Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut.

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.

Chromosome-Based blaOXA-48-Like Variants in Shewanella Species Isolates from Food-Producing Animals, Fish, and the Aquatic Environment.

Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based blaOXA-48-like variants in Shewanella spp., including two new alleles, blaOXA-514 and blaOXA-515 Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

Competitive Exclusion Reduces Transmission and Excretion of Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Broilers.

Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of ß-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpC-producing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field.IMPORTANCE Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases are a primary cause of resistance to ß-lactam antibiotics among members of the family Enterobacteriaceae in humans, animals, and the environment. Food-producing animals are not exempt from this, with a high prevalence being seen in broilers, and there is evidence pointing to a possible foodborne source for human contamination. We investigated the effect of administration of a commercial probiotic product as an intervention to reduce the amount of ESBL-producing Escherichia coli in broilers. Our results showed a substantial reduction in the level of colonization of broiler intestines by ESBL-producing E. coli after administration of commercial probiotic product. The protective effect provided by these probiotics could be implemented on a larger scale in poultry production. Reductions in the levels of ESBL-producing Enterobacteriaceae in the food chain would considerably benefit public health.

Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK.

OBJECTIVES: This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. METHODS: A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. RESULTS: The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. CONCLUSIONS: ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans.

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum ß-lactamase (ESBL) or AmpC ß-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

The IncI1 plasmid carrying the blaCTX-M-1 gene persists in in vitro culture of a Escherichia coli strain from broilers.

BACKGROUND: Commensal bacteria are a reservoir for antimicrobial-resistance genes. In the Netherlands, bacteria producing Extended Spectrum Beta-Lactamases (ESBL) are found on chicken-meat and in the gut of broilers at a high prevalence and the predominant ESBL-gene is the bla(CTX-M-1) located on IncI1 plasmids. We aim to determine the fitness costs of this plasmid for the bacterium.We investigated the conjugation dynamics of IncI1 plasmids carrying the bla(CTX-M-1) gene in a batch culture and its impact on the population dynamics of three E. coli populations: donors, recipients and transconjugants. The intrinsic growth rate (ψ), maximum density (K) and lag-phase (λ) of the populations were estimated as well as the conjugation coefficient. Loss of the plasmid by transconjugants was either assumed constant or depended on the effective growth rate of the transconjugants.Parameters were estimated from experiments with pure culture of donors, recipients and transconjugants and with mixed culture of donors and recipients with a duration of 24 or 48 hours. Extrapolation of the results was compared to a 3-months experiment in which a mixed culture of recipient and transconjugant was regularly diluted in new medium. RESULTS: No differences in estimated growth parameters (ψ, K or λ) were found between donor, recipient and transconjugant, and plasmid loss was not observed. The conjugation coefficient of transconjugants was 104 times larger than that of the donor. In the 3-months experiment, the proportion of transconjugants did not decrease, indicating no or very small fitness costs. CONCLUSIONS: In vitro the IncI1 plasmid carrying the blaCTX-M-1 gene imposes no or negligible fitness costs on its E. coli host, and persists without antimicrobial usage.

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Transmission of Brucella canis in a canine kennel following introduction of an infected dog.

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

Extended-spectrum-ß-lactamase- and AmpC-ß-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers.

OBJECTIVES: The aim of this study was to establish the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli at Dutch broiler farms and in farmers and to compare ESBL/AmpC-producing isolates from farmers and their animals. METHODS: Twenty-five to 41 cloacal swabs collected from broilers at each of 26 farms and 18 faecal samples from 18 broiler farmers were analysed for determination of the presence of ESBL/AmpC-producing E. coli. ESBL/AmpC genes were characterized by microarray, PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing or restriction fragment length polymorphism. E. coli genotypes were determined by multilocus sequence typing. RESULTS: Birds from all farms were positive for ESBL/AmpC-producing E. coli, and on 22/26 farms the within-farm prevalence was ≥ 80%. Six of 18 farmers carried isolates containing ESBL/AmpC genes bla(CTX-M-1), bla(CMY-2) and/or bla(SHV-12), which were also present in the samples from their animals. In five of these isolates, the genes were located on identical plasmid families [IncI1 (n = 3), IncK (n = 1) or IncN (n = 1)], and in isolates from two farmers the genes were carried on identical plasmid subtypes (IncI1 ST12 and IncN ST1, where ST stands for sequence type) as in the isolates from their animals. CONCLUSIONS: This study shows a high prevalence of birds carrying ESBL/AmpC-producing E. coli at Dutch broiler farms and a high prevalence of ESBL/AmpC-producing E. coli in farmers. This is undesirable due to the risk this poses to human health. Future research should focus on identification of the source of these isolates in the broiler production chain to make interventions resulting in reduction of these isolates possible.

