【作者】 高锦；

【导师】 王中新；

【作者基本信息】 安徽医科大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 背景近年来,随着广谱抗生素、免疫抑制剂的广泛应用,器官移植、骨髓移植、介入性诊疗的开展以及艾滋病患者的增多,深部真菌感染的发生率逐年增加,其中以白假丝酵母菌最为常见,其次就是光滑假丝酵母菌。三唑类代表性药物氟康唑因具有良好的生物利用度和安全性,被广泛用于治疗侵袭性假丝酵母菌感染。光滑假丝酵母菌对氟康唑存在固有的低敏感率,在氟康唑的长期治疗下更易发展为氟康唑耐药株。目前,唑类药物主要的耐药机制在白假丝酵母菌和酿酒酵母中研究的较多的包括:14DM的过表达;唑类药物与靶酶结合位点的改变;麦角固醇生物合成途径中其他基因的突变以及由于ATP-结合盒(ABC)和主要易化扩散载体超家族基因过表达而导致的假丝酵母菌属药物外排的增加。国内尚未见光滑假丝酵母菌对唑类抗真菌药物耐药机制的报道。本课题就2007年7月至2008年7月安徽医科大学第一附属医院临床分离的光滑假丝酵母菌的耐药性及对氟康唑的耐药机制进行研究。目的了解临床分离的光滑假丝酵母菌的耐药性及其对氟康唑的耐药机制。方法运用ATB Fungus3药敏板条检测2007年7月至2008年7月安徽医科大学第一附属医院临床分离的190株光滑假丝酵母菌对抗真菌药物的敏感性;从中随机选取氟康唑耐药和敏感各5株,采用微量肉汤稀释法进一步检测对抗真菌药物的最低抑菌浓度;PCR扩增光滑假丝酵母菌靶酶基因CgERG11并测序分析;测定荧光染料罗丹明123在敏感与耐药菌株中的外排情况;RT-PCR检测光滑假丝酵母菌外排泵基因的表达水平。结果190株光滑假丝酵母菌中有23株对氟康唑耐药,耐药率为12.1%;10株光滑假丝酵母菌的MIC值显示,一株氟康唑耐药株对伊曲康唑剂量依赖性敏感,一株氟康唑敏感株对伊曲康唑耐药;靶酶基因的扩增测序结果显示,一株光滑假丝酵母菌氟康唑耐药株的靶酶基因CgERG11出现了一处点突变,并未引起氨基酸改变;光滑假丝酵母菌氟康唑耐药株与敏感株对荧光染料罗丹明123外排检测结果显示,耐药株和敏感株中外排存在显著的差异;RT-PCR检测氟康唑耐药株与敏感株中外排泵基因结果显示,CgCDR1与CgCDR2基因在耐药株与敏感株之间表达水平有显著性差异。结论光滑假丝酵母菌对氟康唑的耐药形势严峻;氟康唑和伊曲康唑在光滑假丝酵母菌中存在交叉耐药现象;荧光染料罗丹明123可作为耐药光滑假丝酵母菌外排情况的指示剂;光滑假丝酵母菌对氟康唑耐药的主要机制系外排泵基因的过表达,未发现靶酶基因的有意义突变。更多还原

【Abstract】 Background:In recent years, following the widespread use of wide-spectrum antibiotics, immunosuppressive ,the application of organ transplantation,marrow transplantation, interventional therapy and the rise of the acquired immunodeficiency syndrome(AIDS), the incidence of fungous infections had increased year by year. C. albican is the most prevalent opportunistic pathogen of candida species in human, C. glabrata ranks the second. Being one of triazole antifungal agents, fluconazole has becoming the treatment of choice for invasive candidiasis because of its biological availability and safety. C. glabrata exhibits intrinsically low susceptibility to fluconazole, and resistant strains of this species occurs easily after prolonged therapy with fluconazoleThe major mechanisms of azoles resistance described to date have been primarily based on studies done in C. albicans and Saccharomyces cerevisiae include over expression of C14-lanosterol demethylase, alteration of the azole-binding site of C14-lanosterol demethylase, mutation in other ergosterol biosynthetic genes, and increased drug efflux. Increased drug efflux in Candida species is mainly due to increased expression of ATP-binding cassette (ABC) and major facilitator superfamily transporters. There is no report about the mechanisms of C. glabrata to azoles antifungal agents in China. The subject is to analyze the drug resistance of C. glabrata isolated from the First Affiliated Hospital of Anhui Medical University from Jul 2007 to Jul 2008 and to investigate the molecular mechanisms of fluconazole resistance in clinical isolates of C. glabrata. Objective: To analyze the drug resistance of C. glabrata isolated from clinical and to investigate the molecular mechanisms of fluconazole resistance in clinical isolates of C. glabrata.Methods: The ATB Fungus 3 reagents board was performed to determine the drug susceptibilities of antifungal agents to 190 strains of C. glabrata isolated from the First Affiliated Hospital of Anhui Medical University from Jul 2007 to Jul 2008.The broth microdilution method was performed to determine the minimal inhibitory concentration of four sorts of antifungal agents to 5 strains of fluconazole-resistace C. glabrata and 5 strains of fluconazole-susceptible C. glabrata which were selected randomly from 190 strains of C. glabrata isolates. The CgERG11 genes of 5 strains of FCA-susceptible and 5 strains of FCA-resistant isolated C. glabrata were amplified by PCR and were sequenced ; Detected the efflux of fluromchrome Rhodamine 123 between FCA-susceptible and FCA-resistant strains; Reverse transcriptase-poly- -merase chain reaction (RT-PCR) was used to measure the mRNA level of active efflux pump gene CgCDR1 and CgCDR2 .Results: 23 of the 190 isolates were resistant to fluconazole,the rate of resistance is 12.1%;The MIC results of 10 strains of C. glabrata showed that one strain of FCA- resistance C. glabrata was S-DD to itraconazole, one strain of FCA-susceptible isolate was resistance to itraconazole. The CgERG11 genes of all C. glabrata isolates were amplified and sequenced; the results showed that there is a mutation in one strain of FCA-resistant C. glabrata but no varian chang of amino acid. The results of the efflux of fluromchrome Rhodamine 123 between FCA-resistance isolates and FCA-susceptible isolates showed that there were significant difference between FCA-resistance isolates and FCA-susceptible C. glabrata isolates; and the gene expression levels of CgCDR1 and CgCDR2 were also significant difference between FCA-resistance and FCA-susceptible C. glabrata isolates by RT-PCR.Conclusions: The resistance of C. glabrata to fluconazole is severe; There is cross-resistance between fluconazole and itraconazole in C. glabrata; Fluorochrome Rhodamine 123 can use as an indicator of the efflux experiment of FCA- resistance C. glabrata isolates; The main mechanisms of FCA-resistance in clinical isolates of C. glabrata is the over-express of efflux pump genes, no found the missense mutation of the target enzyme genes.更多还原