| Development of healthy breeding is the only way to ensure the healthy growth of livestock,to reduce the dependence of animals on antibiotics and to stabilize the economic benefits of livestock breeding industry.Dairy industry is the symbol of the moderni zation level of animal husbandry.Mastitis is one of the most important diseases restricting the healthy breeding of dairy industry.Staphylococcus aureus is one of the most important pathogenic bacteria causing clinical and subclinical mastitis in dairy cows.With strong environmental adaptability and antibiotic tolerance,Staphylococcus aureus can invade the cell interior,making it very difficult to be eradicated,and finally bringing great difficulties for the treatment.In this study,the methicillin resistant Staphylococcus aureus(MRSA)BA01611 related to cow mastitis was used as the research object,and the molecular biological method was used to explore the the effect of transcription factor sigB on antibiotic resistance and virulence.The main results are as follows:(1)Construction of gene knockout vector and complement vector.The shuttle plasmid p KZ2 was linked with the homologous arm of the target gene to construct knockout vector(p KZ2-? sigB)and complement vector(p KZ2-? sigB COMP).The vector was transferred into E.coli by chemical transformation,and the vector was verified by enzyme digestion and sequencing.(2)Construction of gene knockout and complement strain.The sigB knockout strain(BA01611-?sigB)and complement strain(BA01611-?sigB::sigB)of Staphylococcus aureus were successfully constructed by temperature sensitive plasmid and homologous recombination method.(3)Effects of transcription factor sigB on growth,hemolysis,virulence and antibiotic resistance of staphylococcus aureus.Transcription factor sigB had no significant effect on the growth ability and hemolysis ability of staphylococcus aureus BA01611(P > 0.05).After sigB knockout,the golden pigment formation ability of Staphylococcus aureus BA01611 was losed,and it was restored to the wild type level after sigB gene supplementation;sigB had an extensive effect on the antibiotic resistance of Staphylococcus aureus BA01611.Under the condition of solid agar plate,the antibiotic resistance of knockout strains to chloramphenicol(9.8 ?g/ml),ciprofloxacin(12 ?g/ml),cefazolin(40 ?g/ml),oxacillin(16 ?g/ml),penicillin(7 ?g/ml),teicoplanin(0.25 ?g/ml)and vancomycin(0.6 ?g/ml)was significantly lower than that of wild-type strains,and the antibiotic resistance of complement strains returned to the wild-type level;When growth under the condition of liquid culture,the growth ability of sigB knockout strains in the medium containing cefazolin(6 ?g/ml),oxacillin(15 ?g/ml),teicoplanin(0.8 ?g/ml)and vancomycin(1 ?g/ml)was significantly weaker than that of wild type.(4)Mechanism of sigB affecting Staphylococcus aureus resistance.The results of trition X-100 induced autolysis experiment showed that sigB had an effect on the cell wall biosynthesis of Staphylococcus aureus BA01611,and the autolysis rate of sigB knockout strain was significantly higher than that of wild strain and co mplement strain(P<0.01).q RT-PCR results showed that the knockout of sigB gene would significantly reduce the expression of bla Z,fstQ,fstW,fstY and fstZ at m RNA level(P<0.05).To sum up,this study used the gene editing method to knock out and complement the sigB gene,then explored the effect of sigB on the antibiotic resistance and virulence of Staphylococcus aureus.Although sigB knockout did not lead to significant changes in the growth and hemolysis phenotypes of Staphylococcus aureus,after sigB knockout,the bacteria lost the ability to produce golden yellow pigment and their tolerance to a variety of antibiotics decreased.In addition,sigB knockout significantly increased the autolysis activity,and decreased the expression of genes bla Z,fstQ,fstW,fstY and fstZ. |
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