Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    We will mail you new results for this query: keywords==Antibiotic resistance
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Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread : A review
Krzeminski, Pawel ; Tomei, Maria Concetta ; Karaolia, Popi ; Langenhoff, Alette ; Almeida, C.M.R. ; Felis, Ewa ; Gritten, Fanny ; Andersen, Henrik Rasmus ; Fernandes, Telma ; Manaia, Celia M. ; Rizzo, Luigi ; Fatta-Kassinos, Despo - \ 2019
Science of the Total Environment 648 (2019). - ISSN 0048-9697 - p. 1052 - 1081.
Antibiotic resistance - Biological processes - CEC removal - Crop uptake - EU Watch list - Secondary wastewater treatment

Contaminants of emerging concern (CEC) discharged in effluents of wastewater treatment plants (WWTPs), not specifically designed for their removal, pose serious hazards to human health and ecosystems. Their impact is of particular relevance to wastewater disposal and re-use in agricultural settings due to CEC uptake and accumulation in food crops and consequent diffusion into the food-chain. This is the reason why the chemical CEC discussed in this review have been selected considering, besides recalcitrance, frequency of detection and entity of potential hazards, their relevance for crop uptake. Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been included as microbial CEC because of the potential of secondary wastewater treatment to offer conditions favourable to the survival and proliferation of ARB, and dissemination of ARGs. Given the adverse effects of chemical and microbial CEC, their removal is being considered as an additional design criterion, which highlights the necessity of upgrading conventional WWTPs with more effective technologies. In this review, the performance of currently applied biological treatment methods for secondary treatment is analysed. To this end, technological solutions including conventional activated sludge (CAS), membrane bioreactors (MBRs), moving bed biofilm reactors (MBBRs), and nature-based solutions such as constructed wetlands (CWs) are compared for the achievable removal efficiencies of the selected CEC and their potential of acting as reservoirs of ARB&ARGs. With the aim of giving a picture of real systems, this review focuses on data from full-scale and pilot-scale plants treating real urban wastewater. To achieve an integrated assessment, technologies are compared considering also other relevant evaluation parameters such as investment and management costs, complexity of layout and management, present scale of application and need of a post-treatment. Comparison results allow the definition of design and operation strategies for the implementation of CEC removal in WWTPs, when agricultural reuse of effluents is planned.

Dynamics of cefotaxime resistant Escherichia coli in broilers in the first week of life
Dierikx, Cindy M. ; Goot, Jeanet van der; Essen-Zandbergen, Alieda van; Mevius, Dik J. - \ 2018
Veterinary Microbiology 222 (2018). - ISSN 0378-1135 - p. 64 - 68.
AmpC - Antibiotic resistance - Broilers - Cefotaxime - E. coli - ESBL

Extended-spectrum beta-lactamase producing E. coli (ESBL-E) are wide spread among broilers, with the highest prevalence among individual birds at broiler production farms. Previous research describes low prevalences among individual birds at arrival at the farm (below 30%), and a rapid increase up to 100% within the first week. Our goal was to investigate whether this rapid increase was due to latent contamination of ESBL-E or to contamination at the broiler farm. Two broiler groups, one hatched at a conventional hatchery and the other individually hatched in an ESBL-free environment, were housed individually in an experimental ESBL-free environment. A third group was hatched at a conventional hatchery and kept at a conventional broiler farm. The birds were sampled daily during the first week after hatch and tested for the presence of ESBL-E. In addition ESBL-E presence in eggs that were not incubated was investigated. All birds and eggs came from one ESBL-E positive parent flock. ESBL/AmpC genes, plasmids and E. coli sequence types were determined for a selection of isolates. ESBL-E was never found in the two groups kept in the ESBL-free experimental environment or in the sampled eggs, whereas all broilers sampled at the conventional farm became positive for ESBL-E within three days. One dominant E. coli strain (ST88) carrying blaCTX-M-1 gene on an IncI1/pST3 plasmid was found in parent and broiler samples. We conclude that the rapid increase in ESBL-E prevalence in the first week of life is not caused by a latent contamination of the majority of birds at arrival, but that this increase must be caused by other factors.

