本文關(guān)鍵詞：中國多中心連續(xù)五年侵襲性感染熱帶念珠菌流行病學(xué)及唑類耐藥機制研究　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 熱帶念珠菌 侵襲性感染 分子流行病學(xué) 抗真菌藥物敏感性 耐藥機制

【摘要】：背景:熱帶念珠菌是臨床侵襲性真菌感染的重要病原菌,病死率高。在我國,其對唑類藥物的耐藥性已經(jīng)引起了廣泛關(guān)注。本研究對中國侵襲性感染熱帶念珠菌的體外藥物敏感性變遷、分子流行病學(xué)特征及對唑類藥物的耐藥機制進行了系統(tǒng)性的研究。方法:本研究選取了 2009年8月至2014年7月連續(xù)五年參加CHIF-NET的十家監(jiān)測中心收集的所有非重復(fù)侵襲性感染熱帶念珠菌進行研究,總納入菌株數(shù)507株。在菌株準(zhǔn)確鑒定的基礎(chǔ)上,測定菌株9種抗真菌藥物的最小抑菌濃度。同時計算監(jiān)測期內(nèi)氟康唑、伏立康唑抗菌藥物使用強度。利用本研究新建立的針對熱帶念珠菌的微衛(wèi)星分型體系,對全部507株菌進行分子分型,進而進行聚類及同源性分析。耐藥機制研究方面,對氟康唑耐藥組與敏感組菌株的靶位點基因ERG11及泵蛋白基因MDR1、CDR1以及線粒體細(xì)胞色素b基因CYTb進行熒光定量PCR分析,以分析不同表型組別之間基因表達量的差異。此外,對所有507菌株ERG11基因進行擴增測序,篩查堿基突變位點。利用pYES2/CT質(zhì)粒構(gòu)建表達載體,對潛在的唑類耐藥錯義突變進行功能驗證。另外,使用三種唑類對1株臨床分離株進行體外誘導(dǎo)耐藥,觀察不同藥物誘導(dǎo)菌株對唑類藥物的MIC值變化,并檢測其耐藥機制。結(jié)果:侵襲性熱帶念珠菌感染中,最常見的血流感染,占到53.4%(220/507)。體外抗菌藥物敏感性結(jié)果顯示,23.1%和20.7%的菌株對氟康唑及伏立康唑不敏感。同時,有11.4%的菌株呈現(xiàn)氟康唑、伏立康唑交叉耐藥的現(xiàn)象。對于棘白菌素類藥物,99%以上的菌株敏感。唑類藥物非敏感率在監(jiān)測五年期間有顯著增長的趨勢,對氟康唑的非敏感率由11.2%增長至42.7%;對伏立康唑的非敏感率由10.4%增長至39.1%。氟康唑-伏立康唑交叉耐藥的耐藥率也由6.6%上升至21.7%。但是,唑類非敏感率的上升與臨床唑類藥物使用強度沒有相關(guān)性。對比分子分型“金標(biāo)準(zhǔn)”脈沖場凝膠電泳,本研究新建的ctm1-ctm3-ctm24三位點微衛(wèi)星分型體系的分辨力能夠達到0.99。507株熱帶念珠菌中,共檢出296個微衛(wèi)星型,其中最常見的基因型為MT178型,占比4.1%。總體來講,菌株分子型別散在,并沒有發(fā)現(xiàn)地域聚集現(xiàn)象。但是,結(jié)合體外抗真菌藥物敏感性結(jié)果發(fā)現(xiàn),氟康唑非敏感菌株相對集中在四個微衛(wèi)星群中,尤其是Ⅳ群,24株菌全部為氟康唑-伏立康唑交叉耐藥株。綜合分析本研究收集菌株的唑類耐藥機制,ERG11基因的錯義突變是導(dǎo)致唑類耐藥的最主要原因,65株氟康唑耐藥株中,僅有3株未發(fā)現(xiàn)耐藥相關(guān)突變。ERG11基因A395T/WC461T/Y雙突變在氟康唑耐藥菌中檢出頻率最高,能夠占到耐藥菌總數(shù)的76.9%(50/65)。載體構(gòu)建功能驗證發(fā)現(xiàn),A395T單突變即可導(dǎo)致菌株唑類MIC值急劇升高,但相反C461T突變對菌株的唑類藥物敏感性并無影響。同時,ERG11基因T374C、T769C、G1390A均會導(dǎo)致菌株唑類MIC值的大幅升高,但A983G突變與耐藥無關(guān)。熒光定量PCR結(jié)果顯示,氟康唑耐藥組ERG11基因的平均表達水平高于敏感組;耐藥組CYTb基因的表達量低于敏感組。相比,CDR1、MDR1基因平均表達水平不同組間并無明顯差異。結(jié)論:我國侵襲性感染相關(guān)熱帶念珠菌對氟康唑和伏立康唑呈現(xiàn)高度唑類耐藥性,并且近年來呈現(xiàn)明顯的持續(xù)升高的趨勢,尤其是監(jiān)測后兩年耐藥率急劇升高。臨床分離熱帶念珠菌基因背景整體呈現(xiàn)多態(tài)性,但發(fā)現(xiàn)了與氟康唑非敏感表型相關(guān)的克隆群。ERG11基因的錯義突變是引起我國熱帶念珠菌唑類耐藥的最主要原因,絕大多數(shù)的氟康唑耐藥菌是由A395T錯義突變導(dǎo)致。同時,ERG11基因的過表達與線粒體的缺陷也對耐藥有一定的作用,而藥物外排泵的高表達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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R519.3;R446.5
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本文編號：1369427