中国多中心连续五年侵袭性感染热带念珠菌流行病学及唑类耐药机制研究

发布时间：2018-01-02 13:27

  本文关键词：中国多中心连续五年侵袭性感染热带念珠菌流行病学及唑类耐药机制研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

  更多相关文章： 热带念珠菌 侵袭性感染 分子流行病学 抗真菌药物敏感性 耐药机制

【摘要】：背景:热带念珠菌是临床侵袭性真菌感染的重要病原菌,病死率高。在我国,其对唑类药物的耐药性已经引起了广泛关注。本研究对中国侵袭性感染热带念珠菌的体外药物敏感性变迁、分子流行病学特征及对唑类药物的耐药机制进行了系统性的研究。方法:本研究选取了 2009年8月至2014年7月连续五年参加CHIF-NET的十家监测中心收集的所有非重复侵袭性感染热带念珠菌进行研究,总纳入菌株数507株。在菌株准确鉴定的基础上,测定菌株9种抗真菌药物的最小抑菌浓度。同时计算监测期内氟康唑、伏立康唑抗菌药物使用强度。利用本研究新建立的针对热带念珠菌的微卫星分型体系,对全部507株菌进行分子分型,进而进行聚类及同源性分析。耐药机制研究方面,对氟康唑耐药组与敏感组菌株的靶位点基因ERG11及泵蛋白基因MDR1、CDR1以及线粒体细胞色素b基因CYTb进行荧光定量PCR分析,以分析不同表型组别之间基因表达量的差异。此外,对所有507菌株ERG11基因进行扩增测序,筛查碱基突变位点。利用pYES2/CT质粒构建表达载体,对潜在的唑类耐药错义突变进行功能验证。另外,使用三种唑类对1株临床分离株进行体外诱导耐药,观察不同药物诱导菌株对唑类药物的MIC值变化,并检测其耐药机制。结果:侵袭性热带念珠菌感染中,最常见的血流感染,占到53.4%(220/507)。体外抗菌药物敏感性结果显示,23.1%和20.7%的菌株对氟康唑及伏立康唑不敏感。同时,有11.4%的菌株呈现氟康唑、伏立康唑交叉耐药的现象。对于棘白菌素类药物,99%以上的菌株敏感。唑类药物非敏感率在监测五年期间有显著增长的趋势,对氟康唑的非敏感率由11.2%增长至42.7%;对伏立康唑的非敏感率由10.4%增长至39.1%。氟康唑-伏立康唑交叉耐药的耐药率也由6.6%上升至21.7%。但是,唑类非敏感率的上升与临床唑类药物使用强度没有相关性。对比分子分型“金标准”脉冲场凝胶电泳,本研究新建的ctm1-ctm3-ctm24三位点微卫星分型体系的分辨力能够达到0.99。507株热带念珠菌中,共检出296个微卫星型,其中最常见的基因型为MT178型,占比4.1%。总体来讲,菌株分子型别散在,并没有发现地域聚集现象。但是,结合体外抗真菌药物敏感性结果发现,氟康唑非敏感菌株相对集中在四个微卫星群中,尤其是Ⅳ群,24株菌全部为氟康唑-伏立康唑交叉耐药株。综合分析本研究收集菌株的唑类耐药机制,ERG11基因的错义突变是导致唑类耐药的最主要原因,65株氟康唑耐药株中,仅有3株未发现耐药相关突变。ERG11基因A395T/WC461T/Y双突变在氟康唑耐药菌中检出频率最高,能够占到耐药菌总数的76.9%(50/65)。载体构建功能验证发现,A395T单突变即可导致菌株唑类MIC值急剧升高,但相反C461T突变对菌株的唑类药物敏感性并无影响。同时,ERG11基因T374C、T769C、G1390A均会导致菌株唑类MIC值的大幅升高,但A983G突变与耐药无关。荧光定量PCR结果显示,氟康唑耐药组ERG11基因的平均表达水平高于敏感组;耐药组CYTb基因的表达量低于敏感组。相比,CDR1、MDR1基因平均表达水平不同组间并无明显差异。结论:我国侵袭性感染相关热带念珠菌对氟康唑和伏立康唑呈现高度唑类耐药性,并且近年来呈现明显的持续升高的趋势,尤其是监测后两年耐药率急剧升高。临床分离热带念珠菌基因背景整体呈现多态性,但发现了与氟康唑非敏感表型相关的克隆群。ERG11基因的错义突变是引起我国热带念珠菌唑类耐药的最主要原因,绝大多数的氟康唑耐药菌是由A395T错义突变导致。同时,ERG11基因的过表达与线粒体的缺陷也对耐药有一定的作用,而药物外排泵的高表达的作用并不十分重要。
[Abstract]:Background: Candida tropicalis is an important pathogen in patients with invasive fungal infection, the mortality rate is high. In our country, the azole resistance has attracted wide attention. The study of in vitro drug sensitivity changes of Candida tropicalis China invasive infection, molecular epidemiology characteristics and mechanisms of resistance to azoles were a systematic study. Methods: this study selected ten monitoring center from August 2009 to July 2014 for five consecutive years to participate in the CHIF-NET collection of all non repeated invasive infection of Candida tropicalis, the total number of 507 strains into strains. Based on the accurate identification of strains, minimal inhibitory concentration of 9 strains of antifungal agents at the same time. The calculation in the monitoring period, the use of antibacterial drugs fluconazole, voriconazole. Using the new strength of this study for Candida tropicalis microsatellite genotyping system for all 507 Strains of molecular typing, and clustering and homology analysis. Research on the resistant mechanism, target genes ERG11 and pump MDR1 gene on strains of fluconazole resistance group and sensitive group, CDR1 and mitochondrial cytochrome b gene CYTb were analyzed by fluorescence quantitative PCR, the analysis between different groups of gene expression differences in phenotype. In addition, all 507 strains of ERG11 gene was amplified and sequenced. Screening mutation expression vector was constructed using pYES2/CT plasmid of azole resistance potential missense functional verification mutation. In addition, the use of three kinds of azoles against 1 strains of clinical isolates were resistant in vitro, observation of different drug induced strains to azole drugs the changes of MIC value, and test