Optimisation du protocole de recherche des Escherichia coli producteurs de Shiga-toxines (STEC) dans les aliments

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des agents pathogènes importants émergents en santé publique. Ces bactéries sont à l origine des épidémies de colites hémorragiques et de syndrome hémolytique et urémique (SHU). La grande majorité des cas est liée à la consommation d aliments contaminés par un type particulier de STEC appelés E. coli entérohémorragiques (EHEC) parmi lesquels on trouve les sérogroupes définis comme pathogènes par l AFSSA : O26, O103, O111, O145 et O157.Les bovins ont depuis longtemps été identifiés comme un important réservoir de STEC. Bien que la transmission des STEC à l'homme soit fréquemment associée à la consommation de viande, les produits laitiers ont également été impliqués dans les cas humains. Le développement de méthodes rapides pour la détection des STEC les plus impliqués en pathologie humaine est donc essentiel pour assurer la sécurité des produits alimentaires. Cependant, la détection des STEC dans les aliments est problématique principalement en raison de la diversité des sérogroupes de STEC et de l'absence de caractéristiques biochimiques communes permettant de les distinguer des autres E. coli.L'objectif de cette thèse était d optimiser le protocole de détection des STEC dans les aliments de manière à pouvoir proposer aux industriels des protocoles leur permettant une réelle maîtrise du danger STEC dans leur filière.Pour se faire, nous sommes intervenus à différentes étapes du protocole. Nous avons notamment sélectionné un milieu d enrichissement permettant la détection des E. coli O26 dans les fromages au lait cru : EPT+ acriflavine + CT. Nous avons également évalué les performances d une méthode de détection des E. coli O157 :H7 (VIDAS ECPT UP) dans la viande de boeuf. Cette nouvelle technique est basée sur l utilisation de protéines recombinante de phage qui ciblent spécifiquement les E. coli O157. A travers cette étude, nous avons démontré la possibilité d avoir des temps d enrichissement minimes pour l analyse d échantillon de 25g (6h) et la faisabilité d analyser des échantillons de 375g avec un ratio de dilution de et un enrichissement de 24h. Enfin nous nous sommes intéressés à l étape de confirmation en mettant au point un test d immuno-concentration automatisé (utilisant des protéines de phages spécifiques de nos bactéries cibles) permettant d immuno-concentrer les 5 sérogroupes les plus connus de STEC (O26, O103, O111, O145 et O157) en une seule foisEnterohemorrhagic Escherichia coli (EHEC) are an important subset of Shiga toxin producing E. coli (STEC) associated with foodborne infections. These bacteria can cause hemorrhagic diarrhoeal disease and Haemolytic and Uremic Syndrome which are associated with some predominant serotypes defined by AFSSA institute: O26, O103, O111, O145 and O157.Cattle are the primary reservoir of STEC. Although STEC transmission to humans is frequently associated with consumption of meat, dairy products have also been implicated in human cases. Sensitive and rapid detection methods for STEC are essential for the food industry to ensure a safe food supply. However, the detection of STEC in foods is problematic because it s an heterogeneous group which displays a broad range of both genotypic and phenotypic differences.The aim of this thesis was to optimize the research protocol of STEC strains in food in order to provide some protocols to the industries for improving risk management protocol.So, we involved in different steps of the protocol: the enrichment of E. coli O26 allowing their detection in raw milk cheeses after 24 h enrichment in EPT+ acriflavine + cefixime-tellurite. Then, we evaluated the VIDAS ECPT UP performances for the detection of E. coli O157: H7 in raw ground beef. Through this study, we showed the ability to reduce enrichment time for sample analysis of 25g (6h) and the feasibility of analyzing samples of 375g with a sample to broth ratio of after 24h enrichment. Thereafter we looked at the confirmation step. We developed and optimized automated VIDAS immuno-concentration of E. coli O157, O26, O103, O111 and O145 (VIDAS ESPT) with the use of recombinant phage proteins for captureDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Analyse du danger lié à Listeria monocytogènes dans la saucisson sec

