体外诱导耐万古霉素金黄色葡萄球菌及分子机制  

Mechanism of vancomycin intermediate resistance development in Staphylococcus aureus in vitro

作　　者：王玉冰[1] 李晓丽[1] 王伟轩[1] 陈南[1] 何晓青[1] 金一[1] 谢响明[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《微生物学通报》2016年第11期2473-2479,共7页Microbiology China

基　　金：中央高校基本科研业务费专项资金项目(No.TD2012-03)~~

摘　　要：【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] In order to study the mechanism of vancomycin intermediate resistance Staphylococcus aureus development, strains were treated with vancomycin in vitro and mutations in critical genes were analyzed. [Methods] Thirteen vancomycin susceptible S. aureus were treated by low dose of vancomycin in vitro. Minimum inhibitory concentrations were determined by the agar dilution and E-test methods. The complete sequences of rpo B, vra S, gra R and gra S associated with drug-resistance development were compared with those in the parental strains. [Results] Through a 60 d vancomycin treatment in vitro, 6 vancomycin intermediate resistance strains were generated, whereas 7 strains remained vancomycin susceptible. Sequence analysis revealed that 3 vancomycin intermediate resistance strains contained L466 S and H481 N mutations in rpo B gene and R232 K mutation in gra S gene. [Conclusion] Evolution of vancomycin intermediate resistance S. aureus could be achieved by long-term vancomycin treatment in vitro. Our results suggest that rpo B and gra S play crucial roles in vancomycin non-susceptibility development, whereas vra S and gra R are less important.

关 键 词：金黄色葡萄球菌 万古霉素 耐药性 基因突变 

分 类 号：R378.11[医药卫生—病原生物学]