Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

European-wide antimicrobial resistance monitoring in commensal Escherichia coli isolated from healthy food animals between 2004 and 2018.

OBJECTIVES: To describe the susceptibility of Escherichia coli to medically important antibiotics, collected over four periods (2004-2006, 2008-2009, 2013-2014, 2017-2018), from food-producing animals at slaughter. METHODS: Intestinal contents from cattle, pigs and broilers were randomly sampled (5-6 countries/host; ≥4 abattoirs/country; one sample/animal/farm) for isolation of Escherichia coli; antimicrobial susceptibilities were centrally determined by CLSI agar dilution. Clinical breakpoints (CLSI) and epidemiological cut-off values (EUCAST) were applied for data interpretation. RESULTS: In total, 10 613 E. coli strains were recovered. In broilers, resistance percentages were the lowest (P ≤ 0.01) in the latest time period. A significant decrease in MDR over time was also observed for broilers and a tendency for a decrease for pigs. Resistance to meropenem and tigecycline was absent, and resistance to azithromycin was 0.2%-2.0%. Also, low resistance to third-generation cephalosporins (1.1%-7.4%) was detected in broilers. Resistance to colistin varied between 0.1%-4.8%. E. coli from broilers showed high resistance to ciprofloxacin (7.3%-23.3%), whereas for cattle and pigs this was 0.2%-2.5%. Low/moderate resistance to chloramphenicol (9.3%-21.3%) and gentamicin (0.9%-7.0%) was observed in pigs and broilers. The highest resistance was noted for ampicillin (32.7%-65.3%), tetracycline (41.3%-67.5%), trimethoprim (32.0%-35.7%) and trimethoprim/sulfamethoxazole (27.5%-49.7%) from pigs and broilers, with marked country differences. MDR peaked in pigs and broilers with 24 and 26 phenotypes, with 21.9%-26.2% and 18.7%-34.1% resistance, respectively. CONCLUSIONS: In this pan-EU survey antibiotic susceptibility of commensal E. coli varied largely between antibiotics, animal species and countries. Resistance to critically important antibiotics for human medicine was absent or low, except for ciprofloxacin in broilers and ampicillin in pigs and broilers.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Prevalence of mcr-1 in commensal Escherichia coli from French livestock, 2007 to 2014.

Colistin resistance was investigated in 1,696 isolates collected from 2007 to 2014 within the frame of the French livestock antimicrobial resistance surveillance programme. The mcr-1 gene was detected in all commensal Escherichia coli isolates with a minimum inhibitory concentration to colistin above the 2 mg/L cut-off value (n=23). In poultry, mcr-1 prevalence was 5.9% in turkeys and 1.8% in broilers in 2014. In pigs, investigated in 2013, this prevalence did not exceed 0.5%. These findings support that mcr-1 has spread in French livestock.

Low or high doses of cefquinome targeting low or high bacterial inocula cure Klebsiella pneumoniae lung infections but differentially impact the levels of antibiotic resistance in fecal flora.

The combination of efficacious treatment against bacterial infections and mitigation of antibiotic resistance amplification in gut microbiota is a major challenge for antimicrobial therapy in food-producing animals. In rats, we evaluated the impact of cefquinome, a fourth-generation cephalosporin, on both Klebsiella pneumoniae lung infection and intestinal flora harboring CTX-M-producing Enterobacteriaceae. Germfree rats received a fecal flora specimen from specific-pathogen-free pigs, to which a CTX-M-producing Escherichia coli strain had been added. K. pneumoniae cells were inoculated in the lungs of these gnotobiotic rats by using either a low (10(5) CFU) or a high (10(9) CFU) inoculum. Without treatment, all animals infected with the low or high K. pneumoniae inoculum developed pneumonia and died before 120 h postchallenge. In the treated groups, the low-inoculum rats received a 4-day treatment of 5 mg/kg of body weight cefquinome beginning at 24 h postchallenge (prepatent phase of the disease), and the high-inoculum rats received a 4-day treatment of 50 mg/kg cefquinome beginning when the animals expressed clinical signs of infection (patent phase of the disease). The dose of 50 mg/kg targeting the high K. pneumoniae inoculum cured all the treated rats and resulted in a massive amplification of CTX-M-producing Enterobacteriaceae. A dose of 5 mg/kg targeting the low K. pneumoniae inoculum cured all the rats and averted an outbreak of clinical disease, all without any amplification of CTX-M-producing Enterobacteriaceae. These findings might have implications for the development of new antimicrobial treatment strategies that ensure a cure for bacterial infections while avoiding the amplification of resistance genes of human concern in the gut microbiota of food-producing animals.

