白念珠菌论文：念珠菌ERG3基因突变对抗真菌药物耐药的作用

白念珠菌论文：念珠菌ERG3基因突变对抗真菌药物耐药的作用 白念珠菌论文:念珠菌ERG3基因突变对抗真菌药物耐药的作用 【中文摘要】背景近年来，侵袭性真菌感染发病率不断上升，以念珠菌感染为主，其中白念珠菌为主。但由于预防性使用抗真菌药物，非白念珠菌尤其是对氟康唑天然耐药的克柔念珠菌感染出现上升趋势，但白念珠菌感染仍占优势。随着白念珠菌和克柔念珠菌对唑类抗真菌药物的耐药现象越来越严重，深入研究其耐药机制迫在眉睫。调查本院住院患者侵袭性真菌感染的病原学特征及相关危险因素。了解白念珠菌和克柔念珠菌对常用抗真菌药物的敏感性，对白念珠菌耐药菌株和敏感菌株ERG3基因扩增、测序、生物信息学分析，明确白念珠菌ERG3基因突变与抗真菌药物耐药之间的关系。方法收集2006-2010年山西医科大学第二临床医学院452例住院患者无菌体液标本，使用沙氏培养基、玉米吐温80琼脂培养基、CHROMagar念珠菌显色培养基和API 20C AUX鉴定系统进行念珠菌的分离和鉴定。从中分别选取35株白念珠菌和20株克柔念珠菌进行体外药物敏感性试验，确立耐药菌株和敏感菌株。对白念珠菌ERG3基因进行PCR扩增、测序，最后应用BLAST软件进行比对分析。结果(1)检出的163株真菌中，白念珠菌54.61%，非白念珠菌42.94%，霉菌2.45%。其中非白念珠菌从高到低依次为克柔念珠菌(16.57%)、光滑念珠菌(14.72%)、热带念珠菌(4.91%)、近平滑念珠菌(4.29%)和葡萄牙念珠 菌(2.45%);Logistic多因素回归分析结果显示:性别、病程、体温、预防性使用抗真菌药物、免疫抑制剂、有创伤的检查和治疗、患者预后和地域因素是住院患者无菌体液标本中侵袭性真菌感染的独立危险因素。(2)从中选取的35株白念珠菌体外药物敏感性试验结果显示:白念珠菌对5-氟胞嘧啶和两性霉素B敏感性为100%，对伊曲康唑、伏立康唑和氟康唑有不同程度的耐药，耐药率分别是17.14%、11.43%、8.57%，而且存在交叉耐药和多重耐药情况;20株克柔念珠菌体外药物敏感性试验结果显示:克柔念珠菌对伏立康唑和两性霉素B有较高的敏感性，5-氟胞嘧啶、氟康唑和伊曲康唑有不同程度的耐药，耐药率分别是20.00%、50.00%、45.00%，也有交叉耐药和多重耐药现象。(3)本研究显示白念珠菌临床耐药菌株和敏感菌株的ERG3基因均存在突变位点，共检出9个点突变，其中耐药菌株A17号同时存在1个同义突变和4个错义突变。A17号菌株同义突变位点为1080bp。突变前碱基所在的密码子为GGC，所编码的氨基酸为甘氨酸，未引起氨基酸的改变。4个错义突变位点分别为I340L、V374G、P902L、V1076A，突变发生氨基酸置换。第100位碱基由A突变为C，导致第340位氨基酸由异亮氨酸转变为亮氨酸;第134位碱基由T突变为G，导致第374位氨基酸由缬氨酸转变为甘氨酸，第662位碱基由C突变为T，导致第902位氨基酸由脯氨酸转变为亮氨酸;第836位碱基由T突变为C，导致第1076位氨基酸由缬氨酸转变为丙氨酸，这4个点突变在其它文献中尚未见报道，是本实验新发现的突变位点。其余菌 株均为同义突变，突变位点分别是602bp、795bp、969bp、1080bp、 1110bp，未引起氨基酸改变。结论(1)白念珠菌感染仍占优势，但非 白念珠菌感染明显上升，尤其是对氟康唑天然耐药的克柔念珠菌，临 床医师应重视发生的这一流行病学变化。(2)体外药敏试验结果提示: 白念珠菌对唑类药物有不同程度的耐药，并且对伊曲康唑的耐药率高 于氟康唑，但大多数菌株是敏感菌株，所以唑类抗真菌药物仍然可以 用于临床上白念珠菌感染的治疗。(3)白念珠菌部分耐药菌株存在麦 角甾醇合成途径中酶编码基因ERG3的错义突变，并且为多位点突变， 但突变的数目和分布模式没有明显的规律性，所以ERG3基因突变在 耐药机制中的作用尚需进一步证实。实验中发生的错义突变是单独突 变还是同时突变才可以导致耐药，也需要进一步研究。 【英文摘要】BackgroundIn recent years invasive fungal infections have increased obviously, Candida albicans accountsfor the first number of conditional pathogenic fungi isolated,however,there has been a hugeincreases in the frequency of non-albicans Candida krusei due to the use of antifungal agents.Candida albicans and Candida krusei have increased as a consequence of resistance to azoleantifungal agents.Therefore, researches on the mechanism of resistance in Candida albicans andCandida krusei have been highlighted.To investigate the etiology features and relevant risk factors of invasive fungal infections fromclinically used abacterial body in hospital. To know the drug of azole antifungal agents ofsensitivity of Candida albicans and Candida krusei . To study the mechanism of azole antifungalagents resistance in clinical isolates of Candida albicans and Candida krusei, may be associatedwith the mutations in gene of ERG3.MethodsThe abacterial body fluid of invasive fungal infections during 2006 to 2010 from the clinicallaboratory of the Second Hospital of Shanxi Medical University were enrolled and analyzed.Theidentification of all isolates were confirmed by CHROMagar、CMA and API 20C AUX.A total of163 clinical isolates of Candida species were collected . So we can get 35 strains of azoleantifungal agents resistent and susceptible of Candida albicans and 20 strains of azole antifungalagents resistent and susceptible of Candida krusei respectively.ERG3 gene was amplified by PCRusing Candida albicans genomic DNA , and compared the DNA sequences of the PCR productsthrough BLAST softwares.Results(1) Most of 163 fungal isolates was Candida albicans(54.61%).Non-albicans Candidaaccounted for 42.94% and Aspergillus spp. was 2.45%. The analysis showed that sex, course ofdisease, temperature, prophylaxisly antifungal drugs using,immunosuppressants using, invasiveexamination and treatment, prognosis, sample originals were the independent risk factors forinvasive fungal infections.(2) Of the 35 Candida albicans, the rate of strains susceptible to 5-flucytosine andamphotericine B was 100%,the rate of strains resistant to itraconazole, voriconazole andfluconazole was 17.14%、11.43%、8.57%. Of the 20 Candida krusei, the rate of strains susceptibleto voriconazole and amphotericine B was 95%,the rate of strains resistant to 5-flucytosine, fluconazole and itraconazole was 20.00%、50.00%、45.00%. Candida albicans and Candida kruseihave the phenomenon of multiple drug resistance.(3) The comparison of ERG3 gene sequences identified mutations at 9 sites in Candidaalbicans strains.A17 of drug-resistant strain harbored 4 misssense mutations and 1 samesensemutations in ERG3,4 misssense mutations including I340L、V374G、P902L、V1076A respectively,the samesense mutation is 1080bp.The other strain harbored 5 samesense mutations, including602bp、795bp、969bp、 1080bp、1110bp.ConclusionsMost of 163 fungal isolates was Candida albicans,but non- Candida albicans to rise espicallyCandida krusei.Although Candida albicans have resistance to azole antifungal agents ,but the rateof strains susceptible to itraconazole, fluconazole and voriconazole was 71.43%、85.72%、88.57%,so azole antifungal agents also useful drugs in clinical.The results suggest that missensemutations in ERG3 of Candida albicans have contribute to the mechanism of azole antifungalagents resistance,but the numble and pattern of those mutations have no any regular, thosemutations at the same time or at the different time should not be discounted. 【关键词】白念珠菌 克柔念珠菌 耐药机制 ERG3基因 【英文关键词】Candida krusei Candida albicans drug resistance ERG3 gene 【目录】念珠菌ERG3基因突变对抗真菌药物耐药的作用 中文 摘要 5-7 Abstract 7-8 英文缩略词语表 9-10 前 言 10-12 第一部分 临床菌株分离、鉴定与耐药性分析 12-19 1.1 材料和方法 12-15 2.1 结果 15-17 3.1 讨论 17-19 第二部分 念珠菌临床分离株 ERG3 基因突变分析 19-31 1.1 材料和方法 19-23 2.1 结果 23-28 3.1 讨论 28-31 结论 31-32 参考文 献 32-35 综述 35-40 参考文献 38-40 个人简历 40-41 致谢 41