotics.Increased infection prevention and control including the increased use of vaccination to prevent disease, with increased funding for new vaccine development.Global application of EU guidelines, limiting antibiotic use for treatment.A ban on antibiotic use in horticulture, with more research on containment systems and other means of control of plant pathogens.More research to assess whether bacteriocin use promotes cross-resistance to antibiotics used in human medicine, potentially followed by restricting their use to one area only.Increased monitoring of antibiotic residues in alcohol production, with appropriate penalties when antibiotics are found.Increased monitoring of antibiotic residues on boat hull surfaces, with appropriate penalties if residues are found.Withdrawal of triclosan from all domestic applications globally.Better disposal of triclosan waste.Greater investment in sewage and water distribution systems, especially in low-income countries, to prevent antibiotic- and/or bacteria-contaminated water leeching into the environment.Development of improved methods to remove antibiotic residues, antibiotic-resistant bacteria, and antibiotic resistance genes from wastewater treatment plant effluent and drinking water sources.


Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties. Pemphigus vulgaris is a potentially life threatening autoimmune blistering disease caused by IgG autoantibodies against Dsg3 +/- Dsg1, desmosomal adhesion proteins of keratinocytes –4. EpitoAlthough monovalent anti-Dsg antibody variable region fragments are sufficient to cause skin blisters in the absence of the constant region , prefereWhile it is clear that IgG4 is associated with pathogenicity in PV, it is unknown whether the same variable region of a pathogenic IgG4 antibody, expressed on an IgG1 constant region, directly affects antibody function, for example antibody affinity, epitope binding characteristics, Dsg3 internalization, or pathogenicity. In the study presented here, we selected three representative pathogenic PV mAbs cloned from 3 different PV patients: F706, an anti-Dsg3 IgG4 isolated by heterohybridoma, F779, an anti-Dsg3 IgG1 isolated by heterohybridoma, and Px43, an anti-Dsg3/Dsg1 monovalent IgG antibody of unknown subclass isolated by phage display, and generated pairs of recombinant IgG1 and IgG4 antibodies expressing the identical variable region in order to assess the effects of the constant region on antibody function.The three patients from whom the antibodies were cloned had active, extensive PV involving both mucosa and skin. The diagnosis of PV was confirmed by histology and immunofluorescence. All patients were off systemic therapies at the time of blood draw. F706 IgG4 and F779 IgG1 were isolated from two different patients by heterohybridoma and their desmoglein binding specificities were previously described ,12. Px43Primers were designed to amplify the F706 and F779 heavy chain (HC) and light chain (LC) variable domains for subcloning into the pHC or pLC vector ,14. PrimGCTAGCCGCCACCATGGACTGGACCTGGAGGGTCTT5’ F706HC NheI: TCTAAAGCTTGCTGAGGAGACGGTGACCAGG3’ F706HC HindIII: TAGGGCGCTAGCCGCCACCATGAGGCTCCTTGCTCAGCT5’ F706LC NheI: TCTAGCGGCCGCAGTTCGTTTGATTTCCACCTT3’ F706LC NotI: TAGGGCGCTAGCCGCCACCATGGACTGGACCTGGAGGGTCTT5’ F779HC NheI: TCTAAAGCTTGCTGAGGAGACGGTGACCAGG3’ F779HC HindIII: TAGGGCGCTAGCCGCCACCATGAGGCTCCTTGCTCAGCT5’ F779LC NheI: TCTAGCGGCCGCAGTTCGTTTGATCTCCAGCTT3’ F779LC NotI: TAGGGCin vitro experiments. Px43 IgG1 and IgG4 were produced as described [Recombinant DNA plasmids were co-transfected into CHO-K1 cell lines using Lipofectamine 2000 , and transfectants were cloned by limiting dilution in selective media  to establish permanent cell lines. The antibody supernatant was collected and purified by affinity chromatography on a Sepharose protein G column with acid glycine elution. F71 IgG1 mAb (targeting an unknown antigen) and anti-HIV b12 IgG4 mAb were used as isotype antibody controls for some escribed ,16.For epitope mapping, extracellular full length and domain-swapped Dsg1 and Dsg3 molecules were produced in baculovirus and the epitope determined by immunoprecipitation-immunoblot as previously described . For epi2 and 5% dry milk. After washing, bound antibody was detected using HRP-conjugated mouse monoclonal anti-human IgG F(ab')2  or mouse monoclonal anti-human lambda . After 1 hour incubation at room temperature, plates were washed, developed with 3,3’,5,5’ tetramethylbenzidine substrate, and optical density determined at 450 or 490 nm using an automated plate reader.Dsg1 or Dsg3 ELISA  was used to measure relative binding affinities of IgG subclasses. Serial dilutions of IgG1 and IgG4 antibodies were incubated with Dsg1 or Dsg3 coated plates for 1 hour at room temperature using kit sample buffer or TBS buffer containing 1mM CaClRelative affinity was determined using regression analysis of double-reciprocal plots of optical density versus antibody concentration. The relative constant was calculated as (1/y intercept)*slope.2 and 1% BSA and visualized using an Olympus BX61 microscope.Direct immunofluorescence (DIF) was performed on frozen human skin sections after mAb injection. Bound antibody in human skin was detected with Alexa Fluor 594 anti-human IgG  in TBS buffer containing 1mM CaCl2 and 1% BSA. Bound antibody was subsequently detected with FITC-conjugated anti-human lambda light chain  and visualized using an Olympus BX61 microscope. The titer is reported as the last dilution at which epidermal cell surface staining is clearly positive.Indirect immunofluorescence (IIF) was performed by incubating serial dilutions of IgG1 or IgG4 mAbs on frozen normal human skin sections (obtained through the Penn Skin Disease Research Center) in TBS buffer containing 1mM CaClHuman oropharynx epithelial tumor cell line, UM-SCC-47 (SCC-47), was obtained from Dr. Thomas Carey, University of Michigan. Cell line was grown in 10% FBS-DMEM medium containing 10% heat inactivated FBS, 2 mM L-glutamine, 0.1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 10 μg/ml gentamicin. A live cell based ELISA was employed using SCC-47 cells seeded in a 96-well flat bottom plate for two days. Serial dilutions of PV IgG1 and IgG4 mAbs and isotype control antibodies were added to the cells for one hour. After washing, bound antibody was detected using biotin-conjugated goat anti-kappa chain antibody (1:5000)  for one hour. The plate was washed and HRP-conjugated streptavidin  (SouthernBiotech) was added and incubated for 45 minutes. After a final wash, o-phenylenediamine (OPD) substrate  was added, and the plate was incubated for 20 min and read at 490nm.Staphylococcus aureus exfoliative toxin A  to inactivate Dsg1, was injected into skin sections, followed by incubation of skin pieces on transwell inserts , with defined keratinocyte serum free media  containing 1.2 mM CaCl2 in the outer compartment. Skin was harvested at 16–24 hours for DIF by embedding in OCT compound  and for histology by fixation in 10% phosphate buffered formalin.Fresh normal human skin specimens were obtained from the Penn Skin Disease Research Center. The protocol was reviewed by the University of Pennsylvania Institutional Review Board, who conferred a human subjects research exemption, since the samples were obtained for clinical purposes, otherwise discarded, and de-identified, and therefore did not qualify as human subjects research. Skin was trimmed of fat and cut into 5 mm sections. 25–50 μg of purified isotype mAb, with or without 100 ng of Primary human keratinocytes obtained from the Penn Skin Disease Research Center were treated with 50 μg/ml anti-Dsg3 mAbs for the indicated amount of time during calcium-induced  desmosome assembly as described . At 8 hoBecause the hinge region of IgG1 differs from IgG4, creating differences in the orientation of the variable regions , Dsg3 afTo determine whether the IgG1 or IgG4 constant region affects epitope specificity, we first tested F706 and F779 IgG1 and IgG4 mAbs against recombinant purified Dsg1 and Dsg3 antigens. ELISA showed that F706 and F779 IgG1 and IgG4 mAbs specifically bound to Dsg3 but not Dsg1 , indicatTo determine whether IgG1 or IgG4 constant regions affect antigen affinity, we evaluated IgG1 and IgG4 variants of pathogenic PV mAbs by ELISA and keratinocyte cell surface staining. IgG1 and IgG4 variants of F706 and F779 show a similar level of binding to Dsg3 by ELISA . The appWe next investigated the pathogenicity of IgG1 and IgG4 recombinant mAbs, to determine whether IgG subclass could play a role in modulating the pathogenic effects of Dsg3-reactive variable regions. In skin, Dsg1 can provide compensatory adhesion if Dsg3 is inactivated ,22. ConsPreviously, pathogenic but not non-pathogenic human PV mAbs were shown to cause Dsg3 internalization in primary human keratinocytes . ConsistThis study demonstrates that epitope specificity, antigen affinity, and pathogenicity of PV mAbs are not directly affected by the presence of the IgG1 versus IgG4 constant region. Although our conclusions are limited by the study of only three mAbs, these mAbs were cloned from 3 different patients and represent three distinct types of pathogenic PV mAbs that have been described in the literature, including anti-Dsg3 IgG1, anti-Dsg3 IgG4, and a cross-reactive anti-Dsg3/Dsg1 