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【目的】通过前期体外诱导获得耐万古霉素金黄色葡萄球菌,从基因突变方面对万古霉素耐药性菌株进行研究。【方法】通过低浓度万古霉素逐步诱导13株敏感性金黄色葡萄球菌,用琼脂稀释法和E-test法检测所有菌株对万古霉素的耐药性(最低抑菌浓度,MIC),PCR扩增与万古霉素耐药性密切相关的4个重要基因:rpo B、vra S、gra R和gra S,并测序分析,比较诱导前后不同菌株的基因序列。【结果】通过60 d的体外诱导实验,13株对万古霉素敏感性金黄色葡萄球菌中有6株被诱导为中介耐药金黄色葡萄球菌(Vancomycin intermediate Staphylococcus aureus,VISA),7株菌被诱导之后对万古霉素仍处于敏感状态,MIC<4 mg/L。检测诱导前后所有菌株的rpo B、vra S、gra R和gra S基因发现:有3株VISA的rpo B基因同时有L466S和H481N的突变,gra S基因同时有R232K的突变。【结论】对万古霉素敏感的金黄色葡萄球菌经过较长时间的体外诱导可发展为VISA。在已检测的重要基因中,rpo B和gra S的突变对耐药性的发展很可能起关键作用,而vra S和gra R对这一过程没有显著影响。
【OBJECTIVE】 Vancomycin-resistant Staphylococcus aureus was obtained through in vitro induction in vitro and vancomycin-resistant strains were studied in terms of gene mutation. 【Method】 Thirteen strains of susceptible Staphylococcus aureus were gradually induced by low concentration vancomycin. All strains were tested for resistance to vancomycin (minimum inhibitory concentration, MIC) by agar dilution method and E-test, and PCR Four important genes closely related to vancomycin resistance were amplified: rpo B, vra S, gra R and gra S, and sequenced to compare the gene sequences of different strains before and after induction. 【Result】 Six of 13 strains of vancomycin-susceptible Staphylococcus aureus were induced into Vancomycin intermediate Staphylococcus aureus (VISA) by 60-day in vitro induction experiment. Vancomycin is still sensitive after induction with MIC <4 mg / L. The detection of rpo B, vra S, gra R and gra S genes of all strains before and after induction showed that there were three mutations in the rpo B gene of VISA with L466S and H481N, and the mutation of R232K in gra S gene. 【Conclusion】 Vancomycin-sensitive Staphylococcus aureus can be developed into VISA after prolonged in vitro induction. Among the important genes tested, the mutations of rpo B and gra S are likely to play a key role in the development of drug resistance, while vra S and GR R have no significant effect on this process.