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The diversity of O-polysaccharides (O-antigens) among 28 Aeromonas salmonicida strains isolated from ill fish has been determined by using high-resolution magic angle spinning (HR MAS) NMR spectroscopy. The new O-polysaccharide has been identified in two isolates. This new structure was investigated by 1 H and 13 C NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The following structure of the linear hexasaccharide repeating unit of A. salmonicida O-antigen has been established: →3)-α-L-Rha p -(1→3)-α-D-Man p NAc-(1→2)-β-D-Glc p -(1→3)-α-L-Rha p 2OAc4OAc-(1→3)-β-D-Man p NAc-(1→3)-α-D-Glc p -(1→. This new A. salmonicida O-polysaccharide was detected among two isolates collected from trout and turbot fish in 2010 and 2011, respectively. Further investigations should be conducted to evaluate the distribution of this new O-polysaccharide among a larger collection of isolates, depending on their geographic origin, the species of fish, and the health status of the fish.

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

Introduction: Within 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., Methods: Antimicrobial 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 Discussion: In 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Perrin-Guyomard, Houée, Lucas, Felten, Le Devendec, Chauvin and Kempf.)

Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 10 7 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity.We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19-381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19-381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1. RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis.A bivalent autogenous vaccine was produced and a passive immunization trial was carried out.Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge).Further work is needed in particular to evaluate the protection against heterologous challenge.