Use of accuracy profile procedure to validate a real-time PCR method to quantify bacteria in feces.

This study describes a novel validation procedure of real-time PCR based on accuracy profile to estimate bacterial concentrations in fecal samples. To assess the performance of the method, measurements of axenic fecal samples spiked with a measured quantity of known bacterial species (Bacteroides fragilis, Bifidobacterium adolescentis, Enterococcus faecium, and Escherichia coli) were performed under repeatability and intermediate precision conditions. Data collected were used to compute a tolerance interval that was compared to a defined acceptance interval. It is concluded that the method is valid and relevant for the studied validation range of 8.20-10.24 and 7.43-9.47 log10 CFU/g of feces to ensure proper measurement of B. fragilis and E. coli, respectively. The LOQ is 8.20 and 7.43 log10 CFU/g of feces. In contrast, the method is not valid for the quantification of E. faecium and B. adolescentis, but by applying a correction factor of +0.63 log10 CFU/g, it can be considered valid for E. faecium. This correction is included in the final results. In conclusion, the accuracy profile is a statistical tool that is easy to use and totally adapted to validate real-time PCR.

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

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.

Selection of antibiotic resistance in pigs after exposure to feed cross-contaminated with oxytetracycline.

Due to possible cross-contamination of animal feedstuff with antibiotics, food-producing animals may be exposed to undesirable low concentrations of antimicrobials. These sub-therapeutic levels of antibiotics can lead to the selection of resistant bacteria in the animal gut. The goal of this study was to assess, through analysis of the faeces of treated and control pigs, the risk of resistant E. coli being selected after daily exposure for three weeks to feed contaminated with oxytetracycline at 1% of the therapeutic dose. Liquid Chromatography coupled to tandem Mass Spectrometry was used to determine the oxytetracycline concentrations in faecal samples. In the treated group, concentrations were in the range of 4481.9 - 8671.2 µg/kg. In the control group, these concentrations were either below the method's limit of quantification or up to 60.5 µg/kg. After a transient increase in resistance in both groups, microbiological analysis showed that the treated group had a significantly higher oxytetracycline resistance rate by the end of the study than the control group (p < 0.001). Furthermore, the treated animals were found to select co-resistances to nalidixic acid and ampicillin. Finally, at tolerated antibiotic contamination levels of feed, the treated group had a higher proportion of multidrug-resistant isolates at the end of the study than the control one (p < 0.05). The present study demonstrates that, at the tolerated contamination rates, both antimicrobial resistance and multidrug-resistant bacteria can be selected and evidenced in the gut microbiota.

Tell me if you prefer bovine or poultry sectors and I'll tell you who you are: Characterization of Salmonella enterica subsp. enterica serovar Mbandaka in France.

Introduction: In north-western France, Salmonella enterica susp. enterica serovar Mbandaka (S. Mbandaka) is most frequently isolated from bovine and dairy samples. While this serovar most often results in asymptomatic carriage, for a number of years it has caused episodes of abortions, which have serious economic consequences for the sector. Interestingly, this serovar is also isolated from Gallus gallus in the same geographic zone. Despite its prevalence in bovines in north-western France, S. Mbandaka has not been broadly studied at the genomic level, and its prevalence and host adaptation are still not fully understood. Methods: In this study, we analyzed the genomic diversity of 304 strains of S. Mbandaka isolated from the bovine and poultry sectors in this area over a period of 5 years. A phylogenetic analysis was carried out and two approaches were followed to identify conserved genes and mutations related to host associations. The first approach targeted the genes compiled in the MEGARESv2, Resfinder, VFDB and SPI databases. Plasmid and phage contents were also investigated. The second approach refers to an in-house algorithm developed for this study that computes sensitivity, specificity, and accuracy of accessory genes and core variants according to predefined genomes groups. Results and discussion: All the analyzed strains belong to the multi-locus sequence type profile ST413, and the phylogenomic analysis revealed main clustering by host (bovine and poultry), emphasizing the circulation of 12 different major clones, of which seven circulate in poultry and five in the bovine sector in France and a likely food production chain adaptation of these clones. All strains present resistance determinants including heavy metals and biocides that could explain the ability of this serovar to survive and persist in the environment, within herds, and in food processing plants. To explore the wild animal contribution to the spread of this serovar in north-western France, we retrieved S. Mbandaka genomes isolated from wild birds from EnteroBase and included them in the phylogenomic analysis together with our collection. Lastly, screening of accessory genes and major variants allowed us to identify conserved specific mutations characteristic of each major cluster. These mutations could be used to design useful probes for food safety surveillance.

