Faecal carriage, risk factors, acquisition and persistence of ESBL-producing Enterobacteriaceae in dogs and cats and co-carriage with humans belonging to the same household.

BACKGROUND: ESBL-producing Enterobacteriaceae (ESBL-E) are observed in many reservoirs. Pets might play an important role in the dissemination of ESBL-E to humans since they live closely together. OBJECTIVES: To identify prevalence, risk factors, molecular characteristics, persistence and acquisition of ESBL-E in dogs and cats, and co-carriage in human-pet pairs belonging to the same household. METHODS: In a nationwide study, one person per household was randomly invited to complete a questionnaire and to submit a faecal sample. Dog and cat owners were invited to also submit a faecal sample from their pet. Repeated sampling after 1 and 6 months was performed in a subset. ESBL-E were obtained through selective culture and characterized by WGS. Logistic regression analyses and random forest models were performed to identify risk factors. RESULTS: The prevalence of ESBL-E carriage in these cohorts was 3.8% (95% CI: 2.7%-5.4%) for human participants (n=550), 10.7% (95% CI: 8.3%-13.7%) for dogs (n=555) and 1.4% (95% CI: 0.5%-3.8%) for cats (n=285). Among animals, blaCTX-M-1 was most abundant, followed by blaCTX-M-15. In dogs, persistence of carriage was 57.1% at 1 month and 42.9% at 6 months. Eating raw meat [OR: 8.8, 95% CI: 4.7-16.4; population attributable risk (PAR): 46.5%, 95% CI: 41.3%-49.3%] and dry food (OR: 0.2, 95% CI: 0.1-0.5; PAR: 56.5%, 95% CI: 33.2%-66.6%) were predictors for ESBL-E carriage in dogs. Human-dog co-carriage was demonstrated in five households. Human-cat co-carriage was not observed. CONCLUSIONS: ESBL-E prevalence was higher in dogs than in humans and lowest in cats. The main risk factor for ESBL-E carriage was eating raw meat. Co-carriage in dogs and household members was uncommon.

Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals: a systematic review.

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.

Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae.

Bacterial antimicrobial resistance (AMR) is constantly evolving and horizontal gene transfer through plasmids plays a major role. The identification of plasmid characteristics and their association with different bacterial hosts provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of AMR determinants. Molecular identification of plasmid and strain genotypes elicits a distinction between spread of AMR genes by plasmids and dissemination of these genes by spread of bacterial clones. For this reason several methods are used to type the plasmids, e.g. PCR-based replicon typing (PBRT) or relaxase typing. Currently, there are 28 known plasmid types in Enterobacteriaceae distinguished by PBRT. Frequently reported plasmids [IncF, IncI, IncA/C, IncL (previously designated IncL/M), IncN and IncH] are the ones that bear the greatest variety of resistance genes. The purpose of this review is to provide an overview of all known AMR-related plasmid families in Enterobacteriaceae, the resistance genes they carry and their geographical distribution.

Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut.

Animal health benefits from a stable intestinal homeostasis, for which proper development and functioning of the intestinal microbiota and immune system are essential. It has been established that changes in microbial colonization in early life (the first 2 wk post hatch) impacts the functioning of the adult gut and the associated crosstalk between microbiota and intestinal mucosal cells. The aim of the present study was to study the effect of the administration of antibiotics later in life (d 15 to 20 post hatch) on microbiota and immune parameters. For this purpose, chickens received from 15 d post hatch during 5 d amoxicillin or enrofloxacin through their drinking water. Before and at 6, 16, and 27 d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. At 6 d after the start of the administration of the antibiotics, the composition and diversity of the microbiota were affected significantly (P < 0.05), but this change was small and observed only temporarily since differences disappeared at 16 d after initiating treatment with amoxillin and at 27 d after starting treatment with enrofloxacin. Intestinal morphology and development were not visibly affected since there were no differences between villus/crypt ratios and numbers of PAS+ and PCNA+ cells in the duodenum and jejunum at any time point. At 16 d after the start of antibiotic administration, the number of CD4+ T-cells and CD8+ T-cells in the duodenum was lower compared to the control animals; however, this difference was not significant. At some time points, significant differences (P < 0.05) were observed among the groups to locally expressed IL-8, IL-1ß, IFN-γ, IL-2, and IL-4 mRNA. However, this effect was not long lasting, as differences that were observed at 16 d after starting the treatment had disappeared at 27 d after treatment was started. The results of this study indicate that later in the broiler's life, antibiotics only temporarily affect intestinal microbial and immune parameters.

