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Abstract

The antimicrobial resistance remains an important threat to the public and animal health. The current trends of antimicrobial resistance reveal an alarming and ongoing dissemination of associated genes to different bacteria. With the aim of preserving important antimicrobials for human treatment, certain antimicrobial classes, such as carbapenems are categorized as critically important antimicrobials and are not licensed for use in foodproducing animals. However, in recent years, reports of carbapenemase-producing bacteria in livestock animals started to emerge. In Germany, several studies reported detection of carbapenemase-producing enterobacteria in wild birds, livestock, meat and seafood products. An important observation was association of New Delhi Metallo (NDM-1) and Verona Integron Metallo (VIM-1) carbapenemases to different Salmonella serovars, suggesting their role as reservoirs of these carbapenemases. Motivated by the detection of carbapenemase-producing Salmonella serovars in a wild bird and poultry production, in this doctoral thesis scenarios of avian native NDM-1-producing Salmonella enterica subsp. enterica serovar (S.) Corvallis and VIM-1-producing S. Infantis entering a broiler flock were investigated. The aim of the in vivo studies was to understand behavior of these strains and their plasmids in the absence of antibiotic pressure. Particular focus was on the in vivo spread and stability of carbapenemase-encoding blaNDM-1-carrying plasmids belonging to the incompatibility group A/C2 (IncA/C2) and blaVIM-1-carrying plasmids belonging to group HI2 (IncHI2). The insight into transfer and structural alteration of plasmids was obtained by an in-depth molecular analysis with S1-pulsed-field gel electrophoresis plasmid profiling and subsequent short and long-read whole genome sequencing analysis. The in vivo studies showed that in a scenario of NDM-1-producing S. Corvallis entering a broiler flock, the blaNDM-1-carrying IncA/C2 plasmid disseminates to different gut enterobacteria, such as Escherichia (E.) coli and Klebsiella (K.) pneumoniae, in the absence of antibiotic pressure. The acquisition of this plasmid was not restricted to a particular E. coli clone, as this plasmid was detected in different E. coli ST-types and a K. pneumoniae strain. The blaNDM-1-carrying IncA/C2 plasmid remained detected in donor and E. coli strains until the end of trial, revealing stable persistence of this plasmid also in new hosts. Although less frequent in contrast to gut enterobacteria, acquisition of the blaNDM-1-carrying IncA/C2 plasmid in broiler-associated S. Paratyphi B (dTartrate-positive) and S. Infantis strains was detected. Acquisition of the blaVIM-1-carrying IncHI2 plasmid was not detected in other bacteria during the course of in vivo study. As for the in vivo stability of blaNDM-1-carrying IncA/C2 and blaVIM-1-carrying IncHI2 plasmid, stable persistence of these plasmids in their native hosts was observed. In the case of blaNDM-1-carrying IncA/C2 plasmid a smaller (~10 kb) and larger (~70 kb) structural deletion was identified, along with formation of a newly emerged blaNDM-1-carrying IncA/C2-IncHI2 multireplicon megaplasmid. Despite structural alterations, the blaNDM-1 gene was maintained in these variants of the blaNDM-1-carrying IncA/C2 plasmid. In the selection of blaVIM-1-carrying IncHI2 plasmids, from donor reisolates, smaller (~10 kb) structural deletions were observed. In conclusion, the in vivo broiler chicken infection studies revealed stable persistence of the blaNDM-1-carrying IncA/C2 and blaVIM-1-carrying IncHI2 plasmids, despite sporadic structural alterations, in their hosts in a scenario of entry into a broiler flock. As for blaNDM-1-carrying IncA/C2 plasmid, a worrying observation is the rapid and broad dissemination of the blaNDM-1-carrying IncA/C2 plasmid to gut enterobacteria and Salmonella recipients in the absence of selective pressure. Although here primarily investigating the behavior of NDM-1-producing S. Corvallis and VIM-1-producing S. Infantis strains, the results can serve as a model for the potential dissemination of other MDR-encoding broad host range plasmids in a broiler flock. Therefore, important preventive measure should be achieved to avoid such bacteria entering a broiler flock, as their entrance could lead to the spread of MDR-encoding plasmids even in the absence of antibiotic pressure.