Notre étude a permis de dresser un état des lieux de la contamination par L. monocytogenes des entreprises de charcuteries-salaison jugées représentatives de la production française. Le sérotypage des 1028 souches collectées au sein de ces entreprises et dans les produits qu elles fabriquent a mis en évidence la présence de cinq différents serotypes : 1/2a (49,9%), 1/2c (20,4%), 1/2b (12%), 4b (8%) et 4e (0,1%). La présence de souches de sérotypes 1/2a, 1/2b et 4b dans les ateliers, et plus précisément dans les saucissons est préoccupante pour le gestionnaire du risque car la majorité des cas de listériose sont associés à ces sérotypes. Elle s avère d autant plus préoccupante que le pulsotypage a révélé la présence dans les ateliers de souches génétiquement très proches d une souche impliquée dans une listériose humaine. Par ailleurs, Le pulsotypage a révélé que ces souches sont majoritairement introduites dans les ateliers de fabrication par le biais de matières premières contaminées. En outre cette contamination est amplifiée par des contaminations croisées impliquant la matière première elle-même mais aussi des surfaces de travail encore souillées en dépit des procédures de nettoyage désinfection appliquées. En effet, la présence de souches persistantes a été démontrée dans le cadre de notre travail. En conséquence, une attention toute particulière devrait être portée à l application de procédures de nettoyage et désinfection adéquates et efficaces (élimination de la matière organique, utilisation des désinfectants à des concentrations appropriées), l implantation de chaînes de travail simples et à la conception de machines compatibles avec le nettoyage et la désinfection. Par ailleurs, notre travail a montré la présence de "clones circulants" dans différentes entreprises, suggérant le caractère ubiquiste ou de "supériorité" écologique de certaines .Enfin, l étude de la prévalence de Listeria monocytogenes a montré que les taux de contamination des produits diminuent de manière significative au cours de la fabrication suggérant que les conditions dysgénésiques pour le pathogène sont de plus en plus marquées au fur et à mesure de la fabrication du saucisson sec. Par ailleurs, l inoculation expérimentale de saucissons a montré que cette réduction est souche dépendante (P < 0,001). En effet, les souches adaptées aux stress induits par le procédé de fabrication sont plus difficilement éliminées que les souches non adaptées. Ces résultats suggèrent que la fabrication de saucissons secs exempts de L. monocytogenes semble difficile, mais que les professionnels pourraient s en approcher en appliquant des principes simples d hygiène générale. La rédaction d un guide de Bonnes Pratiques d Hygiène pour les entreprises artisanales et l application pertinente de la méthode HACCP et dans les salaisons industrielles apporterait des stratégies efficaces pour maîtriser le danger L. monocytogenesLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness

Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses

International audienceShiga toxin producing Escherichia coli (STEC) are an important cause of human foodborne outbreaks. The consumption of raw milk dairy products may be an important route of STEC infection. For successful foodborne transmission, STEC strains must survive stress conditions met during gastrointestinal transit in humans. The aim of this study was to evaluate the survival of two STEC strains of serotypes O157:H7 and O26:H11 during simulated human digestion in the TNO gastro-Intestinal tract Model (TIM) of contaminated uncooked pressed cheeses. The survival of cheese microflora during in vitro gastrointestinal transit was also determined for the first time. The level of STEC increased from 2 log(10) CFU/ml to 4 log(10) CFU/g during the first 24 h of cheese making and remained stable at around 4 log(10) CFU/g during cheese ripening and conservation. During transit through the artificial stomach and duodenum, levels of STEC decreased: O2% of E. coil O157:H7 and 1.8% of E. coil O26: H11 were recovered at 150 min in the gastric compartment, compared with 14.3% for the transit marker. Bacterial resumption was observed in the jejunum and ileum: 35.8% of E. coil O157:H7 and 663.2% of E. coil O26:H11 were recovered at 360 min in the ileal compartment, compared with 12.6% for the transit marker. The fate of STEC was strain-dependent, the survival of E. coli O26:H11 being 13 times greater than that of E. coli 0157: H7 at the end of digestion in the cumulative ileal deliveries. These data provide a better understanding of STEC behavior during gastrointestinal transit in humans after ingestion of contaminated cheese. (C) 2013 Elsevier B.V. All rights reserved

Strain- and serotype-dependent affinity of Shiga toxin-producing Escherichia coli for bovine milk fat globules

