白念珠菌对氟康唑耐药机制的研究  

Study on the mechanism of fluconazole resistance in Candida albicans

作　　者：封小川 元航 郑莹莹 白江苗[1] 张欠欠 FENG Xiaochuan;YUAN Hang;ZHENG Yingying;BAI Jiangmiao;ZHANG Qianqian(Medical College of Yan’an University,Yan’an 716000,China;Yan’an People’s Hospital,Yan’an 716000,China;Yanan University Affiliated Hospital,Yan’an 716000,China)

机构地区：[1]延安大学医学院,延安716000 [2]延安市人民医院,延安716000 [3]延安大学附属医院,延安716000

出　　处：《中国真菌学杂志》2023年第6期481-486,共6页Chinese Journal of Mycology

基　　金：陕西省重点研发计划项目(2021SF-230);榆林市科技项目(CXY-2020-064);延安大学校产学研项目(CXY202121)。

摘　　要：目的通过研究白念珠菌对氟康唑耐药相关基因ERG11、CDR1、CDR2和MDR1的表达水平及其与耐药的关系,旨在对本地区以白念珠菌感染为主的侵袭性念珠菌病的防治提供依据。方法选取延安地区两家三甲医院临床确诊的对氟康唑耐药及敏感的白念珠菌各10株,扩增其靶酶编码基因ERG11,分析突变位点;使用荧光染料罗丹明6G分析耐药组与敏感组菌株的外排情况;采用荧光定量PCR检测白念珠菌外排泵编码基因的表达水平。结果ERG11扩增测序结果显示,耐药菌株C3号虽发生了碱基位点突变,但并未引起编码氨基酸的改变,耐药菌株C7号及敏感菌株C12号发生了碱基位点的突变,引起编码氨基酸的改变;荧光染料罗丹明6G检测白念珠菌外排实验结果显示,耐药菌和敏感菌无论加入葡萄糖与否外排均存在显著差异,且耐药组加入葡萄糖较未加入葡萄糖外排量明显增加,差异有统计学意义(P<0.01);RT-PCR检测外排泵基因结果显示,CDR1与CDR2外排泵基因表达水平在耐药菌与敏感菌之间差异有统计学意义(P<0.05)。结论研究基因表达及外排泵功能检测结果均表明,本地区白念珠菌对氟康唑的耐药机制主要与外排泵基因过度表达相关,未发现靶酶编码基因有意义的突变。Objective To study the expression levels of fluconazole resistant genes ERG11,CDR1,CDR2,and MDR1 in Candida albicans and their relationship with drug resistance,and to provide a basis for the prevention and treatment of invasive candidiasis mainly caused by Candida albicans infection in this region.Methods Ten fluconazole resistant strains and 10 fluconazole sensitive strains were selected from each hospital in two tertiary and first-class hospitals in Yan'an area.Amplifying the target enzyme coding gene ERG11,analyzing the mutation sites,fluorescent dye using rhodamine 6G were used to analyze the efflux of strains from drug resistant and sensitive groups.Then,fluorescence quantitative PCR was used to detect the expression level of the gene encoding the efflux pump of Candida albicans.Results ERG11 amplification and sequencing results showed that although the resistant strain C3 had a base site mutation,it did not cause changes in the coding amino acid.The resistant strain C7 and the sensitive strain C12 had a base site mutation,causing changes in the coding amino acid;fluorescent dye rhodamine 6G was used to detect the efflux of Candida albicans.The results showed that the efflux of resistant isolate and sensitive isolate were significantly different whether glucose was added or not,and the efflux of drug resistant isolate with glucose was significantly higher than that without glucose,and the difference was statistically significant(P<0.05);the RT-PCRdetection of efflux pump genes showed a statistically significant difference in the expression levels of CDR1 and CDR2 efflux pump genes between drug-resistant and sensitive isolate(P<0.05).Conclusion The results of gene expression and efflux pump function testing in this study indicate that the resistance mechanism of Candida albicans to fluconazole in this region was mainly related to overexpression of efflux pump genes,and no significant mutations were found in the target enzyme coding genes.

关 键 词：氟康唑 耐药机制 ERG11 CDR1 CDR2 

分 类 号：R379.4[医药卫生—病原生物学]