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

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

Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut

<p>Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.</p

Correction: Time to acquire and lose carriership of ESBL/pAmpC producing E. coli in humans in the Netherlands.

[This corrects the article DOI: 10.1371/journal.pone.0193834.]

Competition between Escherichia coli populations with and without plasmids carrying a gene encoding extendedspectrum beta-lactamase in the broiler chicken gut

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1. At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro. Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro. Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation

ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae carriage among veterinary healthcare workers in the Netherlands.

The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4-12.7). The most frequently occurring ESBL genes were blaCTX-M-15, blaCTX-M-14 and blaDHA-1. The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker

Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.</p

Time to acquire and lose carriership of ESBL/pAmpC producing E. coli in humans in the Netherlands.

A subset of the study population from a cross-sectional study of carriership of ESBL/pAmpC-producing E. coli (ESBL-E) in the general population was followed up by five successive samples over an approximate half year period, leading to six samples in 333 persons. Fecal samples were cultured and analyzed for the presence of E. coli types as characterized by MLST, and ESBL/pAmpC genes were analysed by PCR and sequencing. The study included 255 persons who had a negative first sample, to allow observations of acquiring carriership of ESBL-E. Any individual record thus consisted of a series of snapshots of episodes of presence and absence of ESBL-E carriage. A survival model was built to estimate times to acquire or lose carriership, allowing for any combination of ESBL/pAmpC gene and E. coli MLST type. In carriers, the mean time to lose carriership was 1.1 (95% range 0.8-1.6) years. The estimated mean time to acquire carriership was 3.0 (95% range 1.6-6.3) years. Analysis of these times by ESBL/pAmpC gene found substantial variation among resistance genes both in persistence of carriership and in rates of acquiring carriership: blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27 and blaSHV-12 were easily acquired, but blaCTX-M-1 and blaSHV-12 were also easily lost, while blaCTX-M-15, blaCTX-M-27 and blaCMY-2 were more likely to persist. When in addition bacterial host types were included, some combinations appeared more persistent than others (blaCTX-M-1 in ST10 and ST58; blaCTX-M-14, blaCMY-2, and blaSHV-12 in ST69), or were acquired with higher frequency (blaCTX-M-14 in ST38, ST69, and ST131; blaCTX-M-15 and blaCTX-M-27 in ST131; blaSHV-12 in ST69). The relatively short duration of carriership means that when an intervention drastically reduces the exposure of humans to ESBL-E, the prevalence will be halved in 0.66 years. The observed differences between carriage rates of ESBL/pAmpC genes and E. coli strains need further investigation