Longitudinal Study of Dynamic Epidemiology of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Pigs and Humans Living and/or Working on Pig Farms

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales have been increasingly isolated from pigs, highlighting their potential for transmission to humans living and/or working within pig farms. As longitudinal data on the prevalence and the molecular characteristics of such isolates from the high-risk farming population remain scarce, we performed a long-term study on 39 Dutch pig farms. Fecal samples from pigs, farmers, family members, and employees were collected during four sampling occasions with a 6-month period. The presence of ESBL-producing Enterobacterales and their molecular characteristics (ESBL gene, plasmid, and sequence types) were determined by standard methods. Data on personal and farm characteristics were collected using questionnaires. ESBL-producing Escherichia coli was present in pigs at least once for 18 of 39 farms and in 17 of 146 farmers, family members, and/or employees. Among these 417 E. coli isolates, bla CTX-M-1 was the most frequently observed ESBL gene in pigs ( n  = 261) and humans ( n  = 25). Despite the great variety in plasmid (sub)types and E. coli sequence types (STs), we observed genetic similarity between human- and pig-derived isolates in (i) ESBL gene, plasmid (sub)type, and ST, suggesting potential clonal transmission in seven farms, and (ii) only ESBL gene and plasmid (sub)type, highlighting the possibility of horizontal transfer in four farms. Five pig farmers carried ESBL producers repeatedly, of whom two carried an identical combination of gene, plasmid (sub)type, and ST over time. Human ESBL carriage was associated with both presence of ESBL producers in pigs and average number of hours working on the pig farm per week, while prolonged human carriage was observed only incidentally. IMPORTANCE Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a public health hazard due to reduced therapeutic options for the treatment of infections. Although direct contact with pigs is considered a risk factor for human ESBL-producing E. coli carriage through occupational exposure, nationwide data regarding the occurrence of such isolates among pigs and humans living and/or working on farms remain scarce. Therefore, we determined (i) the longitudinal dynamics in prevalence and molecular characteristics of ESBL-producing E. coli in Dutch pig farmers and their pigs over time and (ii) the potential transmission events between these reservoirs based on genetic relatedness and epidemiological associations in longitudinal data. Our data suggesting the possibility of clonal and horizontal dissemination of ESBL-producing Escherichia coli between pigs and pig farmers can be used to inform targeted intervention strategies to decrease the within-farm human exposure to ESBL-producing E. coli

The shufflon of IncI1 plasmids is rearranged constantly during different growth conditions

One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifies the pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur in in vitro conditions, little is known about the regulation and the extent of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflon region and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon and allow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and after addition of cefotaxime. Furthermore, analysis was performed in different E. coli host cells to determine if recombination is likely to be influenced. Recombination of the shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at which novel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to the conclusion that the shufflon is continuously recombining at a constant rate, or that the method used here was not sensitive enough to detect differences in this rate. For one of the plasmids, the host cell appeared to have an effect on the specific shufflon variants that were formed which were not predominant in another host, indicating that host factors may be involved. As previously reported, the pilV-A and pilV-A' ORFs are formed at higher frequencies than other pilV ORFs. These results demonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by these in vitro conditions could not be revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.</p

Genomic Characterization of Extended-Spectrum Cephalosporin-Resistant Salmonella enterica in the Colombian Poultry Chain

Salmonella enterica serovars have been isolated from Colombian broilers and broiler meat. The aim of this study was to investigate the diversity of ESBL/pAmpC genes in extended-spectrum cephalosporin resistant Salmonella enterica and the phylogeny of ESBL/pAmpC-carrying Salmonella using Whole Genome Sequencing (WGS). A total of 260 cefotaxime resistant Salmonella isolates, obtained between 2008 and 2013 from broiler farms, slaughterhouses and retail, were included. Isolates were screened by PCR for ESBL/pAmpC genes. Gene and plasmid subtyping and strain Multi Locus Sequence Typing was performed in silico for a selection of fully sequenced isolates. Core-genome-based analyses were performed per ST encountered. blaCMY−2−like was carried in 168 isolates, 52 carried blaCTX−M−2 group, 7 blaSHV, 5 a combination of blaCMY−2−like-blaSHV and 3 a combination of blaCMY−2−like-blaCTX−M−2 group. In 25 isolates no ESBL/pAmpC genes that were screened for were found. WGS characterization of 36 selected strains showed plasmid-encoded blaCMY−2 in 21, blaCTX−M−165 in 11 and blaSHV−12 in 7 strains. These genes were mostly carried on IncI1/ST12, IncQ1, and IncI1/ST231 plasmids, respectively. Finally, 17 strains belonged to S. Heidelberg ST15, 16 to S. Paratyphi B variant Java ST28, 1 to S. Enteritidis ST11, 1 to S. Kentucky ST152 and 1 to S. Albany ST292. Phylogenetic comparisons with publicly available genomes showed separate clustering of Colombian S. Heidelberg and S. Paratyphi B var. Java. In conclusion, resistance to extended-spectrum cephalosporins in Salmonella from Colombian poultry is mainly encoded by blaCMY−2 and blaCTX−M−165 genes. These genes are mostly associated with IncI1/ST12 and IncQ1 plasmids, respectively. Evolutionary divergence is observed between Colombian S. Heidelberg and S. Paratyphi B var. Java and those from other countries