Keywords

Animal models ; Broilers ; Chickens ; Enterobacteriaceae ; Salmonella ; Drug resistance ; Carbapenems ; Antimicrobial properties

http://dx.doi.org/10.17169/refubium-28307

Abstract

Background: By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques.

Results: Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, β-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms – of which also farm livestock resistomes were analysed – showed positive associations between the number of on-farm working hours and human faecal AMR loads.

Conclusion: We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.

Keywords

Resistome; Microbiome; Antimicrobial resistance; Occupational exposure; Farmers; Slaughterhouse workers

 

Environment International, Volume 143, October 2020, 105939; https://doi.org/10.1016/j.envint.2020.105939

Abstract

As a consequence of human and animal antimicrobial use (AMU), antimicrobial resistance (AMR) has emerged. Antimicrobial resistant bacteria – containing antimicrobial resistance genes (ARGs) – are ubiquitous in our living environment. Not only can humans be directly exposed to AMR bacteria in livestock, livestock bacteria could potentially also serve as a reservoir for horizontal gene transfer through which human pathogenic bacteria can acquire ARGs. Studies investigating determinants for livestock and human AMR carriage should therefore also include the study of ARGs to enable the estimation of the overall exposure of humans to – and the risk posed by – AMR. This thesis concerns the epidemiological analysis of AMR in the European food chain. It describes how AMU and AMR in livestock affect AMR in humans and focuses on identifying determinants for AMR at the genomic level. It elaborates on how the pig and poultry gut resistome, i.e. the full collection of ARGs in the gut, is affected by AMU, livestock farm management and on-farm biosecurity practices. Molecular techniques like qPCR and next-generation sequencing techniques (i.e. metagenomic sequencing) are applied to identify an umbrella of on-farm AMR mitigation measures targeted at the livestock resistome, while generating data regarding the genetic characteristics of resistance. This work also analyses AMR in, and the faecal resistome and bacteriome of, persons occupationally exposed to AMR in livestock (i.e. farmers and slaughterhouse workers), who might not only be highly exposed but also serve as entry points for the spread of AMR bacteria and ARGs to the general population. Finally, the strengths and limitations of the application of metagenomic methods within AMR surveillance networks are discussed. A more detailed summary can be found in the addendum to this thesis (page 256, in English and Dutch).

 

Doctoral Thesis, Utrecht University, 2020-05-27, ISBN 9789464022117

Abstract

The aim of the present study was to explore the relation between both farm performance and antimicrobial use (AMU) of broiler farms. Farm performance was expressed as technical efficiency, obtained by using a bootstrap data envelopment analysis.AMU was expressed as treatment incidence. Cluster analysis is used to obtain groups of farms with similar characteristics regarding technical farm performance and AMU. Results indicate that the farms within the different clusters combine different technical farm performance and different levels of AMU. Between the clusters, significant differences were found in technical farm performance, AMU, the resource intensity of the number of animals at set-up, the number of antimicrobial treatments, the number of antimicrobial treatments related to either gut health or combined problems, and the number of antimicrobial treatments with either yellow or orange active substances. Farmers who combine high levels of AMU with high technical farm performance are likely to overestimate the real economic value of AMU. Proper coordination between the farmer and the veterinarian can be crucial in that case for reducing AMU. Farms with low performance are likely to have poor farm conditions. Improving those farm conditions can help reducing the need for AMU on this kind of farms. The farm-specific conditions have to be considered in future policies aimed at reducing AMU in livestock production.

