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Human 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

The influence of five technological parameters selected amongst uncooked semi-hard cheese-making practices, i. e. milk maturation temperature and time, stirring during maturation, curd stirring time and pressing time was examined on Staphylococcus aureus growth, enterotoxin gene expression and enterotoxin (SE) production during cheese manufacture. A fractional factorial experimental design was applied to perform 32 cheese batches independently inoculated at 10 3 cfu/ml of milk using four strains producing SEA, SEB, SEC or SED. The S. aureus population was found to exceed 10 5 cfu/g of cheese four hours after molding. SED was the only enterotoxin detected. It was produced in very low quantities that varied with the parameters studied. Early sed gene expression during cheese processing was correlated with SED detection in curd and cheese. Milk maturation temperature and time emerge as key technological parameters that control SED production. A response surface methodology was then carried out to further characterize the relationships between both factors and SE production in cheese and whey. Two SED-producing strains were used to perform two sets of ten cheese batches based on a central composite design of experiments at five levels. Predictive mathematical models were established. Increasing the temperature at the beginning of the cheese-making process was shown to increase SED production. Furthermore, we determined that the proportion of SED drained after molding from the curd in the whey depended only on the technological parameters. The two SED-producing strains showed similar trends of behavior but specific level of gene expression and enterotoxin production in response to the same set of milk maturation parameters. [ABSTRACT FROM AUTHOR]