多药耐药外排泵ABC基因对白色假丝酵母菌及克柔假丝酵母菌耐药性产生的研究

Possible contribution of multidrug resistant efflux pump ABC gene to the drug resistance of Candida albicans and Candida krusei

目的探讨多药耐药外排泵基因在白色假丝酵母菌和克柔假丝酵母菌耐唑类药物中的作用,对假丝酵母菌属的耐药研究提供理论基础。方法通过体外药敏试验筛选出临床白色假丝酵母菌和克柔假丝酵母菌对伊曲康唑的耐药菌株和敏感菌株,使用实时荧光定量PCR(FQ-RT-PCR)方法检测耐药和敏感菌株多药耐药外排泵基因(CDR1/CDR2/ABC1)的mRNA表达水平,使用罗丹明6G为示踪剂了解白色假丝酵母菌和克柔假丝酵母菌的药物外排情况。结果临床分离的白色假丝酵母菌和克柔假丝酵母菌对氟胞嘧啶、两性霉素B的敏感率均较高,均>80.00%,白色假丝酵母菌对伊曲康唑的耐药率较高,>70.00%;根据实时荧光定量RT-PCR实验获得的ΔCt值,经统计分析发现,白色假丝酵母菌耐药菌株CDR1、CDR2mRNA的表达量高于敏感菌株的表达量,差异有统计学意义(P<0.05),但克柔假丝酵母菌中仅有一株耐药菌株出现高表达,敏感菌株和耐药菌株ABC1mRNA的表达量差异无统计学意义;通过罗丹明6G外排试验测定,随时间延长罗丹明6G外排明显增加,在CDR1/CDR2mRNA高表达的耐药菌株明显增高。结论外排泵基因CDR1、CDR2在白色假丝酵母菌耐药性的产生中可能起到一定作用,但不能说明外排泵基因ABC1在克柔假丝酵母菌耐药性的产生中无作用,因样本量较小,需要扩大样本量进一步研究。

OBJECTIVE To investigate the possible contribution of multidrug resistant efflux pump gene to Candida albicans and Candida krusei azole resistance drugs,so as to provide theory basis for the drug resistance research of Candida spp.METHODS The drug resistant strains and sensitive strains of C.albicans and C.krusei to itraconazole were screened by drug sensitivity test in vitro,using real-time fluorescent quantitative PCR（FQ-RTPCR）method to detect the mRNA expression level of multi-resistant efflux pump gene（CDR1/CDR2/ABC1）in resistant and sensitive strains,and using rhodamine 6Gas a tracer to test the efflux status in C.albicans and C.krusei.RESULTS The sensitive rates of C.albicans and C.kruseiisolated from clinic to flucytosine and amphotericin B were relatively higher,all〉80.00%,and the drug resistant rate of C.albicans to itraconazole was relatively higher〉70.00%.After the statistical analysis toΔCt numbers gotten from real-time fluorescent quantitative RTPCR,it showed the expression quantity of CDR1,CDR2 mRNA from the drug resistant strains of C.albicans was significantly higher than that in sensitive strains（P〈0.05）.However only one drug-resistant strain of C.krusei appeared,expression quantity of ABC1 mRNA had significant difference between sensitive strains and resistant strains.After the rhodamine 6Gefflux test,the drug resistant strains would have significant increase in the expression of CDR1/CDR2 mRNA,as the efflux of rhodamine 6Gincreased as time goes.CONCLUSION Efflux pump gene CDR1 and CDR2in C.albicans may play a role in the mechanism of drug resistance,but it cannot prove that there is no contribution of efflux pump gene ABC1 to the drug resistance in C.krusei,as the sample amount is not enough,which needs to expand the sample amount and give further study.