Isolation,Identification And Biological Characteristics Of Staphylococcus Aureus From Yak

In this experiment,45 nasal swabs and 56 yak milk samples collected aseptically from clinical yaks in different regions of Tibet were isolated and cultured.the isolated bacteria were identified by Gram staining,biochemical test and 16 S r DNA sequencing,and Staphylococcus aureus was identified.Drug resistance gene and virulence gene were detected by PCR technique.At the same time,drug sensitivity test and mouse challenge test were carried out to study its drug resistance and pathogenicity,and the whole genome of the bacteria was detected,and its biological characteristics were analyzed based on the above experimental data.in order to provide some basis and reference for the treatment of yak Staphylococcus aureus infection.The specific experimental studies are as follows:1.Isolation and identification of Staphylococcus aureus from yaks in TibetThe bacteria from 101 yak samples were isolated and cultured.After Gram staining,biochemical test and 16 S rDNA sequencing,7 strains with high similarity to Staphylococcus aureus were obtained after NCBI comparison,and the similarity was more than 98%.Combining the above factors,it can be further identified as yak Staphylococcus aureus,named ZJ1～ZJ7,and the isolation rate is about 6.93%.All the 7 isolates showedβ hemolysis in 5% sheep blood Agar plate,indicating that these strains have a certain pathogenicity and can make the blood plate produce hemolysis ring.2.Analysis of drug resistance of Staphylococcus aureus from yaks in TibetFive kinds of drug resistance genes related to Staphylococcus aureus were selected,and the corresponding primers were synthesized.fexA,cfr,mecA,tetM and tetK 5 drug resistance genes were detected by PCR amplification technique.The results showed that 7 strains of Staphylococcus aureus from yak carried tetM,tetK and mec A3 drug resistance genes,while the other two drug resistance genes were not detected.16 common antibiotics were selected,and drug sensitivity test was carried out by K-B disk diffusion method.The results showed that 7strains were highly resistant to penicillin and ampicillin,and the resistance rate was 100%.7 strains were sensitive to 6 antibiotics such as amikacin and gentamicin,except ZJ3 to neomycin,the other 6 strains were sensitive to neomycin,and the sensitive rate was about 85.71%.Except for ZJ5 to clindamycin,ZJ1～ZJ4,ZJ6 and ZJ7 were sensitive to clindamycin.3.Study on the pathogenicity of Staphylococcus aureus from yaks in TibetThe corresponding primers of 13 virulence genes of Staphylococcus aureus,including nuc,clf A,sea,seb,sec,eta,etb and hla,were synthesized.The 13 virulence genes were amplified by PCR.The results showed that ZJ1～ZJ7 carried seb,clfA,hla,hlb and sec 5 virulence genes,only ZJ7 did not detect nuc gene,ZJ1～ZJ6PCR results showed that all carried nuc gene.The challenge test of mice showed that the bacteria could cause pathological changes in the internal organs of mice and had a lethal effect on mice.The above factors showed that the pathogenic ability of Staphylococcus aureus isolated from yak was strong.4.Genome analysis of Staphylococcus aureus from yakZJ6 was selected and sent to the sequencing company for whole-genome sequencing analysis using the second-generation and third-generation sequencing technology.The results showed that most of the products encoded by ZJ6 gene were related to biological processes.There were five gene islands,and a total of 461 viral factor proteins,12 drug-resistant genes,and 1 prophage were annotated,which further showed that ZJ6 had strong pathogenicity and drug resistance.To sum up,7 strains of Staphylococcus aureus from yak were isolated from 101 samples,and the isolation rate was 6.93%.5 drug resistance genes and 13 virulence genes were detected by conventional PCR,and tetM,tetK and mecA 3 drug resistance genes mediated tetracycline resistance were detected.All the isolates carried seb,clf A,hla,hlb and sec5 virulence genes.The results of drug sensitivity test showed that the resistance rate of 7 strains to penicillin and ampicillin was 100%.In the challenge test of mice,ZJ6 had lethal effect on mice.