育龄妇女VVC光滑假丝酵母菌基因多态性与药物敏感性相关性研究

Gene polymorphism and drug sensitivity of Candida glabrata associated with vulvovaginal candidiasis in women of childbearing age

目的 分析100例育龄妇女（其中50例为妊娠妇女）外阴阴道假丝酵母菌病（VVC）感染光滑假丝酵母菌的基因分型和药物敏感性结果,以了解贵阳地区育龄妇女VVC感染光滑假丝酵母菌的基因多态性和药物敏感性特征,并探讨基因多态性与妊娠状态、耐药性的相关性.方法 运用多位点序列分型（MLST）技术,对100株光滑假丝酵母菌的6个管家基因进行测序分析,确定菌株序列型（ST）.采用MEGA6.0软件中的加权配对组平均法和最小生成树法,比较分析不同菌株间的微进化及亲缘关系;采用ATB FUNGUS药物敏感性分析系统进行药物敏感性试验;运用Fisher和Ridit分析方法探究光滑假丝酵母菌的基因型与妊娠状态、耐药性的关系.结果 100株光滑假丝酵母菌经MLST分型,得到34个ST型,其中ST-753株（来自妊娠妇女26株）,ST-37株（来自妊娠妇女3株）,ST-196株（来自妊娠妇女5株）,ST-153株（来自妊娠妇女1株）,ST-102株（来自妊娠妇女2株）,其余ST型各为1株.100株光滑假丝酵母菌对两性霉素B和氟胞嘧啶均敏感,伊曲康唑抑菌效果较差,仅20%为敏感,对氟康唑和伏立康唑的耐药率为4%和1%;伏立康唑耐药组中,仅ST-X1对伏立康唑耐药,其他基因型均敏感;不同基因型对伊曲康唑耐药性的相关性研究中,以ST-7为标准组,ST-15、ST-19及其他ST组均不包含标准组的平均Ridit值（0.5）.使用邻接法（NJ）构建系统进化树,将所有基因型分为3群,记作Ⅰ、Ⅱ、Ⅲ.其中Ⅰ群44株,Ⅱ群53株,Ⅲ群3株.所有收集的临床菌株在分子进化中遗传距离较小.结论 ST-7为贵阳地区VVC光滑假丝酵母菌中优势菌株;不同基因型与妊娠状态没有相关性;ST-7与ST-15、ST-19及其他ST组的光滑假丝酵母菌对伊曲康唑的耐药性具有差异性;贵阳地区VVC光滑假丝酵母菌呈现出基因的高度多态性,基因多态性的ST之间的遗传距离小.

Objective To investigate genotypes and drug susceptibility of the 100 strains of Candida glabrata isolated from 100 women （ including 50 pregnant women ） in order to study the drugresistance and gene polymorphism, and to investigate the correlation of drug-resistance, gestation and gene polymorphism. Methods Multilocus sequence typing （MLST） technique was introduced to identity sequences of 6 housekeeping genes from 100 isolates of Candida glabrata. The results were compared with sequence information in MLST databases by Clustalx software to determine a strain allelic profile and sequence type （ST）. Drawing the phylogenetic tree by weighted paired group average method and the minimal spanning tree method of MEGA6. 0 software, the microevolution and relationship between different strains were analyzed. ATB FUNGUS semi-automatic system was used to test the drug susceptibility. Fisher analysis method was used to analyze the correlation between the genotypes and pregnancy. Ridit analysis method was used to analyze the correlation between the genotypes and drug susceptibility. Results The 100 isolates belonged to 34 clone sequences. There were 53 isolates belonged to ST-7（ 26 isolates of pregnancy）, 7 isolates belonged to ST-3 （3 isolates of pregnancy）, 6 isolates belonged to ST-19 （5 isolates of pregnancy ）, 3 isolates belonged to ST-15 （1 isolate of pregnancy）, 2 isolates belonged to ST-10 （2 isolates of pregnancy）, 1 isolate belonged to the other types of ST. Total of 100 isolates of Candida glabrata were 100% sensitive to fluorocytosine and amphotericin. The effect of itraconazole was poor with the sensitive rate of 20%. The resistance rates of flucouazole and voriconazole were 4% and 1% respectively. All genotypes were sensitive to voriconazole except ST-X1. In the correlation between genotype and itraconazole resistance, ST-7 as the standard group, the Ridit values in the group of ST-15, ST-19 and other types of ST were not included the mean Ridit value of the standard group in itraconazole （0. 5 ）. The system evolution tree was built using the neighbor-joining method （NJ） . All genotypes could be divided into 3 groups, as Ⅰ , Ⅱ , Ⅲ. Group Ⅰ had 44 cases, group Ⅱ had 53 cases , group Ⅲ had 3 cases . All the collected clinical strains had small genetic distances during molecular evolution. Conclusions ST-7 was the dominant genotype in Guiyang. No correlation between different STs and patients＇ pregnancy was found. The different drug susceptibility in itraconazole between ST-7, ST-15, ST-19 and other STs were found. The Candida glabrata associated with VVC showed highly discrimative diversity. However, the phylogenic analysis exhibited genetic similarity among the strains studied.