Enterobacter cloacae complex isolated from shrimps from Vietnam carrying blaIMI-1 resistant to carbapenems but not cephalosporins
Brouwer, Michael S.M. ; Rapallini, Michel ; Geurts, Yvon ; Harders, Frank ; Bossers, Alex ; Mevius, Dik J. ; Wit, Ben ; Veldman, Kees T. - \ 2018
Antimicrobial Agents and Chemotherapy 62 (2018)7. - ISSN 0066-4804
Antibiotic resistance - Carbapenems - Elements - Enterobacter - Mobile genetic
Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals : a systematic review
Köck, R. ; Daniels-Haardt, I. ; Becker, K. ; Mellmann, A. ; Friedrich, A.W. ; Mevius, D. ; Schwarz, S. ; Jurke, A. - \ 2018
Clinical Microbiology and Infection 24 (2018)12. - ISSN 1198-743X - p. 1241 - 1250.
Antibiotic resistance - Carbapenemase - Enterobacteriales - Epidemiology - Livestock - Zoonosis
Objectives: The spread of carbapenem-resistant Enterobacteriaceae (CRE) in healthcare settings challenges clinicians worldwide. However, little is known about dissemination of CRE in livestock, food, and companion animals and potential transmission to humans. Methods: We performed a systematic review of all studies published in the PubMed database between 1980 and 2017 and included those reporting the occurrence of CRE in samples from food-producing and companion animals, wildlife, and exposed humans. The primary outcome was the occurrence of CRE in samples from these animals; secondary outcomes included the prevalence of CRE, carbapenemase types, CRE genotypes, and antimicrobial susceptibilities. Results: We identified 68 articles describing CRE among pigs, poultry, cattle, seafood, dogs, cats, horses, pet birds, swallows, wild boars, wild stork, gulls, and black kites in Africa, America, Asia, Australia, and Europe. The following carbapenemases have been detected (predominantly affecting the genera Escherichia and Klebsiella): VIM, KPC, NDM, OXA, and IMP. Two studies found that 33–67% of exposed humans on poultry farms carried carbapenemase-producing CRE closely related to isolates from the farm environment. Twenty-seven studies selectively screened samples for CRE and found a prevalence of <1% among livestock and companion animals in Europe, 2–26% in Africa, and 1–15% in Asia. Wildlife (gulls) in Australia and Europe carried CRE in 16–19%. Conclusions: The occurrence of CRE in livestock, seafood, wildlife, pets, and directly exposed humans poses a risk for public health. Prospective prevalence studies using molecular and cultural microbiological methods are needed to better define the scope and transmission of CRE.
Adaptive benefits from small mutation supplies in an antibiotic resistance enzyme
Salverda, Merijn L.M. ; Koomen, Jeroen ; Koopmanschap, Bertha ; Zwart, Mark P. ; Visser, J.A.G.M. De - \ 2017
Proceedings of the National Academy of Sciences of the United States of America 114 (2017)48. - ISSN 0027-8424 - p. 12773 - 12778.
Adaptation - Antibiotic resistance - Experimental evolution - Fitness landscape - Mutation supply
Populations with large mutation supplies adapt via the “greedy” substitution of the fittest genotype available, leading to fast and repeatable short-term responses. At longer time scales, smaller mutation supplies may in theory lead to larger improvements when distant high-fitness genotypes more readily evolve from lower-fitness intermediates. Here we test for long-term adaptive benefits from small mutation supplies using in vitro evolution of an antibiotic-degrading enzyme in the presence of a novel antibiotic. Consistent with predictions, large mutant libraries cause rapid initial adaptation via the substitution of cohorts of mutations, but show later deceleration and convergence. Smaller libraries show on average smaller initial, but also more variable, improvements, with two lines yielding alleles with exceptionally high resistance levels. These two alleles share three mutations with the large-library alleles, which are known from previous work, but also have unique mutations. Replay evolution experiments and analyses of the adaptive landscape of the enzyme suggest that the benefit resulted from a combination of avoiding mutational cohorts leading to local peaks and chance. Our results demonstrate adaptive benefits from limited mutation supplies on a rugged fitness landscape, which has implications for artificial selection protocols in biotechnology and argues for a better understanding of mutation supplies in clinical settings.
A probabilistic approach to assess antibiotic resistance development risks in environmental compartments and its application to an intensive aquaculture production scenario
Rico, Andreu ; Jacobs, Rianne ; Brink, Paul J. Van den; Tello, Alfredo - \ 2017
Environmental Pollution 231 (2017). - ISSN 0269-7491 - p. 918 - 928.
Antibiotic resistance - Antibiotics - Aquaculture - Environment - Risk assessment
Estimating antibiotic pollution and antibiotic resistance development risks in environmental compartments is important to design management strategies that advance our stewardship of antibiotics. In this study we propose a modelling approach to estimate the risk of antibiotic resistance development in environmental compartments and demonstrate its application in aquaculture production systems. We modelled exposure concentrations for 12 antibiotics used in Vietnamese Pangasius catfish production using the ERA-AQUA model. Minimum selective concentration (MSC) distributions that characterize the selective pressure of antibiotics on bacterial communities were derived from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Minimum Inhibitory Concentration dataset. The antibiotic resistance development risk (RDR) for each antibiotic was calculated as the probability that the antibiotic exposure distribution exceeds the MSC distribution representing the bacterial community. RDRs in pond sediments were nearly 100% for all antibiotics. Median RDR values in pond water were high for the majority of the antibiotics, with rifampicin, levofloxacin and ampicillin having highest values. In the effluent mixing area, RDRs were low for most antibiotics, with the exception of amoxicillin, ampicillin and trimethoprim, which presented moderate risks, and rifampicin and levofloxacin, which presented high risks. The RDR provides an efficient means to benchmark multiple antibiotics and treatment regimes in the initial phase of a risk assessment with regards to their potential to develop resistance in different environmental compartments, and can be used to derive resistance threshold concentrations.
Characterization of Enterococcus isolates colonizing the intestinal tract of intensive care unit patients receiving selective digestive decontamination
Bello Gonzalez, Teresita D.J. ; Pham, Phu ; Top, Janetta ; Willems, Rob J.L. ; Schaik, Willem van; Passel, Mark W.J. van; Smidt, Hauke - \ 2017
Frontiers in Microbiology 8 (2017). - ISSN 1664-302X - 10 p.
Antibiotic prophylactic therapy - Antibiotic resistance - Enterococcus - Intestinal colonization - Selective digestive decontamination - Virulence factors

Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5)Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU patients.

Buwchitin : A ruminal peptide with antimicrobial potential against Enterococcus faecalis
Oyama, Linda B. ; Crochet, Jean Adrien ; Edwards, Joan E. ; Girdwood, Susan E. ; Cookson, Alan R. ; Fernandez-Fuentes, Narcis ; Hilpert, Kai ; Golyshin, Peter N. ; Golyshina, Olga V. ; Privé, Florence ; Hess, Matthias ; Mantovani, Hilario C. ; Creevey, Christopher J. ; Huws, Sharon A. - \ 2017
Frontiers in Chemistry 5 (2017)JUL. - ISSN 2296-2646
Antibiotic resistance - Antimicrobial activity - Antimicrobial peptides - Enterococcus faecalis - Metagenomics - Microbiome - Rumen bacteria
Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.
Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges
Versluis, Dennis ; McPherson, Kyle ; Passel, Mark W.J. van; Smidt, Hauke ; Sipkema, Detmer - \ 2017
Marine Biotechnology 19 (2017)5. - ISSN 1436-2228 - p. 454 - 468.
Antibiotic resistance - Cultivation - Environmental resistance - Sponge bacteria

Sponges often harbour a dense and diverse microbial community. Presently, a large discrepancy exists between the cultivable bacterial fraction from sponges and the community in its natural environment. Here, we aimed to acquire additional insights into cultivability of (previously uncultured) bacteria from three sponge species, namely Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis, by studying bacterial growth on five media in the form of 60 communities scraped from plates without antibiotics, as well as in the form of individual isolates that were grown on these media supplemented with antibiotics. We applied (double-)barcoded 16S ribosomal RNA (rRNA) gene amplicon sequencing for species identification. We show that previously uncultured bacteria can be cultivated using conventional plating and that application of antibiotics in the media can serve to capture a greater bacterial diversity. Moreover, we present criteria to address an important caveat of the plate scraping method whereby bacteria may be detected that did not actually grow. Fourteen out of 27 cultivated novel taxa (<95% identity of the 16S rRNA gene amplicon to reported species) belong to Actinobacteria, which indicates the presence of a large untapped reservoir of bioactive compounds. Three Flavobacteriaceae spp. were isolated that potentially constitute two new genera and one new species.