Manure is a major source of antimicrobial-resistant bacteria and resistance genes carried by mobile genetic elements such as plasmids. In France, the number of on-farm biogas plants has increased significantly in recent years. Our study investigated the impact of mesophilic anaerobic digestion (AD) and the post-treatment of digestates on the fate of conjugative plasmids, along with their potential transfer of antimicrobial resistance. Samples of raw manure, digestates and post-treated digestates were collected from three on-farm biogas plants. Conjugative plasmids were captured using the Escherichia coli CV601 recipient strain and media supplemented with rifampicin and kanamycin - to which the recipient strain is resistant - and tetracycline, sulfamethoxazole, gentamicin, trimethoprim, amoxicillin, cefotaxime, ciprofloxacin or colistin. Putative transconjugants were identified and characterised by disc diffusion and whole genome sequencing. The results showed that the antimicrobial resistance genes transferred from the different matrices conferred resistance to tetracyclines, sulphonamides, trimethoprim, and/or streptomycin. Transconjugants were obtained from raw manure samples but not from digestates or post-digestates, suggesting that mesophilic AD processes may produce fewer conjugative plasmids potentially able to be transferred to Enterobacterales., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Avian 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, frz orf4, 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Colistin 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mobile genetic elements (MGEs) such as plasmids or integrative conjugative elements (ICEs) are widely involved in the horizontal transfer of antibiotic resistant genes (ARGs), but their environmental host-range and reservoirs remain poorly known, as mainly assessed through the analysis of culturable and clinical bacterial isolates. In this study, we used a gradual approach for determining the environmental abundance and host-range of ICEs belonging to the SXT/R391 family, otherwise well known to bring ARGs in Vibrio spp. epidemic clones and other pathogens. First, by screening a set of aquatic bacteria libraries covering 1794 strains, we found that almost 1% of the isolates hosted an SXT/R391 element, all belonging to a narrow group of non-O1/non-O139 Vibrio cholerae. However, when SXT/R391 ICEs were then quantified in various aquatic communities, they appeared to be ubiquitous and relatively abundant, from 10 -6 to 10 -3 ICE copies per 16 S rDNA. Finally, the molecular exploration of the SXT/R391 host-range in two river ecosystems impacted by anthropogenic activities, using the single-cell genomic approach epicPCR, revealed several new SXT/R391 hosts mostly in the Proteobacteria phylum. Some, such as the pathogen Arcobacter cryaerophilus (Campylobacteraceae), have only been encountered in discharged treated wastewaters and downstream river waters, thus revealing a likely anthropogenic origin. Others, such as the non-pathogenic bacterium Neptunomonas acidivorans (Oceanospirillaceae), were solely identified in rivers waters upstream and downstream the treated wastewaters discharge points and may intrinsically belong to the SXT/R391 environmental reservoir. This work points out that not only the ICEs of the SXT/R391 family are more abundant in the environment than anticipated, but also that a variety of unsuspected hosts may well represent a missing link in the environmental dissemination of MGEs from and to bacteria of anthropogenic origin., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Avian colibacillosis is the most common bacterial disease affecting broilers. To better evaluate the diversity and the origin of the causative Escherichia coli strains infecting birds, we conducted a study on 80 broiler flocks. Just before the arrival of chicks on the farm, samples were collected in the farm environment (walls, feeders, air inlets, etc.) and, upon delivery, day-old chicks (DOCs) and the transport boxes were also sampled. Isolates were obtained from these samples, and from organs of chickens exhibiting typical colibacillosis symptoms. The isolates were characterized using high-throughput qPCR to detect a range of genetic markers (phylogroups, main serogroups virulence markers, etc.). A total of 967 isolates were studied, including 203 from 28 colibacillosis episodes, 484 from DOCs, 162 from transport boxes and 118 from the farm environment. These isolates yielded 416 different genetic profiles, of which 267 were detected in single isolates, and the others were observed in up to 44 isolates from nine farms. The distributions of isolates across phylogroups and the main serogroups varied with the origin of isolation. The isolates obtained from colibacillosis cases either shared a single genetic profile or were different. In a few cases, we observed the same profile for isolates obtained from DOCs and colibacillosis lesions in the same flock or different flocks. However, some flocks receiving DOCs contaminated with isolates bearing the genetic profile of colibacillosis cases identified in other flocks remained healthy. This study highlights the huge diversity among avian E. coli isolated from diseased and non diseased birds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We explored the between-group and temporal variations in the intestinal Escherichia coli populations of broilers under experimental conditions, taking both antimicrobial resistance and virulence into consideration. Four replicates of 45 commercial chicks were reared in four animal facilities. On their first day of life (Day 0), they were orally inoculated with two extended-spectrum-cephalosporin-resistant (ESCR) E. coli (2.72 log 10 CFU of a bla CMY-2- and 2.55 log 10 CFU of a bla CTX-M- carrying E. coli ). Faecal samples were then collected weekly and caecal samples were obtained from birds sacrificed on Days 21 or 42. The total, ESC-, ciprofloxacin- and gentamicin-resistant E. coli populations were enumerated on MacConkey (MC) and MC-supplemented media, and eight virulence-associated genes (VAGs) ( iroN, iutA, iss, ompT, hlyF, vat , frz orf4 , and fyuA ) were sought by PCR on isolates obtained on MC agar. The results showed significant between-group differences in the size of the resistant sub-populations and the presence of VAGs. Contrary to bla CTX-M -positive strains, bla CMY -positive strains persisted up to Day 42, but represented only a minor fraction of the total E. coli population. The ESC-, gentamicin- and ciprofloxacin-resistant populations decreased over time. Isolates obtained during the first week contained a mean of 5.1 VAGs. The percentages of some VAG profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. The fluctuations or differences between E. coli isolates according to group, age, and faecal or caecal origin need to be considered when designing experimental protocols and seeking to improve colibacillosis control. RESEARCH HIGHLIGHTS Temporal variations in the intestinal E. coli populations of broilers was studied. The antibiotic-resistant populations decreased over time. Virulence profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. Strains with the highest numbers of virulence genes were present during the first days.

