Characterization of multidrug-resistant, qnrB2-positive and extended-spectrum-b-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 b-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 b-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

Comparative Analysis of ESBL-Positive Escherichia coli isolates from Animals and Humans from the UK, the Netherlands and Germany

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans

Attributable sources of community-acquired carriage of Escherichia coli containing β-lactam antibiotic resistance genes: a population-based modelling study

Background: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), plasmid-mediated AmpC-producing E coli (pAmpC-EC), and other bacteria are resistant to important β-lactam antibiotics. ESBL-EC and pAmpC-EC are increasingly reported in animals, food, the environment, and community-acquired and health-care-associated human infections. These infections are usually preceded by asymptomatic carriage, for which attributions to animal, food, environmental, and human sources remain unquantified. Methods: In this population-based modelling study, we collected ESBL and pAmpC gene data on the Netherlands population for 2005–17 from published datasets of gene occurrences in E coli isolates from different sources, and from partners of the ESBL Attribution Consortium and the Dutch National Antimicrobial Surveillance System. Using these data, we applied an established source attribution model based on ESBL-EC and pAmpC-EC prevalence and gene data for humans, including high-risk populations (ie, returning travellers, clinical patients, farmers), farm and companion animals, food, surface freshwater, and wild birds, and human exposure data, to quantify the overall and gene-specific attributable sources of community-acquired ESBL-EC and pAmpC-EC intestinal carriage. We also used a simple transmission model to determine the basic reproduction number (R0) in the open community. Findings: We identified 1220 occurrences of ESBL-EC and pAmpC-EC genes in humans, of which 478 were in clinical patients, 454 were from asymptomatic carriers in the open community, 103 were in poultry and pig farmers, and 185 were in people who had travelled out of the region. We also identified 6275 occurrences in non-human sources, including 479 in companion animals, 4026 in farm animals, 66 in wild birds, 1430 from food products, and 274 from surface freshwater. Most community-acquired ESBL-EC and pAmpC-EC carriage was attributed to human-to-human transmission within or between households in the open community (60·1%, 95% credible interval 40·0–73·5), and to secondary transmission from high-risk groups (6·9%, 4·1–9·2). Food accounted for 18·9% (7·0–38·3) of carriage, companion animals for 7·9% (1·4–19·9), farm animals (non-occupational contact) for 3·6% (0·6–9·9), and swimming in freshwater and wild birds (ie, environmental contact) for 2·6% (0·2–8·7). We derived an R0 of 0·63 (95% CI 0·42–0·77) for intracommunity transmission. Interpretation: Although humans are the main source of community-acquired ESBL-EC and pAmpC-EC carriage, the attributable non-human sources underpin the need for longitudinal studies and continuous monitoring, because intracommunity ESBL-EC and pAmpC-EC spread alone is unlikely to be self-maintaining without transmission to and from non-human sources. Funding: 1Health4Food, Dutch Ministry of Economic Affairs, and the EU's Horizon-2020 through One-Health European Joint Programme.</p

Samenvatting ESBL-Attributieanalyse (ESBLAT) : Op zoek naar de bronnen van antibioticaresistentie bij de mens

Samenvatting over een project rondom ESBL's. Doelstellingen waren: vaststellen wat de bijdrage is van alle ESBL-reservoirs aan het dragerschap en infecties bij mensen en vaststellen wat de transmissieroutes zijn vanuit deze reservoirs naar de (geïnfecteerde) mens

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses

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 10(2) and 10(5)CFU/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

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.</p

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.</p

High prevalence of fecal carriage of extended spectrum β-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs

Extended-spectrum-β-lactamase (ESBL)/AmpC producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing animals, companion animals, and environmental sources. However, data on prevalence of fecal carriage of ESBL/AmpC producing Enterobacteriaceae in healthy companion animals is limited. This pilot study describes the prevalence of ESBL/AmpC encoding genes in healthy cats and dogs, and cats and dogs with diarrhea. Twenty fecal samples of each group were cultured on MacConkey agar supplemented with 1 mg/L cefotaxime and in LB-enrichment broth supplemented with 1 mg/L cefotaxime, which was subsequently inoculated on MacConkey agar supplemented with 1 mg/L cefotaxime. ESBL/AmpC genes were identified using the Check-Points CT103 micro array kit and subsequently by sequencing analysis. Chromosomal ampC promoter mutations were detected by PCR and sequencing analysis. From the healthy and diarrheic dogs, respectively 45 and 55% were positive for Escherichia coli with reduced susceptibility for cefotaxime. From the healthy and diarrheic cats, the estimated prevalence was respectively 0 and 25%. One diarrheic cat was positive for both reduced susceptible E. coli and Proteus mirabilis. The ESBL/AmpC genes found in this study were mainly bla CTX-M-1, but also bla CTX-M-14, bla CTX-M-15, bla TEM-52-StPaul, bla SHV-12, and bla CMY-2 were detected. This pilot study showed that the prevalence of ESBL/AmpC producing Enterobacteriaceae in healthy and diarrheic dogs, and diarrheic cats was relatively high. Furthermore, the genes found were similar to those found in isolates of both human and food-producing animal origin. However, since the size of this study was relatively small, extrapolation of the data to the general population of cats and dogs should be done with great care