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International audience; Streptococcus suis is a zoonotic pathogen causing important economic losses in swine production. The most commonly used antibiotics in swine industry are tetracyclines, beta-lactams, and macrolides. Resistance to these antibiotics has already been observed worldwide (reaching high rates for macrolides and tetracyclines) as well as resistance to aminoglycosides, fluoroquinolones, amphenicols, and glycopeptides. Most of the resistance mechanisms are encoded by antibiotic resistance genes, and a large part are carried by mobile genetic elements (MGEs) that can be transferred through horizontal gene transfer. This review provides an update of the resistance genes, their combination in multidrug isolates, and their localization on MGEs in S. suis. It also includes an overview of the contribution of biofilm to antimicrobial resistance in this bacterial species. The identification of resistance genes and study of their localization in S. suis as well as the environmental factors that can modulate their dissemination appear essential in order to decipher the role of this bacterium as a reservoir of antibiotic genes for other species.

Highlights • mcr-1-positive ETEC and STEC strains persisted up to four weeks after inoculation. • O149-F4 ETEC inoculation resulted in hyperthermia and moderate diarrhea. • Fewer positive samples were obtained from pigs with the F4 resistant genotype. • An mcr-1 gene transfer to other commensal isolates was observed for O139-F18 STEC., 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.

International audience; Livestock data on antimicrobial resistance (AMR) are commonly collected from bacterial populations of clinical and non-clinical isolates. In contrast to data on non-clinical isolates from livestock, data on clinical isolates are not harmonized in Europe. The Normalized Resistance Interpretation (NRI) method was applied to overcome the lack of harmonization of laboratory methods and interpretation rules between monitoring systems. Statistical analyses were performed to identify associations between the isolate type (clinical vs. non-clinical) and resistance to four antimicrobials (ampicillin, tetracycline, gentamicin, and nalidixic acid) per animal category in Germany and France. Additional statistical analyses comparing clinical and non-clinical isolates were performed with the available data on the same antimicrobial panel and animal categories from the UK and Norway. Higher resistance prevalence was found in clinical isolates compared to non-clinical isolates from calves to all antimicrobials included in Germany and France. It was also found for gentamicin in broilers from France. In contrast, in broilers and turkeys from Germany and France and in broilers from the UK, a higher resistance level to ampicillin and tetracycline in non-clinical isolates was encountered. This was also found in resistance to gentamicin in isolates from turkeys in Germany. Resistance differed within countries and across years, which was partially in line with differences in antimicrobial use patterns. Differences in AMR between clinical and non-clinical isolates of Escherichia coli are associated with animal category (broiler, calf, and turkey) and specific antimicrobials. The NRI method allowed comparing results of non-harmonized AMR systems and might be useful until international harmonization is achieved.

By 2010, systems set up to monitor the antimicrobial resistance of pathogenic bacteria and antimicrobial usage identified a sustained increase regarding third‐ and fourth‐generation cephalosporin resistance in French pig production. This sector mobilised and collectively committed to responsible action in the following months. This led to a multi‐professional voluntary stewardship programme that was started in 2011. A consensus of veterinary opinion led to the definition of restrictive rules on the prescription of the third‐ and fourth‐generation cephalosporins targeted by the antimicrobial stewardship programme (ASP). All pig sector professionals, including farmers, were informed. Existing monitoring systems for usage and resistance were supplemented by data from the records of veterinarians' cephalosporin deliveries and from individual pig farm surveys investigating antimicrobial usage. The second step, from 2014, entailed regulatory measures that consolidated the programme by setting quantitative reduction objectives and specifying the terms and conditions for prescribing and dispensing a list of critical antimicrobial molecules including cephalosporins. All the data sources confirmed a significant fall of more than 90% in cephalosporin usage in the French pig production sector between 2010 and 2016. Monitoring systems recorded that the resistance of commensal and pathogenic Escherichia coli isolates also tended to decrease over the same period. The stewardship programme proved highly effective in reducing usage and containing resistance, illustrating the efficiency of a well‐defined multi‐professional strategy., This study details success conditions of an antimicrobial stewardship program in animal agriculture. Based on data provided by complementary and independent monitoring tools, success could be assessed on both usage and resistance. Results highlight the strong relationship between cephalosporins usage and resistance in pig production, supporting a containment perspective through usage restriction.