Validation of a LC-MS/MS method for the quantitative analysis of four antibiotics in pig tissues and plasma to assess the risk of transfer of residues to edible matrices after exposure to cross-contaminated feed.

Cross-contamination between medicated and non-medicated feed can occur during production, processing, transport or storage of animal feed. This may lead to the presence of low concentrations of antibiotics in supposedly drug-free feed for food production animals, which potentially could also harm consumers due to residues. In addition, consumption of sub-therapeutic concentrations of antibiotics may increase the risk of emergence of resistant bacteria. In this study, LC-MS/MS methods were developed to quantify four antibiotics (sulfadimethoxine, oxytetracycline, trimethoprim and amoxicillin) in several pig matrices, i.e. plasma, muscle, liver, kidneys and faeces. All methods were validated using the accuracy profile, except for amoxicillin in faeces, for which extraction could not be optimised for low concentrations. These methods were then applied as part of an animal study during which several pigs received contaminated feed at a concentration corresponding to 2% of therapeutic dose, in order to evaluate the risk of the presence of residues in animal faeces and tissues. The results showed that sulfadimethoxine is well absorbed and accumulates in the muscle, kidneys and liver, where concentrations were higher than the maximum residue limits (MRLs) authorised in EU legislation. Conversely, oxytetracycline was mostly found in faeces as its oral absorption is very low. Trimethoprim concentrations were slightly higher than the tolerated MRL in the kidneys, but they were below this level in the other tissues. Finally, amoxicillin concentrations remained below the lower limit of quantification of the methods in all matrices.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

International collaborative study on the occurrence of plasmid-mediated quinolone resistance in Salmonella enterica and Escherichia coli isolated from animals, humans, food and the environment in 13 European countries.

OBJECTIVES: This study was initiated to collect retrospective information on the occurrence of plasmid-mediated quinolone resistance (PMQR) in Salmonella enterica and Escherichia coli isolates in Europe and to identify the responsible genes. METHODS: Databases of national reference laboratories containing MIC values for Salmonella and E. coli isolated between 1994 and 2009 in animals, humans, food and the environment from 13 European countries were screened for isolates exhibiting a defined quinolone resistance phenotype, i.e. reduced susceptibility to fluoroquinolones and nalidixic acid. PCR and sequence analysis were performed to identify the responsible PMQR genes. RESULTS: Screening of databases of 13 European countries resulted in a selection of 1215 Salmonella and 333 E. coli isolates. PMQR genes were identified in 59% of the Salmonella isolates and 15% of the E. coli isolates selected. In Salmonella, qnrS1 (n = 125) and variants of qnrB (n = 138) were frequently identified, whereas qnrA1 (n = 3) and aac(6')-1b-cr (n = 3) were rarely found. qnrD was detected in 22 Salmonella isolates obtained from humans and animals. In E. coli, qnrS1 was identified in 19 isolates and qnrB19 was found in one isolate. No qnrC or qepA genes were detected in either Salmonella or E. coli. CONCLUSIONS: This study shows the occurrence and dissemination of PMQR genes in Salmonella and E. coli in Europe with a defined quinolone resistance phenotype. We also report the first detection of qnrD in Salmonella collected in Europe.

An efficient cephalosporin stewardship programme in French swine production.

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.

Comparison of Phenotypical Antimicrobial Resistance between Clinical and Non-Clinical E. coli Isolates from Broilers, Turkeys and Calves in Four European Countries.

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.

Emergence of resistant Klebsiella pneumoniae in the intestinal tract during successful treatment of Klebsiella pneumoniae lung infection in rats.

