title: Tool for quantification of staphylococcal enterotoxin gene expression in cheese.
abstract: 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.
authors.affiliation: UMR 1319, Institut Micalis, Microbiologie de l'Alimentation au Service de la Santé, INRA, AgroParisTech, Jouy en Josas, France.
authors.forename: Manon
authors.initials: M
authors.lastname: Duquenne
authors.forename: Isabelle
authors.initials: I
authors.lastname: Fleurot
authors.forename: Marina
authors.initials: M
authors.lastname: Aigle
authors.forename: Claire
authors.initials: C
authors.lastname: Darrigo
authors.forename: Elise
authors.initials: E
authors.lastname: Borezée-Durant
authors.forename: Sylviane
authors.initials: S
authors.lastname: Derzelle
authors.forename: Marielle
authors.initials: M
authors.lastname: Bouix
authors.forename: Véronique
authors.initials: V
authors.lastname: Deperrois-Lafarge
authors.forename: Agnès
authors.initials: A
authors.lastname: Delacroix-Buchet
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D002611:Cheese / Q000382:microbiology*; D004768:Enterotoxins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D008892:Milk / Q000382:microbiology; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D012329:RNA, Bacterial