热带念珠菌多位点序列分型及其临床重要性

发布时间：2019-03-07 12:51

【摘要】：目的:用基于6对等位基因的多位点序列分型,分析在北京大学深圳医院诊断为外阴阴道念珠菌病的36位患者分离出38株热带念珠菌的亲缘关系、体外比较其对常见抗真菌药物的敏感性、平板法检测热带念珠菌酯酶、磷脂酶及溶血酶分泌及耐药基因在热带念珠菌中的表达水平。方法:通过对6对等位基因进行聚合酶链反应、扩增产物进行测序、拼接、检查出突变位点,截取需要的片段,与热带念珠菌MLST数据库进行对比,从而获得DST,用e BRUST软件进行分组,UPGMA方法构建系统发育树,比较菌株的进化和变异。应用肉汤稀释法抗真菌药物敏感试验参考方法M27-S4,进行体外药物敏感试验,平板法检测热带念珠菌酯酶、磷脂酶及溶血毒素的分泌、通过RT-q PCR评估外排基因CDR1,CDR2和靶酶基因ERG11基因及ERG基因家族(包括ERG1、ERG3、ERG6、ERG9和ERG11)表达水平,用△CT法计算相对表达量。结果:38株热带念珠菌中获得30个DST,其中14新DSTs。根据e BURST软件进行分组,结果示:属于1组有8个菌株5个DSTs,DST分别为:330、333、426、532和724。属于2组有3个菌株,DST分别是114、139和321,其他DSTs属于单体型。根据bootstrap值≥70%对系统发育树进行分组,总共分为18个分化枝。阿尼芬净、米卡芬净、卡泊芬净、氟康唑、咪康唑、两性霉素B、布康唑、伏立康唑、克霉唑、伊曲康唑、特康唑、氟胞嘧啶、特比萘芬和制霉菌素14种常见抗真菌药物的MIC 90分别是:0.030μg/ml、0.015μg/ml、0.500μg/ml、1.000μg/ml、2.000μg/ml、0.125μg/ml、0.250μg/ml、0.250μg/ml、0.250μg/ml、0.250μg/ml、256.000μg/ml和4.000μg/ml。所有实验菌株均能产生酯酶和溶血毒素,阳性率100%,其中9株未显示出磷脂酶活性,阳性率为76.3%。耐药基因实验中,每组ΔCT的最大值和最小值分别在分化枝分组中如下:ERG1、ERG3、ERG6、ERG9、ERG11、CDR1、CDR2和ERG11分别为分化枝18和11;分化枝16和15:分化枝18和11;分化枝16和11:分化枝18和14;分化枝7和11:分化枝16和14:分化枝1和14。ΔCT值越小,表示基因表达量相对就越高,绝大多数基因的表达量较高的菌株在分化枝11和14。结论:本研究热带念珠菌MLST变异较大,菌株之间遗传距离较远。热带念珠菌对特比萘芬普遍耐药,对目前常用的抗真菌药物大多数是敏感的,出现极少数的耐药菌株。热带念珠菌对14种常见抗真菌药物的几何均数均高于白念珠菌。所有菌株均能产生酯酶和溶血酶,绝大多数菌株表现出磷脂酶活性阳性。耐药基因表达较高的菌株并未产生耐药。
[Abstract]:Objective: to analyze the phylogenetic relationship of 38 strains of Candida tropicalis isolated from 36 patients with vulvovaginal candidiasis diagnosed as vulvovaginal candidiasis in Peking University Shenzhen Hospital based on 6 pairs of alleles. The susceptibility to common antifungal drugs was compared in vitro. The expression levels of esterase, phospholipase, lyase secretion and drug resistance genes in Candida tropicalis were detected by flat plate method. Methods: six pairs of alleles were amplified by polymerase chain reaction (PCR), the amplified products were sequenced, spliced, the mutation sites were detected, the required fragments were intercepted and compared with the MLST database of Candida tropicalis to obtain DST,. E-BRUST software was used to group and UPGMA method was used to construct phylogenetic tree, and the evolution and variation of the strains were compared. Using broth dilution method as reference method for antifungal drug sensitivity test, the in vitro drug sensitivity test was carried out, and the secretion of Candida tropicalis esterase, phospholipase and hemolysin was detected by flat plate method. The efflux gene CDR1, was evaluated by RT-q PCR. The expression levels of CDR2, target enzyme gene ERG11 gene and ERG gene family (including ERG1,ERG3,ERG6,ERG9 and ERG11) were calculated by CT method. Results: 30 DST, were obtained from 38 strains of Candida tropicalis, of which 14 new DSTs. were obtained. According to e-BURST software, the results showed that there were 8 strains belonging to group 1 and 5 DSTs,DST were 330333426532 and 724 respectively. There were 3 strains in two groups, DST was 114139 and 321 respectively. Other DSTs belonged to haplotype. Phylogenetic trees were divided into 18 branches according to bootstrap 鈮，

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