Antibiotic treatment of lung infections may lead to the emergence of resistance in the gut flora. Appropriate dosing regimens could mitigate this adverse effect. In gnotobiotic rats harboring intestinal Escherichia coli and Enterococcus faecium populations, a lung infection by Klebsiella pneumoniae was instigated with two different sizes of inoculum to represent an early or a late initiation of antibiotic treatment. The rats were treated with marbofloxacin, an expanded-spectrum fluoroquinolone, by a single-shot administration or a fractionated regimen over 4 days. Intestinal bacterial populations were monitored during and after treatment. At the infection site, bacterial cure without any selection of resistance was observed. Whatever the dosage regimen, fluoroquinolone treatment had a transient negative impact on the E. coli gut population but not on that of E. faecium. The intestinal flora was colonized by the pathogenic lung bacteria, and there was the emergence of intestine-resistant K. pneumoniae, occurring more often in animals treated with a single marbofloxacin dose than with the fractionated dose. Bacterial cure without resistance selection at the infection site with fluoroquinolone treatment can be linked to colonization of the digestive tract by targeted pulmonary bacteria, followed by the emergence of resistance.

Decrease in fluoroquinolone use in French poultry and pig production and changes in resistance among E. coli and Campylobacter.

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.

Influence of inoculum size and marbofloxacin plasma exposure on the amplification of resistant subpopulations of Klebsiella pneumoniae in a rat lung infection model.

We tested the hypothesis that the bacterial load at the infection site could impact considerably on the pharmacokinetic/pharmacodynamic (PK/PD) parameters of fluoroquinolones. Using a rat lung infection model, we measured the influence of different marbofloxacin dosage regimens on selection of resistant bacteria after infection with a low (10(5) CFU) or a high (10(9) CFU) inoculum of Klebsiella pneumoniae. For daily fractionated doses of marbofloxacin, prevention of resistance occurred for an area-under-the-concentration-time-curve (AUC)/MIC ratio of 189 h for the low inoculum, whereas for the high inoculum, resistant-subpopulation enrichment occurred for AUC/MIC ratios up to 756 h. For the high-inoculum-infected rats, the AUC/MIC ratio, C(max)/MIC ratio, and time within the mutant selection window (T(MSW)) were not found to be effective predictors of resistance prevention upon comparison of fractionated and single administrations. An index corresponding to the ratio of the time that the drug concentrations were above the mutant prevention concentration (MPC) over the time that the drug concentrations were within the MSW (T(>MPC)/T(MSW)) was the best predictor of the emergence of resistance: a T(>MPC)/T(MSW) ratio of 0.54 was associated with prevention of resistance for both fractionated and single administrations. These results suggest that the enrichment of resistant bacteria depends heavily on the inoculum size at the start of an antimicrobial treatment and that classical PK/PD parameters cannot adequately describe the impact of different dosage regimens on enrichment of resistant bacteria. We propose an original index, the T(>MPC)/T(MSW) ratio, which reflects the ratio of the time that the less susceptible bacterial subpopulation is killed over the time that it is selected, as a potentially powerful indicator of prevention of enrichment of resistant bacteria. This ratio is valid only if plasma concentrations achieve the MPC.

Impact of therapeutic treatment with beta-lactam on transfer of the bla(CTX-M-9) resistance gene from Salmonella enterica serovar Virchow to Escherichia coli in gnotobiotic rats.

The conjugative transfer of the plasmid carrying the bla(CTX-M-9) gene from Salmonella enterica serovar Virchow isolated from a chicken farm to a recipient Escherichia coli strain was evaluated in vitro and in axenic rats inoculated with both strains, with or without selective pressure due to therapeutic doses of cefixime. The transfer of the bla(CTX-M-9) gene of S. enterica serovar Virchow to E. coli was confirmed in vitro, at a low frequency of 5.9 x 10(-8) transconjugants/donors. This transfer rate was higher in gnotobiotic rats and reached approximately 10(-5) transconjugants/donors without selective pressure. This frequency was not affected by the addition of therapeutic doses of cefixime. Thus, estimates of in vitro transfer underestimated potential transfer in the digestive tract, and therapeutic doses of cefixime did not increase the selection for transconjugants.

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

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.