Prevalence and molecular epidemiology of mcr-mediated colistin-resistance Escherichia coli from healthy poultry in France after national plan to reduce exposure to colistin in farm

IntroductionWithin the 2007–2014 programme for the surveillance of antimicrobial resistance (AMR) in livestock in France, mcr-1 prevalence average in commensal Escherichia coli was found to be 5.9% in turkeys and 1.8% in broilers, indicating that mobile colistin resistance had spread in farm animals. In 2017, the French national Ecoantibio2 plan was established to tackle AMR in veterinary medicine, with the objective of a 50% reduction in exposure to colistin in farm animals within 5 years (from 2014–2015 to 2020). Our objective was to update data concerning the prevalence and molecular epidemiology of colistin resistance, in consideration of colistin sales in poultry production in France.MethodsAntimicrobial susceptibility of commensal E. coli isolated from broilers and turkeys at slaughterhouse was determined by broth micro-dilution. The mcr genes were screened by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was used to investigate the genetic diversity of colistin-resistant isolates. Transformation experiments enabled identification of the mcr-bearing plasmid replicon types. The correlation between prevalence of colistin resistance and colistin usage data was explored statistically.Results and discussionIn 2020, in France, the resistance prevalence to colistin in poultry production was 3% in turkeys and 1% in broilers, showing a significant highly positive correlation with a −68% decrease of poultry exposure to colistin since 2014. Only the mcr-1 gene was detected among the colistin-resistant E. coli. More than 80% of isolates are multi-drug resistant with 40% of isolates originating from turkeys and 44% originating from broilers co-resistant to the critically important antimicrobial ciprofloxacin. Most of the strains had no clonal relationship. The mcr gene was located in different plasmid types, carrying various other AMR genes. The decrease in colistin resistance among poultry in France can be considered a positive outcome of the national action plans for reduced colistin usage

Characterization of Colistin-Resistant Escherichia coli Isolated from Diseased Pigs in France

We studied a collection of 79 colistin-resistant Escherichia coli isolates isolated from diseased pigs in France between 2009 and 2013. We determined a number of phenotypic and genetic characters using broth microdilution to characterize their antimicrobial susceptibility. We performed pulse field gel electrophoresis (PFGE) to assess their genetic diversity and assign them to phylogroups. High-throughput real-time PCR micro-array was used to screen for a selection of genetic markers of virulence, and PCR and sequencing of the main recognized resistance genes allowed us to investigate the mechanisms of colistin resistance. Results showed that isolates belonged to several phylogroups and most had a unique PFGE profile. More than 50% of the isolates were also resistant to sulfonamides, trimethoprim, tetracycline, ampicillin or chloramphenicol. The mcr-1 gene was detected in 70 out of 79 isolates and was transferred by conjugation in 33 of them, sometimes together with resistance to sulfonamides, trimethoprim, tetracycline, ampicillin, chloramphenicol, cefotaxime, or gentamicin. Mutations in the amino-acid sequences of proteins MgrB, PhoP, PhoQ, PmrB, but not PmrA, were detected in isolates with or without the mcr-1 gene. More than one-third of the isolates harbored the F18, F4, astA, hlyA, estI, estII, elt, stx2e, iha, orfA, orfB, paa, terE, ecs1763, or ureD virulence markers. In conclusion, although most isolates had a unique PFGE profile, a few particular combinations of phylogenetic groups, virulence genes and mutations in the sequenced genes involved in colistin resistance were identified on a number of occasions, suggesting the persistence of certain isolates over several years

Impact of colistin and colistin-loaded on alginate nanoparticles on pigs infected with a colistin-resistant enterotoxigenic Escherichia coli strain

International audienceColistin is frequently used for the control of post-weaning diarrhoea in pigs. Colistin resistance caused by plasmidic genes is a public health issue. We evaluated, in experimental animal facilities, whether free colistin or colistin-loaded on alginate nanoparticles (colistin/Alg NPs) could select a colistin-resistant Enterotoxigenic Escherichia coli. The Alg NPs were produced by a simple top-down approach through ball milling of sodium alginate polymer precursor, and colistin loading was achieved through physical adsorption. Colistin loading on Alg NPs was confirmed using various tools such Fourier transform infrared spectroscopy and dynamic light scattering measurements. Thirty-four piglets were orally inoculated or not with a mcr-1-positive, rifampicin-resistant enterotoxigenic E. coli strain, and the inoculated pigs were either treated or not during five days with commercial colistin (100,000 IU/kg) or colistin/Alg NPs (40,415 IU/kg). Clinical signs were recorded. Fecal and post-mortem samples were analyzed by culture. The result clearly indicated that colistin/Alg NPs had a slightly better therapeutic effect. Both treatments led to a transitory decrease of the total E. coli fecal population with a majority of colistin-resistant E. coli isolates during treatment, but the dominant E. coli population was found susceptible at the end of the trial. Further studies are needed to evaluate, in diverse experimental or field conditions, the therapeutic efficacy of colistin/Alg NPs for post-weaning diarrhoea

