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

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：rpoB、vraS、graR和graS，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rpoB、vraS、graR和graS基因发现：有3株VISA的rpoB基因同时有L466S和H481N的突变，graS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，rpoB和graS的突变对耐药性的发展很可能起关键作用，而vraS和graR对这一过程没有显著影响。

Abstract:

[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 rpoB, vraS, graR and graS 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 L466S and H481N mutations in rpoB gene and R232K mutation in graS gene. [Conclusion] Evolution of vancomycin intermediate resistance S. aureus could be achieved by long-term vancomycin treatment in vitro. Our results suggest that rpoB and graS play crucial roles in vancomycin non-susceptibility development, whereas vraS and graR are less important.