临床烟曲霉菌株的生物特性及唑类耐药机制  

Biological characteristics and drug resistance mechanism of a clinical Aspergillus fumigatus strain

作　　者：曾猛 周雪 王睿 刘方燕 宋章永 ZENG Meng;ZHOU Xue;WANG Rui;LIU Fangyan;SONG Zhangyong(Department of Clinical Laboratory,Yongchuan Hospital of Chongqing Medical University,Chongqing 402160,China;School of Basic Medical Sciences,Public Center of Experimental Technology,Southwest Medical University,Luzhou 646000,Sichuan,China)

机构地区：[1]重庆医科大学附属永川医院检验科,重庆402160 [2]西南医科大学基础医学院公共实验技术中心,泸州646000

出　　处：《医学研究与战创伤救治》2023年第11期1188-1194,共7页Journal of Medical Research & Combat Trauma Care

基　　金：四川省自然科学基金(2023NSFSC0529,2022NSFSC1539);泸州市人民政府-西南医科大学战略合作项目(2020LZXNYDJ23)。

摘　　要：目的 探究临床分离丝状真菌(命名AF70)的菌株生物学地位、生物学特性及其耐唑类药物的分子机制。方法 通过形态学和分子生物学技术明确AF70菌株的生物学地位。采用稀释点板法、甲基四氮盐(XTT)还原法、高效液相色谱法和大蜡螟幼虫存活试验探究AF70菌株的产孢能力、生物膜形成能力、麦角甾醇含量和毒力。通过微量液基稀释法和E-test法测定AF70菌株对三唑类,棘白菌素和两性霉素B的敏感性。Sanger测序法明确唑类药物靶基因cyp51A的突变类型,并通过Discovery Studio软件对CYP51A蛋白的三维结构进行分析。荧光定量PCR技术检测麦角甾醇合成和药物外排泵相关基因mRNA表达水平。结果 形态学和分子生物学鉴定AF70菌株为烟曲霉菌株。与标准菌株AF293相比,AF70菌株生物膜形成和产孢能力增加(P<0.05),但麦角甾醇含量差异无统计学意义(P>0.05)。药敏分析结果表明AF70菌株对伏立康唑和艾沙康唑耐药,对伊曲康唑、泊沙康唑、两性霉素B、卡泊芬净、米卡芬净和阿尼芬净敏感。AF70菌株的cyp51A基因突变类型为TR46/Y121F/T289A,并存在CYP51A蛋白的支链空间结构改变。同时,AF70菌株cyp51A和外排泵相关基因显著上调表达。结论 烟曲霉AF70菌株是由cyp51A基因突变、过表达以及外排泵基因过表达共同介导对VRC耐药。Objective With the increasing number of immunocompromised patients and resistant strains of Aspergillusfu-migatus,A.fumigatusinfection has become one of the global public health problems.The biological characteristics and molecular mechanism of resistance to azole drugs of a clinically isolated filamentous fungus(named AF70)were investigated.Methods The biological status of AF70 strain was determined by morphological and molecular biology techniques.The sporulation ability,biofilm for-mation ability,ergosterol content,and virulence of AF70 strain were evaluated by spot dilution assays,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazoliumhydroxide(XTT)reduction assay,high performance liquid chromatography,and survival analysis of Galleriamellonellalarva,respectively.The susceptibility of AF70 strain to triazoles,echinocandins and ampho-tericin B was determined by broth microdilution technique and E-test method.The mutation types of cyp51A,the target gene of azole drugs,were determined by Sanger sequencing.And the three-dimen-sional structure of cyp51A protein was analyzed by means of the Dis-covery Studio software.The mRNA expression levels of genes related to ergosterol synthesis and drug efflux pump were detected by Reverse-transcription quantitative Polymerase Chain Reaction(RT-qPCR).Results The AF70 strain was identified as A.fumigatusstrain by morphology and molecular biology.Compared to the AF293 strain,the AF70 strain exhibited a significant increase in biofilm forma-tion ability and conidial yield,while no significant differences were observed in ergosterol content.The results of antifungal susceptibil-ity testing revealed that the AF70 strain exhibited resistance to voriconazole(VRC)and isavuconazole and sensitivity towards itracon-azole,posaconazole,amphotericin B,caspofungin,micafungin,and anidulafungin.The cyp51Agene mutation type of AF70 strain was TR46/Y121F/T289A,and the branch chain spatial structure of CYP51A protein was changed.Moreover,the expression of cyp51Aand eff

关 键 词：烟曲霉 唑类药物 耐药 外排泵基因 cyp51A 

分 类 号：R379[医药卫生—病原生物学]