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty-one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22 ± 2℃ and for the 177 Yersinia ruckeri they were read after 24-28 hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44-48 hr. Applying epidemiological cut-off values to the susceptibility data generated in these tests, the isolates were categorized as wild-type or non-wild-type. The agent-specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation.

Objectives To characterize and compare resistance trends in clinical Escherichia coli isolates from humans, food-producing animals (poultry, cattle and swine) and pets (dogs and cats). Methods Antibiogram results collected between January 2014 and December 2017 by MedQual [the French surveillance network for antimicrobial resistance (AMR) in bacteria isolated from the community] and RESAPATH (the French surveillance network for AMR in bacteria from diseased animals) were analysed, focusing on resistance to antibiotics of common interest to human and veterinary medicine. Resistance dynamics were investigated using generalized additive models. Results In total, 743 637 antibiograms from humans, 48 170 from food-producing animals and 7750 from pets were analysed. For each antibiotic investigated, the resistance proportions of isolates collected from humans were of the same order of magnitude as those from food-producing animals or pets. However, resistance trends in humans differed from those observed in pets and food-producing animals over the period studied. For example, resistance to third-generation cephalosporins and fluoroquinolones was almost always below 10% for both humans and animals. However, in contrast to the notable decreases in resistance observed in both food-producing animals and pets, resistance in humans decreased only slightly. Conclusions Despite several potential biases in the data, the resistance trends remain meaningful. The strength of the parallel is based on similar data collection in humans and animals and on a similar statistical methodology. Resistance dynamics seemed specific to each species, reflecting different antibiotic-use practices. These results advocate applying the efforts already being made to reduce antibiotic use to all sectors and all species, both in human and veterinary medicine.

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

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.

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 blaCMY-2 gene, and most of them had no other resistance genes. All blaCMY-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 blaCTX-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 blaCMY-2 plasmids, the epidemic nature of the IncI1 ST3 blaCTX-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.

For ethical, technical, regulatory, and cost reasons, in vitro methods are increasingly used as an alternative to in vivo experimentations. The aim of the present study was to validate, according to in vivo data in living animals, a new in vitro model of the piglet colon, the PigutIVM, under both control conditions and antibiotic disturbance by the widely used colistin. The PigutIVM reproduces the main biotic and abiotic parameters of the piglet colon: temperature, pH, retention time, supply of ileal effluents, complex, and metabolically active microbiota and self-maintained anaerobiosis. Under both control and antibiotic-treated conditions, qPCR analyses showed that the main bacterial populations of piglet gut microbiota were similar in vitro and in vivo, with Pearson correlation coefficient higher than 0.9. During colistin administration, both in piglets and in the in vitro model, a significant decrease in Escherichia coli populations was observed together with changes in microbial composition of subdominant populations. SCFA concentrations were similar in vitro and in vivo and were not modified by colistin. Interestingly, the administration of the probiotic Saccharomyces cerevisiae var. boulardii CNCM I-1079 led in vitro to a decrease in E. coli levels, as previously observed when the antibiotic treatment was applied. This new in vitro model of the piglet colon provides a flexible, reproducible, and cost-effective tool for the screening of drugs or new dietary compounds, such as pre- or probiotics. It will be helpful for researchers, feed producers, or veterinarians when developing innovative non-antibiotic strategies.

International audience; Objectives: To examine the relevance of co-resistance to amoxicillin and tetracycline as an indicator of multidrug resistance (MDR) in animal health. Methods: Escherichia coli isolates collected between 2012 and 2016 by the French surveillance network for antimicrobial resistance in diseased animals (RESAPATH) were analyzed. The proportions of MDR isolates and the proportions of isolates presenting co-resistance to amoxicillin and tetracycline were calculated for seven animal species (cattle, horse, dog, swine, poultry, duck, and turkey). The degree of agreement between these two proportions was estimated by calculating the kappa value. Results: In total, 55,904 isolates were analyzed. MDR proportions were variable among animal species, ranging from 21.9% [20.2; 23.7] in horses to 56.0% [55.4; 56.7] in cattle. A similar situation was observed for proportions of isolates with co-resistance to amoxicillin and tetracycline, with the highest value for cattle 65.0% [64.3; 65.6]. This co-resistance was also most often associated with resistance to other antibiotics, regardless of the animal species considered. Comparative analysis showed substantial agreement between MDR and this co-resistance, with a kappa value of 0.75, all animal species considered. Conclusion: Given the widespread use of penicillins and tetracyclines in animal health, co-resistance to amoxicillin and tetracycline could be an efficient indicator of MDR in E. coli isolates. Based on a specific resistance profile and not an arbitrary number of resistances compared with MDR, this potential indicator is also precise, convenient and suitable for routine use.

