摘要目的 了解PDR1基因调节光滑念珠菌对唑类药物耐药的机制.方法 收集5家医院的38株光滑念珠菌,采用微量稀释法检测氟康唑、伊曲康唑和伏立康唑对光滑念珠菌的最低抑菌浓度(MIC).实时荧光定量PCR扩增PDR1基因并测序,构建含PDR1突变位点的表达质粒,转化入光滑念珠菌,罗丹明6G外排实验、CDR1和CDR2基因表达水平和药物敏感试验验证突变位点的功能.结果 38株光滑念珠菌中,有17株至少对一种唑类药物耐药,且每株耐药株的PDR1基因均存在突变.表型实验证实,P927S使光滑念珠菌CDR1和CDR2基因表达分别上调20.53倍和4.03倍,外排后罗丹明6G的荧光强度下降至0.62.结论 PDR1基因P927S突变可诱导光滑念珠菌CDR1和CDR2的表达,使外排泵的作用增强,从而导致菌株耐药.

abstractsObjective To investigate the role of PDR1 gene in azole-resistant Candida glabrata (C.glabrata).Methods Thirty-eight clinical isolates of C.glabrata were collected from five different hospitals.The minimal inhibitory concentrations (MIC) of azole antifungals including fluconazole,itraconazole and voriconazole against C.glabrata were determined by broth microdilution.Sequencing and amplification of PDR1 gene was achieved by real-time quantitative polymerase chain reaction (PCR).The mutation was cloned into an expression plasmid and then transferred into C.glabrata.The efflux of rhodamine 6G and drug sensitivity test were performed,and expressions of CDR1 and CDR2 were examined to verify function of mutation.Results Among these 38 isolates of C.glabrata,17 were resistant to at least one of azole antifungals.Moreover,mutations of PDR1 gene existed in every resistant isolates.Results of phenotyping test showed that in the isolate that expressed PDR1P927S,the expression of CDR1 and CDR2 were increased by 20.53 and 4.03 fold,respectively.And the fluorescence intensity of rhodamine 6G was decreased to 0.62 in efflux experiment.Conclusion P927S mutation of PDR1 gene could induce azole resistance of C.glabrata by increasing the expressions of CDR1 and CDR2,which results in drug resistance due to enhanced effect of efflux pump.