白念珠菌临床株氟康唑敏感性试验与ERG11基因突变检测

目的 了解白念珠菌临床株对氟康唑的敏感性,筛查临床菌株的ERG11突变,探讨其与耐药的关系.方法 收集鉴定白念珠菌临床株,采用微量稀释法和药敏纸片法体外测定试验菌株对氟康唑的敏感性.分段扩增ERG11基因的第295～777bp(483bp)、第723～1204bp(482bp)和第1179～1667bp(489bp)等3个片段,并测序.结果 共收集念珠菌80株,分离白念珠菌52株,52株白念珠菌均为敏感株.经测序的5株敏感白念珠菌ERG11基因共出现19处突变,其中14处为同义突变,其余5处错义突变(T495A、A530C、G640A、A945C、G1609A)分别可引起羊毛甾醇14α-去甲基酶的下列氨基酸改变:D116E、K128T、E165K、E266D、V488I.结论 白念珠菌临床株对氟康唑敏感性较高;E165K和V488I可能与耐药无关.

Correlation between antifungal susceptibility of clinical isolates of Candida albicans to fluconazole and ERG11 mutations

Objective To analyze the sensitivity of clinical isolates of Candida albicans to fluconazole, to detect mutations in their ERG11 genes, and to investigate the correlation between ERG11 gene mutation and resistance to fluconazole. Methods Candida albicans was identified from clinical isolates of Candida spp.. The sensitivity to fluconazole was detected in vitro by microdilution-basesd method and Rosco tablets method. Three pairs of primers were designed to amplify three fragments of ERG11 gene （483 bps, from 295 bp to777 bp; 482bps, from 723 bp to 1204 bp; 489 bps, from 1179 bp to 1667 bp） after the extracting of genomic DNA. PCR products were sequenced. Results Eighty clinical isolates of Candida spp. were collected, which included 52 isolates of Candida albicans , all of which were sensitive to fluconazole. Nineteen mutations were detected in ERG11 gene of 5 fluconazole-sensitive clinical isolates. Of the 19 mutations, 14 were samesense mutations, and the remaing 5 missense mutations （T495A, A530C, G640A, A945C and G1609A）, resulting in amino acid substitution D116E, K128T, E165K, E266D and V488I, respectively in lanosterol 14 alpha-demethylase. E165K was a novel mutation. Conclusions The clinical isolates of Candida albicans were highly sensitive to fluconazole; E165K and V488I might not lead to the resistance of Candida albicans to fluconazole.