Dynamics of CMY-2 producing E. coli in a broiler parent flock
Dame-Korevaar, Anita ; Fischer, Egil A.J. ; Stegeman, Arjan ; Mevius, Dik ; Essen-Zandbergen, Alieda van; Velkers, Francisca ; Goot, Jeanet van der - \ 2017
Veterinary Microbiology 203 (2017). - ISSN 0378-1135 - p. 211 - 214.
Antibiotic resistance - Broiler parent stock - CMY - Escherichia coli - Poultry

Extended-spectrum β-lactamase and plasmid mediated AmpC β-lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC-Escherichia coli in a broiler parent flock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen (n = 228), and in the laying period two groups of 33 chickens were individually sampled (n = 66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC-E. coli presence was determined by selective culturing. In the samples of week 16–19 the concentration of ESBL/pAmpC-E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene blaCMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2-E. coli prevalence decreased from 91% (95%CI 86–94%) at day 7 (week 1) to 0% (95%CI 0–5%) in week 21. However, CMY-2-E. coli remained present in the environmental samples during the whole study. CMY-2-E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2-E. coli in this broiler parent flock in absence of antibiotics suggests a selective disadvantage of blaCMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain.

Sponge microbiota are a reservoir of functional antibiotic resistance genes
Versluis, Dennis ; Evgrafov, Mari Rodriguez de; Sommer, Morten O.A. ; Sipkema, Detmer ; Smidt, Hauke ; Passel, Mark W.J. van - \ 2016
Frontiers in Microbiology 7 (2016)NOV. - ISSN 1664-302X
Antibiotic resistance - Functional metagenomics - Microbiota - Resistance gene - Sponge

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis, and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n = 6), gentamicin (n = 1), amikacin (n = 7), trimethoprim (n = 17), chloramphenicol (n = 1), rifampicin (n = 2) and ampicillin (n = 3). Fifteen of 37 inserts harbored resistance genes that shared <90% amino acid identity with known gene products, whereas on 13 inserts no resistance gene could be identified with high confidence, in which case we predicted resistance to be mainly mediated by antibiotic efflux. One marine-specific ampicillin-resistance-conferring β-lactamase was identified in the genus Pseudovibrio with 41% global amino acid identity to the closest β-lactamase with demonstrated functionality, and subsequently classified into a new family termed PSV. Taken together, our results show that sponge microbiota host diverse and novel resistance genes that may be harnessed by phylogenetically distinct bacteria.

Transmission dynamics of extended-spectrum β-lactamase and AmpC β-lactamase-producing Escherichia coli in a broiler flock without antibiotic use
Huijbers, Patricia M.C. ; Graat, E.A.M. ; Hoek, Angela H.A.M. van; Veenman, Christiaan ; Jong, Mart C.M. de; Duijkeren, Engeline van - \ 2016
Preventive Veterinary Medicine 131 (2016). - ISSN 0167-5877 - p. 12 - 19.
Antibiotic resistance - ESBL - Organic farm - Phylogenetic group - Reproduction ratio

