Delphine Thevenot-Sergentet

Behavior of Different Shiga Toxin-Producing Escherichia coli Serotypes in Various Experimentally Contaminated Raw-Milk Cheeses

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log10 CFU · g−1) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

Fate of Escherichia coli O26 in corn silage experimentally contaminated at ensiling, at silo opening, or after aerobic exposure, and protective effect of various bacterial inoculants.

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are responsible for human illness. Ruminants are recognized as a major reservoir of STEC, and animal feeds, such as silages, have been pointed out as a possible vehicle for the spread of STEC. The present study aimed to monitor the fate of pathogenic E. coli O26 strains in corn material experimentally inoculated (105 CFU/g) during ensiling, just after silo opening, and after several days of aerobic exposure. The addition of 3 bacterial inoculants, Propionibacterium sp., Lactobacillus buchneri, and Leuconostoc mesenteroides (106 CFU/g), was evaluated for their abilities to control these pathogens. The results showed that E. coli O26 could not survive in corn silage 5 days postensiling, and the 3 inoculants tested did not modify the fate of pathogen survival during ensiling. In the case of direct contamination at silo opening, E. coli O26 could be totally eradicated from corn silage previously inoculated with Leuconostoc mesenteroides. The combination of proper ensiling techniques and the utilization of selected bacterial inoculants appears to represent a good strategy to guarantee nutritional qualities of cattle feed while at the same time limiting the entry of pathogenic E. coli into the epidemiological cycle to improve the microbial safety of the food chain.

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

Shiga 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 log₁₀ CFU/ml to 4 log₁₀ CFU/g during the first 24h of cheese making and remained stable at around 4 log₁₀ CFU/g during cheese ripening and conservation. During transit through the artificial stomach and duodenum, levels of STEC decreased: 0.2% of E. coli O157:H7 and 1.8% of E. coli 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. coli O157:H7 and 663.2% of E. coli 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 O157: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.

Increased EHEC survival and virulence gene expression indicate an enhanced pathogenicity upon simulated pediatric gastrointestinal conditions

Background:Enterohemorrhagic Escherichia coli (EHEC) are major foodborne pathogens that constitute a serious public health threat, mainly in young children. Shiga toxins (Stx) are the main virulence determinants of EHEC pathogenesis but adhesins like intimin (eae) and Long polar fimbriae (Lpf) also contribute to infection. The TNO GastroIntestinal Model (TIM) was used for a comparative study of EHEC O157:H7 survival and virulence under adult and child digestive conditions.Methods:Survival kinetics in the in vitro digestive tract were determined by plating while bacterial viability was assessed by flow cytometry analysis. Expression of stx, eae, and lpf genes was followed by reverse transcriptase-quantitative PCR (RT-qPCR) and Stx production was measured by ELISA (enzyme-linked immunosorbent assay).Results:Upon gastrointestinal passage, a higher amount of viable cells was found in the simulated ileal effluents of children compared to that of adults (with 34 and 6% of viable cells, respectively). Expression levels of virulence genes were up to 125-fold higher in children. Stx was detected only in child ileal effluents.Conclusion:Differences in digestive physicochemical parameters may partially explain why children are more susceptible to EHEC infection than adults. Such data are essential for a full understanding of EHEC pathogenesis and would help in designing novel therapeutic approaches.

Genome Sequence and Annotation of a Human Infection Isolate of Escherichia coli O26:H11 Involved in a Raw Milk Cheese Outbreak

ABSTRACT The consumption of raw milk cheese can expose populations to Shiga toxin-producing Escherichia coli (STEC). We report here the genome sequence of an E. coli O26:H11 strain isolated from humans during the first raw milk cheese outbreak described in France (2005).

Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpsons discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

Although serotype O157:H7 remains the pathogenic Shiga toxin-producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Lactobacillus reuteri suppresses E. coli O157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy to improve food safety?

The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants.

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

BackgroundEnterohemorrhagic 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.ResultsBased 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 O2621765 and O157EDL933 strains by comparison with their behavior in beef meat without microbiota. In strain O2621765, 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 O157EDL933, 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.ConclusionIn 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.