Molecular typing of Staphylococcus aureus isolated from Italian dairy products on the basis of coagulase gene polymorphism, multiple-locus variable-number tandem-repeat and toxin genes Morandi Stefano 1 *, Brasca Milena 1 , Lodi Roberta 1 and Brusetti Lorenzo 2 1 Institute of Sciences of Food Production, Italian National Research Council, via Celoria 2, 20133 Milan, Italy 2 Department of Food Science and Microbiology (DISTAM), Universita ` degli Studi di Milano, via Celoria 2, 20133, Milan, Italy Received 18 June 2007 ; accepted for publication 12 May 2008 Coagulase gene restriction fragment length polymorphism (RFLP), six-locus variable-number tandem-repeat analysis patterns (MLVA) and detection of enterotoxin genes (se)(sea, sec, sed, seg, seh, sei, sej and sel) were used to determine the phylogenetic relationship among isolates of Staphylococcus aureus isolated from dairy products from different regions of Italy. A total of 25 Staph. aureus were subtyped into 16 coagulase genotypes by RFLP, and MLVA revealed marked genomic variability. Furthermore, 17 of the isolates harboured at least one toxin gene, with the predominance of sea, sed and sej among cow isolates and sec-sel among the goat and sheep strains. Combined RFLP, MLVA polymorphism and se genes were found to be useful techniques for discriminating several genetic variants in Staph. aureus isolates. Keywords : Staphylococcus aureus, enterotoxins, coagulase, molecular typing, MLVA, RFLP. Staphylococcus aureus is an ubiquitous bacteria com- monly isolated from bulk raw milk, and from the milk of dairy cattle suffering from mastitis (Jørgensen et al. 2005). Its presence in raw milk is a major concern for the safety and quality of traditionally produced cheeses. Some Staph. aureus strains in the natural population can produce staphylococcal enterotoxins (SEs) (Balaban and Rasooly 2000), and these were recently reported to be responsible for food poisoning associated with reconstituted milk, in Japan (Ikeda et al. 2005), and cheese consumption, in European countries (Le Loir et al. 2003) and Brazil (do Carmo et al. 2002). A number of typing techniques are available to help trace the source and transmission rates of Staph. aureus from foods and clinical specimens : restriction fragments length polymorphism analysis (RFLP), pulse-field gel elec- trophoresis (PFGE), multilocus sequence typing (MLST) and multiple-locus variable-number tandem-repeat (MLVA). RFLP analysis of the coagulase (coa) is a useful method for typing Staph. aureus isolates for epidemiological study : amplified DNA fragments of different size can be further discriminated by digestion with AluI. This geno- typing method is an easy and useful tool that provides epidemiological information about Staph. aureus (Hookey et al. 1998; Moon et al. 2007). The MLVA approach is based on the detection of the number of tandem repeats (TRs) at a specific locus in the genome of a microorganism. These can vary as a conse- quence of DNA polymerase enzyme slippage during rep- lication, and these differences can be detected using PCR primers designed to anneal to the flanking regions (Keim et al. 2000). The complete TR is amplified and sized using a conventional agarose gel. MLVA has proved very efficient, not only for Staph. aureus but also for numerous bacterial pathogens such as Bacillus anthracis and Yersinia pestis (Lindstedt, 2005 ; Merabishvili et al. 2006). It was recently applied to human Staph. aureus isolates and its discriminatory power was comparable to PFGE and MLST (Sabat et al. 2003; Francois et al. 2005 ; Malachowa et al. 2005). However, in contrast to the human isolates, data available in the literature about the spreading and typing of Staph. aureus isolated from bovine and from dairy products is quite limited (Vimercati et al. 2006; Gilbert et al. 2006). In the present study, coagulase gene (coa) RFLP analy- sis, considered easy to perform and having high levels of specimen typeability and reproducibility (Chiou et al. 2000) and MLVA typing (a technique not yet used for typing Staph. aureus isolated from food) were used to *For correspondence ; e-mail : stefano.morandi@ispa.cnr.it Journal of Dairy Research, Page 1 of 6. f Proprietors of Journal of Dairy Research 2008 1 doi:10.1017/S0022029908003476 Printed in the United Kingdom