A Polyphasic Approach To Detect Enterotoxigenic Staphylococcus aureusand Diarrheagenic Escherichia coliin Raw Milk Italian Cheeses by Multiplex PCR

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)-encoding genes (sea, sec, sed, seg, seh, sei, sej, sel)and the Escherichia coligenes most commonly associated with virulence factors (eae, elt, ipaH, stx)in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture- independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community (“bulk”). Genes associated with enteroinvasive E. coli, ipaH,and Shiga toxin-producing E. coli, stx,were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (seain all three samples, associated with sedand sejin two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel,and sei.No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.