IgG.All PV mAbs tested in this study bind the Dsg3 EC1-2 domains that are crucial for cis- and trans-adhesive interactions of the Dsgs. This result agrees with a previous large scale mapping study of PV sera in which 91% of PV sera mapped to the Dsg3 EC1 domain .Although F706, F779, and Px43 all demonstrated comparable antigen affinity of IgG1 and IgG4 variants by ELISA and/or IIF staining, Px43 IgG1 and IgG4 were notable for demonstrating differing results by ELISA and IIF. Px43 was previously shown to recognize only mature conformational Dsg3  and by sNo change of pathogenicity among PV IgG1 and IgG4 mAb variants was observed, in regard to Dsg3 internalization or the ability to cause suprabasal blisters in human skin. These data are consistent with the finding that the constant region is not required for autoantibody pathogenicity in PV  or for DWith no difference in pathogenicity, the question arises as to why IgG4 is preferentially associated with active disease. IgG4 is typically observed in conditions of chronic antigen stimulation, such as in individuals undergoing allergic desensitization or beekeepers . Class sIn summary, we have found that subclass-switching between IgG1 and IgG4 has no significant effect on epitope specificity, antigen binding affinity, or pathogenicity in 3 human PV mAbs isolated from 3 different PV patients. Although limited by the study of only three antibodies, as well as the study of only pathogenic antibodies and not non-pathogenic antibodies, this study provides the first direct evidence that the immunochemical and pathogenic properties of PV pathogenic variable regions are not significantly modulated by antibody subclass, supporting the conclusion that the antibody variable region is most important for determining pathogenicity in PV. Our data also suggest that pathogenic PV mAbs cannot be engineered to be non-pathogenic agents for drug delivery based on modulation of the constant region. Instead, only antibodies with non-pathogenic anti-Dsg3 variable regions should be used as a tool for targeting drug delivery, for example to Dsg3+ head and neck squamous cell carcinomas  or infla


Portulaca quadrifida, Dioscorea abyssinica, Abelmoschus esculentus, and Oxytenanthera abyssinica, were evaluated for their minerals composition and bioavailability. Mineral elements, namely, Ca, Fe, Zn, and Cu, were analyzed using Shimadzu atomic absorption spectrophotometer. Effects of household processing practices on the levels of mineral elements were evaluated and the bioavailability was predicted using antinutrient-mineral molar ratios. Fe, Zn, Ca, Cu, P, Na, and K level in raw edible portions ranged in (0.64 ± 0.02–27.0 ± 6.24), (0.46 ± 0.02–0.85 ± 0.02), (24.49 ± 1.2–131.7 ± 8.3), (0.11 ± 0.01–0.46 ± 0.04), (39.13 ± 0.34–57.27 ± 0.94), (7.34 ± 0.42–20.42 ± 1.31), and (184.4 ± 1.31–816.3 ± 11.731) mg/100 g FW, respectively. Although statistically significant losses in minerals as a result of household preparation practices were observed, the amount of nutrients retained could be valuable especially in communities that have limited alternative sources of these micronutrients. The predicted minerals' bioavailability shows adequacy in terms of calcium and zinc but not iron.Edible parts of some wild and traditional vegetables used by the Gumuz community, namely, Numerous wild edible plant species have been used by different communities in Ethiopia, mainly as supplement to conventional foods –8. Howevβ-carotene that could help in overcoming micronutrient malnutrition and easily accessed by the community at a low cost. Because micronutrient deficiencies (such as vitamin A) are associated with low intake of foods such as vegetables, as opposed to starchy (energy rich) staples which provide the majority of energy intake in typical African diets, increment in energy production and consumption will likely do little to ameliorate the problem of micronutrient deficiency unless identification, proper evaluation, and domestication of nutritionally potential lesser known vegetables are integrated into the diets of the population. Moreover, the roles of edible wild plants and lesser known crops in human nutrition are potentially valuable to maintain a balance between population growth and agricultural productivity, particularly in the tropical and subtropical areas of the world [Micronutrient deficiencies affect billions of people globally. Although less prevalent in higher-income populations, these deficiencies do occur in such groups, especially among premature infants, children, and the elderly. Several sources revealed that edible wild plant and traditional vegetable species increase the nutritional quality by providing minerals, fiber, vitamins, and essential fatty acids and enhance taste and color in rural diets . Underuthe world . Hence, he world , the preThe study plants were collected from Agalo Meti district in Kemash zone of Benishangul Gumuz Regional state located at 530 km west of the capital Addis Ababa. Dioscorea abyssinica (tuber), Oxytenanthera abyssinica Munro (young shoot), Abelmoschus esculentus (L.) Moench (immature pod), and Portulaca quadrifida  were selected based on their wider utilization by the community in the study area. The first two species are wild and the last two exist both under cultivation and wild stand. The selected vegetables were recommended for domestication according to an association in Benishangul Gumuz called “Tikuret le Gumuz Limat Mahiber” for the reason that they are widely consumed by the community and because they are most suited for domestication. Each species was collected at the time when edible parts are acceptable for consumption by the local community. Voucher specimens of the selected edible plants were collected and processed for verification. The specimens were identified by a botanist from Bioversity International and Ethiopian Public Health Institute (EPHI) using standard procedures and deposited in the National Herbarium of Addis Ababa University (AAU).Selection of species for our investigation was based on ethnobotanical study findings in the study area . DioscorEdible parts of the selected vegetables were harvested manually from their natural habitat in different areas of Agalo Meti woreda. Five-kilogram edible parts of the respective species were collected from 20 different plants of the same species to ensure the representativeness. Sample preparation for laboratory analyses is illustrated in As is the absorbance of sample, AB is the absorbance of blank, and Slope is calculated from the calibration curve.The methods of  were use3·6H2O and 0.3% sulfosalicylic acid) was added to 3 mL of the supernatant sample solution. The solution mixture was homogenized and centrifuged (3000 rpm/10 minutes). The absorbance was measured at 500 nm using UV-visible spectrophotometer. The amount of phytic acid was calculated using phytic acid standard curve prepared in the same condition and the result was expressed as phytic acid mg/100 g dry weight.The phytate content was determined as described by . Briefly4OH solution (drop wise) until the solution was changed from pink to yellow color. Each portion was then heated to 90°C, cooled, and filtered to remove the precipitate containing ferrous ion. Each of the filtrates was again heated to 90°C and 10 mL of 5% CaCl2 solution was added to each of the samples with stirring consistently. After cooling, the samples were left overnight. The solutions were then centrifuged at 2500 rpm for 5 min. The supernatants were decanted and the precipitates were completely dissolved in 10 mL 20% H2SO4. The total filtrates resulting from digestion of 2 g of each of the samples were made up to 200 mL. Aliquots of 125 mL of the filtrates were heated until near boiling and then titrated against 0.02 M standardized KMnO4 solutions to a pink color which persisted for 30 sec. The oxalate contents of each sample were calculated [The oxalate content of the samples was determined using titration method , 17. Twolculated . The anaMolar ratios of antinutrient/minerals were used to predict the minerals bioavailability . The sugP < 0.05) were applied to compare the means of each parameter between different household preparation practices using SPSS version 16 software. Paired comparison t-test was used to determine if there was a significant mean difference between raw and processed vegetables for each parameter.Descriptive statistics such as means and standard deviation were calculated using SPSS version 16 software. One-way analysis of variance (ANOVA) was used to see the effect of household processing methods on mineral contents and the bioavailability of minerals in selected vegetables. Multiple comparison tests using least significant difference technique  were 2.3 mg/100 g and 0.68 mg/100 g, respectively. Iron content in immature pods of raw A. esculentus obtained in the present study was higher than findings of [ A. esculents varieties reported within the range of 0.87–0.97 mg/100 g FW but the zinc content (0.68 ± 0.04 mg/100 g FW) obtained in this study was slightly lower compared to the value reported (1.29–1.37 mg/100 g FW) in their study. The variations in the contents of the minerals in A. esculentus may be due to the varietal difference or genetic factor, environmental factor, and their interactions [ A. esculentus