Comparison of molecular Staphylococcus aureus virulence factors among a large world isolates collection

Aim of study: Staphylococcus aureus is one of the most common causative agents of bovine mastitis, responsible for up to 40% of all mastitis cases in some geographical areas (Tenhagen et al., 2006). S. aureus is recognized worldwide as one of the major agent of dairy cow intra-mammary infections and causes subclinical and clinical intrammamary infections (IMI), which may persist through successive lactations and that are transmitted from cow to cow during the milking process. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host (Foster T.J., 2005). The present paper aimed to compare the molecular-epidemiologic profiles of a large world collection of S. aureus isolates to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis. Materials and methods: In this study, a total of 120 isolates from eight different countries (Argentina, Brazil, Colombia, Germany, Italy, New York State, South Africa, Tunisia) were collected from clinical mastitis quarters or composite samples or high somatic cell count samples; they were selected to investigate the diffusion of 27 virulence factors, related to S. aureus pathogenicity. In particular, these isolates were analyzed, by using primers and protocols described in literature, for genes encoding extracellular proteins, such as cell-associated proteins and other extracellular enzymes (cna, scn, chp, sak, fmtB, coa, clfA, spa, nuc) (Zecconi et al., 2006; Sung et al., 2008; Akineden et al., 2001; Cremonesi et al., 2005), leukotoxins (lukS-lukF/PV, lukE-lukD, lukE, lukM) (McClure et al., 2006; Jarraud et al., 2001; Fournier et al., 2008), exfoliatin toxins (eta, etb) (Akineden et al., 2001), superantigens, included enterotoxins (from sea to sel) (Cremonesi et al., 2005; Pinto et al., 2005; Monday & Boach, 1999) and toxic shock syndrome toxin (tsst) (Akineden et al., 2001), but also the acquisition of methicillin resistance (mecA) (McClure et al., 2006). The investigation of these isolates was based on the analysis by PCR and their DNA was amplified to verify the presence of these factors. Main results and Conclusions: At first, all the isolates were positive for polymorphic (coa, spa) genes and thermonuclease (nuc) gene, but negative for SEB and SEE enterotoxins and for an exfoliative toxin (etb) gene. Only one isolate from South Africa was positive for the other exfoliative toxin (eta) gene. Among the virulence factors, fmtB, cna, clfA and leukotoxins genes were the most frequent. Indeed, except for the American isolates, in the other seven countries fmtB and clfA genes were widely present in the isolates, particularly in Brazilian and Tunisian ones. About other extracellular proteins and enzymes, besides Brazil, Germany and USA, the remaining countries showed (27%) isolates encoding at least 2 virulence factors out of scn, chp and sak. Concerning leukotoxins, the presence of lukS/F-PV gene, encoding Panton-Valentine leucocidin (PVL), was lower than 20% in isolates from South Africa and New York State, while it was higher than 80% in those collected in Argentina, Colombia and Brazil. In Germany, Italy and Tunisia none of the isolates carried this gene. Additionally, gene encoding another bicomponent leucotoxin (lukE-lukD) was observed in all isolates, except for one from Germany and another from Tunisia. Furthermore, except for South Africa with only 2 isolates, most (71%) of the others harboured also lukM. Superantigens, especially enterotoxins, were identified in the most (86%) all of isolates: particularly, except for Tunisia, sea and sei were the main enterotoxins genes present in seven countries, with a prevalence between 50 and 90%. On the other hand, seh gene was widespread in Sout