Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/l) than using a fluorigenic TaqMan probe (6 nuc gene copies/l). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 ؋ 10 2 to 6.4 ؋ 10 2 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR. Food poisoning caused by the enterotoxins produced byStaphylococcus aureus is of major concern in food hygiene. Many outbreaks involving contaminated milk and milk products have been reported (2,8,12,13,21). The natural habitat of S. aureus is warm-blooded animals, including humans. Ten to 40% of people are asymptomatic carriers of S. aureus, mostly at the mucosal membrane (20). Food handlers with infected skin lesions and asymptomatic carriers are often sources of food contamination, although with milk and milk products the raw milk itself can be contaminated with S. aureus.Once introduced into food-processing plants, bacteria can persist for a long time. CFU of S. aureus numbering 10 6 to 10 8 per g or ml of food must be present in order to produce sufficient amounts of toxins for poisoning (13). The bacteria are readily destroyed by heat, but the toxins are thermostable. Austrian national regulations concerning the presence of S. aureus in milk and milk products apply three-class plans that define m values to separate acceptable from marginally acceptable products and M values to separate marginally acceptable from defective-quality products; when numbers of bacteria are higher than M values, the presence of enterotoxins has to be investigated. The International Standardization Organization and International Dairy Federation methods (3, 4, 5, 6) apply conventional microbiological quantification techniques such as the plate count method and the most-probable-number (MPN) method. Some of these techniques require up to 6 days for quantification and detection, thus being time-consuming. There are rapid alternative methods for quantification of S. aureus cells. These comprise the application of MPN-PCR (17), a rather laborious procedure, and the quantification of the enzymatic activity of the bacterial phosphatase (10), which is not specific and can therefore be applied only to pure cultures. Another rapid alternative is real-time quantitative PCR (RTQ-PCR) (11), which quantifies DNA and th...

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We report the first documented Campylobacter jejuni outbreak in an Austrian youth centre. Sixty-four children were involved of which 38 showed classical signs of campylobacter gastroenteritis. Since unpasteurized milk distributed by a local dairy was suspected to be the source of infection, stool samples were collected from 20 cows providing the milk. Five of the cows tested positive for C. jejuni. These isolates together with 37 clinical samples were compared by pulsed-field-gel electrophoresis (PFGE). The PFGE patterns, using the restriction endonucleases SmaI and SalI, were identical for the human and bovine isolates. This finding confirmed that the outbreak was caused by the consumption of unpasteurized milk contaminated with C. jejuni.