| Staphylococcus aureus(referred to as S.aureus)as the main pathogenic bacteria causing disease infection,not only threat to human and animal health,but also has effects on food health and safety.S.aureus is the main pathogen causing infection disease in dairy farming-bovine mastiti.As a special group of S.aureus,small colony variants(SCVs)can not only survive in host cells for a long time,but also cause persistent infection.In this study,milk samples from some large-scale dairy farms in southern Xinjiang were collected,and S.aureus was isolated and identified.S.aureus was typed using multilocus sequence typing(MLST)and Staphylococcal protein A(spa)methods.At the same time,the isolates were tested for their antimicrobial sensitivity and virulence gene.Meanwhile,analysising the biological characteristics and genome of SCVs to provide theoretical basis for the subsequent prevention and treatment of infection.In this study,71 strains of S.aureus were subjected to identification by routine biochemical and 16 S r RNA and nuc gene sequencing was used to confirmation of identification.All isolates was typed using multilocus sequence typing(MLST)and Staphylococcal protein A(spa)methods,and was tested for 12 kinds of antimicrobial susceptibility by K-B method.The molecular typing results showed that 71 isolates belonged to 18 ST types(Sequence typing)(including 11 news ST-types and 5 Clonal complex(CC)(includng CC188,CC22,CC398,CC352 and CC5406.All strains belong to 6 spa types(including t2846,t12527,t309,t705 and t2876).This result indicated the population diversity of S.aureus in southern Xinjiang.The results showed that all strains were resistant to 5 drugs,including gentamicin(9.9%,7/71),norfloxacin(12.7%,9/71)and erythromycin(30.9%,22/71).In addition,nine multidrug-resistan(MDR)strains were found.At the same time,the resistance spectrum of S.aureus is different because of its CC and spa types.Except CC398,the other clones were resistant to erythromycin.The MDR strain belong to ST398 and CC398-t2876.All isolates were tested 20 virulence genes,including enterotoxin gene SEs etc,The clf A and clf B have the highest detection rate of all genes(98.6%,70/71).The hemolytic genes hla and hlb(73.3%,52/71)and(90.1%,64/71)were found in majority strains,and had high detection rates in CC188,CC352,and CC5406.fnb A and fnb B only were not detected in strain CC5406.The exfoliative toxin genes(eta and etb)and some SEs genes(see,seh,sej)were not detected in all strains.The biological characteristics of the SCV strains was found that the SCV were transparent,needle-like,less pigment,and the mannitol fermentation ability was obviously reduced.The SCV strain also showed resistance to aminoglycosides.The expression of mannitol operon(mtl ARFD)in wild-type and SCV strains was detected,and the related genes were found in both wild-type and SCV strain.But at the transcription level,the expression of mtl A,mtl F,and mtl D were found down-regulated in the SCV.While the expression of mtl R gene was up-regulated.Which Explained the molecular mechanism of SCV strain not fermenting mannitol.At the same time,the aminoglycoside resistance gene detection revealed that aph(3'),ant(6'),and ant(4')were not detected in wild-type and SCV.Aac(6')/aph(2')were detected in wild-type and SCV.At the same time,the ant(3')was not detected in the wild-type,but it was detected in the SCV.The result indicated that ant(3')could be the reason of resistant for gentamicin and extremely MIC for multiple aminoglycoside antibiotics.By whole-genome sequencing and functional element annotation,the result found that wild-type and SCV had obvious difference in metabolism and antibiotic resistance.The number and type of t RNA predicted in SCV were different from those in wild strains,and no TRNA was found to transport valine in SCV strains,these results suggest that there may be significant differences in the efficiency of transcription and the types of translated proteins.The results of GO,egg NOG,and KEGG annotations indicated that there are differences in functional genes involved in metabolism and antibiotic resistance among the SCV and wild-type,which are closely related to phenotype and biochemical characteristics.In this study,molecular typing,virulence genes,and drug sensitivity analysis found that some of the virulence carrying and drug resistance were related to clone complexes.For example,fnb A and fnb B were not detected in the CC5406 type,while the CC398 was closely related to Multi-drug resistant strains.And the spread of drug resistance is not only related to the clonal population,but also to the survival mode of pathogenic bacteria.The whole genome sequencing of the parental strains and SCV strains obtained revealed that there are significant differences in metabolism and antibiotic resistance between the two different phenotypes and survival modes of the strains,which are reflected at the gene level and transcription level. |
PDF Full Text Request