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.

Surveillance of Pasteurella multocida resistance in food-producing animals is essential to guide the first-line treatment of respiratory diseases and to limit economic losses. Since Pasteurella are the most common bacteria isolated from dog and cat bites, this surveillance is also needed to guide treatment in humans in case of bites. The aim of this study was to characterize the phenotypic resistance of P. multocida strains isolated from respiratory infections in animals, including both food-producing animals and pets. Data collected between 2012 and 2017 by the French national surveillance network for antimicrobial resistance referred to as RESAPATH were analyzed. The proportions of resistance to antimicrobials of relevance in veterinary and human medicines were estimated for each animal species. For cattle, resistance trends over the period were investigated using non-linear analysis applied to time-series. In total, 5356 P. multocida isolates were analyzed. Proportions of resistance of P. multocida were almost all below 20% over the period, and, more precisely, all resistance proportions were below 10% for rabbits, sheep and dogs. The highest resistance proportions to enrofloxacin were identified for cattle (4.5%) and dogs (5.2%). Despite its frequent use in livestock, resistance to florfenicol was less than 1% in P. multocida strains, regardless of the animal species considered. Time series analyses revealed continuous increases in resistance to tetracycline, tilmicosin, flumequine and fluoroquinolones in P. multocida strains isolated from cattle. These trends contrast with the decrease in use of antibiotics in cattle in France and with the decrease in resistance observed in E. coli isolated from diseased cattle.

Resistance to extended-spectrum cephalosporins (ESCs) is a matter of considerable concern for public health. Here, we studied the spontaneous loss of an extended-spectrum beta-lactamase (ESBL)-encoding plasmid from a rifampin-resistant Escherichia coli isolate orally inoculated into pigs under controlled conditions. Fecal samples were collected and cultured on rifampin-supplemented medium, and the resistance of the E. coli isolates to ESCs was studied by phenotypic tests, PCR detection of plasmid genes, and complete sequencing. The results showed that only 3 out of 353 rifampin-resistant E. coli isolates were ESC susceptible, and PCR and bioinformatics analysis confirmed the loss of the plasmid. These in vivo experiments indicate that the loss of an ESBL-encoding plasmid seems a rare event in gut microbiota.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli . All isolates were negative for the major virulence factors/toxins tested ( ehxA , cdt , heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

This paper presents the impact on antimicrobial resistance (AMR) in poultry and pig bacteria of the French EcoAntibio plan, a public policy to reduce antimicrobial use in animals. The analysis was performed using sales data of veterinary antimicrobials and AMR data from bacteria obtained at slaughterhouse and from diseased animals. From 2011-2018, fluoroquinolones exposure decreased by 71.5 % for poultry and 89.7 % for pigs. For Campylobacter jejuni isolated from broilers at slaughterhouses, ciprofloxacin resistance increased from 51 % in 2010 to 63 % in 2018, whereas for turkeys the percentages varied from 56 % in 2014 to 63 % in 2018. For commensal E. coli isolated from the caecal content of broilers at slaughterhouses, the resistance to ciprofloxacin - assessed using an epidemiological cut-off value - increased in broiler isolates from 30.7 % in 2010 to 38.1 % in 2018. In turkeys, the percentage of resistant E. coli isolates decreased from 21.3 % in 2014 to 15.2 % in 2018, whereas in pigs, it increased from 1.9 % in 2009 to 5.5 % in 2017. However, for E. coli isolated from diseased animals, when the breakpoints of 2018 were applied, resistance to fluoroquinolones significantly decreased between 2010 and 2018 from 9.0%-5.4% for broilers/hens, from 7.4 % to 3.4 % for turkeys and from 9.4 % to 3.6 % for pigs. These data show that the major, rapid decrease in the exposition to fluoroquinolones had contrasting effects on resistance in the diverse bacterial collections. Co-selection or fitness of resistant strains may explain why changes in AMR do not always closely mirror changes in use.

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 × 105 CFU/pig for the low-dose group and 2.5 × 108 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.

International audience; 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 bla CTX-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 bla CTX-M-1-containing plas-mid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the bla CMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The bla CMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a bla CTX-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 bla CMY-2 plasmid. In conclusion , our results show the dominance and high similarity of bla CTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a bla CMY-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, 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.

SUMMARY 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 blaCTX-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.

