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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.

A multi-laboratory study was performed to investigate the most suitable incubation time for susceptibility tests of fish pathogens Vibrio anguillarum and Vibrio vulnificus performed at 28 degrees C. An isolate set consisting of 30 V. anguillarum and 26 V. vulnificus was used by four participating laboratories in Denmark, France, Sweden, and the Netherlands. Inhibition zone diameters were determined by agar disc diffusion for eight agents and Minimum Inhibitory Concentration (MIC) values were determined for seven agents using the standard CLSI testing protocols for non-fastidious organisms that specify 24-28 h incubation. In this work an additional set of readings was made after 48 h incubation. In total, 1120 paired zone sizes and 399 paired MIC observations were made at the two incubation times. Examination of the data demonstrated that incubation time had a small but statistically significant effect on the numerical values of susceptibility measures. However, the effects of incubation time on the precision of the data sets and the categorisation of isolates based on the application of epidemiological cutoff values were slight and statistically non significant. These analyses suggest that the susceptibility of these Vibrio species could be established using protocols that specify either 24-28 h or 44-48 h incubation.This study does not provide evidence that prolonged incubation to 48 h improves the quality of data generated by the tests. Therefore, it is recommended that the existing standard CLSI protocols with 24-28 h at 28 degrees C should be adopted for susceptibility testing of V. anguillarum and V. vulnificus. This study was performed in the frame of CoVetLab partner Insitutes https://www.covetlab.org/c5/ peer-reviewed

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.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5 µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum. There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30 µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.

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.

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 blaCTX-M-1 and sul2 genes, and some of them also had the tet(A), aadA5 or dfrA17 genes. The blaCTX-M-1 gene was also detected on an IncN plasmid. Five plasmids obtained from four rivers contained blaCTX-M-14, either on IncI1 or on IncFII plasmids. Two strains from two rivers contained blaCTX-M-15 on IncN pMLST7 plasmids, with qnrS1 and dfrA14 genes. One plasmid contained the blaCTX-M-55, a blaTEM-1B-like, and fosA genes. One plasmid contained the blaCMY-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.

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.

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.

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.