Les facteurs déclenchants de la colibacillose du poulet de chair : étude épidémiologique dans 80 élevages en France

International audienceAvian colibacillosis is a multifactorial disease caused by the bacterium Escherichia coli, a common host in the digestive tract of poultry. E. coli has the particularity of having a great diversity of strains: while most are nonpathogenic, some have virulence factors capable of triggering colibacillosis. The objective of the Colisée study was to highlight avenues for the prevention of this disease by characterizing virulence markers of E. coli strains and identifying particularly risky breeding practices. This study was carried out in 80 broiler farms between 2017 and 2019, with two visits: one during installation (cloths in the building before the arrival of the chicks, sampling of chicks and bottom of the box cloth. at the arrival of the chicks, rating of their quality, their behavior, environmental measurements), and the other at three weeks of age (ambient measurements, rating of the behavior of the chickens). If the breeder suspected colibacillosis, chickens were sent to the laboratory for bacteriological diagnosis. Almost a hundred virulence markers have been searched for on strains isolated from the farming environment or from clinical cases of colibacillosis. The results indicate that E. coli was identified in 47% of the buildings before the arrival of the chicks, on the bottom of the box upon delivery of the animals in 95% of the lots and on the chick in 79% of the lots. Colibacillosis was reported in 39% of herds. The study of risk factors made it possible to identify breeding factors linked to biosecurity (passage of the rendering truck, rodent control, practice of disinfection of the building and water treatment) and reception of chicks (hygrometry, homogeneity of chicks and unloading method). A predictive model also demonstrated a greater influence of husbandry practices compared to that of the virulence of E. coli strains present at start-up on the onset of colibacillosis.La colibacillose aviaire est une maladie multifactorielle provoquée par la bactérie Escherichia coli, un hôte commun du tube digestif des volailles. E. coli a la particularité de présenter une grande diversité de souches : si la plupart sont non pathogènes, certaines possèdent des facteurs de virulence susceptibles de déclencher une colibacillose. L’étude Colisée avait pour objectif de mettre en évidence des pistes de prévention de cette maladie par la caractérisation de marqueurs de virulence des souches d’E.coli et l’identification de pratiques d’élevage particulièrement à risque. Cette étude a été conduite dans 80 élevages de poulets de chair entre 2017 et 2019, avec deux visites : l’une à la mise en place (chiffonnettes dans le bâtiment avant l’arrivée des poussins, prélèvement de poussins et chiffonnette de fond de boite à l’arrivée des poussins, notation de leur qualité, de leur comportement, mesures d’ambiance), et l’autre à trois semaines d’âge (mesures d’ambiance, notation du comportement des poulets). En cas de suspicion d’une colibacillose par l’éleveur, des poulets ont été envoyés au laboratoire pour diagnostic bactériologique. Près d’une centaine de marqueurs de virulence ont été recherchés sur les souches isolées d’environnement d’élevage ou de cas cliniques de colibacillose. Les résultats indiquent qu’ E. coli a été identifié dans 47% des bâtiments avant l’arrivée des poussins, sur fonds de boîte à la livraison des animaux dans 95% des lots et sur poussin dans 79% des lots. Des colibacilloses ont été signalées dans 39% des élevages. L’étude des facteurs de risque a permis d’identifier des facteurs d’élevages liés à la biosécurité (passage du camion d’équarrissage, lutte contre les rongeurs, pratique de désinfection du bâtiment et traitement de l’eau) et à l’accueil des poussins (hygrométrie, homogénéité des poussins et méthode de déchargement). Un modèle prédictif a également mis en évidence une influence plus importante des pratiques d’élevage par rapport à celle de la virulence des souches d’E. coli présentes au démarrage sur le déclenchement de la colibacillose

Les facteurs déclenchants de la colibacillose du poulet de chair : étude épidémiologique dans 80 élevages en France