International audience; Antimicrobial resistance (AMR) among bacteria isolated from food-producing animals is a growing concern with implications for public health. AMR surveillance is essential to identify resistance trends and help in the design of effective and efficient control strategies. The aim of the study was to describe the antimicrobial susceptibility of pathogenic Escherichia coli isolated from three livestock productions in France (cattle, swine and poultry). The trend in resistance to the most commonly prescribed antibiotics in animal health was analysed as follows: amoxicillin (penicillin), spectinomycin or streptomycin (aminoglycoside), tetracycline and trimethoprim-sulfamethoxazole/Enrofloxacin and ceftiofur were also taken into account as members of critically important antimicrobial families in human and veterinary medicine, that is fluoroquinolones and third-generation cephalosporins, respectively. Data collected between 2002 and 2015 by the French national surveillance network of AMR referred to as RESAPATH were analysed. Resistance trends were investigated using non-linear analysis (generalized additive models) applied to time-series stratified by livestock production and antibiotic. Irrespective of the species and the antibiotic considered, resistance signals over time showed no significant annual cycle. Resistance to third-generation cephalosporins emerged during the period of the study, with a peak at 22% [20.5; 24.0] in poultry in 2010, decreasing afterwards, while it remained consistently below 10% for the other species. The proportion of resistance to fluoroquinolones was broadly similar between species and remained under 30%, with a slight decreasing trend after 2009. Resistances to tetracycline and amoxicillin remained high, between 90% and 40% over time in cattle and swine. After 2010, there was a decrease in resistance to these antibiotics for all species, especially to tetracycline for poultry with a drop from 84% in 2009 to 43% in 2015. These results contribute to risk assessment and constitute objective evidence on which to evaluate the efficacy of control measures implemented to limit AMR occurrence.

The importance of the role of environment in the dissemination of antimicrobial resistant bacteria is now well recognized. Thus, bacterial indicators to monitor the phenomena are required. The Aeromonas genus is autochthonous in the aquatic environment and easy to detect in any water type, such as freshwater, or wastewater. These microorganisms are also causing infections in humans and animals (including fish). Furthermore, as Aeromonas spp. is able to acquire antimicrobial resistance mechanisms, it is candidate for indicator bacteria to follow antimicrobial resistance dissemination in aquatic environments. Unfortunately, to date, interpretation criteria for Aeromonas spp. for antimicrobial susceptibility tests are scarce in the literature. No epidemiological cut-off values for Aeromonas are currently available at EUCAST to interpret Minimum Inhibitory Concentrations (MIC). The only interpretation criteria available are clinical breakpoints from CLSI that are adapted from Enterobacteriaceae. Based on the results of MIC distributions obtained for a collection of environmental isolates of Aeromonas, this study aimed at proposing tentative epidemiological cut-off values (COWT) for Aeromonas spp. assessing whether the genus is an acceptable level of definition. Thus, 233 isolates collected from 16 rivers were identified at species level using Maldi-Tof (Bruker). Eleven different species were identified, the most abundant were A. bestiarum (n = 54), A. salmonicida (n = 45), A. sobria (n = 41), and A. eucrenophila (n = 37). 96-well micro-plates containing different concentrations of 15 antimicrobials, namely cefotaxime, ceftazidime, chloramphenicol, colistin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, nalidixic acid, oxolinic acid, streptomycin, temocillin, tetracycline, and trimethoprim-sulfamethoxazole, were prepared. The broth micro-dilution method was used to determine the antimicrobial susceptibility of each isolate. The estimation of COWT values was satisfactory obtained at genus level for all antimicrobials except cefotaxime and erythromycin. This first step is an invitation for other research teams to increase the amount of antimicrobial resistance data collected. Then, robustness of our proposed provisional generic epidemiological cut-off values could be assessed by testing antimicrobial susceptibility of various Aeromonas collections.

International audience; The early 2000s marked the end of the Golden age of the antibiotics and the beginning of the awareness on the potential threat to human health due to the dissemination of antimicrobial resistance. As a base-line study, we investigated the antimicrobial susceptibility of 99 strains of non-O1/non-O139 Vibrio cholerae isolated fromwastewater and shellfish in 2000/2001 within La Rance estuary (Brittany, France). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, nalidixic acid, ciprofloxacin, norfloxacin, amikacin, gentamicin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and erythromycin. The only resistances were to streptomycin, sulfonamides and ampicillin: 54.6% of the isolates had acquired resistance to at least one antimicrobial agent among them and only six isolates from cockles were multidrug resistant. On the basis of the distribution of a limited selection of resistance associated genes, our study shows that V. cholerae can constitute an environmental reservoir for these genes. However, none of our isolates harbored integron. This result casts doubt on the capacity of non-O1/non-O139 V. cholerae to acquire resistance-associated genes in such context, and on its potential role of indicator of the dissemination of antimicrobial resistance in the aquatic environment.