Keywords

Broiler production, antimicrobial use, technical farm performance

2020 Poultry Science Volume 99, Issue 3, March 2020:1349–1356; https://doi.org/10.1016/j.psj.2019.10.054

Abstract

The aim of this article is to report on antimicrobial resistance (AMR) in commensal Escherichia coli  from livestock from several European countries. The relationships with antimicrobial usage (AMU) at country level and harmonized indicators to cover the most relevant AMR aspects for human health in animal production were also investigated. E. coli  were isolated in faeces from broilers and fattening pigs (from nine countries), and fattening turkeys and veal calves (from three countries) and screened against a fixed antimicrobial panel. AMU data were collected at farm and average treatment incidences stratified by antimicrobial class, country and livestock species were calculated. Associations between AMR and AMU at country level were analysed. Independent of animal species, the highest resistance was observed for ampicillin, sulphamethoxazole, tetracycline and trimethoprim. E. coli  from broilers showed the highest resistance level for (fluoro)quinolones, and multidrug resistance peaked in broilers and fattening turkeys. Colistin resistance was observed at very low levels with the exception of fattening turkeys. High resistance to third- and fourth-generation cephalosporins was detected in broilers and fattening turkeys. The lowest levels of resistance were for meropenem, azithromycin and tigecycline (<1 %). Significant correlations between resistance and usage at country level were detected in broilers for polymyxins and aminoglycosides, and in fattening pigs for cephalosporins, amphenicols, fluoroquinolones and polymyxins. None of the correlations observed between AMR and AMU were statistically significant for fattening turkey and veal calves. The strength of the analysis performed here is the correlation of aggregated data from the same farms at country level for both AMU and AMR within antimicrobial classes.

Keywords

Escherichia coli, antimicrobial resistance, antimicrobial usage, MIC, Europe, livestock. ​

 

Journal of Medical Microbiology Volume 69, Issue 4, First Published: 18 March 2020 https://doi.org/10.1099/jmm.0.001176

Abstract

Objectives: Slaughterhouse staff is occupationally exposed to antimicrobial resistant bacteria. Studies reported high antimicrobial resistance gene (ARG) abundances in slaughter pigs. This cross-sectional study investigated occupational exposure to tetracycline (tetW) and macrolide (ermB) resistance genes and assessed determinants for faecal tetW and ermB carriage among pig slaughterhouse workers.

Methods: During 2015–2016, 483 faecal samples and personal questionnaires were collected from workers in a Dutch pig abattoir, together with 60 pig faecal samples. Human dermal and respiratory exposure was assessed by examining 198 carcass, 326 gloves, and 33 air samples along the line, next to 198 packed pork chops to indicate potential consumer exposure. Samples were analyzed by qPCR (tetW, ermB). A job exposure matrix was created by calculating the percentage of tetW and ermB positive carcasses or gloves for each job position. Multiple linear regression models were used to link exposure to tetW and ermB carriage.

Results: Workers are exposed to tetracycline and macrolide resistance genes along the slaughter line. Tetw and ermB gradients were found for carcasses, gloves, and air filters. One packed pork chop contained tetW, ermB was non-detectable. Human faecal tetW and ermB concentrations were lower than in pig faeces. Associations were found between occupational tetW exposure and human faecal tetW carriage, yet, not after model adjustments. Sampling round, nationality, and smoking were determinants for ARG carriage.

Conclusion: We demonstrated clear environmental tetracycline and macrolide resistance gene exposure gradients along the slaughter line. No robust link was found between ARG exposure and human faecal ARG carriage.

Keywords

Air, dermal exposure, faecal carriage, gloves, macrolide resistance, respiratory exposure, retail meat, tetracycline resistance

 

Annals of Work Exposures and Health, Volume 64, Issue 2, March 2020, Pages 125–137, https://doi.org/10.1093/annweh/wxz098.

