【摘要】：目的:用基于6對等位基因的多位點(diǎn)序列分型,分析在北京大學(xué)深圳醫(yī)院診斷為外陰陰道念珠菌病的36位患者分離出38株熱帶念珠菌的親緣關(guān)系、體外比較其對常見抗真菌藥物的敏感性、平板法檢測熱帶念珠菌酯酶、磷脂酶及溶血酶分泌及耐藥基因在熱帶念珠菌中的表達(dá)水平。方法:通過對6對等位基因進(jìn)行聚合酶鏈反應(yīng)、擴(kuò)增產(chǎn)物進(jìn)行測序、拼接、檢查出突變位點(diǎn),截取需要的片段,與熱帶念珠菌MLST數(shù)據(jù)庫進(jìn)行對比,從而獲得DST,用e BRUST軟件進(jìn)行分組,UPGMA方法構(gòu)建系統(tǒng)發(fā)育樹,比較菌株的進(jìn)化和變異。應(yīng)用肉湯稀釋法抗真菌藥物敏感試驗(yàn)參考方法M27-S4,進(jìn)行體外藥物敏感試驗(yàn),平板法檢測熱帶念珠菌酯酶、磷脂酶及溶血毒素的分泌、通過RT-q PCR評估外排基因CDR1,CDR2和靶酶基因ERG11基因及ERG基因家族(包括ERG1、ERG3、ERG6、ERG9和ERG11)表達(dá)水平,用△CT法計(jì)算相對表達(dá)量。結(jié)果:38株熱帶念珠菌中獲得30個(gè)DST,其中14新DSTs。根據(jù)e BURST軟件進(jìn)行分組,結(jié)果示:屬于1組有8個(gè)菌株5個(gè)DSTs,DST分別為:330、333、426、532和724。屬于2組有3個(gè)菌株,DST分別是114、139和321,其他DSTs屬于單體型。根據(jù)bootstrap值≥70%對系統(tǒng)發(fā)育樹進(jìn)行分組,總共分為18個(gè)分化枝。阿尼芬凈、米卡芬凈、卡泊芬凈、氟康唑、咪康唑、兩性霉素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種常見抗真菌藥物的MIC 90分別是: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。所有實(shí)驗(yàn)菌株均能產(chǎn)生酯酶和溶血毒素,陽性率100%,其中9株未顯示出磷脂酶活性,陽性率為76.3%。耐藥基因?qū)嶒?yàn)中,每組ΔCT的最大值和最小值分別在分化枝分組中如下:ERG1、ERG3、ERG6、ERG9、ERG11、CDR1、CDR2和ERG11分別為分化枝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,表示基因表達(dá)量相對就越高,絕大多數(shù)基因的表達(dá)量較高的菌株在分化枝11和14。結(jié)論:本研究熱帶念珠菌MLST變異較大,菌株之間遺傳距離較遠(yuǎn)。熱帶念珠菌對特比萘芬普遍耐藥,對目前常用的抗真菌藥物大多數(shù)是敏感的,出現(xiàn)極少數(shù)的耐藥菌株。熱帶念珠菌對14種常見抗真菌藥物的幾何均數(shù)均高于白念珠菌。所有菌株均能產(chǎn)生酯酶和溶血酶,絕大多數(shù)菌株表現(xiàn)出磷脂酶活性陽性。耐藥基因表達(dá)較高的菌株并未產(chǎn)生耐藥。
[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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