Four trials were conducted to evaluate the impact of Escherichia coli probiotic strain ED1a administration to pigs on the gut carriage or survival in manure of extended-spectrum-β-lactamase-producing E. coli . Groups of pigs were orally inoculated with strain E. coli M63 carrying the bla CTX-M-1 gene ( n = 84) or used as a control ( n = 26). In the first two trials, 24 of 40 E. coli M63-inoculated pigs were given E. coli ED1a orally for 6 days starting 8 days after oral inoculation. In the third trial, 10 E. coli M63-inoculated pigs were given either E. coli ED1a or probiotic E. coli Nissle 1917 for 5 days. In the fourth trial, E. coli ED1a was given to a sow and its 12 piglets, and these 12 piglets plus 12 piglets that had not received E. coli ED1a were then inoculated with E. coli M63. Fecal shedding of cefotaxime-resistant Enterobacteriaceae (CTX-RE) was studied by culture, and bla CTX-M-1 genes were quantified by PCR. The persistence of CTX-RE in manure samples from inoculated pigs or manure samples inoculated in vitro with E. coli M63 with or without probiotics was studied. The results showed that E. coli M63 and ED1a were good gut colonizers. The reduction in the level of fecal excretion of CTX-RE in E. coli ED1a-treated pigs compared to that in nontreated pigs was usually less than 1 log 10 CFU and was mainly observed during the probiotic administration period. The results obtained with E. coli Nissle 1917 did not differ significantly from those obtained with E. coli ED1a. CTX-RE survival did not differ significantly in manure samples with or without probiotic treatment. In conclusion, under our experimental conditions, E. coli ED1a and E. coli Nissle 1917 could not durably prevent CTX-RE colonization of the pig gut.

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.

Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 μg/ml. When nisin A (1.70 μg/ml) or pediocin PA-1/AcH (1.56 μg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 μg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 μm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.

We investigated the antimicrobial susceptibility of 50 environmental isolates of Vibrio cholerae non-O1/non-O139 collected in surface waters in Haiti in July 2012, during an active cholera outbreak. A panel of 16 antibiotics was tested on the isolates using the disk diffusion method and PCR detection of seven resistance-associated genes (strA/B, sul1/2, ermA/B, and mefA). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, ciprofloxacin, norfloxacin, amikacin, and gentamicin. Nearly a quarter (22.0%) of the isolates were susceptible to all 16 antimicrobials tested and only 8.0% of the isolates (n = 4) were multidrug-resistant. The highest proportions of resistant isolates were observed for sulfonamide (70.0%), amoxicillin (12.0%), and trimethoprim-sulfamethoxazole (10.0%). One strain was resistant to erythromycin and one to doxycycline, two antibiotics used to treat cholera in Haiti. Among the 50 isolates, 78% possessed at least two resistance-associated genes, and the genes sul1, ermA, and strB were detected in all four multidrug-resistant isolates. Our results clearly indicate that the autochthonous population of V. cholerae non-O1/non-O139 found in surface waters in Haiti shows antimicrobial patterns different from that of the outbreak strain. The presence in the Haitian aquatic environment of V. cholerae non-O1/non-O139 with reduced susceptibility or resistance to antibiotics used in human medicine may constitute a mild public health threat.

International audience; 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.

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.

Colistin has been used in veterinary medicine for decades, mainly for the prevention and treatment of Enterobacteriaceae infections. However, data regarding colistin resistance in bacteria from animals and food of animal origin are relatively scarce, partly because there are methodological difficulties hampering the analysis of susceptibility to colistin. Most data regarding clinical isolates are related to enteropathogenic Escherichia coli and Salmonella. The resistance percentages are sometimes high for pathogenic strains, and the mcr-1 gene has been detected in pathogenic E. coli isolates from pigs, cattle and poultry in different countries. The prevalence of colistin resistance in Salmonella from healthy animals is usually low but depends on the proportion of intrinsically colistin-resistant serotypes. For indicator E. coli, the resistance levels are often very low, although higher levels have been observed in Asia. The mcr-1 gene has been detected in indicator E. coli from pigs, cattle, poultry and their products. Thus, there is an urgent need to re-assess the use of colistin in livestock throughout the world to ensure a global strategy for preserving this last-resort antimicrobial.

