Impact of cleaning and disinfection procedures on microbial ecology and Salmonella antimicrobial resistance in a pig slaughterhouse

International audienceTo guarantee food safety, a better deciphering of ecology and adaptation strategies of bacterial pathogens such as Salmonella in food environments is crucial. The role of food processing conditions such as cleaning and disinfection procedures on antimicrobial resistance emergence should especially be investigated. In this work, the prevalence and antimicrobial resistance of Salmonella and the microbial ecology of associated surfaces communities were investigated in a pig slaughterhouse before and after cleaning and disinfection procedures. Salmonella were detected in 67% of samples and isolates characterization revealed the presence of 15 PFGE-patterns belonging to five serotypes: S.4,5,12:i:-, Rissen, Typhimurium, Infantis and Derby. Resistance to ampicillin, sulfamethoxazole, tetracycline and/or chloramphenicol was detected depending on serotypes. 16S rRNA-based bacterial diversity analyses showed that Salmonella surface associated communities were highly dominated by the Moraxellaceae family with a clear site-specific composition suggesting a persistent colonization of the pig slaughterhouse. Cleaning and disinfection procedures did not lead to a modification of Salmonella susceptibility to antimicrobials in this short-term study but they tended to significantly reduce bacterial diversity and favored some genera such as Rothia and Psychrobacter. Such data participate to the construction of a comprehensive view of Salmonella ecology and antimicrobial resistance emergence in food environments in relation with cleaning and disinfection procedures

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

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

La méthodologie d'étude de la sensibilité aux antibiotiques par diffusion et par dilution

National audienc

Étude de l influence du nettoyage et de la désinfection et des procédés d abattage en abattoir de volailles sur le niveau de résistance aux antibiotiques des campylobacters

Les campylobacters sont des bactéries responsables d entérites chez l homme. La viande de volaille est une source de contamination. Les stress subis par les bactéries au cours des procédures de nettoyage et désinfection et des procédés d abattage des volailles pourraient favoriser la sélection de gènes de résistance aux antibiotiques. Afin d explorer cette hypothèse, des prélèvements dans 4 abattoirs de volailles ont été réalisés. Les niveaux de résistance des campylobacters isolés ont été déterminés pour 6 antibiotiques et 2 substances actives entrant dans la composition de désinfectants. Des souches isolées dans l environnement des abattoirs après nettoyage et désinfection et sur les carcasses de volailles avant l entrée en salle de ressuage, ont été génotypées avec la technique de PCR-RFLP des gènes Pfla/gyrA et flaA. Nos résultats montrent d une part que les campylobacters survivent aux opérations de nettoyage et de désinfection. D autre part, les opérations d abattage et les procédures de nettoyage et désinfection dans les abattoirs de volailles ne semblent pas favoriser la sélection de souches de campylobacter résistantes aux antibiotiques.Campylobacter is a common cause of human bacterial enteritis and consumption of poultry products is a major source of human infection. It has been suggested that disinfectants and environmental stress can lead to changes in the nature and scale of antibiotic resistance expressed by bacteria. In order to investigate this hypothesis, sampling of poultry and environment in 4 unrelated slaughterhouses were conducted. Minimal inhibitory concentrations (MIC) against 6 antibiotics and 2 disinfectants were measured for all the isolates collected. Genotype profile of selected isolates collected after cleaning and disinfection and from poultry carcasses was done by PCR-restriction fragment length polymorphism of P/fla///gyr/A and /fla/A genes. Our results indicate that campylobacter is able to survive overnight on surfaces of poultry slaughterhouses and that these strains may contaminate carcasses during the slaughter process. Our results also suggest that cleaning and disinfection procedures and shlaughter procces don t lead to increase antibiotic resistance in campylobacter.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

Multiplex immunoassay based on biochip technology for the screening of antibiotic residues in milk: validation according to the European guideline

International audienceThe Infiniplex for milk(R) (IPM) kit is a quick method for the simultaneous and qualitative detection of more than 100 molecules including antibiotic residues, mycotoxins, anti-inflammatories and antiparasitic drugs into a single test that does not require milk treatment. The IPM(R) kit was validated according to the European decision EC/2002/657 and according to the European guideline for the validation of screening methods (2010). Our validation was focused only on antibiotic residues. The washing step was identified as the most critical step of the assay. Insufficient washes could cause a significant background noise that prevents imaging. Positive controls have to be freshly prepared each day (insufficient stability). The method was specific with a low false-positive rate of 1.7% on 5 discrete test regions (DTR) ((beta-lactams, lincomycin, virginiamycin, quinolones and sulphonamides)) and a false-positive rate of 0% on the 26 other DTR. During our validation, the 42 determined detection capabilities CCbeta for 12 antibiotic families (aminoglycosides, cephalosporins, lincosamides, macrolides, miscellaneous antibiotics, penicillins, phenolated polymixins, polypeptide antibiotics, quinolones, sulphonamides, tetracyclines) were at between once and twice the decision levels stated by the manufacturer. Forty CCbeta determined were lower than the respective regulatory limits (i.e. MRL, RC, MRPL) in milk, except for tilmicosin (1.5 times the MRL) and neospiramycin (>1.25 times the MRL). The estimated CCbeta of thiamphenicol, cloxacillin, danofloxacin, sulphathiazol, ceftiofur and sulphamonomethoxine were lower than or at the MRL. However, it was difficult to approach an accurate CCbeta with only qualitative results. It is impossible to know whether or not we were close to the cut-off value. The software could be improved by differentiating between low-positive and high-positive results. The results of our participation in three qualitative proficiency tests in 2016 and 2017 for the detection of quinolones, tetracyclines and sulphonamides in cows' milk were very satisfactory

La définition de la résistance aux antibiotiques dans le cadre de la surveillance épidémiologique de ce risque en santé publique

International audienc

Evaluation and validation of a multi-residue method based on biochip technology for the simultaneous screening of six families of antibiotics in muscle and aquaculture products

International audienceThe Evidence Investigator™ system (Randox, UK) is a biochip and semi-automated system. The microarray kit II (AM II) is capable of detecting several compounds belonging to different families of antibiotics: quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines. The performance of this innovative system was evaluated for the detection of antibiotic residues in new matrices, in muscle of different animal species and in aquaculture products. The method was validated according to the European Decision No. EC/2002/657 and the European guideline for the validation of screening methods, which represents a complete initial validation. The false-positive rate was equal to 0% in muscle and in aquaculture products. The detection capabilities CCβ for 12 validated antibiotics (enrofloxacin, difloxacin, ceftiofur, desfuroyl ceftiofur cysteine disulfide, thiamphenicol, florfenicol, tylosin, tilmicosin, streptomycin, dihydrostreptomycin, tetracycline, doxycycline) were all lower than the respective maximum residue limits (MRLs) in muscle from different animal origins (bovine, ovine, porcine, poultry). No cross-reactions were observed with other antibiotics, neither with the six detected families nor with other families of antibiotics. The AM II kit could be applied to aquaculture products but with higher detection capabilities from those in muscle. The detection capabilities CCβ in aquaculture products were respectively at 0.25, 0.10 and 0.5 of the respective MRL in aquaculture products for enrofloxacin, tylosin and oxytetracycline. The performance of the AM II kit has been compared with other screening methods and with the performance characteristics previously determined in honey