Antimicrobial resistance is a "One Health" issue that requires improved knowledge of the presence and abundance of resistant bacteria in the environment. Extended-spectrum cephalosporins (ESCs) are critically important antibiotics (CIAs), and resistance to these CIAs is often encoded by beta-lactamase genes borne on conjugative plasmids. We thus decided to characterise 21 plasmids of ESC-resistant Escherichia coli randomly selected from isolates previously obtained from river water collected in a rural area in western France. The plasmids encoding ESC resistance were sequenced to investigate the diversity of the genes encoding ESC resistance and their genetic context. Sequences revealed that eleven IncI1 pMLST3 plasmids carried the bla CTX-M-1 and sul2 genes, and some of them also had the tet(A), aadA5 or dfrA17 genes. The bla CTX-M-1 gene was also detected on an IncN plasmid. Five plasmids obtained from four rivers contained bla CTX-M-14 , either on IncI1 or on IncFII plasmids. Two strains from two rivers contained bla CTX-M-15 on IncN pMLST7 plasmids, with qnrS1 and dfrA14 genes. One plasmid contained the bla CTX-M-55 , a bla TEM-1B -like, and fosA genes. One plasmid contained the bla CMY-2 gene. The diversity of the genes and plasmids of the resistant bacteria isolated from French rivers is probably related to the various animal and human origins of the isolated bacteria., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Colistin resistance associated with plasmidic resistance genes is a serious public health issue. We aimed at studying the transmission of an mcr-1 colistin- and rifampicin-resistant Escherichia coli strain between inoculated pigs and sentinels in different controlled conditions. Three groups of four pigs were bred in separated animal rooms and inoculated on Day 0 (D0). In each inoculated group, six contact pigs were introduced on D2. The first inoculated-and-contact group was left untreated. The ten pigs in the second inoculated-and-contact group received colistin (100 000 IU/kg) before inoculation or contact (D-7 to D-5), simulating prophylactic administration. Pigs in the third inoculated-and-contact group were treated just after inoculation or before transfer (D0 to D2), simulating metaphylactic administration. Faecal samples were regularly collected and segments of intestinal tracts were obtained at necropsy, on D20-D22. Samples were cultured on rifampicin-supplemented media, and PCR was used to detect the mcr-1 gene. The kinetics of infection, based on culture results, were analysed using an SIR model. The inoculated strain was detected in all inoculated and contact pigs. The SIR model showed that one infected pig could transmit the resistant bacteria to one susceptible individual in less than 3 h on average. Prophylactic administration significantly enhanced the transmission rate and resulted in more samples containing the mcr-1 resistance gene at necropsy. No effect of metaphylactic administration could be detected on the transmission rate, nor on the carriage of the resistant strain. Our study confirms that colistin should not be used in a prophylactic manner., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Colistin-resistant Escherichia coli are isolated from pigs suffering from post-weaning diarrhea (PWD). This study was designed to develop an experimental model of PWD using mcr-1-carrying shiga toxin-producing E. coli (STEC) or enterotoxigenic E. coli (ETEC), for the future evaluation of control measures. Three groups of eight piglets, kept in high biosecurity units, were orally inoculated with mcr-1-positive STEC or ETEC, and one unchallenged group was used as a control. Clinical signs were recorded. Regularly-collected fecal samples and samples obtained from the digestive tract of animals sacrificed one month after inoculation were cultured in selective media and isolates were characterized. Blood samples were used to genotype the polymorphisms of the pigs' intestinal receptors for F4 and F18 E. coli adhesins. Diarrhea was more frequent and more fecal samples contained the inoculated strain in the group inoculated with the O149-F4 ETEC strain than with the O141-F18 or O139-F18 STEC strains. However, fewer positive samples were obtained from the two pigs with the F4 resistant genotype. The three inoculated strains could be re-isolated up to the end of the experiment. Excretion peaked on the first week after inoculation with the O149-F4 ETEC strain, and later for the other two. An mcr-1 gene transfer to other commensal isolates was observed only for O139-F18 STEC, while the loss of mcr-1 from the inoculated strain occurred in all groups. The O149-F4 ETEC challenge may be used to evaluate alternative solutions to combat PWD caused by colistin-resistant E. coli in pigs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Resistance 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 