but not statistically significant between raw and sun dried young pods of A. esculentus. This could be due to leaching of the minerals into the cooking water. Sun drying is the gradual loss of water through evaporation and cannot support leaching. It has to also be noted that minerals are not volatile.As presented in dings of  for diffractions . Signifi Dioscorea abyssinica was 27 mg/100 g FW. However, cooking and peeling the bark of the tuber significantly reduced the iron content to 1.79 mg/100 g FW. The iron content of D. abyssinica obtained in this study in the raw sample was higher when compared with the value reported for Dioscorea pentaphylla (8 mg/100 g FM) [ D. abyssinica was also significantly reduced during cooking and debarking from 0.5 mg/100 g to 0.3 mg/100 g FW. The statistically significant (P < 0.05) reduction of iron and zinc concentration upon cooking and peeling of the tuber may be because of the presence of minerals in the outer nonedible part of the tuber which was removed by peeling after boiling the tuber. The same reason may apply to the minerals which showed enormous reduction in the boiled and peeled D. abyssinica. Reduction in zinc content was also reported in peeled and boiled tubers of Dioscorea cayenensis [ D. abyssinica was 341.16 mg/100 g. Statistically significant losses were observed when the tubers were cooked compared to the raw samples. This loss may be attributed to the leaching out of minerals including potassium into the cooking water [Iron level in raw (whole)00 g FM) . Zinc coyenensis . The potng water . The hig Oxytenanthera abyssinica is presented in  O. abyssinica were 0.6 mg/100 g and 0.9 mg/100 g, respectively. The zinc content was higher in raw bamboo shoots compared to other study vegetables. All minerals tested were decreased in the cooked samples with the reduction being statistically significant in Zn, Cu, P, Na, and K but Fe and Ca were not significantly (P < 0.05) lost in the cooked samples. The variation in the degree of loss might be related to the chemical forms of minerals in the food matrix. The respective potassium contents determined in raw and cooked samples of O. abyssinica were 456.2 mg/100 g and 273.2 mg/100 g on fresh weight basis. The potassium content of raw O. abyssinica obtained in the current study is well agreed with the value reported for other bamboo species such as Bambusa tulda  and Dendrocalamus hamiltonii  [ O. abyssinica had the highest phosphorus content compared to other study vegetables. However, cooking has significantly reduced the phosphorus level. Phosphorus is a major component of bones and teeth [The minerals content of raw and cooked juvenile shoots of0 g, FW) . Raw shond teeth .Studies confirmed that there is no known benefit of high sodium consumption. Sodium intakes more than 1 g per day tend to aggravate a genetically determined susceptibility to hypertension, and intakes above 7 g/day may induce hypertension even in individuals who have no specific genetic susceptibility . In this A. esculentus could provide 116.5% of the RDA requirement for iron if 500 g fresh vegetable is consumed per day, considering it would be fully bioavailable. The cooked and sun dried A. esculentus could provide 34% and 129% RDA requirement for iron from 500 g FW meal. Cooked D. abyssinica and bamboo shoots can also provide 89.5% and 30% RDA requirement of iron from fresh 500 g meal, respectively, and only 125 g fresh leaves and steams of Portulaca quadrifida can provide 100% RDA requirement for iron. This RDA calculation did not consider the inhibitory effect of different antinutritional factors that affect the bioavailability of iron. The RDA for zinc is 12 mg/day for lactating woman [ A. esculentus could provide 23% of the zinc RDA requirement for lactating mothers. Similarly, cooked D. abyssinica and juvenile shoots of O. abyssinica in the cooked form could provide 12.1 and 22.5% RDA requirement for zinc, respectively. Portulaca quadrifida could provide 35% of RDA for zinc considering the requirement for lactating woman. Calcium content ranged from 22.9 mg/100 g FW in cooked bamboo shoot to 140.4 mg/100 g FW in sun dried A. esculentus. Raw immature pods of A. esculentus contained 131.7 mg/100 g fresh weight basis. The calcium level obtained in this study for A. esculentus grown in Ethiopia was higher than the values reported for different A. esculentus varieties in Nigeria [ P. quadrifida contained higher (118 mg/100 g FW) calcium content next to sun dried and raw A. esculentus. The calcium content in raw and cooked O. abyssinica was 24.5 mg/100 g and 22.9 mg/100 g FW, respectively. The calcium content reported in India for the different bamboo species was within the range of 21.17–180.69 mg/100 g (29); the present value obtained for O. abyssinica was similar to Bambusa balcooa (24.01 mg/100 g). The calcium content shows no significant (P > 0.05) variation between raw and boiled D. abyssinica and O. abyssinica . Phosphorus content in this study ranged from 23.7 mg/100 g to 57.3 mg/100 g FW. Raw A. esculentus, D. prehensilis, and O. abyssinica contained 39.1, 40.7, and 57.3 mg/100 g phosphorus, respectively. Cooking resulted in significant losses (7.3–41.76%) of phosphorus with variable degree of proportion among the vegetables  for iron is 10 mg/day for adults . The rawng woman . Five hu Nigeria . Raw aeryssinica . Howeverr adults . Consumpgetables . SimilarAntinutritional components such as oxalates, tannins, polyphenols, and phytic acid (myoinositol hexaphosphate) present in plant foods are known to have adverse effects on human nutrition by inhibiting iron  and zinc A. esculentus and P. quadrifida had moderate zinc bioavailability, whereas the molar ratios suggested that high zinc bioavailability could be achieved from D. abyssinica and juvenile shoots of O. abyssinica. However, the critical phytate : zinc molar ratio may also depend on dietary calcium levels because of the kinetic synergism between calcium and zinc ions resulting in Ca : Zn : Phy complex which is less soluble than phytate complexes formed by either of the ions alone [The calculated phytate/zinc molar ratios for raw and processed WEPs were within the range of 2.03–14.22, which were in the range of the suggested critical level (<15 regarded as favorable for zinc absorption) . Ratios ns alone , suggest A. esculents and P. quadrifida were above the critical level (0.5 mol/Kg) as indicated in  A. esculentus varieties in Nigeria was within the range of 0.293–0.436 [ A. esculentus that grows in Ethiopia compared to Nigeria could be attributed to genetic differences, environmental variability, and interaction of the genetic factor with the environment. Considering both Phy/Zn and Ca × Phy/Zn molar ratios, zinc could adequately be absorbed in the body from D. abyssinica and shoots of O. abyssinica.Calcium × phytate/zinc molar ratios of cooked and sun dried93–0.436 . Higher  D. abyssinica. This might be due to the reported higher phytate content and insufficient phytic acid degradation (4.5–27.5%) by boiling alone. Phytate reduction (13–33%) after boiling Dioscorea sp. was reported by [ Dioscorea abyssinica could be a better source of bioavailable iron.As indicated in orted by . DioscorThe calcium/phytate molar ratios in all the raw and processed study plants were >6, which is regarded as favorable for calcium absorption , predict Abelmoschus esculentus, aerial parts of Portulaca quadrifida, and juvenile shoots of Oxytenanthera abyssinica consumed by the Gumuz community indicated that they are appreciably important sources of essential minerals. Except D. abyssinica, the wild and traditional plant parts have significant micronutrient compositions. However, cooking significantly reduced some of the minerals. Reducing duration of cooking time and using other processing methods such as fermentation (D. abyssinica and O. abyssinica) might alleviate the deterioration of the nutrients. The predicted mineral bioavailability shows adequacy in terms of calcium and zinc (moderately bioavailable) but not in iron. Hence, there is a need for some enhancers to increase iron absorption in all species. The study results further revealed that A. esculentus and P. quadrifida are rich sources of bioavailable calcium. With the exception of D. abyssinica, the vegetables are rich in potassium and can contribute in maintaining normal blood pressure and its heart protective role.Immature pods ofTo be able to justify the overall nutritional value of the wild and semiwild edible vegetables, proper assessment of the type and concentration of their antinutrients is necessary. The rate in reduction of antinutritional factors depended upon the type of processing (cooking and sun drying) and vegetable. However, the reduction of phytic acid, oxalate, and tannins by traditional cooking methods alone was not adequate to the level that could improve iron and zinc bioavailability. The vegetables have higher moisture content and are mostly used after cooking as side dishes by the community. The combination effects of these factors might reduce the actual impact of antinutritional factors in impairing bioavailability of nutrients and/or causing ill health. However, appropriate processing methods that are known to reduce the antinutritional factors (such as fermentation) could be encouraged in the community.