Escherichia coli strains from swine origin, either susceptible or resistant to colistin, were grown under planktonic and biofilm cultures. After which, they were treated with antibacterial agents including nisin and enterocin DD14 bacteriocins, colistin and their combinations. Importantly, the combination of colistin, enterocin DD14 and nisin eradicated the planktonic and biofilm cultures of E. coli CIP54127 and the E. coli strains with colistin-resistance phenotype such as E. coli 184 (mcr-1(+)) and E. coli 289 (mcr-1(-)), suggesting therefore that bacteriocins from lactic acid bacteria could be used as agents with antibiotic augmentation capability.

Summary Aims: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 104 colony-forming units per animal, and three naive pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naive contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 104 colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

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.

This study was conducted to determine the occurrence of zinc and copper resistances in methicillin-resistant Staphylococcus aureus (MRSA) from swine and veal calves in a global strain collection. The test population consisted of 476 porcine MRSA isolates from ten European countries, 18 porcine MRSA isolates from Canada and seven MRSA from China, 92 MRSA and 60 methicillin-susceptible S. aureus (MSSA) isolates from veal calves in the Netherlands and 88 porcine MSSA isolates from four European countries. Most porcine MRSA (n=454) and all bovine MRSA belonged to clonal complex (CC) 398 whereas 37 of the pig MRSA from Europe and the seven Chinese isolates belonged to other CCs and 3 isolates were not classified into a CC. All isolates were tested for susceptibility to zinc chloride and copper sulphate using agar dilution and tested by PCR for the czrC gene encoding zinc resistance. Phenotypic zinc resistance (MIC>2mM) was observed in 74% (n=324) and 42% (n=39) of European MRSA CC398 from pigs and veal calves, respectively, and in 44% of the Canadian isolates (n=8), but not among the Chinese isolates. Almost all (99%) zinc-resistant MRSA carried czrC. Of the 37 European non-CC398 MRSA, 62% were resistant to zinc, but only 46% of them carried czrC. The MICs of the MSSA isolates to zinc chloride ranged from 1 to 4mM and none carried czrC. The MICs of copper sulphate were associated neither with methicillin resistance nor with the detection of czrC. This study showed that zinc resistance and the czrC gene are widespread among CC398 MRSA isolates. This suggests that the use of zinc in feed might have contributed to the emergence of MRSA.

The aim of the experiment was to evaluate under controlled conditions the impact on the excretion of 3GC-resistant Escherichia coli of the injection of one-day-old chicks with ceftiofur, a third-generation cephalosporin (3GC). Three isolators containing specific-pathogen-free chicks were used. In the first one, 20 birds were injected with ceftiofur then ten of them were orally inoculated with a weak inoculum of a 3GC-resistant E. coli field isolate containing an IncI1/ST3 plasmid encoding a blaCTX-M-1 beta-lactamase. The other chicks were kept as contact birds. None of the 20 birds in the second isolator were injected with ceftiofur, but ten of them were similarly inoculated with the 3GC-resistant strain and the others kept as contact birds. A third isolator contained ten non-injected, non-inoculated chicks. Fecal samples were collected regularly over one month and the E. coli isolated on non-supplemented media were characterized by antimicrobial agar dilution, detection of selected resistance genes and determination of phylogenetic group by PCR. The titers of 3GC-resistant E. coli in individual fecal samples were evaluated by culturing on 3GC-supplemented media. Results showed that the inoculated strain rapidly and abundantly colonized the inoculated and contact birds. The ceftiofur injection resulted in significantly higher percentages of 3GC-resistant E. coli isolates among the analyzed E. coli. No transfer of the 3GC-encoding plasmid to other isolates could be evidenced. In conclusion, these results highlight the dramatic capacity of 3GC-resistant E. coli to colonize and persist in chicks, and the selecting pressure imposed by the off-label use of ceftiofur.