bla CTX-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 bla CTX-M-27 and bla CTX-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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Colistin resistance in Enterobacteriaceae is a public health problem. The present study was designed to evaluate the dissemination of a colistin-resistant Escherichia coli strain and its resistance gene, mcr-1, between orally inoculated pigs and their contacts. A non-inoculated control group, one low-dose and one high-dose group-both including two pens of two inoculated and three contact pigs-were raised in separate rooms. After inoculation of a colistin- and rifampicin-resistant E. coli suspension (2.5 × 10 5 CFU/pig for the low-dose group and 2.5 × 10 8 CFU/pig for the high-dose group), feces from inoculated and non-inoculated contact pigs were collected and inoculated on colistin- and rifampicin-supplemented media directly or after enrichment in rifampicin-supplemented media, then the isolates were characterized. PCR was used to detect the mcr-1 gene in lysates from feces cultivated in colistin-supplemented broth and DNA prepared from feces. Results showed that the low-dose inoculum was probably insufficient to obtain durable colonization, but could lead to the temporary presence of mcr-1-positive E. coli strains. The high-dose inoculum resulted in durable colonization of both inoculated and contact animals. In all groups, the mcr-1 gene was also detected in rifampicin-susceptible strains, suggesting its transfer to several commensal strains. A comparison of detection methods showed that more positive samples were obtained with cultures in rifampicin-supplemented media and suggests that current methods to evaluate the prevalence of colistin resistance in fecal samples suffer from poor sensitivity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antimicrobial-resistant Escherichia coli may be present in various foods. The aim of this study was to evaluate the impact of heat treatment, simulating food preparation, on the possibility of antimicrobial resistance genes being transferred from E. coli cells. The study was performed on antimicrobial-resistant E. coli cells in suspension in a sterile saline solution. The stability of resistance genes and the possibility of their transfer by transformation or conjugation were analyzed. Results showed that antimicrobial-resistant E. coli cells managing to survive after a few minutes at 60 °C retained their antimicrobial resistance. No plasmid could be transferred by conjugation from antimicrobial-resistant E. coli cells heated to 60 °C for ten or more minutes. Twelve electroporation experiments were performed using a bacterial suspension heated to 70 °C for 30 min. Genes coding for resistance to extended-spectrum cephalosporins, tetracycline or sulfonamides were transferred to an E. coli DH5α recipient on two occasions. In conclusion we showed that heat-treated E. coli may occasionally transfer resistance genes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Resistance to extended-spectrum cephalosporins (ESCs) is mostly borne by conjugative plasmids. The aim of the present study was to evaluate the characteristics and diversity of ESC resistance plasmids in Escherichia coli from different free-range broiler flocks in France, and their persistence in flocks during rearing. Two hatcheries were selected. Faecal samples from 11 flocks were collected from before their arrival on the broiler production farm up to their slaughter at the end of the rearing period. A selection of 25 E. coli isolates obtained at different times from different flocks but all harbouring an ESC resistance gene was characterised. The plasmids coding for ESC resistance were sequenced using Mi-seq Illumina technology or the ion proton system (Ion Torrent). Ten IncI1 ST12 plasmids carried the bla CMY-2 gene, and most of them had no other resistance genes. All bla CMY-2 plasmids were obtained from day-old to 7-day-old chicks from four flocks hatched at the same hatchery and sent to three different farms. Sequence comparisons showed identity percentages higher than 99%. Fifteen IncI1 ST3 plasmids were obtained from day-old to 77-day-old broilers from seven flocks on six farms. These plasmids harboured the bla CTX-M-1 gene, and most also had the tet(A) and sul2 genes, with sequence identity higher than 99%. For both types of plasmid, very high identity percentages were also obtained with published sequences of plasmids isolated from broilers in other countries or from other animal species. Thus, unlike the IncI1 ST12 bla CMY-2 plasmids, the epidemic nature of the IncI1 ST3 bla CTX-M-1 plasmids in the French poultry production makes it difficult to determine the origin of a contamination which may persist for weeks in a flock., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