International audienceAvian colibacillosis is a multifactorial disease caused by the bacterium Escherichia coli, a common host in the digestive tract of poultry. E. coli has the particularity of having a great diversity of strains: while most are nonpathogenic, some have virulence factors capable of triggering colibacillosis. The objective of the Colisée study was to highlight avenues for the prevention of this disease by characterizing virulence markers of E. coli strains and identifying particularly risky breeding practices. This study was carried out in 80 broiler farms between 2017 and 2019, with two visits: one during installation (cloths in the building before the arrival of the chicks, sampling of chicks and bottom of the box cloth. at the arrival of the chicks, rating of their quality, their behavior, environmental measurements), and the other at three weeks of age (ambient measurements, rating of the behavior of the chickens). If the breeder suspected colibacillosis, chickens were sent to the laboratory for bacteriological diagnosis. Almost a hundred virulence markers have been searched for on strains isolated from the farming environment or from clinical cases of colibacillosis. The results indicate that E. coli was identified in 47% of the buildings before the arrival of the chicks, on the bottom of the box upon delivery of the animals in 95% of the lots and on the chick in 79% of the lots. Colibacillosis was reported in 39% of herds. The study of risk factors made it possible to identify breeding factors linked to biosecurity (passage of the rendering truck, rodent control, practice of disinfection of the building and water treatment) and reception of chicks (hygrometry, homogeneity of chicks and unloading method). A predictive model also demonstrated a greater influence of husbandry practices compared to that of the virulence of E. coli strains present at start-up on the onset of colibacillosis.La colibacillose aviaire est une maladie multifactorielle provoquée par la bactérie Escherichia coli, un hôte commun du tube digestif des volailles. E. coli a la particularité de présenter une grande diversité de souches : si la plupart sont non pathogènes, certaines possèdent des facteurs de virulence susceptibles de déclencher une colibacillose. L’étude Colisée avait pour objectif de mettre en évidence des pistes de prévention de cette maladie par la caractérisation de marqueurs de virulence des souches d’E.coli et l’identification de pratiques d’élevage particulièrement à risque. Cette étude a été conduite dans 80 élevages de poulets de chair entre 2017 et 2019, avec deux visites : l’une à la mise en place (chiffonnettes dans le bâtiment avant l’arrivée des poussins, prélèvement de poussins et chiffonnette de fond de boite à l’arrivée des poussins, notation de leur qualité, de leur comportement, mesures d’ambiance), et l’autre à trois semaines d’âge (mesures d’ambiance, notation du comportement des poulets). En cas de suspicion d’une colibacillose par l’éleveur, des poulets ont été envoyés au laboratoire pour diagnostic bactériologique. Près d’une centaine de marqueurs de virulence ont été recherchés sur les souches isolées d’environnement d’élevage ou de cas cliniques de colibacillose. Les résultats indiquent qu’ E. coli a été identifié dans 47% des bâtiments avant l’arrivée des poussins, sur fonds de boîte à la livraison des animaux dans 95% des lots et sur poussin dans 79% des lots. Des colibacilloses ont été signalées dans 39% des élevages. L’étude des facteurs de risque a permis d’identifier des facteurs d’élevages liés à la biosécurité (passage du camion d’équarrissage, lutte contre les rongeurs, pratique de désinfection du bâtiment et traitement de l’eau) et à l’accueil des poussins (hygrométrie, homogénéité des poussins et méthode de déchargement). Un modèle prédictif a également mis en évidence une influence plus importante des pratiques d’élevage par rapport à celle de la virulence des souches d’E. coli présentes au démarrage sur le déclenchement de la colibacillose

Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs

International audienceResistance to extended-spectrum cephalosporins is prevalent in French pig E. coli isolates. The aim of this study was to characterize the plasmids and genes present in pathogenic and commensal extended-spectrum cephalosporins -resistant isolates. The resistance plasmids of 26 strains were sequenced and then analyzed to identify resistance and virulence genes. Results showed that resistance to extended-spectrum cephalosporins in French pig E. coli isolates is-as in other food animals in France-mainly carried by highly similar blaCTX-M-1 IncI1/ST3 plasmids. These plasmids very often bear other resistance genes such as resistance to sulphonamides (sul2), trimethoprim (dfrA17) and aminoglycosides (aadA5), and occasionally to tetracycline (tet(A)), macrolides (mph(A) and erm genes), phenicols (floR) or streptomycin (strA, strB). Few virulence genes were detected, including colicins, heat-stable enterotoxins, adhesins or temperature-sensitive hemagglutinins. The other cefotaximases detected were blaCTX-M-27 and blaCTX-M-14, the latter being on an IncF plasmid which showed very close identity to a human epidemic plasmid. Importantly, resistance genes for quinolones or polymyxins were never detected on the extended-spectrum cephalosporins resistance plasmids. These results are helpful to evidence the risk of co-selecting cephalosporins -resistance using antibiotics outside this group. They also highlight the occasional presence in pigs of human epidemic plasmids

Immunisation passive des poussins par l'administration d'un autovaccin aux poules reproductrices