Increasing prevalence and diversity of ESBL/AmpC-type ß-lactamase genes in Escherichia coli isolated from veal calves from 1997 to 2010.

OBJECTIVES: Several studies on faecal carriage of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli have been performed in cattle, but little is known about faecal carriage in veal calves. This study describes the prevalence and molecular characteristics of ESBL/AmpC genes in E. coli isolated from faecal samples of veal calves from 1997 to 2010. METHODS: Pooled faecal samples were inoculated using selective enrichment broth and subsequently selective MacConkey agar. All isolates with reduced susceptibility to cefotaxime were screened by PCR and sequencing analysis for the presence of ESBL/AmpC genes. RESULTS: The prevalence of E. coli with reduced susceptibility to cefotaxime showed a discontinuous increasing trend, ranging from 4% in 1998 and 1999 to 39% in 2010. Promoter mutations of the chromosomal ampC gene were present in all years. In 2000, ESBL genes blaCTX-M-1, blaTEM-52 and blaTEM-20 were first observed. Before 2005 the majority of E. coli with reduced susceptibility to cefotaxime harboured ampC promoter mutations. From 2005 onwards the majority harboured blaCTX-M genes, of which blaCTX-M-1 was the most abundant, followed by blaCTX-M-14 and blaCTX-M-15. The diversity of blaCTX-M genes gradually increased from one variant in 2000 to six variants in 2010. The prevalence of blaTEM-52 was relatively low, but it was detected from 2000 onwards. blaCMY and blaSHV were found sporadically. CONCLUSIONS: The prevalence and molecular diversity of genes encoding cefotaxime resistance in E. coli isolated from veal calves over a 14 year period showed an increasing trend. From 2005 onwards, blaCTX-M genes were most abundant, especially blaCTX-M-1.

One Health surveillance-A cross-sectoral detection, characterization, and notification of foodborne pathogens.

Introduction: Several Proficiency Test (PT) or External Quality Assessment (EQA) schemes are currently available for assessing the ability of laboratories to detect and characterize enteropathogenic bacteria, but they are usually targeting one sector, covering either public health, food safety or animal health. In addition to sector-specific PTs/EQAs for detection, cross-sectoral panels would be useful for assessment of the capacity to detect and characterize foodborne pathogens in a One Health (OH) perspective and further improving food safety and interpretation of cross-sectoral surveillance data. The aims of the study were to assess the cross-sectoral capability of European public health, animal health and food safety laboratories to detect, characterize and notify findings of the foodborne pathogens Campylobacter spp., Salmonella spp. and Yersinia enterocolitica, and to develop recommendations for future cross-sectoral PTs and EQAs within OH. The PT/EQA scheme developed within this study consisted of a test panel of five samples, designed to represent a theoretical outbreak scenario. Methods: A total of 15 laboratories from animal health, public health and food safety sectors were enrolled in eight countries: Denmark, France, Italy, the Netherlands, Poland, Spain, Sweden, and the United Kingdom. The laboratories analyzed the samples according to the methods used in the laboratory and reported the target organisms at species level, and if applicable, serovar for Salmonella and bioserotype for Yersinia. Results: All 15 laboratories analyzed the samples for Salmonella, 13 for Campylobacter and 11 for Yersinia. Analytical errors were predominately false negative results. One sample (S. Stockholm and Y. enterocolitica O:3/BT4) with lower concentrations of target organisms was especially challenging, resulting in six out of seven false negative results. These findings were associated with laboratories using smaller sample sizes and not using enrichment methods. Detection of Salmonella was most commonly mandatory to notify within the three sectors in the eight countries participating in the pilot whereas findings of Campylobacter and Y. enterocolitica were notifiable from human samples, but less commonly from animal and food samples. Discussion: The results of the pilot PT/EQA conducted in this study confirmed the possibility to apply a cross-sectoral approach for assessment of the joint OH capacity to detect and characterize foodborne pathogens.