Pseudomonas aeruginosa (PA) biofilms, which are majorly associated with chronic lung infection, contain extracellular DNA (eDNA) as a major component. Herein, we report for the first time that L-Methionine (L-Met) at 0.5 μM inhibits Pseudomonas aeruginosa (PA) biofilm formation and disassembles established PA biofilm by inducing DNase expression. Four DNase genes  were highly up-regulated upon L-Met treatment along with increased DNase activity in the culture supernatant. Since eDNA plays a major role in establishing and maintaining the PA biofilm, DNase activity is effective in disrupting the biofilm. Upon treatment with L-Met, the otherwise recalcitrant PA biofilm now shows susceptibility to ciprofloxacin. This was reflected in vivo, in the murine chronic PA lung infection model. Mice treated with L-Met responded better to antibiotic treatment, leading to enhanced survival as compared to mice treated with ciprofloxacin alone. These results clearly demonstrate that L-Met can be used along with antibiotic as an effective therapeutic against chronic PA biofilm infection.Bacterial biofilms are associated with 80–90% of infections. Within the biofilm, bacteria are refractile to antibiotics, requiring concentrations >1,000 times the minimum inhibitory concentration. Proteins, carbohydrates and DNA are the major components of biofilm matrix.  Most of the microorganisms form micro-colonies and produce extracellular matrix to form biofilm. Biofilm formation is a lifestyle commonly adopted by a variety of microorganisms whether in the environment or in clinical settings. These biofilms are formed upon adherence to a living or non-living surface, formation of micro-colonies and finally the production of a resilient extracellular matrix. While the composition of this extracellular matrix might vary slightly in different microbes, biofilms endow the bacteria a unique resistance against antibiotics and other anti-microbial agents. This reduced susceptibility may be due to the presence of extracellular polymer matrix which acts as a physical barrier to diffusion (intrinsic) or due to the transfer of extrachromosomal DNA from resistant organisms to susceptible organisms (acquired)Pseudomonas aeruginosa (PA), a Gram-negative bacterium can form biofilms on a variety of surfaces such as lungs, contact lenses and contaminated catheters256756756789111210Pseudomonas quinolone signaling (PQS) molecule, are known to regulate eDNA release. QS independent mechanism of eDNA secretion via type IV pili and flagella has also been demonstrated5PA, an opportunistic human pathogen, can cause life-threatening infections in cystic fibrosis (CF) patients and individuals with a compromised immune system15Bacillus subtilis and Staphylococcus aureus23DNase secretion and eDNA degradation might be the mechanism underlying biofilm inhibition and disassembly. In a mouse model of chronic PA lung infection, L-Met treatment in combination with antibiotics could rescue the mice whereas antibiotic therapy alone was ineffective. To our knowledge, this is the first report demonstrating the therapeutic potential of L-Met through its inhibitory effect on the formation and maintenance of PA biofilm.Apart from these molecules, D-amino acids , produced at the late phase of biofilm development, have been shown to trigger the disassembly of the existing biofilms of Effect of exogenous addition of L/D-amino acids, at different concentrations, on the growth of PA biofilms was tested, of which L-Methionine (L-Met) was found to inhibit PA biofilm at 0.5 μM concentration at 72 h. PA biomass reduced by 75% when incubated with 0.5 μM L-Met for 72 h . InteresNext we examined whether L-Met could disassemble the established PA biofilms. CV staining revealed that L-Met treatment could disassemble the established PA biofilms . InitialFormation of PA biofilm, seen after 24 h of incubation, was accompanied by a marked increase in the viscosity of the medium. This viscosity was visibly absent upon treatment with L-Met. Earlier reports have shown that PA biofilms have extracellular DNA as a major component. We hypothesized that the observed decrease in PA biofilm upon treatment was due to a reduction in the eDNA component of the matrix. 72 h old PA biofilm culture supernatant was loaded in a 1.0% agarose gel and it was visible that L-Met treated PA biofilm culture media (L-Met-Sup) lacked DNA whereas, high concentration of DNA was present in the untreated PA culture media (U-Sup) . This DNStudies have shown that eDNA, while being a structural component of the PA biofilm, can also serve as an alternative nutrient source. Upon biofilm maturation and in nutrient limiting conditions, the bacteria are known to secrete DNase (EddB/PA3909) which aids in eDNA degradation which is used for bacterial growth and eventual dispersalDNase genes in PA (sbcB (Exodeoxyribonuclease I), endA (DNA-specific endonuclease I), eddB or PA3909  and recJ  showed 3 to 4 fold higher expression compared to the untreated PA biofilm (DNase genes including xthA (Exodeoxyribonuclease III), nth (Endonuclease III) and sbcD (Exonuclease SbcD) showed no difference. This suggests that L-Met treatment results in transcriptional up-regulation of four of the DNase genes were in PA biofilm. Sub-cellular localization of these DNases was predicted using an online prediction tool, PredictProteinIn order to gain an insight into the regulation of DNase activity in the L-Met-Sup, QPCR was performed for different es in PA . In L-Me biofilm . The oth4) overnight PA culture was added to 0.5% M9 agar plate with different concentrations of L-Met, swarming motility was decreased by approximately 30% at 0.5 μM L-Met  chronic pneumonia infection modelin vivo. Mice were infected with 1 × 107 PA per mouse intranasally. The mice were sacrificed 3 days after the infection and the number of bacteria in the lung was calculated by plating the homogenized lung . It is already known that in PA, extracellular DNA is required for the biofilm formation88D-amino acids are known to disassemble the established biofilmsDNase genes at mRNA level. Hence, the DNase are not only transcriptionally up-regulated but are active enough to degrade the eDNA in the outside environment. The presence of a smear in the L-Met-Sup /U-Sup were incubated with the L-Met-Sup, a smear was observed and there was a slight increase in the mobility. This indicated the presence of DNase activity in the L-Met-Sup. QPCR results confirmed the up-regulation of four -Met-Sup  confirmsDNase genes were highly up-regulated. Though DNase activity was observed and confirmed in the culture supernatant, the mechanism of DNase secretion is not known. There are 3 different possibilities of DNase activity in the culture supernatant. In our first hypothesis, DNase may be secreted by unknown mechanism using the secretion system present in PA. Only EddB, which plays an important role in eDNA degradation, was predicted as a secretory protein using bioinformatics tool. The other DNases are predicted as cytoplasmic and their functions with respect to eDNA are not studied. In the second hypothesis, DNase is up-regulated in protein level inside the bacteria and upon autolysis or other lysis mechanism of PA, DNase are released into the culture media and show their activity. It is also possible that L-Met increase activity of DNases released from lysed bacteria. We have tested this hypothesis by incubating the L-Met treated and untreated culture supernatant with L-Met and we did not find any significant difference in the DNase activity , DNaseI treatment is already establishedPseudomonas aeruginosa biofilm formation. We also found that L-Met could disassemble the established 48 h and 72 h old PA biofilms. L-Met induced DNase secretion may be the possible mechanism for the biofilm inhibition and disassembly. Importantly, we have shown that intranasal L-Met treatment along with antibiotics can cure chronic PA lung infection in mice. Hence, the nasal spray or drop formulations with L-Met along with antibiotic treatment can open up better avenues to treat CF cases or PA induced lung infections.In the present study, we found for the first time that L-Methionine at low concentrations (~0.5 μM) and not at higher concentrations inhibited the Pseudomonas aeruginosa strain PA014 stationary phase cells were inoculated into 2 ml of Luria broth without NaCl and kept at 25 °C in static condition in a 24-well plate in the presence or absence of different concentrations of L-Methionine for different duration.545).24-well plate containing biofilm on the inner walls and the bottom of the plate was confirmed by CV staining4 stationary phase PA were inoculated into 10 ml of Luria broth without NaCl or M9 minimal media in the presence (0.5 μM) or absence of L-Met and kept at 37 °C in shaking condition (180 rpm) and at different time points optical density was determined at 600 nm (OD600) .1 × 10A sterile cover slip was placed in a 24-well plate and the PA biofilm was formed as described before. Cover slips with biofilm were fixed with 2.5% (v/v) glutaraldehyde and the samples were dehydrated with increasing concentrations of ethanol for 2 min each. The samples were stored in vacuum until use. Prior to analysis by Field emission SEM , the samples were subjected to gold sputtering (JEOL JFC 1100E Ion sputtering device).Different concentrations of L-Met  was added to 48 h and 72 h old biofilm in a 24-well plate and incubated for 24 h at 25 °C static condition. CV staining was performed as described before to measure the biomass.Agarose gel (1%) electrophoresis was performed to detect the presence of extracellular DNA (eDNA) in the biofilm culture media. 