Extended-spectrum and AmpC β-lactamase-producing Escherichia coli (ESBL/AmpC-EC) are found throughout the broiler production pyramid. Transmission of resistance between broilers and humans could occur at any point, representing a potential public health issue. Insight in farm transmission dynamics could provide a basis for control, leading to fewer contaminated broilers. The aim was quantifying transmission rates and routes of ESBL/AmpC-EC, and specific phylogenetic groups, in an organic broiler flock without antibiotic use. In each of two consecutive production rounds, 80 randomly chosen broilers were followed individually. Cloacal swabs from these, 20 other randomly chosen broilers, and 11 environmental samples were taken at several moments from arrival till slaughter. ESBL/AmpC-EC were isolated by selective pre-enrichment, and ESBL/AmpC-genes and E. coli phylogenetic groups were determined. Transmission parameters (β) were estimated using a Generalised Linear Model with a susceptible-infectious-susceptible model. Effect of direct broiler contact as compared to contact through the environment and previous carriage c.q. infectious status and their interaction were included as explanatory variables. Multiplying β by the length of the infectious period gives the reproduction ratio (R). On day 1, prevalence was 28.8% (95%CI 19.2–40.0%) and 0.0% (95%CI 0.0–4.5%) among individually followed broilers, in round 1 and 2 respectively. In round 2, the environment was positive before arrival of day-old chicks. After 3 days, almost 100% of broilers and environmental samples were positive in both rounds. Most samples were positive for CTX-M-1 group genes, and A1 and B1 were predominant phylogenetic groups. From day 3 there was a shift towards more phylogenetic groups. R was 1.70 (95%CI 0.55–5.25) for total ESBL/AmpC-EC. Risk for broilers to become infectious was lower if previously infectious (βpreviously infectious = 0.02 vs. βnot previously infectious = 3.41; P <0.0001). For phylogenetic groups separately, R was 0.88 (95%CI 0.38–2.07), 0.51 (95%CI 0.27–0.98), 0.99 (95%CI 0.65–1.51) for A1, B1 and rest (i.e. A0, B2, D1, D2) groups, respectively. The interaction effect for A1 and B1 was reflected in the fact that when broilers were previous infectious, the environment was relatively more important for transmission of the A1 group, while this was direct contact between broilers for the B1 group. Positive day-old chicks and the environment both play a role in introduction and transmission of ESBL/AmpC-EC in flocks. These results suggest that, even without selective pressure from antibiotics, total ESBL/AmpC-EC persistence, and resulting endemic situation, seem to be caused by shifts in carriage of different phylogenetic groups. It implies that contaminated broilers enter the slaughterhouse.

Study of the aminoglycoside subsistence phenotype of bacteria residing in the gut of humans and zoo animals
Bello Gonzalez, Teresita ; Zuidema, Tina ; Bor, Gerrit ; Smidt, Hauke ; Passel, M.W.J. van - \ 2016
Frontiers in Microbiology 6 (2016)JAN. - ISSN 1664-302X - 7 p.
Aminoglycosides - Antibiotic resistance - Antibiotic subsistence - Antibiotic subsistence phenotype - Single carbon source

Recent studies indicate that next to antibiotic resistance, bacteria are able to subsist on antibiotics as a carbon source. Here we evaluated the potential of gut bacteria from healthy human volunteers and zoo animals to subsist on antibiotics. Nine gut isolates of Escherichia coli and Cellulosimicrobium sp. displayed increases in colony forming units (CFU) during incubations in minimal medium with only antibiotics added, i.e., the antibiotic subsistence phenotype. Furthermore, laboratory strains of E. coli and Pseudomonas putida equipped with the aminoglycoside 3' phosphotransferase II gene also displayed the subsistence phenotype on aminoglycosides. In order to address which endogenous genes could be involved in these subsistence phenotypes, the broad-range glycosyl-hydrolase inhibiting iminosugar deoxynojirimycin (DNJ) was used. Addition of DNJ to minimal medium containing glucose showed initial growth retardation of resistant E. coli, which was rapidly recovered to normal growth. In contrast, addition of DNJ to minimal medium containing kanamycin arrested resistant E. coli growth, suggesting that glycosyl-hydrolases were involved in the subsistence phenotype. However, antibiotic degradation experiments showed no reduction in kanamycin, even though the number of CFUs increased. Although antibiotic subsistence phenotypes are readily observed in bacterial species, and are even found in susceptible laboratory strains carrying standard resistance genes, we conclude there is a discrepancy between the observed antibiotic subsistence phenotype and actual antibiotic degradation. Based on these results we can hypothesize that aminoglycoside modifying enzymes might first inactivate the antibiotic (i.e., by acetylation of amino groups, modification of hydroxyl groups by adenylation and phosphorylation respectively), before the subsequent action of catabolic enzymes. Even though we do not dispute that antibiotics could be used as a single carbon source, our observations show that antibiotic subsistence should be carefully examined with precise degradation studies, and that its mechanistic basis remains inconclusive.