ESBL carriage in pig slaughterhouse workers is associated with occupational exposure.

We investigated the prevalence of extended-spectrum ß-lactamase (ESBL) carriage in slaughterhouse workers and the association with occupational exposure to slaughter animals and products. Stool samples from 334 employees in a Dutch pig slaughterhouse were obtained. Presence of ESBL was determined by selective plating, microarray analysis, and gene sequencing. Questionnaires were used to collect personal and occupational information. The overall prevalence of ESBL carriage was 4·8% (16/334). All ESBL-producing isolates were Escherichia coli. The ESBL genes detected were bla CTX-M-1 (n = 8), bla CTX-M-15 (n = 3), bla CTX-M-27 (n = 2), bla CTX-M-24 (n = 1), bla CTX-M-55 (n = 1), and bla SHV-12 (n = 1). A higher prevalence of ESBL was seen in workers in jobs with as tasks 'removal of lungs, heart, liver, tongue' (33%), and 'removal of head and spinal cord' (25%). For further analysis, participants were divided in two groups based on potential exposure to ESBL as related to their job title. One group with an assumed higher exposure to ESBL (e.g. stable work, stabbing, dehairing, removal of organs) and another group with an assumed lower exposure to ESBL (e.g. refrigeration, packaging and expedition). In the 'higher exposure' group, ten out of 95 (10·5%) were carrying ESBL vs. six out of 233 (2·6%) in the 'lower exposure' group. Human ESBL carriage was significantly associated with job exposure in the slaughterhouse (OR 4·5, CI 1·6-12·6). Results suggest that ESBL carriage in slaughterhouse workers overall is comparable with the Dutch population. Within the slaughterhouse population a difference in carriage exists depending on their position along the slaughter line and tasks involved.

ESBL/AmpC-producing Enterobacteriaceae in households with children of preschool age: prevalence, risk factors and co-carriage.

OBJECTIVES: ESBL/AmpC-producing Enterobacteriaceae are an emerging public health concern. As households with preschool children may substantially contribute to the community burden of antimicrobial resistance, we determined the prevalence, risk factors and co-carriage of ESBL/AmpC-producing bacteria in preschool children and their parents. METHODS: From April 2013 to January 2015, each month 2000 preschool children were randomly selected from Dutch population registries. The parents were invited to complete an epidemiological questionnaire and to obtain and send a faecal sample from the selected child and from one parent. Samples were tested for ESBL/AmpC-producing bacteria. Logistic regression was used to identify risk factors for ESBL/AmpC carriage in children and parents, and findings were internally validated by bootstrapping. RESULTS: In total, 1016 families were included and ESBL/AmpC prevalence was 4.0% (95% CI 3.2%-5.0%); 3.5% (95% CI 2.5%-4.8%) in children and 4.5% (95% CI 3.4%-6.0%) in parents. Attending a daycare centre (DCC) was the only significant risk factor for children (OR 2.1, 95% CI 1.0-4.3). For parents, the only significant risk factor was having one or more children attending DCCs (OR 2.2, 95% CI 1.2-4.8). For parents of ESBL/AmpC-positive children the OR for ESBL/AmpC carriage was 19.7 (95% CI 9.2-42.4). Co-carriage of specific ESBL/AmpC genotypes in child and parent occurred more often than expected by chance (14.6% versus 1.1%, P < 0.001). CONCLUSIONS: In this study, intestinal carriage with ESBL/AmpCs was detected in ∼4% of households with preschool children. DCC attendance was a risk factor in both children and parents and co-carriage of specific genotypes frequently occurred in child-parent pairs. These findings suggest household transmission or/and family-specific exposure to common sources of ESBL/AmpC-producing bacteria.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee.

