Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.Staphylococcus aureus is a significant bacterial pathogen producing a variety of proteins and toxins that contribute to its ability to colonize and cause diseases (15). Some S. aureus strains are able to produce staphylococcal enterotoxins (SEs) in food matrices and are responsible for food poisoning, characterized by such symptoms as nausea, vomiting, abdominal cramps, and diarrhea (2). In France S. aureus is reported as the most frequent pathogen involved in food-borne diseases associated with dairy products (12) and especially with raw milk cheeses (9). It is generally accepted that SE production constitutes a risk when S. aureus bacteria exceed a threshold of 10 5 S. aureus CFU per gram of cheese during manufacture. Numerous studies have reported on S. aureus behavior during cheese manufacturing, focusing only on S. aureus growth and enterotoxin production (1, 8, 11, 18, 21, 22, 31, 32, 34-36, 42, 43, 49). To our knowledge, no study has investigated the expression of the genes encoding SEs in foods or during food production. Numerous parameters, such as pH, aeration, or temperature, could, indeed, affect the expression of these genes. During the cheese-making process, natural staphylococcal contamination is minor in the total microbial population. The initial S. aureus contamination is usually below 10 3 CFU/ml of raw milk, while bacterial starters are inoculated at least at 10 6 CFU/ml of milk. Analysis of SE gene expression in situ thus requires an efficient method of extracting bacterial RNA from cheese to ensure recovery of quantifiable amounts of staphylococcal RNA. A precise method is also needed to quantify minor transcripts in the extracted bact...
cHuman intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.
Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway.
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