假丝酵母菌ERG11和ERG3基因突变与氟康唑耐药的研究

Study on ERG11 and ERG3 gene mutations and fluconazole resistance in Candida

目的 分析假丝酵母菌ERG11和ERG3基因突变情况及其对氟康唑耐药的机制。方法 100株念球菌,体外药敏试验获得耐氟康唑的热带假丝酵母菌5株和白色假丝酵母菌9株,提获DNA,基于聚合酶链式反应(PCR)分别扩增分为以上假丝酵母菌的ERG11、ERG3基因。比较分析PCR产物和GenBank数据库外供的基因序列,进而探寻到基因突变点。结果 ITS1扩增后形成了片段规格类似的条带,在500~620 bp范围中。鉴定发现,分离到的100株假丝酵母菌内,其中热带假丝酵母菌、白色假丝酵母菌占真菌总数的65%,分别有15株和50株。其中9株白色假丝酵母菌株的氟康唑24 h最小抑菌浓度(MIC)>16 mg/L,确定其是耐药菌株;5株热带假丝酵母菌对氟康唑和伏立康唑双重耐药。PCR对临床分离获得的14株假丝酵母菌进行扩增,都获得了预期产物,规格和DNA Marker相比较,部位吻合,未形成非特异性产物带。基于Blast比较热带假丝酵母菌和白色假丝酵母菌的测序结果 ,将起始密码子ATG的A计数设定成1,基于此探寻到突变基因。5株热带假丝酵母菌ERG11基因存有同义、错义突变分别为5、4个,错义突变分别是Y132H、G487T、A530C、G533C;ERG3基因依次有7个和12个;白色假丝酵母菌ERG11和ERG3基因各发现4个同义突变,前者有3个错义突变,后者为0。分析ERG11基因序列以后,发现其在氟康唑敏感菌株仅是发生了单个突变,康唑剂量自身依赖敏感程度和耐药菌株内ERG11基因突变以多位点错义突变同时出现为主。结论 假丝酵母菌ERG11、ERG3均存在程度不一的突变,其可能参与假丝酵母菌耐药性形成过程。

Objective To analyze the mutations of ERG11 and ERG3 gene in Candida and their mechanism of resistance to fluconazole. Methods The MICs of 100 strains of Candida were detected, and in vitro drug susceptibility tests were performed to obtain 5 strains of Candida tropicalis and 9 strains of Candida albicans resistant to fluconazole, and DNA was extracted and the ERG11 and ERG3 genes of Candida were amplified based on polymerase chain reaction(PCR). The PCR products were compared and analyzed with the gene sequences provided by the GenBank database to detect mutation sites. Results ITS1 amplification resulted in bands with similar fragment specifications in the range of 500-620 bp. The identification revealed that within the 100 Candida strains isolated, Candida tropicalis and Candida albicans accounted for 65% of the total number of fungi, with 15 and 50 strains, respectively. Among them, 9 strains of Candida albicans had fluconazole 24 h minimum inhibitory concentration(MIC) >16 mg/L and were identified as drug-resistant strains;5 strains of Candida tropicalis were doubly resistant to fluconazole and voriconazole. PCR was performed to amplify 14 strains of Candida obtained from clinical isolates, and all obtained the expected products, with the specifications compared with DNA Marker, and the sites matched without forming non-specific product bands. Based on Blast comparing the sequencing results of Candida tropicalis and Candida albicans, the A count of the start codon ATG was set to 1, based on which the mutant gene was probed. 5 strains of Candida tropicalis ERG11 gene had 5 and 4 synonymous and missense mutations,respectively, and the missense mutations were Y132H, G487T, A530C and G533C;ERG3 gene had 7 and 12 mutations, respectively. 4 synonymous mutations were found in ERG11 and ERG3 genes of Candida albicans,the former had 3 missense mutations and the latter was 0. After analyzing the ERG11 gene sequence, it was found that only a single mutation occurred in the fluconazole-susceptible strains, and the ERG11 gene mutations in the conazole dose-dependent and drug-resistant strains were mainly multi-site missense mutations at the same time.Conclusion The ERG11 and ERG3 genes of Candida have different degrees of mutations, which may be involved in the formation of drug resistance in Candida.