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses
Pacholewicz, Ewa ; Liakopoulos, Apostolos ; Swart, Arno ; Gortemaker, Betty ; Dierikx, Cindy ; Havelaar, Arie ; Schmitt, Heike - \ 2015
International Journal of Food Microbiology 215 (2015). - ISSN 0168-1605 - p. 57 - 63.
Antibiotic resistance - Poultry - Slaughter hygiene

Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the concentration of ESBL/AmpC producing E. coli on broiler chicken carcasses through processing. In addition the changes in ESBL/AmpC producing E. coli concentrations were compared with generic E. coli and Campylobacter. In two slaughterhouses, the surface of the whole carcasses was sampled after 5 processing steps: bleeding, scalding, defeathering, evisceration and chilling. In total, 17 batches were sampled in two different slaughterhouses during the summers of 2012 and 2013. ESBL/AmpC producing E. coli was enumerated on MacConkey agar with 1mg/l cefotaxime, and the ESBL/AmpC phenotypes and genotypes were characterised. The ESBL/AmpC producing E. coli concentrations varied significantly between the incoming batches in both slaughterhouses. The concentrations on broiler chicken carcasses were significantly reduced during processing. In Slaughterhouse 1, all subsequent processing steps reduced the concentrations except evisceration which led to a slight increase that was statistically not significant. The changes in concentration between processing steps were relatively similar for all sampled batches in this slaughterhouse. In contrast, changes varied between batches in Slaughterhouse 2, and the overall reduction through processing was higher in Slaughterhouse 2. Changes in ESBL/AmpC producing E. coli along the processing line were similar to changes in generic E. coli in both slaughterhouses. The effect of defeathering differed between ESBL/AmpC producing E. coli and Campylobacter. ESBL/AmpC producing E. coli decreased after defeathering, whereas Campylobacter concentrations increased. The genotypes of ESBL/AmpC producing E. coli (blaCTX-M-1, blaSHV-12, blaCMY-2, blaTEM-52c, blaTEM-52cvar) from both slaughterhouses match typical poultry genotypes. Their distribution differed between batches and changed throughout processing for some batches. The concentration levels found after chilling were between 102 and 105CFU/carcass. To conclude, changes in ESBL/AmpC producing E. coli concentrations on broiler chicken carcasses during processing are influenced by batch and slaughterhouse, pointing to the role of both primary production and process control for reducing ESBL/AmpC producing E. coli levels in final products. Due to similar changes upon processing, E. coli can be used as a process indicator of ESBL/AmpC producing E. coli, because the processing steps had similar impact on both organisms. Cross contamination may potentially explain shifts in genotypes within some batches through the processing.

Role of pleiotropy during adaptation of TEM-1 β-lactamase to two novel antibiotics
Schenk, M.F. ; Witte, Sariette ; Salverda, M.L.M. ; Koopmanschap, Bertha ; Krug, Joachim ; Visser, J.A.G.M. de - \ 2015
Evolutionary Applications 8 (2015)3. - ISSN 1752-4563 - p. 248 - 260.
Antibiotic resistance - Epistasis - Pleiotropy - Protein evolution - TEM-1 β-lactamase - Trade-off

Pleiotropy is a key feature of the genotype-phenotype map, and its form and extent have many evolutionary implications, including for the dynamics of adaptation and the evolution of specialization. Similarly, pleiotropic effects of antibiotic resistance mutations may affect the evolution of antibiotic resistance in the simultaneous or fluctuating presence of different antibiotics. Here, we study the role of pleiotropy during the in vitro adaptation of the enzyme TEM-1 β-lactamase to two novel antibiotics, cefotaxime (CTX) and ceftazidime (CAZ). We subject replicate lines for four rounds of evolution to selection with CTX and CAZ alone, and in their combined and fluctuating presence. Evolved alleles show positive correlated responses when selecting with single antibiotics. Nevertheless, pleiotropic constraints are apparent from the effects of single mutations and from selected alleles showing smaller correlated than direct responses and smaller responses after simultaneous and fluctuating selection with both than with single antibiotics. We speculate that these constraints result from structural changes in the oxyanion pocket surrounding the active site, where accommodation of CTX and the larger CAZ is balanced against their positioning with respect to the active site. Our findings suggest limited benefits from the combined or fluctuating application of these related cephalosporins for containing antibiotic resistance.

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