National audienceAvian Colibacillosis, caused by pathogenic strains of Escherichia coli (APEC) is a major cause of death and antibiotic use in poultry. There is no effective means today to prevent this pathology in chicks. We evaluated the efficacy of an autogenous vaccine in chicks by administration in breeders. Three characterized APEC strains (virulence factors, serogroup and phylogroup) were used to develop in vivo models of colibacillosis in young chicks (phase 1). Animals were inoculated subcutaneously at 1-2 days of age with approximately 3x107 E. coli per animal and mortality was monitored for 10 days. Two APEC strains were then used to produce a bivalent autogenous vaccine which was administered to breeder hens. Chicks from vaccinated and unvaccinated hens were inoculated with each of the APEC strains of the vaccine (homologous challenges) during the phase 2.Two of the strains (AP1 and AP2) were found to be virulent for chicks, the third (AP3) did not cause any mortality nor clinical signs. AP1 causes severe disease with the death of all chicks within 6 days and AP2 causes approximately 50% mortality. Under our conditions, no passive protection was observed against AP1, with at least 90% mortality in chicks from vaccinated or unvaccinated hens. Conversely, those from vaccinated hens and challenged with AP2 had significantly lower mortality rate (40%) than those from unvaccinated hens (80%).This protection, observed during homologous challenges, has to be confirmed taking into account other parameters (heterologous challenges in particular)La colibacillose, due à certaines souches de Escherichia coli (APEC) est une cause majeure de mortalité et d’utilisation d’antibiotiques chez les volailles. Aucun moyen efficace n’existe aujourd’hui pour prévenir cette pathologie chez le poussin. Nous avons évalué l'efficacité d'un autovaccin chez le poussin par administration chez les reproducteurs. Trois souches APEC caractérisées (facteurs de virulence, sérogroupe et phylogroupe) ont été utilisées pour mettre au point des modèles de reproduction de la colibacillose chez le jeune poussin (phase 1). Les animaux ont été inoculés en sous cutané à 1 ou 2 jours d’âge avec environ 3x107 E. coli par animal et la mortalité a été suivie pendant 10 jours. Deux des souches ont ensuite servi à fabriquer un autovaccin bivalent qui a été administré à des poules reproductrices. Les poussins issus de poules vaccinées ou non ont été inoculés avec chacune des souches APEC du vaccin (challenges homologues) lors de la phase 2.Deux des souches (AP1 et AP2) se sont révélées virulentes pour le poussin, la troisième (AP3) n’a entrainé ni mortalité ni signe clinique. AP1 induit une maladie sévère avec la mort de tous les poussins en moins de 6 jours et AP2 une mortalité d’environ 50%. Dans nos conditions, aucune protection passive n’a été observée vis-à-vis de AP1, avec au moins 90% de mortalité chez les poussins issus de poules vaccinées ou non. A contrario, ceux issus de poules vaccinées, et inoculés avec AP2 présentaient un taux de mortalité (40%) significativement moins élevé que ceux issus de poules non vaccinées (80%). Cette protection, observée lors de challenges homologues doit être confirmée en tenant compte d’autres paramètres (challenges hétérologues notamment)

Description and validation of a new set of PCR markers predictive of avian pathogenic Escherichia coli virulence

International audienceAvian colibacillosis is the main bacterial infectious disease in poultry and is caused by avian pathogenic Escherichia coli (APEC). However, E. coli strains are very diverse, and not all are pathogenic for poultry. A straightforward scheme for identifying APEC is crucial to better control avian colibacillosis. In this study, we combined high-throughput PCR and a machine learning procedure to identify relevant genetic markers associated with APEC. Markers related to phylogroup, serotype and 66 virulence factors were tested on a large number of E. coli strains isolated from environmental, faecal or colibacillosis lesion samples in 80 broiler flocks. Nine classification methods and a machine learning procedure were used to differentiate 170 strains presumed non-virulent (obtained from farm environments) from 203 strains presumed virulent (obtained from colibacillosis cases on chicken farms) and to develop a prediction model to evaluate the pathogenicity of isolates. The model was then validated on 14 isolates using a chick embryo lethality assay. The selected and validated model based on the bootstrap aggregating tree method relied on a scheme of 13 positive or negative markers associated with phylogroups (arpA), H4 antigen and virulence markers (aec4, ETT2.2, frzorf4,fyuA, iha, ireA, iroN, iutA1, papA, tsh, and vat). It had a specificity of 84 % and a sensitivity of 85 %, and was implemented as an online tool. Our scheme offers an easy evaluation of the virulence of avian E. coli isolates on the basis of the presence/absence of these 13 genetic markers, allowing for better control of avian colibacillosis