72 h old PA biofilm was established in a 24-well plate as described before. The total number of bacteria (suspension form in the media + adherent biofilm form) in the plate was determined by sonication and plating. The media in the 24-well plate was normalized to total number of bacteria, centrifuged at 2000 g for 5 min and the supernatant [Untreated culture (U-Sup) & L-Met treated culture (L-Met-Sup)] was loaded in the agarose gel and visualized. U-Sup/L-Met-Sup was treated with L-Met , DNase and RNase (Fermentas) for 1 h at 37 °C and then loaded in the agarose gel for visualization.eDNA in the PA culture was determined as described elsewhereViscosities (η) of all samples were determined at 20 °C using a Discovery Hybrid Rheometer-3 (DHR-3)  as per the manufacturers’ protocol. Measurements were taken using an anodized aluminum cone .100 μl of 72 h old PA culture supernatant (U-Sup or L-Met-Sup) were incubated with equal volume of pFPV/mcherry (1 μg/μl) with or without 0.1% EDTA for 1 h at 37 °C. The samples were loaded in 1% agarose gel and visualized. In the other experiment, 100 μl of U-Sup and L-Met-Sup were mixed and incubated for 1 h at 37 °C, loaded in 1% agarose gel and visualized.DNase genes was carried out using QPCR. Briefly, RNA was isolated from 72 h old PA biofilm culture grown at 25 °C static condition with/without L-Methionine using TRIzol (Life Technologies) as per manufacturer’s protocol. The isolated RNA was reverse transcribed using random hexamers (NEB) and Tetro reverse transcriptase (Bioline) as per standard protocol. The cDNA was diluted and analyzed for the presence different DNase genes using specific primers (16s rRNA.Assessment of the expression of different  primers  by QPCR The swarming motility assay was performed on a Petri dish as described elsewherel) and shortest (ds) diameters of each interstitial biofilm and using the following equation:Twitching motility assay was performed as described elsewherePA biofilm after 72 h of incubation with or without L-Met (0.5 μM) in a 24-well plate were exposed to 4 μg/ml of ciprofloxacin under shaking conditions at 180 rpm, 37 °C for 6 h. Then the cells were scraped, homogenized by sonication for 10 minutes in a bath sonicator and plated on LB agarAll the experiments were carried out in accordance with the approved guidelines of institutional animal ethics committee at Indian Institute of Science, Bangalore, India (Registration No: 48/1999/CPCSEA). All procedures with animals were carried out in accordance with the institutional rules for animal experimentation.in vivo experiments. The mice used for the experiments were 6–8 weeks old. All procedures with animals were carried out in accordance with the Institutional rules for animal experimentation. All animal experiments were approved by the Institutional Animal Ethics Committee and the National Animal Care Guidelines were strictly followed. Agarose bead infection method was used to establish chronic pulmonary infection in mice as described elsewhereBALB/c mice bred and housed at the Central Animal Facility of Indian Institute of Science (IISc) were used for all the BALB/c mice were intranasally infected with 50 μl of 1:10 dilution bead slurry. On day 3 post infection, different cohorts of mice were treated with ciprofloxacin alone (2.5 mg/kg – intravenous injection – once per day), L-Met treatment alone , or L-Met  and ciprofloxacin treatment (2.5 mg/kg – intravenous injection – once per day) for 3 days. The mice were sacrificed after 3 days, and the lungs was aseptically removed, weighed and homogenized in sterile PBS. The homogenate was plated using serial dilutions onto LB agar to determine the bacterial load. A small portion of the lung was fixed with 2.5% (v/v) glutaraldehyde and processed with increasing concentrations of alcohol  for 2 min and analysed with SEM as described. For survival assay, same treatment was given (n = 10 mice/group) and the mice were monitored twice daily for 15 days for morbidity and mortality.How to cite this article: Gnanadhas, DP et al. Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy. Sci. Rep.5, 16043; doi: 10.1038/srep16043 (2015).


The increase in antimicrobial-resistant infections has led to significant morbidity, mortality, and healthcare costs. The impact of antimicrobial resistance is greatest on low-income countries, which face the double burden of fewer antibiotic choices and higher rates of infectious diseases. Currently, Guyana has no national policy on rational prescribing. This study aims to characterize antibiotic prescribing patterns in children discharged from the emergency department at Georgetown Public Hospital Corporation (GPHC), as per the World Health Organization (WHO) prescribing indicators.A retrospective chart review of pediatric patients (aged 1 month–13 years) seen in the GPHC emergency department between January and December 2012 was conducted. Outpatient prescriptions for eligible patients were reviewed. Patient demographics, diagnosis, and drugs prescribed were recorded. The following WHO Prescribing Indicators were calculated: i) average number of drugs prescribed per patient encounter, ii) percentage of encounters with an antibiotic prescribed, iii) percentage of antibiotics prescribed by generic name, and iv) percentage of antibiotics prescribed from essential drugs list or formulary.s = 3.98 years). 59.6 % (n = 483) patients were male. An average of 2.5 drugs were prescribed per encounter (WHO standard is 2.0). One or more antibiotic was prescribed during 36.9 % (n = 299) of all encounters (WHO standard is 30 %). 90.83 % of antibiotics were prescribed from the essential drugs formulary list and 30 % of the prescriptions included the drug’s generic name. The average duration of antibiotic therapy was 5.73 days (s = 3.53 days). Of the 360 antibiotics prescribed, 74.7 % (n = 269) were broad-spectrum. B-lactam penicillins were prescribed most frequently (51.4 %), with amoxicillin being the most popular choice (33.9 %). The most common diagnoses were injuries (25.8 %), asthma (20 %), respiratory infections (19.5 %), and gastrointestinal infections (12.1 %).Eight hundred eleven patient encounters were included in the study. The mean patient age was 5.55 years (Per WHO prescribing indicators, the pediatric emergency department at GPHC has higher than standard rates of antibiotic use and polypharmacy. The department excels in adhering to the essential drug formulary. Our findings provide support for investigating drug utilization in other Guyanese settings, and to work towards developing a national rational prescribing strategy. Antibiotics are amongst the most common medications prescribed for children . Recent Guyana is a lower-middle-income country in South America with significant rates of communicable diseases like malaria, HIV and tuberculosis . There iThe WHO has developed prescribing indicators to detect barriers to good antimicrobial stewardship . These iGPHC is a national referral hospital, with a 937-bed capacity making it the country’s largest hospital . The GPHi)average number of drugs prescribed per patient encounter,ii)percentage of encounters with an antibiotic prescribed,iii)percentage of antibiotics prescribed by generic name, andiv)percentage of antibiotics prescribed from essential drugs list or formulary.A retrospective chart review was conducted of pediatric patients (aged 1 month–13 years) seen in the GPHC emergency department in Georgetown, Guyana. Since the treatment of infections in older teenagers can be similar to the approach used in adults, we utilized younger age cut-offs to capture pediatric antimicrobial use. The charts of all eligible patients assessed on the 1st and 15th day of each month from January to December 2012 were included. This sampling method was used to ensure a manageable sample size that was random and accounted for seasonal and temporal variation. If patient charts were missing for the 1st or 15th day of the month, charts from the next closest date were sampled instead . The primary investigator (S.S.) reviewed outpatient prescriptions for these patients. Patients who absconded from hospital prior to assessment by a physician were excluded, as no prescriptions were written in these charts. For each patient encounter, the patient demographics , diagnosis, and drugs prescribed were recorded. If antibiotics were prescribed, the antibiotic class, dose, route, frequency and duration of therapy was recorded. Based on this data, the following WHO Prescribing Indicators were calculated:The distribution of antibiotic prescriptions was sub-divided by class of antibiotic, spectrum of activity, and primary diagnosis. Broad-spectrum antibiotics included second to fourth generation penicillins (i.e. beta-lactam penicillins), sulfonamides, cephalosporins, fluoroquinolones and tetracycline. Narrow-spectrum antibiotics included aminoglycosides, metronidazole, macrolides and first-generation penicillins. All statistical analyses were performed using Microsoft Excel version 12.3.6. Ethical approval was obtained from the Institutional Review Board of the Ministry of Health in Guyana.n = 483) of patients were male (Table Eight hundred and fifty six charts of pediatric patients assessed in the GPHC emergency department were reviewed during the study period. Forty-five patient encounters were excluded because 44 patients absconded prior to receiving therapy and 1 patient did not fit the age criteria. Eight hundred and eleven patient encounters were included in the study. The median patient age was 5.0 years (interquartile range 2.0–9.0) and 59.6 %  of all patient encounters. As a measure of antibiotic overuse, this value is close to the WHO standard, which recommends antibiotic prescriptions not exceed 30 % of patient encounters [n = 52) of all encounters, suggesting that when antibiotics were prescribed in our sample, usually a single antimicrobial was used. The average duration of antibiotic therapy was 5.73 days (s = 3.53 days).One or more antibiotic was prescribed during 36.9 %  were broad-spectrum. B-lactam penicillins were prescribed most frequently (51.4 %), followed by sulfonamides (13 %), cephalosporins (7.7 %) and fluoroquinolones (1.7 %). Of the B-lactam penicillins, amoxicillin was the most popular choice (33.9 %). Fixed-dose combinations of antibiotics were encountered in 12.9 % of all prescriptions. Sulfamethoxazole and trimethoprim