体内外光滑假丝酵母菌氟康唑耐药机制的比较研究

Mechanisms of fluconazole resistance in clinical and experimental induced isolates of Candida glabrata

目的研究比较临床分离和体外诱导的耐氟康唑光滑假丝酵母菌的耐药机制。方法选取临床分离的4对氟康唑敏感一耐药配对的光滑假丝酵母菌，其中4株敏感株在体外氟康唑的作用下均被诱导为耐药株。利用罗丹明6G试验比较敏感株与两种耐药株的外排泵作用，实时荧光定量RT-PCR检测外排相关基因CDRl、CDR2、SNQ2和ERG11的表达。同时，对PDRl基因进行PCR扩增和测序，对比分析临床耐药株和体外诱导耐药株的突变位点。结果临床耐药株和体外诱导耐药株外排泵的功能均明显强于敏感株；两者CDRl表达均显著升高，而CDR2和SNQ2则无明显变化；ERGl1在敏感与耐药菌株之间的表达水平也无显著差别；两种耐药株的PDRl均发现错义突变位点，其中P927S、L543P及$947L突变尚未被报道过。结论光滑假丝酵母菌在体内外氟康唑作用下PDRl均产生突变，引起外排相关基因，尤其是CDRl的表达增加从而增强了外排泵的作用导致耐药。

Objective To investigate the mechanisms of fluconazole resistance in clinical and ex- perimental induced isolates of C. glabrata. Methods Efflux of rhodamine 6G was performed to evaluate the effects of efflux pumps. The expression levels of transporter genes CDR1, CDR2, SNQ2 and ERG11 were examined by real-time RT-PCR. Meanwhile, sequence of PDR1 was determined by PCR based DNA sequen- cing. Results Efflux pumps of all fluconazole-resistant isolates had stronger effects than that of susceptible isolates, consistently with significant upregulation of CDR1, but no obvious difference was found in CDR2 or SNQ2. Also, no notable change in the expression level of ERG11 between susceptible and resistant isolates. PDR1 mutations existed in both clinical and experimental induced isolates of C. glabrata, among which P927S, L543P and S947L haven＇t been reported previously. Conclusion Mutations of PDR1 were induced by fluconazole both in vivo and in vitro, which will result in overexpression of CDR1 and strengthen the effect of efflux pump.