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

Reduction of veterinary antimicrobial use in the Netherlands. The Dutch success model.

Use of antimicrobials in animals poses a potential risk for public health as it contributes to the selection and spread of antimicrobial resistance. Although knowledge of the negative consequences of extensive antimicrobial use in humans and animals accumulated over the decades, total therapeutic antimicrobial use in farm animals in the Netherlands doubled between 1990 and 2007. A series of facts and events formed a window of opportunity to reduce antimicrobial use in farm animals. The recent discovery of significant reservoirs of antimicrobial-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase-producing bacteria (ESBL) in farm animals, with potential public health implications, combined with an increasing lack of confidence of the public in intensive livestock industries, and discrepancy between the very low antimicrobial use in humans and high use in animals, resulted in intensive collaboration between the government, veterinary professional organizations and important stakeholders within the livestock sector. A combination of compulsory and voluntary actions with clear reduction goals resulted in a 56% reduction in antimicrobial use in farm animals in the Netherlands between 2007 and 2012 and aims at accomplishing a 70% reduction target in 2015. This article describes and analyses the processes and actions behind this transition from an abundant antimicrobial use in farm animals towards a more prudent application of antimicrobials in farm animals in the Netherlands.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: prevalence, risk factors and molecular characteristics.

OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK.

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005-2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. ß-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 ß-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

Prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in organic pig herds in The Netherlands.

The prevalence of the methicillin-resistant Staphylococcus aureus (MRSA) among conventional pig herds in the Netherlands is high (around 71%). Nevertheless, information about the prevalence of MRSA among organic pig herds is lacking. Here, we report a study on 24 of the 49 organic pig herds in the Netherlands. The prevalence of MRSA positive herds showed to be 21%. The genetic characteristics of the MRSA isolates were similar to MRSA CC398 described in conventional pigs except one exceptional HA-MRSA CC30 found in one herd, which was presumably caused by human to animal transmission. This resulted in a prevalence of MRSA CC398 in the organic herds of 16.7%.

Antimicrobial resistance in commensal Escherichia coli in veal calves is associated with antimicrobial drug use.

The aim of this study was to determine the association between farm management factors, including antimicrobial drug usage, and resistance in commensal Escherichia coli isolates from the faeces of white veal calves. Ninety E. coli isolates from one pooled sample per farm (n = 48) were tested for their phenotypical resistance against amoxicillin, tetracycline, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole (TMP/SMX). Logistic regression analysis revealed the following risk factors (P < 0·05); farmer wearing the same work clothes for several days [ciprofloxacin, odds ratio (OR) 2·6; tetracycline, OR 2·4], administration of trimethoprim-sulfonamide combinations (TMP/SMX, OR 3·0; amoxicillin, OR 3·1; tetracycline, OR 2·6), ⩾0·3 animal daily dosage per production cycle (ADD/pc), quinolones (ciprofloxacin, OR 2·8), ⩾1·3 ADD/pc, penicillins (ciprofloxacin, OR 3·3; tetracycline, OR 3·4), 20-40 ADD/pc, tetracyclines (tetracycline, OR 3·2) and >40 ADD/pc, tetracyclines (tetracycline, OR 13·1; amoxicillin, OR 6·5). In this study antimicrobial resistance in commensal E. coli was mainly associated with antimicrobial drug use.

Complete sequence of pSAM7, an IncX4 plasmid carrying a novel blaCTX-M-14b transposition unit isolated from Escherichia coli and Enterobacter cloacae from cattle.

The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.

Decreased ciprofloxacin susceptibility in Salmonella Typhi and Paratyphi infections in ill-returned travellers: the impact on clinical outcome and future treatment options.