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, stx 2 e , 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.

We have developed a phenotypic method suited to the systematic screening of resistance to colistin in E. coli, including those with the mcr-1 gene, by the absence of an inhibition zone after an application of a single drop of 8mg/L colistin solution on a previously inoculated Mueller-Hinton agar., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The application of manure may result in contamination of the environment with antimicrobials, antimicrobial-resistant bacteria, resistance genes and plasmids. The aim of this study was to investigate the impact of the administration of colistin and of manure management on (i) the presence of colistin-resistant Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa and (ii) the prevalence of various antimicrobial resistance genes in feces and in composted or stored manure. One flock of chickens was treated with colistin at the recommended dosage and a second flock was kept as an untreated control. Samples of feces, litter and stored or composted manure from both flocks were collected for isolation and determination of the colistin-susceptibility of E. coli, K. pneumoniae and P. aeruginosa and quantification of genes coding for resistance to different antimicrobials. The persistence of plasmids in stored or composted manure from colistin-treated broilers was also evaluated by plasmid capturing experiments. Results revealed that colistin administration to chickens had no apparent impact on the antimicrobial resistance of the dominant Enterobacteriaceae and P. aeruginosa populations in the chicken gut. Composting stimulated an apparently limited decrease in genes coding for resistance to different antimicrobial families. Importantly, it was shown that even after six weeks of composting or storage, plasmids carrying antimicrobial resistance genes could still be transferred to a recipient E. coli. In conclusion, composting is insufficient to completely eliminate the risk of spreading antimicrobial resistance through chicken manure., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Eleven avian pathogenic Escherichia coli (APEC) strains isolated from 2006 to 2010 from different farms in Algeria and resistant to cephalosporins were studied. Their susceptibility to antimicrobials was determined by disk diffusion, and the genes responsible for resistance to critical antimicrobials were studied by PCR, sequencing, and conjugation. Their genetic profiles were compared by pulsed-field gel electrophoresis (PFGE). All strains were resistant to extended-spectrum cephalosporins, ciprofloxacin, tetracycline, trimethoprim-sulfamethoxazole, and neomycin and showed the same PFGE profile. For most of them, resistance was encoded by a nontransferable group 1 bla(CTX-M) gene, and multiple mutations were detected in the quinolone resistance-determining regions. The clonal dissemination of this resistant APEC is worrying for animal and public health.

Resistance of Escherichia coli to third-generation cephalosporin (3GC) in fecal samples representative of French egg production was studied. The susceptibility to cefotaxime of E. coli isolates obtained by culture on nonselective media was determined. Twenty-two nonsusceptible isolates were obtained (7.51%; 95% confidence interval, 4.49 to 10.54%), the majority of which came from young birds. Most isolates carried a blaCTX-M-1 group gene, and a few carried a blaCMY-2-like gene. Control of 3GC resistance in laying hens is needed.