International audienceShiga toxin-producing Escherichia coli (STEC) are widely detected in raw milk products intended for hu-man consumption. Although STEC are a worldwide public health problem, the pathogenicity of STEC in cheese remains unclear. In fact, bacterial association with compounds in raw milk cheeses could reduce their pathogenicity. A previous study showed the association of 2 STEC strains with raw milk cream in a natural creaming assay. Different concentrations of each strain were required to saturate the cream. In this study, we hypothesized that all STEC strains could be associated with milk fat globules (MFG) in raw milk and that the bacterial load required for saturation of the cream is serotype dependent. We evaluated the affinity of STEC strains belonging to the O157:H7, O26: H11, and O103:H2 serotypes for bovine raw milk cream and ana-lyzed saturation of the cream layer by natural creaming assay. We used 12 STEC strains and 3 strains belonging to another pathotype to assess the effects of serotypes on this phenomenon. We performed sucrose density gra-dient centrifugation assays with 2 STEC model strains to confirm the results obtained by natural creaming. The localization of STEC within MFG-enriched creams was observed by confocal and electron microscopy. We recovered approximately 10 times more STEC from the cream layer after natural creaming than from raw bovine milk. The concentration of STEC required to saturate the cream layer (the saturation concentration) was estimated for each strain by nonlinear regression, highlighting a strain and serotype effect. Moreover, the concentration of STEC in the cream was milk fat level dependent. However, even in nonsaturating conditions, a high level of STEC was still present in the aque-ous phase, after fat separation. Thus, natural creaming should not be used as the sole preventive measure to remove STEC from naturally contaminated raw milk. The results of our study suggest that cream saturation is a complex mechanism, most likely involving specific interactions between STEC and raw MFG

Strand-specific transcriptomes of Enterohemorrhagic Escherichia coli in response to interactions with ground beef microbiota: interactions between microorganisms in raw meat

France GenomiqueInternational audienceBackground: Enterohemorrhagic Escherichia coli (EHEC) are zoonotic agents associated with outbreaks worldwide. Growth of EHEC strains in ground beef could be inhibited by background microbiota that is present initially at levels greater than that of the pathogen E. coli. However, how the microbiota outcompetes the pathogenic bacteria is unknown. Our objective was to identify metabolic pathways of EHEC that were altered by natural microbiota in order to improve our understanding of the mechanisms controlling the growth and survival of EHECs in ground beef.Results: Based on 16S metagenomics analysis, we identified the microbial community structure in our beef samples which was an essential preliminary for subtractively analyzing the gene expression of the EHEC strains. Then, we applied strand-specific RNA-seq to investigate the effects of this microbiota on the global gene expression of EHEC O26(21765) and O157(EDL933) strains by comparison with their behavior in beef meat without microbiota. In strain O26(21765), the expression of genes connected with nitrate metabolism and nitrite detoxification, DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous genes involved in amino acid metabolism were down-regulated. Further, the observed repression of ftsL and murF, involved respectively in building the cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell wall peptidoglycan, might help to explain the microbiota's inhibitory effect on EHECs. For strain O157(EDL933), the induced expression of the genes implicated in detoxification and the general stress response and the repressed expression of the peR gene, a gene negatively associated with the virulence phenotype, might be linked to the survival and virulence of O157:H7 in ground beef with microbiota.Conclusion: In the present study, we show how RNA-Seq coupled with a 16S metagenomics analysis can be used to identify the effects of a complex microbial community on relevant functions of an individual microbe within it. These findings add to our understanding of the behavior of EHECs in ground beef. By measuring transcriptional responses of EHEC, we could identify putative targets which may be useful to develop new strategies to limit their shedding in ground meat thus reducing the risk of human illnesses

Comparing Antibiotic Resistance in Free-Ranging vs. Captive African Wild Herbivores

International audienceAntimicrobial resistance (AMR) is a critical challenge of the 21st century for public and animal health. The role of host biodiversity and the environment in the evolution and transmission of resistant bacteria between populations and species, and specifically at the wildlife-livestock-human interface, needs to be further investigated. We evaluated the AMR of commensal Escherichia coli in three mammalian herbivore species-impala (Aepyceros melampus), greater kudu (Tragelaphus strepsiceros), and plains zebra (Equus quagga)-targeting populations living under two conditions: captivity (French zoos) and free ranging (natural and private parks in Zimbabwe). From 137 fecal samples from these three host species, 328 E. coli isolates were isolated. We measured the AMR of each isolate against eight antibiotics, and we assessed the presence of AMR genes and mobile genetic element class 1 integrons (int1). Isolates obtained from captive hosts had a higher probability of being resistant than those obtained from free-ranging hosts (odds ratio, 293.8; confidence interval, 10-94,000). This statistically higher proportion of AMR bacteria in zoos than in natural parks was especially observed for bacteria resistant to amoxicillin. The percentage of int1 detection was higher when isolates were obtained from captive hosts, particularly captive impalas. Ninety percent of bacterial isolates with genes involved in antibiotic resistance also had the int1 gene. The sul1, sul2, blaTEM, and stra genes were found in 14, 19, 0, and 31%, respectively, of E. coli with respective antibiotic resistance. Finally, plains zebra carried AMR significantly more often than the other species

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence

International audienceEnterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness