Colistin-resistant Enterobacterales among veterinary healthcare workers and in the Dutch population.

Objectives: Plasmid-mediated colistin resistance can be transferred from animals to humans. We investigated the prevalence of carriage of mcr-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae (ColR-E/K) in veterinary healthcare workers and in the general population in the Netherlands. Methods: Two cross-sectional population studies were performed: one among veterinary healthcare workers and one in the general population. Participants sent in a faecal sample and filled in a questionnaire. Samples were analysed using selective enrichment and culture. Mobile colistin resistance genes (mcr) were detected by PCR and ColR-E/K were sequenced using Illumina and Nanopore technologies. Results: The prevalence of mcr-mediated ColR-E/K was 0.2% (1/482, 95% CI 0.04%-1.17%) among veterinary personnel and 0.8% (5/660, 95% CI 0.3%-1.8%) in the population sample. mcr-1 was found in E. coli from four persons, mcr-8 in K. pneumoniae from one person and another person carried both mcr-1 and mcr-8 in a K. pneumoniae isolate. mcr-1 was found on different plasmid types (IncX4, IncI1 and IncI2), while mcr-8 was found on IncF plasmids only. Conclusions: mcr-mediated ColR-E/K resistance was uncommon in both populations. Professional contact with animals does not increase the chance of carriage of these bacteria in the Netherlands at present. mcr-8 was found for the first time in the Netherlands. Surveillance of colistin resistance and its underlying mechanisms in humans, livestock and food is important in order to identify emerging trends in time.

Extended-spectrum ß-lactamase- and pAmpC-producing Enterobacteriaceae among the general population in a livestock-dense area.

OBJECTIVES: In the Netherlands there is an ongoing debate regarding environmental health risks of livestock farming for neighbouring residents. This explorative study aims to determine the prevalence of carriage of extended-spectrum ß-lactamase and/or plasmid-mediated AmpC-producing Enterobacteriaceae (ESBL/pAmpC-E) in the general population living in a livestock-dense area, and to study associations between determinants, including exposure through contact with animals and the environment, and human carriage of ESBL/pAmpC-E. METHODS: A cross-sectional study was performed among 2432 adults (aged 20-72 years) in 12 temporary research centres in the south of the Netherlands, consisting of a questionnaire and analysis of a faecal sample to assess carriage of ESBL/pAmpC-E. Risk factors were analysed using logistic regression. RESULTS: The prevalence for carriage of ESBL/pAmpC-E was 4.5% (109/2432; 95% CI 3.7-5.4) ranging from 1.4% to 10.9% among the research centres. ESBL/pAmpC resistance genes were detected in Escherichia coli and Klebsiella pneumoniae isolates obtained from these 109 persons and the most common ESBL-resistance genes were blaCTX-M-15, blaCTX-M-14/17 and blaCTX-M-1, originating from 76 participants. Travel in the previous 12 months to Africa, Asia or Latin America (OR 2.82; 95% CI 1.71-4.63), having kept cows for a hobby in the previous 5 years (OR 3.77; 95% CI 1.22-11.64), usage of proton-pump inhibitors (OR 1.84; 95% CI 1.05-3.23), and living within 1000 m of a mink farm (OR 2.26; 95% CI 1.28-3.98) were identified as risk factors. Exposure to poultry was not identified as a risk factor. CONCLUSIONS: Overall, living in close proximity to livestock animals and farms does not seem to be a risk factor for carriage of ESBL/pAmpC-E.

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.

Quantifying the sources of Salmonella on dressed carcasses of pigs based on serovar distribution.

Salmonella serotyping data, qualitatively described by van Hoek et al. (2012), were used to quantify potential sources of Salmonella in a Dutch pig slaughterhouse. Statistical tests to compare per-day Salmonella prevalence and serotyping data from multiple points in the chain were used to find transmission pathways. A statistical model based on serotyping data was developed to attribute Salmonella on dressed carcasses to the most likely source. Approximately two-third of dressed carcasses carrying Salmonella on the medial surface had been contaminated by house flora. For carcasses carrying Salmonella on the distal surface, transient Salmonella from incoming pigs was a more important source. The relevance of the different sources of Salmonella varied within and between sampling days. Results were compared to those of another modeling approach, in which Salmonella concentration data from the same samples were used (Smid et al., 2012). They mostly agreed. The approach chosen by an individual slaughterhouse depends on the data that are collected.

Prevalence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in humans living in municipalities with high and low broiler density.

Prevalence of, and risk factors for, carriage of extended-spectrum ß-lactamase (ESBL) -producing Enterobacteriaceae were determined for 1025 Dutch adults in municipalities with either high or low broiler densities. Overall prevalence of ESBL carriage was 5.1%. The hypothesis that individuals in areas with high broiler densities are at greater risk for ESBL carriage was rejected, as the risk was lower (OR = 0.45; p 0.009) for these individuals. Owning a horse increased the risk (OR = 4.69; p ≤0.0001), but horse owners often owned multiple species of companion animals. Routes of transmission from animals to humans in the community, and the role of poultry in this process, remain to be elucidated.

Prevalence of livestock-associated MRSA on Dutch broiler farms and in people living and/or working on these farms.

This study aimed to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) on 50 Dutch broiler farms. Of 145 persons living and/or working on these farms, eight tested positive for MRSA (5.5%). Investigation of 250 pooled throat samples of broilers and 755 dust samples resulted in four farms where MRSA-positive samples were present (8.0%). All isolates belonged to the CC398 complex. Living and/or working on a MRSA-positive farm was a risk for MRSA carriage; 66.7% of people on positive farms were MRSA positive vs. 1.5% on negative farms (P<0.0001). Due to the low number of positive farms and persons, and high similarity in farm management, it was impossible to draw statistically valid conclusions on other risk factors. For broiler farming, both farm and human MRSA prevalence seem much lower than for pig or veal farming. However, MRSA carriage in people living and/or working on broiler farms is higher compared to the general human population in The Netherlands (5.5% vs. <0.1%). As broiler husbandry systems are not unique to The Netherlands, this might imply that people in contact with live broilers are at risk for MRSA carriage worldwide.

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

Antimicrobial susceptibility of Lactobacillus rhamnosus.

We aimed to determine the minimum inhibitory concentrations (MICs) of Lactobacillus rhamnosus (n=75) strains, to study their antibiotic resistance genes with microarray, and to assess the microbiological cut-off values of tested antimicrobial agents. L. rhamnosus strains were tested with agar dilution, broth microdilution and Etest methods for ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, and tetracycline using specific LSM medium. Most of the L. rhamnosus strains were found phenotypically susceptible to all six antibiotics tested. Four of the strains were phenotypically multiresistant, three strains to clindamycin, erythromycin and streptomycin and one strain to streptomycin and tetracycline. Some of the resistant (n=8) and susceptible (n=5) strains were further studied with a microarray method to reveal the antibiotic resistance genes behind the phenotypic resistances. From our experience, we recommend that microbiological cut-off values should be proposed according to the method used.