Occurrence of ssl genes in isolates of Staphylococcus aureus from animal infection Davida S. Smyth, 1 3 William J. Meaney, 2 Patrick J. Hartigan 3 and Cyril J. Smyth 1 Correspondence Cyril J. Smyth csmyth@tcd.ie 1 Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Ireland 2 Teagasc, Dairy Production Research Centre, Moorepark, Fermoy, Co. Cork, Ireland 3 Department of Physiology, School of Medicine, Trinity College, University of Dublin, Dublin 2, Ireland Received 6 August 2006 Accepted 26 October 2006 The occurrence of 7 of the 11 known ssl genes that are found within the vSaa genomic island of Staphylococcus aureus and encode the novel Ssl family of exoproteins was examined in isolates from cows (42 isolates), goats (4 isolates), sheep (1 isolate), rabbits (3 isolates) and chickens (2 isolates). Based on seven S. aureus genome sequences for human strains NCTC 8325, N315, Mu50, COL, MRSA 252, MW2 and MSSA-476, and bovine strain RF122, along with the ssl reference gene sequences from strains NCTC 6571, FRI326 and NCTC 8325, ClustalW- generated alignments were used to design PCR primers for unique regions of the ssl genes that are present in the allelic variants of each, except for the ssl4 gene for which specific primers for the set2 and set9 allelic variants were designed individually. The genotypes of isolates were determined using random amplified polymorphic DNA (RAPD) typing. All of the animal-associated S. aureus isolates contained an ssl locus, but there were minor variations in the number of ssl genes present. Forty-nine of the animal isolates possessed a vSaa genomic island containing the ssl3 (set8), ssl5 (set3/set10), ssl7 (set1/set11), ssl8 (set12), ssl9 (set5/set13) and ssl10 (set4/set14) genes. One bovine and one goat isolate lacked the ssl3 gene. The ssl9 gene was absent in one bovine isolate. The goat isolate lacking the ssl3 gene was the only animal isolate that possessed the set2 allele of the ssl4 gene. PCR for the set9 allele of the ssl4 gene was inconclusive. Isolates that showed identical RAPD fingerprints had the same complement of ssl genes, but the ssl gene pattern was not RAPD-type specific. Southern blot hybridization showed similar ssl gene RFLPs in isolates of the same RAPD type. INTRODUCTION Staphylococcus aureus causes a wide variety of clinical syndromes ranging from uncomplicated infections of the skin, such as boils and carbuncles, to life-threatening infections, such as endocarditis and toxic shock syndrome (Murray, 2005; Todd, 2005). S. aureus is also an important pathogen of animals including cows, goats, sheep, rabbits and chickens (Rich, 2005; Barkema et al., 2006). Among the many known virulence factors of S. aureus are three groups of staphylococcal exoproteins: the enzymes such as hyaluro- nidase, proteases and nucleases; the non-enzymic activators of plasma clotting (coagulase) and fibrinolysis (staphyloki- nase); and the exotoxins, namely, cytolytic toxins, exfoliative toxins, leukocidins, enterotoxins, enterotoxin-like proteins and toxic shock syndrome toxin no. 1 (TSST-1) (Ferry et al., 2005). The enterotoxins, enterotoxin-like proteins and TSST-1 belong to the superantigen (SAg) family (Proft & Fraser, 2003). Nineteen staphylococcal enterotoxins (SEs) and staphylococcal enterotoxin-like (SEl) proteins, as well as a number of variants of some of these, have been described (Smyth et al., 2004; Thomas et al., 2006). In addition to these superantigens, S. aureus also produces a family of exoproteins termed the staphylococcal exotoxin- like (Set) proteins (Williams et al., 2000), which have since been renamed staphylococcal superantigen-like (Ssl) pro- teins (Lina et al., 2004). The Ssl proteins are encoded by a cluster of genes (also termed genomic island vSaa; Lindsay & Holden, 2004, 2006) that show sequence similarity of 36–67 % (Williams et al., 2000; Kuroda et al., 2001). The vSaa island is flanked upstream by a putative transposase gene, and downstream by an incomplete restriction and modification system (the hsdS and hsdM genes) forming 3Present address: New York Medical College, Department of Microbiology and Immunology, Valhalla, NY 10595, USA. Abbreviation: RAPD, random amplified polymorphic DNA. 418 46878 G 2007 SGM Printed in Great Britain Journal of Medical Microbiology (2007), 56, 418–425 DOI 10.1099/jmm.0.46878-0