| Staphylococcus aureus is a common zoonotic pathogen,which exists in a variety of environments and is highly contagious.In the past few decades,with the widespread use of penicillin,methicillin-resistant Staphylococcus aureus(MRSA),mediated by chromosome or plasmid can produce penicillinase and hydrolyze ?-lactam ring,showing resistance to penicillins.It has become a pathogen that can cause serious diseases.Therefore,the study of drug resistance and its mechanism of pet source MRSA in Yinchuan area is of great significance for the prevention and treatment of Staphylococcus aureus infection and clinical rational use of antibiotics.1.The nasal swabs of pet dogs and cats from some pet hospitals in Yinchuan,Ningxia from 2019 to 2020 were collected.Staphylococcus aureus and MRSA specific genes were amplified by Staphylococcus aureus chromogenic medium,microscopic examination and triple PCR to identify Staphylococcus aureus and MRSA.A total of 239 strains of Staphylococcus aureus were identified,of which 51 strains were MRSA,positive rate of 21.34%.2.According to the drug sensitivity disk Agar diffusion method recommended by the American Clinical Laboratory Standards Committee(CLSI,2019),the drug sensitivity tests of 51 strains of MRSA were carried out with 16 kinds of antimicrobials.The results showed that the resistance rate of MRSA to sulfamethoxazole was the highest,reaching 94.12%,followed by erythromycin(86.27%),and the resistance rate to tetracycline,doxycycline,clindamycin and ?-lactam was more than 60%.All of them were sensitive to vancomycin and linezolid.The phenomenon of multidrug resistance of MRSA is very serious,the multidrug resistance rate is 96.07%,and the isolated strains are resistant to 14 kinds of antibiotics at most.3.The resistance genes of tetracyclines were detected by PCR method,and the efflux pump inhibitor reserpine was selected for efflux pump inhibition test,and the efflux pump proteins were analyzed by bioinformatics and molecular simulation technology.The results showed that the detection rate of efflux pump gene tetK reached 54.90%,and the existence of this gene was positively correlated with drug resistance phenotype;evolutionary analysis showed that the detected tetK gene was closest to Staphylococcu saureusUP403,JY22 and Staphylococcu ssciuri wo19-3 carrying genes;molecular docking found that tetracycline,doxycycline and hemoptysis could bind to TetK protein to form stable complexes on average.Reserpine has the lowest binding free energy and can inhibit the uptake and efflux of tetracycline and doxycycline.4.The important reaction conditions were determined and optimized by LAMP pre-test,and its specificity and sensitivity were verified.On this basis,the visualization can be achieved by adding HNB dye to avoid gel electrophoresis.The optimized LAMP reaction system is:FIP,BIP16 ? M,F3,B32 ? M,LF,LB4 ? M,a total of 2.5 ? L,Warmstartlamp2 × MasterMix 12.5 ? L,HNB dye 1.0 ? L,bacterial DNA 1.0 ? L,supplement ddH20 to 25 ? L.Reaction at 65? for 30 minutes.The genes related to biofilm formation were detected by visual LAMP method,and the results showed that the detection rate of icaA gene was 26.14%.The detection rate of FnbB gene was 62.35%.The application of this method to the detection of genes related to Staphylococcus aureus and biofilm formation can improve the speed,accuracy and sensitivity of the existing detection methods.To sum up,in this study,through the isolation and identification of MRSA from some pet sources in Yinchuan,analysis of drug resistance and detection of drug resistance genes,combined with bioinformatics technology to analyze the relationship between efflux pump protein characteristics and drug resistance;construct a visual LAMP method for rapid detection of Staphylococcus aureus and biofilm formation related genes.It laid a foundation for elucidating the mechanism of drug resistance of Staphylococcus aureus and the prevention and control of Staphylococcus aureus. |
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