Abstract

One Health surveillance of antimicrobial resistance (AMR) depends on a harmonized method for detection of AMR. Metagenomics-based surveillance offers the possibility to compare resistomes within and between different target populations. Its potential to be embedded into policy in the future calls for a timely and integrated knowledge dissemination strategy. We developed a blended training (e-learning and a workshop) on the use of metagenomics in surveillance of pathogens and AMR. The objectives were to highlight the potential of metagenomics in the context of integrated surveillance, to demonstrate its applicability through hands-on training and to raise awareness to bias factors1. The target participants included staff of competent authorities responsible for AMR monitoring and academic staff. The training was organized in modules covering the workflow, requirements, benefits and challenges of surveillance by metagenomics. The training had 41 participants. The face-to-face workshop was essential to understand the expectations of the participants about the transition to metagenomics-based surveillance. After revision of the e-learning, we released it as a Massive Open Online Course (MOOC), now available at https://www.coursera.org/learn/metagenomics. This course has run in more than 20 sessions, with more than 3,000 learners enrolled, from more than 120 countries. Blended learning and MOOCs are useful tools to deliver knowledge globally and across disciplines. The released MOOC can be a reference knowledge source for international players in the application of metagenomics in surveillance.

Keywords

Surveilance, metagenomics, MOOC, antimicrobial resistance, one health.

 

Front. Public Health, 25 February 2020 | https://doi.org/10.3389/fpubh.2020.00038

Abstract

Companion animals have been described as potential reservoirs of antimicrobial resistance (AMR), however data remain scarce. Therefore, the objectives were to describe antimicrobial usage (AMU) in dogs and cats in three European countries (Belgium, Italy, and The Netherlands) and to investigate phenotypic AMR. A questionnaire and one fecal sample per animal (n = 303) were collected over one year and AMU was quantified using treatment incidence (TI). Phenotypic resistance profiles of 282 Escherichia coli isolates were determined. Nineteen percent of the animals received at least one antimicrobial treatment six months preceding sampling. On average, cats and dogs were treated with a standard daily dose of antimicrobials for 1.8 and 3.3 days over one year, respectively. The most frequently used antimicrobial was amoxicillin-clavulanate (27%). Broad-spectrum antimicrobials and critically important antimicrobials for human medicine represented 83% and 71% of the total number of treatments, respectively. Resistance of E. coli to at least one antimicrobial agent was found in 27% of the isolates. The most common resistance was to ampicillin (18%). Thirteen percent was identified as multidrug resistant isolates. No association between AMU and AMR was found in the investigated samples. The issue to address, regarding AMU in companion animal, lies within the quality of use, not the quantity. Especially from a One-Health perspective, companion animals might be a source of transmission of resistance genes and/or resistant bacteria to humans.

Keywords

Antimicrobial use; antimicrobial resistance; companion animals; critically important antimicrobials; colistin resistance; one health.

 

Antibiotics 2020, 9(2), 87; https://doi.org/10.3390/antibiotics9020087

Abstract

Objectives: Previous studies in food-producing animals have shown associations between antimicrobial use (AMU) and resistance (AMR) in specifically isolated bacterial species. Multi-country data are scarce and only describe between-country differences. Here we investigate associations between the pig faecal mobile resistome and characteristics at the farm-level across Europe.

Methods: A cross-sectional study was conducted among 176 conventional pig farms from nine European countries. Twenty-five faecal samples from fattening pigs were pooled per farm and acquired resistomes were determined using shotgun metagenomics and the Resfinder reference database, i.e. the full collection of horizontally acquired AMR genes (ARGs). Normalized fragments resistance genes per kilobase reference per million bacterial fragments (FPKM) were calculated. Specific farm-level data (AMU, biosecurity) were collected. Random-effects meta-analyses were performed by country, relating farm-level data to relative ARG abundances (FPKM).

Results: Total AMU during fattening was positively associated with total ARG (total FPKM). Positive associations were particularly observed between widely used macrolides and tetracyclines, and ARGs corresponding to the respective antimicrobial classes. Significant AMU-ARG associations were not found for β-lactams and only few colistin ARGs were found, despite high use of these antimicrobial classes in younger pigs. Increased internal biosecurity was directly related to higher abundances of ARGs mainly encoding macrolide resistance. These effects of biosecurity were independent of AMU in mutually adjusted models.