accounted for 5.5 % of all prescriptions and was classified as a sulfonamide for the purposes of this study. Amoxicillin and clavulanic acid was classified as a beta-lactam antibiotic and accounted for 7.4 % of all prescriptions.As outlined in Table The most common diagnoses for which drugs were prescribed were injuries (25.8 %), asthma (20 %), respiratory infections (19.5 %), and gastrointestinal infections (12.1 %), as outlined in Table n = 360). Our chart review recorded only the primary diagnosis listed for each clinical encounter, thus encounters that featured more than one diagnosis  may be appropriate for prescribing an antibiotic. Even still, our estimate of inappropriate use is conservative and likely an underestimate as we assumed that all diagnoses that could theoretically be treated with antibiotics were clinically severe enough to warrant broad-spectrum antibiotic use at the outset, instead of a narrow spectrum agent or a wait-and-see approach. We also excluded unspecified diagnoses  and diagnoses marked as other  from this estimate. This rough estimate of inappropriate antibiotic prescription is in line with previously published estimates, which cite 30–50 % of antibiotic use in hospitals as unnecessary or inappropriate [While it was outside the scope of this study to evaluate drug use based on rational prescribing guidelines, we reviewed the specific diagnoses for which the top three antibiotic classes were prescribed . As outlined in Table ropriate .Table 5DThis is the first study of antibiotic use in children in Guyana, in an outpatient context. In comparing our results to established WHO standards of drug utilization, the pediatric emergency department at GPHC has higher than standard rates of antibiotic use and polypharmacy, albeit lower than pediatric settings in other lower-middle income countries in the world. Adherence to the essential drug formulary is a strength for the department.While it was beyond the scope of this study, our results also suggest that antibiotics are often inappropriately prescribed for non-infectious diagnoses, like asthma and viral infections. Even when used for infectious conditions, broad-spectrum antibiotics were often used as first-line therapy, which highlights the need for judicious use.With respect to limitations, our study was retrospective in design and limited by incomplete and/or missing data. Incomplete charting on weight-based doses per child and duration and frequency of antibiotics prescribed precluded a secondary data analysis to determine if appropriate doses were prescribed for each clinical encounter. Similarly, the diagnoses associated with each encounter were at times vague  or unclear , making it difficult to assess whether the recorded diagnosis accurately described the condition being treated, and whether the appropriate treatment was prescribed. Finally, with respect to generalizability, the results of this study apply only to an outpatient pediatric population in Georgetown.Baseline data on the prevalence of antibiotic use is the first step in encouraging drug monitoring and quality improvement. As a next step, evaluating adherence of prescribing health care providers to clinical criteria for specific diagnoses will be important in assessing rational use. We hope these results provide impetus for examining drug utilization in other settings in Guyana. There is urgent need to improve surveillance of antimicrobial resistance and develop a national rational prescribing strategy in Guyana. A drug use evaluation study is the next step in helping to inform protocols for rational prescribing and antibiotic stewardship efforts at Georgetown Public Hospital.The aggregate data supporting findings contained within this manuscript will be shared upon request submitted to the corresponding author. Identifying patient data will not be shared.As per the Institutional Review Board, written informed consent was not required from the patients involved in this study, as aggregate data was used, with no identifying personal details published.Ethical approval was obtained from the Institutional Review Board of the Ministry of Health in Guyana. The Chief Executive Officer of the Georgetown Public Hospital Corporation granted administrative permission to access medical records.


Erlotinib is a novel therapeutic agent for cancer treatment. This study was performed to investigate the role of c-MET-PI3K-AKT pathway in the regulation of erlotinib-induced radiosensitization.A973 lung adenocarcinoma cells treated with 6 Gy of radiation were incubated in the presence of erlotinib. The apoptotic rate after 24 hours, the colony-formating rate after 14 days, and changes in the c-MET expression levels after 14 days of irradiation were examined. Surviving fractions in different treatment groups  were determined, the survival curves were plotted, and the sensitizer enhancement ratio was calculated using colony formation assays. Expressions of c-MET, p-c-MET, PI3K, AKT, and p-AKT in cells in different treatment groups were examined by Western blot analysis.The apoptotic rate in the combined erlotinib and radiation group was higher than those in single treatment groups; however, the colony-forming rate remained approximately 2.04 ± 1.02%. The expression of c-MET in colony-forming cells in the combined group significantly increased, and the blockade of c-MET activity significantly enhanced the radiosensitizing effect of erlotinib. The expression of c-Met, p-c-MET, PI3K, AKT, and p-AKT among colony-forming cells significantly decreased upon the inhibition of c-MET.Upregulated activity of the c-MET-PI3K-AKT pathway was found to be important for cell survival under combined the treatment with erlotinib and radiation. The blockade of the c-MET-PI3K-AKT signaling pathway enhanced the radiosensitizing effect of erlotinib. A number of previous studies have demonstrated a clear radiosensitizing effect of erlotinib treatment -3. Howev2 incubator used for cell culture was purchased from Heraeus , and the high-speed refrigerated centrifuge was also obtained from Heraeus. The flow cytometer was from Beckman Coulter, Inc. . The A973 lung adenocarcinoma cell line was used in this study. A973 cells expressed high levels of epidermal growth factor receptor (EGFR) and phospho-EGFR, as reported previously [2. Cells in the exponential growth phase were irradiated.RPMI-1640 culture medium was obtained from Gibco , and fetal bovine serum was obtained from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. Monoclonal antibodies targeting c-MET, phosphorylated c-MET, PI3K, AKT, and phosphorylated AKT were purchased from Santa Cruz Biotechnology, Inc. . RNase A and propidium iodide (PI) were obtained from Sigma . The COeviously -7. Cells2, and constant humidity. After 14 days of cell seeding, the culture dishes were collected, and the culture medium was discarded. Cells were fixed and subjected to Giemsa staining. The number of colonies containing more than 50 cells was counted, and the cell survival fraction (SF) was calculated. The experiments were confirmed three times, and each treatment group contained three parallel samples. The single-hit, multitarget model was used to fit the cell survival curves [Colony-forming rates of the tumor cells were determined using the colony formation assay. The experiments on erlotinib-induced radiosensitization included the following treatment groups: blank control group, radiation alone group, erlotinib alone group, anti-c-MET monoclonal antibody alone group, combined erlotinib and radiation group, and combined erlotinib and radiation with anti-c-MET monoclonal antibody group. Cells in the exponential growth phase were trypsinized, counted, diluted, and seeded onto 35-ml flasks. The number of cells seeded onto the flasks was adjusted according to the radiation dose . The concentrations of erlotinib and anti-c-MET monoclonal antibody used were 20 nM. and 10 nM, respectively. A radiation dose of 2 Gy/min was selected, and cells were exposed to 0, 1, 2, 4, 6, 8, or 10 Gy of radiation after the attachment of cells on the plastic at 37°C, 5% CO5 cells were collected. After adding 1 ml of cold phosphate buffered saline (PBS), the cells were centrifuged at 1000 rpm for 10 min at 4°C. The cells were then washed with PBS, centrifuged twice under the above conditions, and resuspended in 200 μl of binding buffer. Ten microliters of Annexin-FITC was added to the cell suspension and mixed well. The cell mixture was incubated at room temperature in the dark for 15 min, and then an additional 300 μl of binding buffer was added. Finally, the cells were analyzed by flow cytometry after adding 5 μl of PI. To detect the expression of c-MET, the cells were incubated with the PE-coupled mouse anti-human c-MET monoclonal antibody, washed, incubated with the FITC-coupled rabbit anti-mouse antibody, washed, and then subjected to flow cytometry analysis [Cell apoptosis and the expression of c-MET were examined by flow cytometry. Experiments included the following treatment groups: blank control group, radiation alone group, erlotinib alone group, and combined erlotinib and radiation group. The concentrations of erlotinib and the anti-c-MET monoclonal antibody were the same as those listed in the preceding section. All irradiated groups were given a dose of 6 Gy. Colony-forming cells from different treatment groups were collected, and the apoptotic rates and expression of c-MET were determined by flow cytometry. Apoptosis was measured experimentally as follows: first, cells were trypsinized, and 5 × 10analysis ,12.The expressions of c-MET, p-c-MET, PI3K, AKT, and p-AKT in the blank control group, the combined erlotinib and radiation group, and the combined erlotinib and radiation with anti-c-MET monoclonal antibody group were examined using Western blotting,. The concentrations of erlotinib and the anti-c-MET monoclonal antibody were the same as those described above. Cells were irradiated at a dose of 6 Gy. The experimental procedures were performed as follows: 14 days