Study On Resistance Mechanism To β-lactams Antibiotics Of Staphylococcus Aureus Isolated From Cow In Ningxia

P-lactams antibiotics is a kind of broad-spectrum,highly effective antibiotics with good activity against Staphylococcus aureus.However,β-lactam antibiotics were used widely,which resulted drug-resistant strains and multiple drug-resistant strains increasing rapidly.It caused serious impact on clinical treatment during Staphylococcus aureus infection.The study of the resistance mechanism of Staphylococcus aureus to β-lactam antibiotics in Ningxia is not only helpful for the rational use of drugs,but also to reduce and control the spread of resistant Staphylococcus aureus.The main materials of this study were collected from subclinical or clinical cases of bovine mastitis of some dairy farms in Ningxia.The methods including gram staining microscopy,CHROMagar Staphylococcus aureus agar and PCR amplification for 16S rDNA and nuc gene were used for the isolation and identification of Staphylococcus aureus.The MICs of 14 antimicrobial agents for Staphylococcus aureus isolates were determined by the broth microdilution method according to the guidelines recommended by the Clinical and Laboratory Standards Institute(CLSI).And the "penicillin zone-edge" determination method was used for β-lactams phenotype confirmation of Staphylococcus aureus isolates.The results showed that 261 strains of Staphylococcus aureus were isolated and identified and the highest proportion of resistance was towards ampicillin(79.74%),followed by penicillin(78.50%),sulfamethoxydiazine(67.43%),lincomycin(45.62%),erythromycin(39.81%),tilmicosin(37.53%).The sensitivity rates of aminoglycosides,cephalosporins and quinolones were above 60%.After the phenotype confirmation test,147 strains of 261 Staphylococcus aureus were detected carrying β-lactams,and the producing-lactamase rate was 56.32%(147/261).23 high resistant strains to penicillin and 10 susceptible strains and 1 strain of Staphylococcus aureus ATCC25923 were determined the accumulation concentration of penicillin in bacteria by the method of High-performance liquid chromatography(HPLC).The determination results showed that the penicillin accumulation of Staphylococcus aureus ATCC25923 and susceptible strains were not influenced seriously while penicillin accumulation of 8 strains penicillin-resistant strains were strongly influenced by CCCP.This result indicates that the active efflux mechanism is one of the resistance mechanisms of Staphylococcus aureus.23 strains of penicillin-resistant Staphylococcus aureus were analyzed the gene expression levels of cell wall biosynthetic genes including pbpB,glmU and murG by qRT-PCR.The results showed that pbpB of 5 penicillin-resistant strains which cultured in the medium with 32μg/mL penicillin was 5 times higer than that strains cultured in the medium with 0μg/mL penicillin.glmU、murG genes expression levels has significantly higher change than Staphylococcus aureus pta reference genes expression.Overexpression of the main synthetic protein gene of cell wall leads to thickening of the cell wall and thus affecting the entry of the drug into the cells.It may be one of the resistance mechanisms of Staphylococcus aureus.In conclusion,we analysed drug resistance of Staphylococcus aureus isolated from cow in Ningxia and researched on resistance mechanism of Staphylococcus aureus to β-lactam antibiotics.It revealed that the drug resistance situation of Staphylococcus aureus in Ningxia and the resistance mechanism of Staphylococcus aureus.Provide theory basis for using antimicrobials rationally and lay the basis for researching the multi-drug resistance mechanisms of Staphylococcus aureus.