| Staphylococcus aureus(S.aureus)can cause many animal diseases,such as mastitis,pneumonia and arthritis in dairy cows,leading to seriously economic losses to the breeding industry and dairy industry.S.aureus can become resistant to a variety of antimicrobial agents,and multidrug resistance is severe,which brings great difficulties to prevention and treatment of diseases caused by S.aureus.This study analyzed the antimicrobial resistance and virulence gene distribution in S.aureus strains isolated from bovine mastitis in Liaoning.This research can provide a basis for clinical prevention and treatment of bovine mastitis in Liaoning area.In this study,S.aureus was isolated from milk samples of bovines with clinical mastitis in Liaoning,and these isolates were identified by biochemical methods.Then the nuc gene was amplified by PCR to further identify S.aureus isolates.The minimal inhibitory concentrations(MICs)of different antimicrobial agents such as beta-lactams,macrolides and aminoglycosides against S.aureus isolates were determined by microbroth dilution method,and their drug resistance phenotypes were analyzed.The results showed that 55 strains of S.aureus were isolated and identified,and antimicrobial resistance varied among different strains.S.aureus isolates showed the highest resistance rates to penicillin and lincomycin,with resistance rates of 90.91%.S.aureus isolates were most sensitive to bacillin and aminoglycosides.The resistance rates to bacillin,gentamicin and amikacin were 14.55%,10.91% and 14.55%,respectively.In addition,the multi-drug resistance of S.aureus isolates was serious.No strains was sensitive to all antibiotic,and all isolate was resistant to at least 5 kinds of drugs.The virulence genes of 55 strains of S.aureus isolated from Liaoning area with clinical mastitis were detected by PCR.The virulence gene of S.aureus isolates from bovines in Liaoning was analyzed.The results showed that three virulence genes were detected,namely hla(58.18%),clf A(36.36%)and fnb A(29.09%).The virulence genes of sea,seb,eta and etb were not detected.The expression of hla m RNA in three groups of S.aureus strains resistant to beta-lactams was determined by real-time fluorescence quantitative PCR.The results showed that the expression of hla gene m RNA decreased with the increase of the number of beta-lactam-resistant strains of S.aureus.Compared with the standard strains,the expression of hla gene in the two beta-lactam-resistant groups and the four beta-lactam-resistant groups increased significantly(P<0.01),but the expression of hla gene in the six beta-lactam-resistant groups did not differ significantly from that in the standard strains.Nine strains of S.aureus with different antimicrobial-resistant phenotypes were selected to carry out pathogenic test in mice,and compared with ATCC29313 which used as a quality control.Pathogenicity test in mice was carried out to analyze the influence of different antimicrobial-resistant phenotypes on pathogenicity.The results showed that the average mortality rates of standard strains,two beta-lactam-resistant groups,four beta-lactam-resistant groups and six beta-lactam-resistant groups were 33.33%,77.78%,50% and 27.78% respectively.With the increase of beta-lactam-resistant strains of S.aureus isolated from bovine mastitis,the mortality rate of mice decreased gradually.This study analyzed the epidemic situation of antimicrobial resistance phenotype and virulence gene of clinical S.aureus isolates from dairy cows in Liaoning province,and the influence of drug resistance on their pathogenicity,providing theoretical basis for antimicrobial use and effective prevention and control of bovine mastitis disease in this area. |
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