Histomoniasis in turkeys can be prevented by administering paromomycin sulfate, an aminoglycoside antimicrobial agent, in feed. The aim of this study was to evaluate the impact of in-feed paromomycin sulfate supplementation on the antimicrobial resistance of intestinal bacteria in turkeys. Twelve flocks of breeder turkeys were administered 100 ppm paromomycin sulfate from hatching to day 120; 12 flocks not supplemented with paromomycin were used as controls. Faecal samples were collected monthly from days 0 to 180. The resistance of Escherichia coli, Enterococcus faecium and Staphylococcus aureus to paramomycin and other antimicrobial agents was compared in paromomycin supplemented (PS) and unsupplemented (PNS) flocks. E. coli from PS birds had a significantly higher frequency of resistance to paromomycin, neomycin and kanamycin until 1 month after the end of supplementation compared to PNS birds. Resistance to amoxicillin or trimethoprim-sulfamethoxazole was also more frequent in PS turkeys. Resistance was mainly due to the presence of aph genes, which could be transmitted by conjugation, sometimes with streptomycin, tetracycline, amoxicillin, trimethoprim or sulfonamide resistance genes. Resistance to kanamycin and streptomycin in E. faecium was significantly different in PS and PNS breeders on days 60 and 90. Significantly higher frequencies of resistance to paromomycin, kanamycin, neomycin and tobramycin were observed in S. aureus isolates from PS birds. Paromomycin supplementation resulted in resistance to aminoglycosides in bacteria of PS turkeys. Co-selection for resistance to other antimicrobial agents was observed in E. coli isolates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An experiment was performed to compare the microbiological efficacy of four treatments (oxytetracycline, trimethoprim-sulphonamide, amoxicillin (AMX) or enrofloxacin (ENR)) to control experimental colibacillosis induced by an avian pathogenic Escherichia coli (APEC) with reduced susceptibility to fluoroquinolones. The protocol was also developed in order to study resistance gene transfer. Broilers were first orally inoculated with multiresistant E. coli bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-M or blaFOX), tetracycline or trimethoprim-sulphonamide. They were then inoculated in their air sacs with the APEC and treated as soon as symptoms appeared. Internal organs from dead or sacrificed birds were cultivated on non-supplemented or supplemented media. The inoculated O78 APEC was recovered significantly less frequently in ENR treated group (26%) compared to untreated group (47%). This was not true for other treated groups. Isolates obtained on non-supplemented media had the same susceptibility profile as the inoculated APEC. However, one isolate from the AMX-treated group obtained on AMX-supplemented media was resistant to AMX only, and one isolate from the same group obtained on ENR-supplemented media, showed a resistance profile suggesting acquisition of one of the multiresistance plasmids present in the intestinal microbiota. Molecular analysis performed on this multiresistant isolate confirmed the presence of a conjugative plasmid with qnr and blaCTX-M resistance genes. Thus, the experiment illustrated the emergence of resistant isolates in internal organs, probably via acquisition of a plasmid from the intestinal microbiota., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments-oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)-on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (bla(CTX-M) or bla(FOX)), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, "old" antimicrobials may coselect antimicrobial resistance to recent and critical molecules.

The clinical and microbial efficacy of antimicrobial treatments of avian colibacillosis was studied, using an experimental model on chickens previously inoculated with multiresistant commensal Escherichia coli strains. One E. coli with pMG252 plasmid containing bla(FOX5) and qnrA1 genes and another E. coli with pMG298 plasmid containing bla(CTX-M15) and qnrB1 genes were first orally inoculated to chickens Both isolates were also resistant to chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin, gentamicin, kanamycin, and tetracycline. The birds were then experimentally infected with an avian pathogenic E. coli (APEC), via the air sac. Treatments (oxytetracycline (OTC), trimethoprim-sulfadimethoxin (SXT), amoxicillin (AMX) or enrofloxacin (ENR) were then offered at the therapeutic doses. Symptoms, lesions in dead or sacrificed birds, and isolation and characterization of APEC from internal organs were studied. Results showed that OTC, SXT or ENR treatments could control the pathology. AMX worsened the disease, possibly due to endotoxin shock. All APEC re-isolated from internal organs showed the same antimicrobial susceptibility as the APEC inoculated strain, except for one APEC isolate from an infected OTC-treated bird, which acquired tetracycline resistance only, and one APEC isolate recovered from the air sacs of a chicken in the infected SXT-treated group, which acquired the pMG252 plasmid and became multi-resistant. Thus three antimicrobials could control the disease but the experimental model enabled, to our knowledge, the first observation of plasmid transfer from a bacterium of the intestinal tract to a pathogenic isolate from the respiratory tract., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of this assay was to develop an experimental model of digestive colonization of chickens with bacteria harboring qnr, extended-spectrum beta-lactamase, or ampC genes. Specific pathogen-free chickens were orally inoculated with two Escherichia coli strains containing either the plasmid pMG252 bearing bla(FOX) and qnrA genes, or pMG298 bearing bla(CTX-M) and qnrB genes. Analysis of strains isolated from fecal samples showed that the two strains were able to persist for several weeks in the digestive flora of inoculated birds and could rapidly spread to noninoculated ones. However, the multi-resistant isolates were maintained as a small proportion of the overall enterobacterial population. The qnr, extended-spectrum beta-lactamase, and ampC resistance genes could be transferred, in vivo, in the absence of selective pressure, to other chicken E. coli or Klebsiella pneumoniae isolates., (© Mary Ann Liebert, Inc.)

Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.