米替福新对耐氟康唑白念珠菌耐药性的体外逆转作用及机制

In vitro reversal effect of miltefosine on fluconazole resistance of Candida albicans and the mechanism

目的探讨米替福新对临床分离耐氟康唑白念珠菌耐药性的影响,对米替福新降低白念珠菌耐药性的作用机制进行初步探究。方法复苏实验室冻存的25株临床分离耐氟康唑白念珠菌,采用棋盘式肉汤微量稀释法测定米替福新与氟康唑联用时的MIC,并以部分抑菌浓度指数评估药物联用的协同效果;通过RT-qPCR检测耐氟康唑白念珠菌外排泵基因CDR1和CDR2的表达水平,并通过罗丹明6G外排实验和ATP含量测定评估米替福新对外排泵活性的影响。结果米替福新和氟康唑对部分耐氟康唑白念珠菌(11株,44%)表现出协同抑菌效果,而且这部分白念珠菌均高表达外排泵基因CDR1和CDR2;米替福新不能抑制CDR1和CDR2的表达,但能抑制白念珠菌的外排能力,并显著降低白念珠菌胞内ATP水平。结论米替福新可体外逆转白念珠菌对氟康唑的耐药性,其机制主要是通过阻断白念珠菌能量ATP的供应而抑制菌株外排泵活性。

Objective To investigate the effect of miltefosine on fluconazole resistance of clinical isolates of Candida albicans and explore the mechanism of miltefosine to reduce the resistance of C.albicans to fluconazole.Methods Twenty-five clinical isolates of fluconazole-resistant C.albicans were revived.The minimum inhibitory concentration(MIC)of fluconazole-miltefosine combination was determined by broth microdilution checkerboard assay.The fractional inhibitory concentration(FIC)index was used to evaluate the synergistic effect of fluconazole-miltefosine combination.The expression level of efflux pump genes CDR1 and CDR2 was determined by RT-qPCR in fluconazole-resistant C.albicans isolates.Rhodamine 6G efflux and ATP content assays were used to evaluate the effect of miltefosine on efflux pump activity.Results Miltefosine-fluconazole combination had synergistic antifungal effect on 44%(11/25)of the fluconazole-resistant C.albicans isolates.These isolates expressed high levels of efflux pump genes CDR1 and CDR2.Miltefosine could not inhibit the expression of efflux pump genes CDR1 and CDR2.Miltefosine could inhibit the efflux pump activity and significantly reduce intracellular ATP level of C.albicans.Conclusions Miltefosine could reverse the resistance of C.albicans to fluconazole in vitro by blocking the ATP supply to inhibit the efflux pump activity.