after treatment, the cells were trypsinized and collected. The total protein was extracted from the cells, and the protein concentration was determined by Coomassie brilliant blue staining. The proteins were separated by polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were then probed with primary antibodies, washed, incubated with horseradish peroxidase–conjugated secondary antibodies, and washed again. Finally, protein signals were visualized ,14.Origin7.5 software was used to fit the cell survival curves. Data were presented as the mean ± standard deviation and were analyzed using SPSS17.0 software. The analysis of variance (ANOVA) tables were used to perform comparisons between multiple groups. P values less than 0.05 were considered statistically significant.The study was not related with patients.Our results showed that the apoptotic rates of cells in the blank control group, erlotinib alone group, radiation alone group, and combined erlotinib and radiation group after 24 hours of treatment were 2.43 ± 1.03%, 11.26 ± 2.14%, 23.45 ± 4.35%, and 47.68 ± 6.73%, respectively. The apoptotic rate in the combined treatment group was significantly higher than those in the other groups . The number of colonies was counted after 14 days of treatment, and the colony-forming rates of cells in the blank control group, erlotinib alone group, radiation alone group, and combined erlotinib and radiation group were 71.45 ± 4.64%, 43.56 ± 3.38%, 15.6 ± 2.26%, and 2.04 ± 1.02%, respectively. Although the colony-forming rate in the combined treatment group was the lowest  alone group, and the combined erlotinib and radiation group after 14 days of treatment were collected, and the results of the flow cytometry analysis showed that the expression rates of c-MET in these groups were 12.25 ± 2.17%, 23.36 ± 3.16%, 27.45 ± 3.79%, and 75.35 ± 6.33%, respectively. Compared to other groups, the expression rate of c-MET in the combined treatment group significantly increased (Figure 0, N, and Dq values for the radiation alone group were 2.51, 2.93, and 1.17, respectively, and for the combined erlotinib and radiation group were 1.54, 1.62, and 0.32, respectively; the sensitizer enhancement ratio was 1.63. The D0, N, and Dq values for the combined erlotinib and radiation with anti-c-MET monoclonal antibody treatment group were 1.01, 1.17, and 0.07, respectively, and the sensitizer enhancement ratio was 2.49. Therefore, the elevated c-MET expression likely acted as a survival factor for the tumor cells treated with a combination of erlotinib and radiation, and the blockade of c-MET signaling significantly enhanced the tumor cytotoxicity effects.The survival curves showed that erlotinib exhibited a clear radiosensitizing effect. Compared to the combined erlotinib and radiation group, the inhibition of the c-MET signaling pathway in these cells using a monoclonal antibody further decreased the SF and enhanced the radiosensitizing effect (ANOVA analysis, P < 0.05, Figure To study the possible survival mechanism utilized by colony-forming cells, cells from the blank control group, the combined erlotinib and radiation group, and the combined erlotinib and radiation with anti-c-MET monoclonal antibody group were collected after 14 days of treatment, and the expression of c-MET, phosphorylated c-MET, PI3K, AKT, and phosphorylated AKT was examined. The results showed that the expression of these proteins significantly increased after 14 days of treatment. However, after treatment with the anti-c-MET monoclonal antibody, the expression of these proteins significantly decreased (Figure The results of the present study indicated that the activation of the c-MET-PI3K-AKT pathway served as an important survival mechanism for cells that received the combined treatment with erlotinib and radiation. However, blockade of the c-MET-PI3K-AKT pathway could further enhance the radiosensitizing effect of erlotinib.Both erlotinib and radiation play important roles in activating the c-MET-PI3K-AKT pathway, which is one of the main causes of acquired resistance to erlotinib. After erlotinib treatment, the PI3K signaling pathway is significantly upregulated in tumor cells. Consequently, downstream pathways and nuclear gene expression are activated, resulting in tumor cell survival and proliferation ,16. In aThe results of the present study support those of previous studies mentioned in the preceding paragraph, because in the present study erlotinib exhibited a clear radiosensitizing effect. However, many cells survived under the combined effect of radiation and erlotinib. The survival of such cells may be because both radiation and erlotinib can promote the expression of c-MET, which subsequently activates downstream pathways and affects their cytotoxic effects. Our finding that the inhibition of c-MET with a monoclonal antibody decreased the SF of tumor cells and downregulated the c-MET-PI3K-AKT pathway activity further confirmed the mechanism of tumor cell survival, and these results may provide a new approach to further enhance the cytotoxic effects of combined therapy with erlotinib and radiation in tumor cells (Figure Previous studies have mainly focused on the molecular mechanism of erlotinib-induced radiosensitization and cytotoxicity ,3,29, anin vitro experiments were performed in this study and many questions remained to be fully explored, we believe that with further in-depth basic research, erlotinib-induced radiosensitization will achieve superior efficacy and possess a broad clinical application.In summary, this study performed constructive investigations of changes in the activity of the c-MET-PI3K-AKT pathway under the combined treatment with erlotinib and radiation and the regulatory effects of blocking the c-MET-PI3K-AKT pathway on the radiosensitizing effect of erlotinib. Our findings revealed an important mechanism for cell survival under the combined treatment with erlotinib and radiation and provided a new experimental basis for further enhancing the radiosensitizing effect of erlotinib. Although only


Pseudomonas aeruginosa PA14 and two pairs of clinically relevant antibiotics (doripenem/ciprofloxacin and cefsulodin/gentamicin). Our results consistently show that the sequential application of two antibiotics decelerates resistance evolution relative to monotherapy. Sequential treatment enhanced population extinction although we applied antibiotics at sublethal dosage. In both experiments, we identified an order effect of the antibiotics used in the sequential protocol, leading to significant variation in the long-term efficacy of the tested protocols. These variations appear to be caused by asymmetric evolutionary constraints, whereby adaptation to one drug slowed down adaptation to the other drug, but not vice versa. An understanding of such asymmetric constraints may help future development of evolutionary robust treatments against infectious disease.Antibiotic resistance is a growing concern to public health. New treatment strategies may alleviate the situation by slowing down the evolution of resistance. Here, we evaluated sequential treatment protocols using two fully independent laboratory-controlled evolution experiments with the human pathogen  Pseudomonas aeruginosa, which evades treatment with any of the currently available antibiotics . Even novel methods seem unable to accelerate antibiotic discovery , causing a surge of infections that resist treatment with standard broad-spectrum antibiotics May . One of y Wright . This leEven if drug discovery can pick up the pace, novel antibiotics are unlikely to solve the conundrum. New antimicrobials will eventually be rendered ineffective by the evolution of resistance, despite their initial success in treating infections. This is illustrated by antimicrobial peptides that were once advocated as invincible hurdles to bacterial evolution Zasloff . Yet bacEscherichia coli  with some of the clinically used antibiotics with different targets. PA14 was isolated from a human burn wound and is also virulent in many animals and plants  or with the same antibiotics throughout the experiment (monotherapy treatment type). Most previous work on sequential therapy only compared regular antibiotic alternations to monotherapy  and 0.1% casamino acids (w/v), according to a standard protocol  of 12 h each. We performed two independent experiments of identical set-up with the antibiotic pairs CIP/DOR and GEN/CEF  and, in each case, a total of eleven treatment protocols that belonged to four treatment types: regular and random sequential treatments (2× four protocols), monotherapy (two protocols) and a no-drug control (see below). Each of the ten treatment protocols with an antibiotic was replicated eight times, while the control included 16 replicate populations, yielding a total of 96 replicate population per experiment . During the experiment, we monitored population growth through continuous absorbance measurements . Thus, we could track the evolutionary dynamics in real time. The data for the two experiments were analysed separately using the statistics software r . At the end of each season, 1% of the culture volume was transferred to fresh media  containing either the preceding or a new antibiotic at IC75 dosage . The choice of IC75 dosage for our experiments represented a compromise to achieve two aims. On the one hand, bacterial cells still survive and can adapt. On the other hand, such dosage still exerts considerable selection pressure. Moreover, antibiotic dosage below the MIC is also clinically relevant, where the applied dosage is usually above MIC, but within the patient often lower due to, for example, incomplete tissue penetrance or drug degradation  or by eight different successions of two antibiotics . In four of the tested sequential treatment protocols, we switched antibiotics regularly, that is at every transfer or at every second transfer (treatment protocols 3–6). In four additional sequential protocols, the alternation of antibiotics followed a random temporal