Farm dust resistomes and bacterial microbiomes in European poultry and pig farms

Background: Livestock farms are a reservoir of antimicrobial resistant bacteria from feces. Airborne dust-bound bacteria can spread across the barn and to the outdoor environment. Therefore, exposure to farm dust may be of concern for animals, farmers and neighboring residents. Although dust is a potential route of transmission, little is known about the resistome and bacterial microbiome of farm dust. Objectives: We describe the resistome and bacterial microbiome of pig and poultry farm dust and their relation with animal feces resistomes and bacterial microbiomes, and on-farm antimicrobial usage (AMU). In addition, the relation between dust and farmers' stool resistomes was explored. Methods: In the EFFORT-study, resistomes and bacterial microbiomes of indoor farm dust collected on Electrostatic Dust fall Collectors (EDCs), and animal feces of 35 conventional broiler and 44 farrow-to-finish pig farms from nine European countries were determined by shotgun metagenomic analysis. The analysis also included 79 stool samples from farmers working or living at 12 broiler and 19 pig farms and 46 human controls. Relative abundance of and variation in resistome and bacterial composition of farm dust was described and compared to animal feces and farmers' stool. Results: The farm dust resistome contained a large variety of antimicrobial resistance genes (ARGs); more than the animal fecal resistome. For both poultry and pigs, composition of dust resistomes finds (partly) its origin in animal feces as dust resistomes correlated significantly with fecal resistomes. The dust bacterial microbiome also correlated significantly with the dust resistome composition. A positive association between AMU in animals on the farm and the total abundance of the dust resistome was found. Occupational exposure to pig farm dust or animal feces may contribute to farmers' resistomes, however no major shifts in farmers resistome towards feces or dust resistomes were found in this study. Conclusion: Poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome

Occurrence of antimicrobial resistance among bacterial pathogens and indicator bacteria in pigs in different European countries from year 2002 – 2004 : the ARBAO-II study

Background: The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–05. The aim of this project was to establish a program for the continuous monitoring of antimicrobial susceptibility of pathogenic and indicator bacteria from food animals using validated and harmonised methodologies. In this report the first data on the occurrence of antimicrobial resistance among bacteria causing infections in pigs are reported. Methods: Susceptibility data from 17,642 isolates of pathogens and indicator bacteria including Actinobacillus pleuropneumoniae, Streptococcus suis and Escherichia coli isolated from pigs were collected from fifteen European countries in 2002–2004.Results: Data for A. pleuropneumoniae from infected pigs were submitted from five countries. Most of the isolates from Denmark were susceptible to all drugs tested with the exceptions of a low frequency of resistance to tetracycline and trimethoprim – sulphonamide. Data for S. suis were obtained from six countries. In general, a high level of resistance to tetracycline (48.0 – 92.0%) and erythromycin (29.1 – 75.0%) was observed in all countries whereas the level of resistance to ciprofloxacin and penicillin differed between the reporting countries. Isolates from England (and Wales), France and The Netherlands were all susceptible to penicillin. In contrast the proportion of strains resistant to ciprofloxacin ranged from 12.6 to 79.0% (2004) and to penicillin from 8.1 – 13.0% (2004) in Poland and Portugal. Data for E. coli from infected and healthy pigs were obtained from eleven countries. The data reveal a high level of resistance to tetracyclines, streptomycin and ampicillin among infected pigs whereas in healthy pigs the frequency of resistance was lower. Conclusion: Bacterial resistance to some antimicrobials was frequent with different levels of resistance being observed to several antimicrobial agents in different countries. The occurrence of resistance varied distinctly between isolates from healthy and diseased pigs, with the isolates from healthy pigs generally showing a lower level of resistance than those from diseased pigs. The study suggests that the choice of antimicrobials used for the treatment of diseased animals should preferably be based on knowledge of the local pattern of resistance

Occurrence of antimicrobial resistance among bacterial pathogens

Background: The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–05. The aim of this project was to establish a program for the continuous monitoring of antimicrobial susceptibility of pathogenic and indicator bacteria from food animals using validated and harmonised methodologies. In this report the first data on the occurrence of antimicrobial resistance among bacteria causing infections in pigs are reported. Methods: Susceptibility data from 17,642 isolates of pathogens and indicator bacteria including Actinobacillus pleuropneumoniae, Streptococcus suis and Escherichia coli isolated from pigs were collected from fifteen European countries in 2002–2004.Results: Data for A. pleuropneumoniae from infected pigs were submitted from five countries. Most of the isolates from Denmark were susceptible to all drugs tested with the exceptions of a low frequency of resistance to tetracycline and trimethoprim – sulphonamide. Data for S. suis were obtained from six countries. In general, a high level of resistance to tetracycline (48.0 – 92.0%) and erythromycin (29.1 – 75.0%) was observed in all countries whereas the level of resistance to ciprofloxacin and penicillin differed between the reporting countries. Isolates from England (and Wales), France and The Netherlands were all susceptible to penicillin. In contrast the proportion of strains resistant to ciprofloxacin ranged from 12.6 to 79.0% (2004) and to penicillin from 8.1 – 13.0% (2004) in Poland and Portugal. Data for E. coli from infected and healthy pigs were obtained from eleven countries. The data reveal a high level of resistance to tetracyclines, streptomycin and ampicillin among infected pigs whereas in healthy pigs the frequency of resistance was lower. Conclusion: Bacterial resistance to some antimicrobials was frequent with different levels of resistance being observed to several antimicrobial agents in different countries. The occurrence of resistance varied distinctly between isolates from healthy and diseased pigs, with the isolates from healthy pigs generally showing a lower level of resistance than those from diseased pigs. The study suggests that the choice of antimicrobials used for the treatment of diseased animals should preferably be based on knowledge of the local pattern of 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

The colistin resistance mcr-1 gene is going wild

Microbial Biotechnolog