Conclusions: Using resistome data in association studies is unprecedented and adds accuracy and new insights to previously observed AMU-AMR associations. Major components of the pig resistome are positively and independently associated with on-farm AMU and biosecurity conditions.

Journal of Antimicrobial Chemotherapy, dky518, https://doi.org/10.1093/jac/dky518

Abstract:

Objectives: To control the emerging threat of antimicrobial resistance, international policy appeals for appropriate monitoring of antimicrobial usage (AMU) at supranational, species and farm level. The aim of this study was to quantify AMU in broilers at farm and flock level in nine European countries.

Methods: Antimicrobial treatment data of one flock and purchased antimicrobials over one year were collected at 181 European broiler farms. Afterwards AMU was quantified using treatment incidence (TI) per 100 days based on Defined Daily Dose (DDDvet), Defined Course Dose (DCDvet) or Used Daily Dose (UDDvet) values. Total AMU at flock level was obtained by summing the TIDDDvet of all treatments in the sampled flock (TIDDDvetFl*).

Results: The median TIDDDvetFl* was 9.0 (95% CI 5.5–10.8), meaning that broilers were treated with antimicrobials during 9% of their rearing period. TIDDDvetFl* varied considerably within and between countries. However, in every country at least one untreated flock was present. Average TIDDDvetFl* at country level ranged from 3.3 to 36.7. Polymyxins, extended-spectrum aminopenicillins and fluoroquinolones were the most used antimicrobials, accounting for 26%, 26% and 18% of total AMU, respectively. Twenty-six percent of the farms started a treatment on day 1 of production, and 49% of overall AMU was administered within the first week.

Conclusions: Results show that rearing broilers without AMU is feasible. However, a huge variation in AMU in terms of amount, moment of administration and antimicrobial classes was observed. This shows that there is still ground to be covered when it comes to AMU on broiler farms.

Journal of Antimicrobial Chemotherapy, dky498, https://doi.org/10.1093/jac/dky498

Abstract:

Objectives: Farm-level quantification of antimicrobial usage (AMU) in pig farms.

Methods: In a cross-sectional study, AMU data on group treatments administered to a single batch of fattening pigs from birth to slaughter (group treatment data) and antimicrobials purchased during 1 year (purchase data) were collected at 180 pig farms in nine European countries. AMU was quantified using treatment incidence (TI) based on defined (DDDvet) and used (UDDvet) daily doses and defined (DCDvet) and used (UCDvet) course doses.

Results: The majority of antimicrobial group treatments were administered to weaners (69.5% of total TIDDDvet) followed by sucklers (22.5% of total TIDDDvet). AMU varied considerably between farms with a median TIDDDvet of 9.2 and 7.1 for a standardized rearing period of 200 days based on group treatment and purchase data, respectively. In general, UDDvet and UCDvet were higher than DDDvet and DCDvet, respectively, suggesting that either the defined doses were set too low or that group treatments were often dosed too high and/or administered for too long. Extended-spectrum penicillins (31.2%) and polymyxins (24.7%) were the active substances most often used in group treatments, with the majority administered through feed or water (82%). Higher AMU at a young age was associated with higher use in older pigs.

Conclusions: Collecting farm-level AMU data of good quality is challenging and results differ based on how data are collected (group treatment data versus purchase data) and reported (defined versus used daily and course doses).

Journal of Antimicrobial Chemotherapy, dky503, https://doi.org/10.1093/jac/dky503

Carbapenems are an important class of β-lactams and one of the last options for treating severe human infections. We present here the complete genome sequence of avian native carbapenemase-producing Salmonella enterica subsp. enterica serovar Corvallis strain 12-01738, harboring a blaNDM-1-carrying IncA/C2 plasmid, isolated in 2012 from a wild bird (Milvus migrans) in Germany.