pattern .As an additional measure for bacterial adaptation, we calculated cumulative OD for the different treatment protocols by summing the endpoint OD measurement of each season across all seasons. This measure specifically summarizes the total bacterial yield during the sixteen seasons of antibiotic treatment, and thus, it is related to above AUC inhibition measures, yet depicts a different aspect of the evolutionary response of the bacterial populations. We compared means of cumulative OD between treatment protocols using nonparametric Welsh P-value indicates the significance of zig-zag growth dynamics due to drug order.We performed frequency tests to assess whether switch to one of the antibiotics generally led to a significant increase in inhibition in the sequential treatment protocols. For each replicate population, we counted the number of antibiotic switches during which inhibition increased relative to the preceding value. If the average was higher than 50% of the total number of switches for a particular replicate population, the score of the respective replicate was set to 1. Otherwise, it was set to 0, indicating either no change or a decrease in inhibition due to the antibiotic switch. Next, the frequency of 0s and 1s were counted for each individual treatment protocol . If the majority were 1s, the treatment protocol received an overall score of 1, otherwise 0. This procedure was done separately for the switch from A → B and B → A. By combining the information across replicate populations for each treatment protocol, we obtained a conservative measure for the effect of a drug switch. For each evolution experiment, we thus obtained 16 count values , which were evaluated using a Fisher’s exact test. The thus obtained 75 of the drug). After 24 h of incubation, we randomly sampled five individual colonies from each replicate population and season and cultured them as in the evolution experiment (i.e. with the same antibiotic). As references, we grew five colonies from the ancestral population in liquid culture without antibiotics. Next, we transferred 1% of the liquid cultures to five concentrations of the same antibiotic and separately to five concentrations of the other antibiotic of the evolution experiment. The cultures of the ancestor were transferred to all four antibiotics and all concentrations. We measured optical density after 12 h of incubation and calculated the area under the dose–response curve. Note that this measure differs from AUC inhibition, which is instead derived from the area under the time-dependent response curve. For statistical analysis, we compared corresponding values for the same antibiotic between evolved clones and ancestral populations using a mixed linear model with colonies nested in replicate populations.After the evolution experiments, we experimentally tested for the evolution of collateral sensitivity and resistance. We focused on evolved material from seasons 6 and 7 from the sequential treatment protocol 4, which showed a particularly pronounced zig-zag pattern (see Results). Seasons 6 and 7 correspond to the valley and peak of the zig-zag curve. For feasibility, we randomly chose three of the originally eight replicate populations of these treatment protocols for further analysis. For these replicate populations, we revived evolved bacterial lines from frozen glycerol stocks by plating them out on agar plates containing the particular antibiotic which was experienced by the line prior to freezing  adaptation over the length of the protocol to only one of the two antibiotics in the sequence , (ii) the starting drug of the sequence, that is, the drug used during the first season alone, or (iii) the cost of evolving resistance to the first-season drug. We also asked whether the observed zig-zag evolutionary dynamics could be explained by (iv) overall variation in the order of antibiotics across the entire sequence or (v) collateral sensitivities between the two antibiotics. Finally, we determined (vi) extinction frequencies of the different types of protocols.Our first analysis assessed whether decelerated adaptation was caused by the sequential treatment protocol itself or merely by the number of seasons during which a particular replicate population was exposed to a certain antibiotic, irrespective of intermittent seasons with the other antibiotic. For this test, we plotted the chronologically obtained AUC inhibition values for the seasons during which only one of the antibiotics was used, thus excluding all AUC inhibition values obtained for intermittent seasons with the other antibiotic. The results are shown in β-lactam DOR in the first season across the various treatments led to lower accumulative OD; Second, we asked which specific aspect of the sequential treatment protocols accounts for decelerated adaptation? Interestingly, cumulative OD varied strongly among regular sequential protocols ,D, indicβ-lactam antibiotics  and CIP- or GEN-adapted populations . Conversely, the opposite switch from CIP to DOR decreased inhibition, thereby forming zig-zag patterns across time. Figureβ-lactam to the non-β-lactam antibiotic – from CEF to GEN – and decreased when antibiotics were changed reversely . These dynamics suggest that adaptation to one antibiotic affects the ability to adapt to another one.Fourth, we investigated the general role of drug order across the entire sequential protocol in determining antibiotic adaptation. Such a role is supported by the exact growth dynamics in the sequential treatment protocols. A switch from the P < 0.001, Experiment 2: P = 0.030, P = 0.069, P = 0.030, Fifth, a more specific explanation for the observed zig-zag pattern could be provided by the evolution of collateral sensitivities. To test this aspect, we measured the dose–response curves against both antibiotics of a particular pair. We focused the analysis on independent clones isolated from three randomly chosen replicate populations of seasons 6 and 7 of the antibiotic alternation treatment protocol 4, which showed a pronounced zig-zag pattern . In bothP = 0.1719, odds ratio = 0.2208). In Experiment 2, suppressed replicate populations were significantly more frequent in the sequential treatment protocols , yet they all recovered in antibiotic-free media, after a lag-phase of approximately 10 h , our study demonstrates that the alternation of antibiotics significantly minimizes adaptation rates  was exposed to regular one-season alternations of rifampicin and streptomycin (Perron et al. For mexAB-oprM operon (Poole P. aeruginosa mostly achieves resistance against the antibiotics used by us through individual mechanisms, which rarely produce cross-resistance (reviewed by Poole β-lactams CIP and GEN, because their overall yield is higher and their AUC inhibition reaches lower values faster than the corresponding values for the β-lactam monotherapies (the two left bars in β-lactams and the peaks for the non-β-lactam (A general drug order effect may alternatively result from the bacteria’s inability to achieve cross-resistance to both drugs in one step, for example through a single molecular mechanism, such as a change or amplification of the multidrug efflux system on Poole . Otherwion Poole , suggestβ-lactam . TherefoE. coli (Imamovic and Sommer S. aureus (Kim et al. β-lactam antibiotic could explain why inhibition usually spiked upon switches to the non-β-lactam. Conversely, exposure to the non-β-lactam was followed by decreased inhibition in a β-lactam environment, possibly indicating evolution of collateral resistance. However, our separate analysis of evolved lines from intermediate seasons of treatment protocol 4 provides only weak support for the importance of asymmetric collateral effects (Resistance mutations that adapt against one antibiotic may also amplify the potency of the other antibiotic. Such collateral sensitivities were recently described for various pairs of antibiotics in  effects .β-lactam-resistant mutants can spread faster than the mutants with resistance to the non-β-lactam, leading to high prevalence of the β-lactam-resistant variants under β-lactam conditions (and thus low AUC inhibition, β-lactam-resistant mutants would only reach intermediate frequencies under the non-β-lactam conditions (resulting in higher values of AUC inhibition, The observed zig-zag pattern thus requires an alternative explanation. Possible factors include the differential speed, with which resistant mutants to either of the antibiotics may be able to spread within the evolving populations. In particular, it is possible that E. coli at hot and cold niche extremes (Mongold et al. Even though the exact underlying process is still unclear, our data clearly demonstrate that adaptation to the antibiotics of a pair is shaped by asymmetric evolutionary constraints. Interestingly, such asymmetric effects are thought to be a common outcome of adaptation to local ecological conditions. Constant local conditions favour the evolution of ecological specialists capable of utilizing the available resources more efficiently than their ancestor Kassen . Their hE. coli lines evolved in alternations of lactose and glucose (Cooper and Lenski In temporally heterogeneous environments, however, reports of such asymmetric evolutionary constraints are rare. Rather, temporal environmental variation most often selects for cost-free generalists that are equally or superiorly fit across the range of environmental variation Kassen . EnvironIn conclusion, we here provide evidence that temporal variation in drug environments minimizes adaptation rates relative to constant single-drug environments. Sequential antibiotic treatment significantly decelerated resistance evolution independent of the temporal regularity by which drugs were switched. Asymmetric evolutionary constraints account for reduced adaptation in the changing environments. Their future analysis, including dissection of the underlying genetic mechanisms, may help to develop evolutionary robust treatments of bacterial infections.