International audience

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli . Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1 . On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli . Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli , probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

We investigated the impact of the hatchery practice of administering third-generation cephalosporin (3GC) on the selection and persistence of 3GC-resistant Escherichia coli in poultry. We studied 15 3GC-treated (TB) and 15 non-3GC-treated (NTB) broiler flocks and 12 3GC-treated (TL) and 10 non-3GC-treated (NTL) future layer flocks. Fecal samples from each flock were sampled before arrival on the farm (day 0), on day 2, on day 7, and then twice more. E. coli isolates were isolated on MacConkey agar without antibiotics and screened for 3GC resistance, and any 3GC-resistant E. coli isolates were further analyzed. 3GC-resistant E. coli isolates were found in all 3GC-treated flocks on at least one sampling date. The percentages of 3GC-resistant E. coli isolates were significantly higher in TB (41.5%) than in NTB (19.5%) flocks and in TL (49.5%) than in NTL (24.5%) flocks. In the day 2 samples, more than 80% of the E. coli strains isolated were 3GC resistant. 3GC-resistant E. coli strains were still detected at the end of the follow-up period in 6 out of 27 3GC-treated and 5 out of 25 non-3GC-treated flocks. Many 3GC-resistant E. coli strains were resistant to tetracycline, and there were significant differences in the percentages of resistance to sulfamethoxazole-trimethoprim, streptomycin, or gentamicin between treated and nontreated flocks. bla CTX-M and bla CMY-2 were the most frequently detected genes. These results clearly demonstrated that 3GC-resistant strains are introduced early in flocks and that the use of 3GC in hatcheries promotes the selection of 3GC-resistant E. coli . Measures must be implemented to avoid the spread and selection of 3GC-resistant strains.

An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

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 bla CTX-M-1 group gene, and a few carried a bla CMY-2 -like gene. Control of 3GC resistance in laying hens is needed.

Colistin is a cyclic decapeptide bound to a fatty acid chain. It is active against many Gram-negative bacteria by destabilising the bacterial outer membrane. Bacteria can become resistant to colistin by modification of their lipopolysaccharide, thereby reducing the affinity of polymyxins. Colistin is often administered orally in poultry and pig production to control colibacillosis. Resistant isolates are sometimes recovered from pathological cases, particularly in piglets. However, in Europe the percentage of resistance to colistin in Escherichia coli strains isolated from the digestive tract microbiota of healthy animals remains

Antimicrobial resistance is of primary importance regarding public and animal health issues. Persistence and spread of resistant strains within a population contribute to the maintenance of a reservoir and lead to treatment failure. An experimental trial was carried out to study the horizontal transmission of a fluoroquinolone-resistant Escherichia coli strain from inoculated to naïve pigs. All naïve contact pigs had positive counts of fluoroquinolone-resistant E. coli after only two days of contact. Moreover, re-infections of inoculated pigs caused by newly contaminated animals were suspected. A maximum likelihood method, based on a susceptible-infectious-susceptible (SIS) model, was used to determine the transmission parameters. Two transmission levels were identified depending on the quantity of bacteria shed by infected individuals: (i) low-shedders with bacterial counts of resistant E. coli in the faeces between 5*103 and 106 CFU/g (βL = 0.41 [0.27; 0.62]), (ii) high shedders with bacterial counts above 106 CFU/g (βH = 0.98 [0.59; 1.62]). Hence, transmission between animals could be pivotal in explaining the persistence of resistant bacteria within pig herds.

This study was designed to determine the genetic basis of florfenicol and ceftiofur resistance in Escherichia coli isolates recovered from French cattle. In these isolates, ceftiofur resistance was conferred by bla(CMY-2) located on three distinct conjugative plasmids on a specific DNA fragment, ISEcp1-bla(CMY-2)-blc- sugE. Two of the plasmids also carried the floR gene conferring resistance to florfenicol. The floR gene was shown to be associated with the insertion sequence ISCR2. Mobile elements appear to contribute to the mobilization of floR and bla(CMY-2) genes in E. coli. The presence of bla(CMY-2) and floR on the same plasmid highlights the potential risk for a co-selection of the bla(CMY-2) gene through the use of florfenicol in food animal production.

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

Clinical Escherichia coli strains with resistance or variable susceptibility to third-generation cephalosporins were detected in cattle, swine and poultry in France. These strains were shown to produce extended-spectrum beta-lactamases (ESBLs), with CTX-M-1- and CTX-M-15-type beta-lactamases being responsible for this phenotype. The bla(CTX-M-1) gene was encountered most commonly and was characterised in seven E. coli strains isolated from cattle, swine and poultry, whereas bla(CTX-M-15) was identified in one E. coli isolated from cattle. These genes were located on a conjugative plasmid and were linked to the insertion sequence ISEcp1, which could have contributed to dissemination of the resistance gene. No epidemiological link between the strains was determined by pulsed-field gel electrophoresis, although two plasmids were identical in two strains isolated from swine and in two strains isolated from cattle and poultry. Thus, this study describes the emergence of ESBLs in animals in France, with a probable similar prevalence rate to that observed in humans. This is a major concern because of the possibility of transfer of these genes between animal species as well as to humans, leading to treatment failures in veterinary and human medicine.

