Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese

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 mic...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2010-03, Vol.76 (5), p.1367-1374
Main Authors: Duquenne, Manon, Fleurot, Isabelle, Aigle, Marina, Darrigo, Claire, Borezée-Durant, Elise, Derzelle, Sylviane, Bouix, Marielle, Deperrois-Lafarge, Véronique, Delacroix-Buchet, Agnès
Format: Article
Language:English
Subjects:
Animals
Bacterial Toxins - biosynthesis
Bacterial Toxins - genetics
Biological and medical sciences
Cheese
Cheese - microbiology
Enterotoxins - biosynthesis
Enterotoxins - genetics
Food Microbiology
Fundamental and applied biological sciences. Psychology
Gene Expression
Genes
Lactobacillus
Microbiology
Microorganisms
Milk - microbiology
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA, Bacterial - genetics
RNA, Bacterial - isolation & purification
Staphylococcus aureus - genetics
Staphylococcus infections
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Summary: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.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.01736-09