The emergence of decreased ciprofloxacin susceptibility (DCS) in Salmonella enterica serovar Typhi and serovar Paratyphi A, B or C limits treatment options. We studied the impact of DCS isolates on the fate of travellers returning with enteric fever and possible alternative treatment options. We evaluated the clinical features, susceptibility data and efficacy of empirical treatment in patients with positive blood cultures of a DCS isolate compared to patients infected with a ciprofloxacin-susceptible (CS) isolate in the period from January 2002 to August 2008. In addition, the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin, levofloxacin and gatifloxacin were determined to assess if increasing the dose would result in adequate unbound fraction of the drug 24-h area under the concentration-time curve/minimum inhibitory concentration (ƒAUC(0-24)/MIC) ratio. Patients with DCS more often returned from the Indian subcontinent and had a longer fever clearance time and length of hospital stay compared to patients in whom the initial empirical therapy was adequate. The mean ƒAUC(0-24)/MIC was 41.3 ± 18.8 in the patients with DCS and 585.4 ± 219 in patients with a CS isolate. For DCS isolates, the mean ƒAUC0-24/MIC for levofloxacin was 60.5 ± 28.7 and for gatifloxacin, it was 97.9 ± 28.0. Increasing the dose to an adequate ƒAUC(0-24)/MIC ratio will lead to conceivably toxic drug levels in 50% of the patients treated with ciprofloxacin. Emerging DCS isolates has led to the failure of empirical treatment in ill-returned travellers. We demonstrated that, in some cases, an adequate ƒAUC(0-24)/MIC ratio could be achieved by increasing the dose of ciprofloxacin or by the use of alternative fluoroquinolones.

Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales.

OBJECTIVES: To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. METHODS: UK CTX-M-14-producing E. coli (n=70) from cattle (n=33), turkeys (n=9), sheep (n=2) and humans (n=26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of the bla(CTX-M-14) gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence. RESULTS: The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 isolates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxigenic E. coli ('VTEC'); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of the bla(CTX-M-14) gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n=9), turkeys (n=8) and humans (n=4). All pCT-like plasmids were conjugative, and most were IncK (n=21) and had the same local genetic environment flanking the bla(CTX-M-14) gene (n=23). RFLP analysis demonstrated ≥ 75% similarity among most plasmids (n=22). CONCLUSIONS: pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla(CTX-M-14) genes to different E. coli isolates from humans, cattle and turkeys.

Occurrence and characteristics of extended-spectrum-ß-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses.

OBJECTIVES: To investigate the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. METHODS: Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤ 25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. RESULTS: Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were bla(CTX-M-1, -2, -9, -14, -15,) bla(TEM-52), bla(CMY-2) and bla(CMY-)(39). bla(CTX-M-1) was predominant (n = 17). bla(CTX-M-9) occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying bla(CTX-M-1) belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. CONCLUSIONS: This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates.

The presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates.

Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains.

Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine milk samples.

The aim of this study was to examine whether antimicrobial resistance profiles of coagulase-negative Staphylococcus (CNS) species isolated from milk of dairy cows differed between bacterial species, and to compare results obtained by phenotypic and genotypic profiling of resistance to penicillin, oxacillin and macrolide-lincosamide (ML) antibiotics. Of 170 CNS isolates, 83 (48.8%) were phenotypically susceptible to all antimicrobial agents tested in minimum inhibitory concentration (MIC) assays, 40.6% expressed resistance to a single compound or a single class of compounds, and 10.6% to multiple drug classes. Nine percent, 68%, 19%, 4% and 1% of isolates were negative for all resistance genes tested by PCR or positive for one, two, three or four resistance genes, respectively. Phenotypic resistance and detection of resistance genes other than blaZ were relatively rare in Staphylococcus chromogenes, which was the most common CNS species (36% of 170 genotypically identified isolates). In Staphylococcus epidermidis, which was the second most common CNS species (14% of isolates), phenotypic penicillin resistance was significantly more common than in other CNS species. Almost half of the S. epidermidis isolates carried multiple resistance genes and 30% carried the methicillin resistance gene mecA. Survival analysis using MIC values showed significant associations between phenotypic and genotypic resistance profiles. We conclude that CNS species from bovine milk differ significantly in phenotypic and genotypic antimicrobial resistance profiles, which has implications for treatment and management decisions.