| Mastitis is one of the most common diseases of dairy cows worldwide,which causes huge economic losses.It also causes food safety problems and has well-recognized detrimental effects on human health.Staphylococcus aureus(S.aureus)is the predominant pathogen associated with bovine mastitis which also constitutes a threat to public health due to the zoonotic potential.The treatment with antibiotics is the major method for controlling S.aureus bovine mastitis;however,S.aureus can easily acquire resistance to various antibiotics under long-term selection pressures.Forming small colony variants(SCVs)is an important strategy used by S.aureus to counteract the action of antimicrobials,which is of great significance for the chronic and persistent S.aureus bovine mastitis.SCVs constitute a subpopulation of bacteria with distinctive phenotypic and pathogenic traits.Clinically,S.aureus SCVs have been associated with persistent and recurrent infections,and the most conspicuous features of these SCVs include:slow growth,decreased oxidative metabolism of sugars,non-pigmented colonies,reduced hemolytic activity,reduced toxin production,delayed coagulase activity,reduced susceptibility to aminoglycosides and β-lactam antibiotics.In order to better control S.aureus SCVs associated with bovine mastitis,S.aureus strains in the milk of cows with a history of chronic S.aureus mastitis were isolated and identified.The infection of S.aureus SCVs in the mammary was gained and several clinical wild-type strains and a SCV strain were obtained.The clinically isolated S.aureus SCVs and their wild-type S.aureus strains were reversed and induced respectively.Then the characteristics of the isolated strains,the induced strains and the revertants were tested.All the wild-type strains showed a typical fast-growing phenotype,while the SC Vs showed a typical phenotype with slow growth and metabolic disorders.At the same time,SCVs showed auxotrophy and increased resistance.Results of whole genome sequencing(WGS)revealed key genes associated with the SCV phenotype and described the adaptation of the SCVs during the reversion.1.Isolation of wild-type S.aureus and SCVWe isolated strains from 5 cows with a history of chronic S.aureus mastitis from a dairy farm located in northern China.As a result,8 clinical wild-type S.aureus strains and a phenotypically stable SCV(106 SCV)were obtained.The SCV(106)and its wild-type S.aureus strain(107)isolated from the same sample were subjected to phenotypic and genotype identification,and the multiple loci VNTR analysis(MLVA)result was obtained.The result showed that the two isolates belonged to the same molecular type.Auxotrophism assay showed that the 106 SCV was menadione auxotroph.This is the first report in China that one S.aureus SCV of menadione auxotroph was isolated from cow milk.2.Induction,reversion and characterization of S.aureus SCVs associated with bovine mastitisThe SCVs of S.aureus are closely associated with chronic and persistent mastitis in dairy cows and are significantly different from their wild-type strains in the characteristics.In this study,SCVs(102,106)were subjected to reversion and their parent strains(101,107)were subjected to artificial induction respectively,then the biological characteristics of strains were tested.Induction test showed that almost all of wild-type strains treated with gentamicin or sulfamethoxazole/trimethoprim(SXT)developed SCVs of menadione auxotrophs,except one SCV induced by SXT was confirmed as thymidine-auxotrophic SCV.The finding that SCV induced by SXT developed menadione auxotroph has not been reported yet.The reversion test showed that both 102 and 106 could be reversed,and the revertants no longer had auxotrophs.All wild-type strains grew rapidly,while SCV strains showed typical phenotypes of SCV,such as slow growth and metabolic disorders.The results of drug susceptibility tests showed that the antimicrobial resistances of the SCV strains to most tested drugs enhanced,especially the resistances to gentamicin sulfate and penicillin G sodium were much higher than that of their parent strains.The induced strains were more resistant to antimicrobials,and their resistance characteristics were consistent with the auxotrophs.The antimicrobial susceptibility of the revertants to a variety of antimicrobial agents was increased.3.Genetic basis of S.aureus SCVs associated with persistent bovine mastitisIt has been reported that the changes in the characteristics and the adaptation in reversion of SCVs of S.aureus are associated with the genome plasticity.The whole genomes of clinical wild type(101)and corresponding clinical SCV(102),induced SCV(SXT-101,G-101),the revertant(102-Reverse),clinical wild type(107)and its corresponding clinical SCV(106),and the revertant(106-Reverse)were sequenced.The results showed that there was a clear correlation between the phenotype and mutation of strains:(1)The deletion of the base of menA gene related to quinol/quinone metabolism in the genome of clinical SCV 102 and 106 might result in the early termination of the protein.(2)Compared with the wild-type strain,G-101 which induced by gentamicin showed a single base change in gene;SXT-101 which induced by SXT showed deletion and insertion of nucleotide fragments in metabolism-related genes MW1485 and aroA,respectively,which was first reported.(3)Most of the SCVs showed mutations in the virulence-related genes spa,clfB and MW2416,while 102 and 106 showed deletion of MW2095,which might be associated with their resistance characteristics.(4)Compared with the wild-type strain,the revertants(102-Reverse,106-Reverse)might completely or partially restore the function of mutated genes causing the SCV phenotype by acquisition of a second-site mutation within the metabolic gene menA and reversion to the wild-type gene sequence within the virulence-related gene MW2416. |