Characterization of multidrug-resistant, qnrB2-positive and extended-spectrum-beta-lactamase-producing Salmonella Concord and Salmonella Senftenberg isolates.

OBJECTIVES: To characterize plasmids and resistance genes of multidrug-resistant (MDR) Salmonella Senftenberg and Salmonella Concord isolated from patients in the Netherlands. METHODS: The resistance genes of four MDR Salmonella isolates (three Salmonella Concord and one Salmonella Senftenberg) were identified by miniaturized microarray, PCR and sequencing. Plasmids were characterized by S1 nuclease-PFGE and PCR-based replicon typing (PBRT). Linkage between plasmids and genes was determined by conjugation experiments and microarray analysis. The genetic relationship between the three Salmonella Concord isolates was determined by XbaI-PFGE. RESULTS: A large variety of resistance genes was detected, including qnrB2 and the beta-lactamase genes bla(TEM-1) and bla(SHV-12) in all isolates; moreover all Salmonella Concord isolates also harboured bla(CTX-M-15). Salmonella Senftenberg harboured a large IncHI2 plasmid. The three Salmonella Concord isolates harboured two large plasmids typed as IncHI2 and IncA/C. CONCLUSIONS: We detected the first plasmid-mediated MDR Salmonella isolates in the Netherlands harbouring both qnr and extended-spectrum beta-lactamase (ESBL) genes. In Salmonella Senftenberg one large plasmid (IncHI2) and in Salmonella Concord two large plasmids (IncHI2 and IncA/C) were responsible for the multidrug resistance.

Diversity of Plasmids and Genes Encoding Resistance to Extended Spectrum Cephalosporins in Commensal Escherichia coli From Dutch Livestock in 2007-2017.

Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin's. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing Escherichia coli isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) E. coli isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 E. coli isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with bla CTX-M-1 being the most prevalent gene in livestock (43.7%), followed by bla CMY -2 and bla SHV -12, independent of the animal source. Prevalence of typically human associated bla CTX-M-15 was highest in cattle. Less than 10% E. coli isolates owed their ESC-R phenotype to promoter mutations of the chromosomal ampC gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1α being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R E. coli isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.

Dynamics of cefotaxime resistant Escherichia coli in broilers in the first week of life.

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.

Occurrence and characterization of extended-spectrum cephalosporin-resistant Enterobacteriaceae in healthy household dogs in Greece.

Extended-spectrum cephalosporin- and/or carbapenem-resistant (ESCR and/or CarbR) Enterobacteriaceae constitute a public health hazard because of limited treatment options and are endemic among humans in Greece. Recently, ESCR and CarbREnterobacteriaceae have been increasingly isolated from companion animals, stressing their potential role as a reservoir for humans. However, the presence of ESCR bacteria in companion animals within Greek households has not been determined yet. Genes conferring the ESCR and CarbR phenotype were detected among canine isolates and their chromosomal or plasmid location was determined. Standard methods were applied for plasmid characterization. The clonal relatedness of the recovered isolates was examined by multilocus sequence typing (MLST). Here, we report the first findings on the presence of ESCREnterobacteriaceae in healthy Greek dogs. ESCREscherichia coli isolates were associated with different sequence types (STs), including the human pandemic ST131 clone. The occurrence of human-related ESBL/pAmpC genes, plasmid types and/or strain STS in this animal reservoir suggests possible bilateral transmission.

Dynamics of CMY-2 producing E. coli in a broiler parent flock.

Extended-spectrum ß-lactamase and plasmid mediated AmpC ß-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.