the resistance mechanism. Results: invasive Candida albicans infection, the most common bloodstream infection, accounted for 53.4% (220/507). In vitro antimicrobial susceptibility. Results show that 23.1% and 20.7% were not sensitive to fluconazole and voriconazole. At the same time, 11.4% of the isolates showed cross resistance to fluconazole, voriconazole. For echinocandin, more than 99% of the strains were sensitive to azoles. Non sensitive rate has a significant growth trend during the five years of monitoring, to fluconazole the non sensitive rate increased from 11.2% to 42.7%; the rate of drug resistance of non sensitive voriconazole increased from 10.4% to 39.1%. and cross resistance rate of fluconazole and voriconazole also increased from 6.6% to 21.7%. but not rising azole sensitive rate and clinical azole drug use intensity had no correlation. Comparison of molecular typing of "gold standard" pulse field gel electrophoresis resolution typing system for the new ctm1-ctm3-ctm24 three microsatellite can reach 0.99.507 strains of Candida tropicalis were detected in 296 microsatellite types, one of the most common gene Type MT178, accounting for 4.1%. in general, molecular type strains scattered, and found no regional aggregation. However, combined with in vitro antifungal susceptibility of fluconazole susceptible strains found non concentrated in four microsatellite groups, especially in group IV, 24 strains to fluconazole voriconazole cross resistance - all comprehensive analysis of strains. This study collected strains of azole resistance mechanisms, missense mutation of the ERG11 gene is the main cause of azole resistance, 65 strains of fluconazole resistant strains, only 3 strains were found resistance related mutations in the.ERG11 gene A395T /WC461T/Y double mutant in fluconazole resistant bacteria were detected in the highest frequency, able to account for the drug-resistant bacteria the total number of 76.9% (50/65). The vector that functional verification of A395T single mutations can lead to strains of azole MIC value increased dramatically, but instead C461T mutation azole drug sensitivity had no effect on the same strain. ERG11, T769C, gene T374C, G1390A strains will lead to azole MIC value significantly increased, but A983G mutation and resistance. Quantitative PCR results showed that the average expression level of ERG11 gene was higher than that of fluconazole sensitive group; low expression of CYTb gene in the sense of resistance group, Yu Min group. Compared to CDR1, MDR1 the average gene expression level among different groups showed no significant difference. Conclusion: China's invasive infection of Candida tropicalis is highly related to azole resistance to fluconazole and voriconazole, and in recent years showed a significant increasing trend, especially after two years of monitoring drug resistance rate increased sharply. Clinical isolates of Candida tropicalis gene background showed polymorphism, but found missense and fluconazole sensitive phenotype related non clonal population.ERG11 gene mutation is the main cause of China's tropical Candida albicans azole resistance, the vast majority of fluoride Conazole resistant bacteria are caused by the missense mutation of A395T. Meanwhile, the overexpression of ERG11 gene and mitochondrial defects also play a role in drug resistance, and the high expression of drug efflux pump is not very important.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R519.3;R446.5
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本文编号：1369427