Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Livestock may serve as a reservoir for extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE). The objectives of this study were to determine the prevalence of carriage with ESBL-PE in pig farmers, family members and employees, and its association with carriage in pigs. Rectal swabs were taken from 2388 pigs (398 pooled samples) on 40 pig farms and faecal samples were obtained from 142 humans living or working on 34 of these farms. Presence of ESBL-PE was determined by selective plating (agar). ESBL genes were analysed by PCR or microarray analysis, and gene sequencing. Genotypes and plasmids were determined by multilocus sequence typing and PCR-based replicon typing for selected isolates. ESBL genes were detected in Escherichia coli from eight humans (6%) (blaCTX-M-1, n = 6; blaTEM-52, n = 1 and blaCTX-M-14, n = 1) on six farms. In 157 pig isolates (107 pooled samples) on 18 farms (45%) ESBL genes were detected (blaCTX-M-1, n = 12; blaTEM-52, n = 6; and blaCTX-M-14, n = 3). Human and pig isolates within the same farm harboured similar ESBL gene types and had identical sequence and plasmid types on two farms (e.g. E. coli ST-453, blaCTX-M-1, IncI1), suggesting clonal transmission. For the remaining farms, sequence types, but not plasmid types, differed. Human ESBL carriage was associated with average number of hours working on the farm per week (OR = 1.04, 95% CI 1.02-1.06) and presence of ESBLs in pigs (OR = 12.5, 95% CI 1.4-111.7). Daily exposure to pigs carrying ESBL-PE is associated with ESBL carriage in humans.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is highly prevalent in pigs and veal calves. The environment and air in pig and veal calf barns is often contaminated with LA-MRSA, and can act as a transmission source for humans. This study explores exposure-response relationships between sequence type 398 (ST398) MRSA air exposure level and nasal ST398 MRSA carriage in people working and/or living on farms. Samples and data were used from three longitudinal field studies in pig and veal calf farm populations. Samples consisted of nasal swabs from the human participants and electrostatic dust fall collectors capturing airborne settled dust in barns. In both multivariate and mutually adjusted analyses, a strong association was found between nasal ST398 MRSA carriage in people working in the barns for >20 h per week and MRSA air levels. In people working in the barns < 20 h per week there was a strong association between nasal carriage and number of working hours. Exposure to ST398 MRSA in barn air seems to be an important determinant for nasal carriage, especially in the highly exposed group of farmers, next to duration of contact with animals. Intervention measures should therefore probably also target reduction of ST398 MRSA air levels.

A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae in feces. Fecal samples from 38 dogs were collected monthly for 6 months. Additional samples were collected from 7 included dogs on a weekly basis for 6 weeks. Numbers of CFU per gram of feces for nonwild-type Enterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for total Enterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence of bla CTX-M , bla CMY , bla SHV , bla OXA , and bla TEM gene families. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. PCR-negative isolates were tested by a double-disk synergy test for enhanced AmpC expression. A total of 259 samples were screened, and 126 samples were culture positive on MCC, resulting in 352 isolates, 327 of which were Escherichia coli. Nine dogs were continuously positive during this study, and 6 dogs were continuously negative. Monthly or weekly shifts in fecal shedding were observed for 23 dogs. Genotyping showed a large variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. The ESBL genes bla CTX-M-1 , bla CTX-M-14 , bla CTX-M-15 , bla SHV-12 , and bla CMY-2 were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 ؋ 10 8 CFU/g feces. This study showed an abundance of ESBL-producing Enterobacteriaceae in dogs in the Netherlands, mostly in high concentrations. Fecal shedding was shown to be highly dynamic over time, which is important to consider when studying ESBL epidemiology.A substantial share of the present global emergence of antimicrobial resistance is represented by extended-spectrumbeta-lactamase (ESBL)-producing Enterobacteriaceae. So far, these bacteria have been isolated from a large variety of sources, including humans, animals, and the environment. Together, these sources seem to form a complex network of reservoirs and transmission routes where ESBL-producing Enterobacteriaceae are circulating (1).High prevalences of ESBL-producing Enterobacteriaceae were found in Dutch poultry, pigs, and cattle (2). High prevalences of ESBL-producing Enterobacteriaceae were also found in Dutch companion animals, i.e., 45% in dogs without clinical signs and 55% in diarrheic dogs (3). As companion animals live in close contact with humans, they might contribute substantially to the exposure of humans to ESBL-producing Enterobacteriaceae. Similar ESBL gene types, i.e., CTX-M-14, CTX-M-15, SHV-12, and CMY-2, were found in strains originating from humans and companion animals (1). Additionally, transmission of CTX-M-15-carrying ST131 and ST648 Escherichia coli stra...