Genetic diversity and virulence characteristics of Staphylococcus aureus isolates from cases of bovine mastitis.

Staphylococcus aureus is one of the major bacterial mastitis pathogens with significant effects on animal and human health. Some studies showed that S. aureus strains that infect different host species are genetically distinct, although most strains can infect a wide range of host species. However, there are no clearly defined clonal patterns of S. aureus strains that are known to infect a specific host. The objectives of this study were to evaluate the clonal diversity and virulence characteristics of S. aureus isolates from cases of bovine mastitis. Bacteriological tests were conducted on milk samples from cases of bovine mastitis from 11 dairy farms including some milk samples from unknown farms in Eastern Tennessee. Overall, a total of 111 S. aureus were isolated and identified, and further evaluated for their genetic diversity by pulsed-field gel electrophoresis (PFGE) and virulence characteristics by PCR. Genotypic virulence factors including staphylococcal enterotoxins, and toxic shock syndrome toxin 1 (tsst-1) were tested by PCR. In addition, the association among several known virulence factors of these isolates based on our current and previous studies in our lab were evaluated. Previously generated data that were included in the analysis of association among virulence factors were the presence of biofilm production associated genes in the ica operon such as icaA, icaD and icaAB, and phenotypic virulence characteristics such as hemolysis on blood agar, slime production and resistance or susceptibility to ten commonly used antimicrobials in dairy farms. The PFGE results showed the presence of 16 PFGE types (designated A - P) throughout farms, of which three pulsotypes, I, M and O were the most frequently isolated PFGE types from most farms. The PFGE type M was the most prevalent of all 16 PFGE types, with 64 isolates being present among nine farms. The PCR results of enterotoxin genes showed that out of the total 111 tested 84 (75.7%) were negative whereas 13 (11.7%), 2 (1.8%), 3 (2.7%), 1 (0.9%) and 8 (7.2%) were positive for seb, seb and sec, sec, see, and tsst-1, respectively. All 111 isolates were negative for sea and sej. Results of the evaluation of I, M and O strains adhesion to and invasion into mammary epithelial cells showed that the total count of each strain of bacteria adhered to and invaded into mammary epithelial cell line (MAC-T cells) was not significantly different (P > 0.05). This may be an indication that there is no significant difference in their ability to establish early host-pathogen interaction and colonization of the host. There were no statistically significant associations among PFGE types and other known virulence factors of these strains. However, PFGE types O and M tend to cluster with β-hemolysin, absence of enterotoxins and susceptibility to antimicrobials. In conclusion, there was not any association between pulsotype and genotypic and phenotypic virulence factors. S. aureus isolates from cases of bovine mastitis had diverse genotypes that possessed variable virulence factors.