Virulence And Resistance Characteristics Of Staphylococcus Aureus Isolated From Bovine Mastitis And Its Pathogenic Difference With SCVs

Bovine mastitis is a common disease in dairy animals and causes heavy losses to the dairy industry worldwide.Staphylococcus aureus,one of the major pathogens of bovine mastitis,is causing clinical,subclinical and recurrent infections indairy animals.Antimicrobial agents are being administered as an important treatment strategy due to lack of effective vaccines against mastitis.Therefore,it is important to know the virulence patterns and antimicrobial susceptibility of this bacteria.Small colony variants(SCVs)are reported to be associated with chronic and recurrent infections in humans and animals.However,the difference with its parent strain in pathogenesis is unknown.Hence,we try to detect the virulence gene patterns,molecular types,phenotype and genotype of antimicrobial resistance in S.aureus isolated from clinical bovine mastitis;and to study the difference in challenging bovine mammary epithelial cells(bMECs)between SCVs and its parent strain.A total of 103 S.aureus isolates were collected from 1021 clinical bovine mastitis milk samples which spread in 19 herds of 9 different provinces.PCR method was used to detect the prevalence of 17 virulence genes and to determine their patterns.Specially,the gene of α-hemolysin is typed according to its gene sequence.In addition,spa typing and MLST typing are determined.The results of this study showed that the prevalence of 17 virulence factors varied from 2%to 100%.The 38 different virulence gene patterns were determined and correlated with MLST types.The hla gene demonstrated high diversity while its amino acid sequence is more conserved.Seven MLST and 24 spa types were obtained,of which ST97 was the most predominant.Phylogenic tree analysis showed that the evolution of hla gene was consistent with MLST classification.In addition,minimum inhibition concentration(MICs)method was used to evaluate the antimicrobial resistant phenotypes.PCR method was carried out to detect the prevalence of antimicrobial resistance genes.Furthermore,survival analysis and index cluster analysis were used to estimate the correlation between antimicrobial resistance phenotypes and genotypes and intll.Results showed that S.aureus were resistant to 11 antibiotics with a range of 1.9-85.5%,and the rate of 15 antimicrobial resistance genes varied from 1 to 82.5%.Index cluster showed that ampicillin-resistance,erythromycin-resistance and penicillin G-resistance were highly related and were mediated by class 1 integron.Survival analysis indicated a significant association between ermC gene and the increasing concentration of erythromycin.In addition,the index cluster showed that ampicillin-resistance,erythromycin-resistance and penicillin G-resistance were highly related and maybe mediated by class 1 integron.Finally,we studied the pathogenicity differences between one isolate of S.aureus and its small colony variant(SCV)by detecting the adhesion,invasion,intracellular survival rate,LDH release level,apoptosis and immune factor mRNA levels.Furthermore,the adhesion and non-adhesion strains were sequenced by transcriptome.The results showed that in comparison with its parent strain,SCV showed stronger ability of the adhesion and invasion,longer intracellular survival time,weaker cytotoxicity,lower cell apoptosis and weaker innate immune response.Transcriptome analysis showed that the differentially expressed genes were mainly related to biological processes,such as localization,transport activity,amino acid metabolism,etcand involved into 176 pathways.Moreover,the differentially expressed genes differed between SA and SCVs during normal conolies and infection,which may be correlated with the different pathogenicity.To sum up,the hla gene evolution is consistent with MLST classification,indicating the potential of being molecular marker for the classification of S.aureus.With a high multiple drug-resistance rate,S.aureus is most resistant to erythromycin,penicillin and ampicillin.Compared with parent strain,SCVs causes weaker apoptosis innate immune response,and the intracellular survival time is longer.Transcriptomic analysis enriched the information of the transcription group of the small colony mutant of S.aureus,and laid a foundation for further research on related genes and its molecular regulation mechanism.