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

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

Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms –a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice

Evaluation of an oral subchronic exposure of deoxynivalenol on the composition of human gut microbiota in a model of human microbiota-associated rats.

BACKGROUND: Deoxynivalenol (DON), a mycotoxin produced by Fusarium species, is one of the most prevalent mycotoxins present in cereal crops worldwide. Due to its toxic properties, high stability and prevalence, the presence of DON in the food chain represents a health risk for both humans and animals. The gastrointestinal microbiota represents potentially the first target for these food contaminants. Thus, the effects of mycotoxins on the human gut microbiota is clearly an issue that needs to be addressed in further detail. Using a human microbiota-associated rat model, the aim of the present study was to evaluate the impact of a chronic exposure of DON on the composition of human gut microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Four groups of 5 germ free male rats each, housed in 4 sterile isolators, were inoculated with a different fresh human fecal flora. Rats were then fed daily by gavage with a solution of DON at 100 µg/kg bw for 4 weeks. Fecal samples were collected at day 0 before the beginning of the treatment; days 7, 16, 21, and 27 during the treatment; and 10 days after the end of the treatment at day 37. DON effect was assessed by real-time PCR quantification of dominant and subdominant bacterial groups in feces. Despite a different intestinal microbiota in each isolator, similar trends were generally observed. During oral DON exposure, a significant increase of 0.5 log10 was observed for the Bacteroides/Prevotella group during the first 3 weeks of administration. Concentration levels for Escherichia coli decreased at day 27. This significant decrease (0.9 log10 CFU/g) remained stable until the end of the experiment. CONCLUSIONS/SIGNIFICANCE: We have demonstrated an impact of oral DON exposure on the human gut microbiota composition. These findings can serve as a template for risk assessment studies of food contaminants on the human gut microbiota

Risk Assessment of Deoxynivalenol by Revisiting Its Bioavailability in Pig and Rat Models to Establish Which Is More Suitable

International audienceDue to its toxic properties, high stability, and prevalence, the presence of deoxynivalenol (DON) in the food chain is a major threat to food safety and therefore a health risk for both humans and animals. In this study, experiments were carried out with sows and female rats to examine the kinetics of DON after intravenous and oral administration at 100 µg/kg of body weight. After intravenous administration of DON in pigs, a two-compartment model with rapid initial distribution (0.030 ˘ 0.019 h) followed by a slower terminal elimination phase (1.53 ˘ 0.54 h) was fitted to the concentration profile of DON in pig plasma. In rats, a short elimination half-life (0.46 h) and a clearance of 2.59 L/h/kg were estimated by sparse sampling non-compartmental analysis. Following oral exposure, DON was rapidly absorbed and reached maximal plasma concentrations (C max) of 42.07 ˘ 8.48 and 10.44 ˘ 5.87 µg/L plasma after (t max) 1.44 ˘ 0.52 and 0.17 h in pigs and rats, respectively. The mean bioavailability of DON was 70.5% ˘ 25.6% for pigs and 47.3% for rats. In the framework of DON risk assessment, these two animal models could be useful in an exposure scenario in two different ways because of their different bioavailability

Time course evolution of <i>Bacteroides-Prevotella</i> normalized concentrations in the 4 isolators during the experimental period.

<p>For the different bacterial groups, results were expressed as the mean of normalized log₁₀ value (n = 5 except for isolator IV where n = 4) ± standard deviation of CFU/g.* P<0.05; ** P<0.01; *** P<0.001.</p

Target organisms, type strains, oligonucleotide primers and probes used in this assay.

<p>Target organisms, type strains, oligonucleotide primers and probes used in this assay.</p

Time course evolution of bacteria levels in the 4 isolators during the experimental period.

<p>Results obtained by qPCR were expressed for all bacteria as the mean of the log₁₀ value (n = 5 except for isolator IV where n = 4) ± standard deviation of CFU/g. Normalization was done by subtracting the log₁₀ CFU/g obtained for the “all bacteria” group from the log₁₀ CFU/g for the other bacterial groups. Results were expressed for the different bacterial groups as the mean of normalized log₁₀ value (n = 5 except for isolator IV where n = 4) ± standard deviation of CFU/g.</p><p>*P<0.05;</p><p>**P<0.01;</p><p>***P<0.001.</p><p>(a)<i>Bifid</i> LOD  =  5.46 log₁₀ CFU/g;</p><p>(b)Lac-Leu-Ped LOD  =  5.03 log₁₀ CFU/g; Bact-Prev: Bacteroides-Prevotella group; C. cocco: Clostridium coccoides group; C. lep: Clostridium leptum group; Bifid: Genus Bifidobacterium; Entero: Genus Enterococcus; E. coli: Escherichia coli; Lac-Leu-Ped: Lactobacillus-Leuconostoc-Pediococcus group.</p

Fecal concentrations of each target bacterial group of the HMA rats and in donors.

<p>(A) Isolator I; (B) Isolator II; (C) Isolator III; (D) Isolator IV; Results obtained by qPCR were expressed as the mean of the log₁₀ value (for rats n = 5 except for isolator IV where n = 4; for humans n = 2 repetitions) of CFU/g. <i>Bact-Prev</i>: <i>Bacteroides-Prevotella</i> group; <i>C. cocco</i>: <i>Clostridium coccoides</i> group; <i>C. lep</i>: <i>Clostridium leptum</i> group; <i>Bifid</i>: Genus <i>Bifidobacterium</i>; <i>Entero</i>: Genus <i>Enterococcus</i>; <i>E. coli</i>: <i>Escherichia coli</i>; <i>Lac-Leu-Ped</i>: <i>Lactobacillus-Leuconostoc-Pediococcus</i> group; LOD: limit of detection.</p

Table_1_Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms.docx

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms –a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.</p