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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. Image 1 • Enterotoxin genes seb, sec, see, and tsst-1 were identified in some tested strains of S. aureus. • Most isolates were negative for enterotoxins and seb was the most prevalent enterotoxin. • A total of 111 S. aureus were grouped into 16 PFGE types, of which I, M, and O were dominant clones. • No significant differences among I, M and O PFGE types in adhesion and invasion of mammary epithelial cells. • We found diverse clonal types, of which M and O clustered with β-hemolysin, weak slime and sensitivity to antibiotics. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a frequent and major contagious mastitis bacterial pathogen. The antibiotic treatment cure rates vary considerably from 4% to 92%. Staphylococcus aureus readily becomes resistant to antibiotics, resulting in persistent noncurable intramammary infection that usually results in culling of infected animals. Because of its notorious ability to acquire resistance to the commonly used as well as last resort antimicrobials such as methicillin and vancomycin and the development of multidrug-resistant strains, antimicrobial resistance (AMR) in S. aureus is of paramount importance in human medicine. The objective of this study was to evaluate the prevalence of AMR and genetic diversity of S. aureus isolates from milk of dairy cattle. Staphylococcus aureus isolates (n = 239) from 33 dairy farms in Tennessee were tested against 10 antimicrobials by broth microdilution method using the Sensititer system. Genetic diversity of resistant isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, AMR of the S. aureus isolates varied from as low as 1.3% for ceftiofur to as high as 25% for sulfadimethoxine. Out of 239 S. aureus isolates, 82 (34.3%) of them were resistant to at least 1 of the 10 antimicrobials. The AMR isolates belonged to two major PFGE types, indicating the presence of dominant clonal patterns among the resistant isolates. In general, there was a variation of prevalence of AMR within and among farms over time, with an increasing trend in tetracycline resistance. Judicious use of antimicrobials in dairy cattle farms can reduce the development of antimicrobial-resistant S. aureus.