Wietske Dohmen

Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages

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.

Transmission through air as a possible route of exposure for MRSA

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.

Carriage of extended-spectrum β-lactamases in pig farmers is associated with occurrence in pigs

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.

Longitudinal Study of Extended-Spectrum-β-Lactamase- and AmpC-Producing Enterobacteriaceae in Household Dogs

ABSTRACT 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 non-wild-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 blaCTX-M, blaCMY, blaSHV, blaOXA, and blaTEM 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 blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaSHV-12, and blaCMY-2 were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 × 108 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.

Dose-response relationship between antimicrobial drugs and livestock-associated MRSA in pig farming.

Decreasing antimicrobial use can lower MRSA prevalence in pigs and subsequently in humans.

Air exposure as a possible route for ESBL in pig farmers

&NA; Livestock can carry extended‐spectrum beta‐lactamase (ESBL)‐producing Enterobacteriaceae, with blaCTX‐M‐1 being most prevalent. ESBL carriage in farmers is associated with ESBL carriage in animals, with direct animal‐human contact considered as the dominant route of transmission. However, inhalation of stable air might represent another route of transmission. We, therefore, quantified presence of blaCTX‐M group 1 genes (CTX‐M‐gr1) in dust and the association with CTX‐M‐gr1 carriage in pig farmers, family members and employees. We included 131 people living and/or working on 32 conventional Dutch pig production farms (farmers, family members and employees) during two sampling moments over a 12‐month interval. Human stool samples, rectal swabs from 60 pigs per farm, and 2–5 dust samples collected using an electrostatic dust collector (EDC) (as a proxy for presence of viable CTX‐M‐gr1 carrying bacteria in air) were obtained per farm. Presence of ESBL‐producing Escherichia Coli (E. coli) in stool samples and rectal swabs was determined by selective plating and CTX‐M‐gr1 was identified by PCR. Dust samples were analyzed directly by PCR for presence of CTX‐M‐gr1. Questionnaires were used to collect information on nature, intensity and duration of animal contact. Overall human prevalence of CTX‐M‐gr1 carriage was 3.6%. CTX‐M‐gr1 was detected in dust on 26% of the farms and in pigs on 35% of the farms, on at least one sampling moment. Human CTX‐M‐gr1 carriage and presence of CTX‐M‐gr1 in dust were associated univariately (OR=12.4, 95% CI=2.7–57.1). In multivariate analysis human CTX‐M‐gr1 carriage was associated with the number of working hours per week (OR=1.03, 95% CI=1.00–1.06), presence of CTX‐M‐gr1 carrying pigs on the farm (OR=7.4, 95% CI=1.1–49.7) and presence of CTX‐M‐gr1 in dust (OR=3.5, 95% CI=0.6–20.9). These results leave open the possibility of airborne CTX‐M‐gr1 transmission from animals to humans next to direct contact. HighlightsCTX‐M‐gr1 carriage in pig farmers and presence of CTX‐M‐gr1 in dust are associated.The number of CTX‐M‐gr1 positive human, pig and dust samples decreased over time.Air transmission of CTX‐M‐gr1 might be possible on pig farms.

Risk factors for ESBL-producing Escherichia coli on pig farms : A longitudinal study in the context of reduced use of antimicrobials

The presence of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) in food animals is a public health concern. This study aimed to determine prevalence of ESBL-E. coli on pig farms and to assess the effect of reducing veterinary antimicrobial use (AMU) and farm management practices on ESBL-E. coli occurrence on pig farms. During 2011–2013, 36 Dutch conventional pig farms participated in a longitudinal study (4 sampling times in 18 months). Rectal swabs were taken from 60 pigs per farm and pooled per 6 pigs within the same age category. Presence of ESBL-E. coli was determined by selective plating and ESBL genes were characterized by microarray, PCR and gene sequencing. An extensive questionnaire on farm characteristics and AMU as Defined Daily Dosages per Animal Year (DDDA/Y) was available for the 6-month periods before each sampling moment. Associations between the presence of ESBL-E. coli-positive pigs and farm management practices were modelled with logistic regression. The number of farms with ESBL-E. coli carrying pigs decreased from 16 to 10 and the prevalence of ESBL-E. coli-positive pooled pig samples halved from 27% to 13%. Overall, the most detected ESBL genes were blaCTX-M-1, blaTEM-52 and blaCTX-M-14. The presence of ESBL-E. coli carrying pigs was not related to total AMU, but it was strongly determined by the presence or absence of cephalosporin use at the farm (OR = 46.4, p = 0.006). Other farm management factors, related with improved biosecurity, were also plausibly related to lower probabilities for ESBL-E. coli-positive farms (e.g. presence of a hygiene lock, pest control delivered by a professional). In conclusion, ESBL-E. coli prevalence decreased in pigs during 2011 and 2013 in the Netherlands. On pig farms, the use of cephalosporins was associated with the presence of ESBL-E. coli carrying pigs.

346 Antimicrobial resistant bacteria in pig farmers in the Netherlands − a study on ESBLs

Objectives Farm animals may serve as a reservoir for (multi)resistant bacteria, such as extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. Animal to man transmission may occur through (in)direct contact during work, which may thus pose an occupational health hazard. In humans, infections with ESBL producing Enterobacteriaceae are associated with high mortality, morbidity and costs. We investigated the prevalence of carriage with ESBL producing Enterobacteriaceae in pig farmers, their family members, and their employees and associations between presence of ESBLs among animals and humans. Methods Rectal swabs were taken from pigs on 40 Dutch conventional pig farms (60 per farm) and stool samples were obtained from 142 humans living and/or working on 34 of these farms (farmers, family members and employees). Sampling was repeated after 6 months. Presence of ESBL-producing bacteria was determined by selective plating and ESBL genes were analysed by microarray analysis and gene sequencing. Questionnaires were used to determine antimicrobial use, hygiene, contact with animals and/or meat, and other relevant determinants. Results ESBL genes, mostly CTX-M-1, TEM-52 en CTX-M-14, were determined in pig isolates on 17 farms (43%) and in isolates from 8 participants (6%). ESBL genes determined in farmers corresponded to those detected in pigs on their farm. ESBL carriage was more likely in farmers working on ESBL positive farms (OR > 10). After 6 months ESBL genes were determined in isolates from 8 farmers (6%). Only 2 of these farmers carried ESBL genes in both stool samples obtained with a 6 months interval. Conclusions We found a strong association between ESBL carriage in farmers and ESBL occurrence on the farm. Repeated sampling indicates that ESBL carriage is not persistent in this human study population.