© 2018 Hadziabdic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Jennie Fischer, Jennie.Fischer@bfr.bund.de.

Genome Announc 6:e00593-18. https://doi.org/10.1128/genomeA.00593-18.

Antimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. The use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock and in humans. We quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, sequencing more than 5,000 Gb of DNA using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a second database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than in poultry. We found higher AMR loads in pigs, whereas poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and between countries. We found that the total acquired AMR level was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. However, functionally determined AMR genes were not associated with total drug use.

Additionally to the publication, you will find here after the blog post related to the article.

Nature Microbiology, 23 July 2018, https://doi.org/10.1038/s41564-018-0192-9

ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

Frontiers in Microbiology, 16 March 2018, https://doi.org/10.3389/fmicb.2018.00469

Abstract:

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S. Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S. Paratyphi B (13-SA01617), referred to here as S. Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S. Corvallis reisolates as well as plasmid acquisition in S. Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S. Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S. Corvallis is significantly higher than that of S. Paratyphi (d-Ta+) and is not hampered by S. Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S. Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

Antimicrobial Agents and Chemotherapy, March 2018, 10.1128/AAC.02128-17

Objectives: Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.

Conclusions: We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.

J Antimicrob Chemother. 2017 Feb;72(2):385-392. doi: 10.1093/jac/dkw415

Abstract:

ColE1 plasmids are small mobilizable replicons that play an important role in the spread of antibiotic resistance in Pasteurellaceae. In this study, we describe how a natural single nucleotide polymorphism (SNP) near the origin of replication of the ColE1-type plasmid pB1000 found in a Pasteurella multocida clinical isolate generates two independent plasmid variants able to coexist in the same cell simultaneously. Using the Haemophilus influenzae Rd KW20 strain as a model system, we combined antibiotic susceptibility tests, quantitative PCRs, competition assays, and experimental evolution to characterize the consequences of the coexistence of the pB1000 plasmid variants. This coexistence produced an increase of the total plasmid copy number (PCN) in the host bacteria, leading to a rise in both the antibiotic resistance level and the metabolic burden produced by pB1000. Using experimental evolution, we showed that in the presence of ampicillin, the bacteria maintained both plasmid variants for 300 generations. In the absence of antibiotics, on the other hand, the bacteria are capable of reverting to the single-plasmid genotype via the loss of one of the plasmid variants. Our results revealed how a single mutation in plasmid pB1000 provides the bacterial host with a mechanism to increase the PCN and, consequently, the ampicillin resistance level. Crucially, this mechanism can be rapidly reversed to avoid the extra cost entailed by the increased PCN in the absence of antibiotics.

Antimicrobial Agents and Chemotherapy, January 2017, 10.1128/AAC.01735-16

Abstract:

The World Health Organization has recognized antimicrobial resistance as one of the top three threats to human health. Any use of antibiotics in animals will ultimately affect humans and vice versa. Appropriate monitoring of antimicrobial use and resistance has been repeatedly emphasized along with the need for global policies. Under the auspices of the European Union research project, EFFORT, we mapped antimicrobial use and resistance monitoring programs in ten European countries. We then compared international and European guidelines and policies. In resistance monitoring, we did not find important differences between countries. Current resistance monitoring systems are focused on food animal species (using fecal samples). They ignore companion animals. The scenario is different for monitoring antibiotics use. Recently, countries have tried to harmonize methodologies, but reporting of antimicrobial use remains voluntary. We therefore identified a need for stronger policies.

Keywords: antimicrobial resistance (AMR) monitoring policies animals antimicrobial use (AMU)

J Public Health Pol (2017). doi:10.1057/s41271-017-0067-y

Abstract:

IncK plasmids are one of the main carriers of blaCTX-M-14 and blaCMY-2 genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2.

Antimicrob Agents Chemother. 2017 Jan 4. pii: AAC.01954-16. doi: 10.1128/AAC.01954-16. [Epub ahead of print]