International audience; Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10$^8$ CFU S. Enteritidis per animal. Anti- lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line ($p = 0.013$, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with $1.7 \times 10^8$ CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM ($p = 0.017$, 0.017 and $p < 0.001$ respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.; Méthodes ELISA permettant de différencier des lignées de poules résistante et sensible à Salmonella Enteritidis inoculées par voie orale. Des méthodes ELISA ont été recherchées pour sélectionner les lignées de poules résistantes à Salmonella Enteritidis produisant des taux élevés d'anticorps. Dans la première expérience, la relation entre la réponse sérologique et les résultats bactériologiques a été évaluée sur les poules des lignées résistante Y11 et sensible L2, inoculées par voie orale avec 10$^8$ UFC S. Enteritidis par animal. Les titres en anticorps IgG anti- lipopolysaccharidiques (LPS) étaient supérieurs mais les foies et rates étaient moins contaminés chez les poules de la lignée Y11 que chez les poules de la lignée L2 ($p = 0.013$, 0.031 et 0.026 respectivement). Dans la seconde expérience, les deux lignées de poules ont été inoculées par voie orale avec $1.7 \times 10^8$ UFC S. Enteritidis par animal afin de sélectionner les méthodes ELISA les plus discriminantes. Les ELISA utilisaient comme antigènes des LPS, flagelles, LPS des souches rough (LPS-R) et smooth (LPS-S), et détectaient des anticorps IgG et IgM dans le sérum et le vitellus. Aucune différence significative n'a été observée à partir du vitellus, ou avec les antigènes LPS-S et R, ainsi qu'avec les IgM anti-LPS dans le sérum. En revanche, les tests ELISA réalisés sur les sérums et basés sur le dépistage des IgG anti-LPS, des IgG ou IgM anti-flagellaires permettent de montrer une différence significative entre les deux lignées ($p = 0.017$, 0.017 et $p < 0.001$ respectivement). La comparaison des cinétiques obtenues avec les tests ELISAs retenus a montré que les IgG anti-LPS permettaient de détecter des différences entre lignées dès 1 semaine jusqu'à 4 semaines après inoculation, alors qu'avec les IgG anti-flagellaires les différences apparaissaient seulement à 1 et 2 semaines, et avec les IgM anti-flagellaires les différences étaient significatives à 1, 2, 4 et 8 semaines après inoculation. En conclusion, les lignées de poules résistantes et produisant des taux d'anticorps plus élevés que les lignées de poules sensibles peuvent être sélectionnées avec ces tests ELISA.

International audience; 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 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.

The susceptibility of 135 Streptococcus suis strains isolated from pigs (n = 110) and from humans (n = 25) to 13 antimicrobial agents was studied by microdilution and disc diffusion methods using Mueller-Hinton Agar II (MH) supplemented with either defibrinated sheep blood (MHSB) or horse serum (MHHS). Results were similar for both methods used except for penicillin G whose zone diameters were reduced with MHSB compared with MHHS. When MH was supplemented with sheep blood, 39% of S. suis strains classified as penicillin susceptible by MHHS microdilution showed intermediate susceptibility. Nearly all strains were susceptible to penicillin G (except by disc diffusion in MHSB), amoxicillin, ceftiofur, florfenicol, gentamicin and bacitracin. The least active antimicrobial agents were doxycycline and macrolides/lincosamides. High-level resistance (MIC > 500 mg/L or zone diameters < 10 mm) to streptomycin and kanamycin was detected in only a few strains. The virulence of strains did not seem to be related to antimicrobial resistance because no statistical difference was reported between the proportion of resistant strains of S. suis isolated from pigs with meningitis, septicaemia and arthritis, and those from tonsils and nasal cavities. However, significant differences were found in the proportions of macrolide- or doxycycline-resistant strains between S. suis serotype 2 and other serotypes. The results of antibiotic susceptibility testing presented in this study indicate that beta-lactams can be used in empirical treatment of human and pig S. suis infections in France.