侵袭性感染白假丝酵母ERG11突变分析与氟康唑耐药关系研究

Mutation of ERG11 in Candida albicans causing invasive infection and its relationship with fluconazole resistance

目的明确医院侵袭性感染的白假丝酵母氟康唑耐药的主要分子机制及其与ERG11突变的关系,以指导临床抗真菌的治疗。方法收集2015年1月-2017年6月医院临床住院患者分离的50株侵袭性感染白假丝酵母,采用VITEK-2COMPACT进行检测及药敏分析,提取其真菌基因组DNA,采用PCR检测ERG11基因并测序分析,同时采用实时荧光定量RT-PCR检测ERG11基因mRNA相对表达量,并采用SPSS19.0统计软件分析氟康唑耐药与ERG11突变及相对表达量之间关系。结果 50株侵袭性白假丝酵母主要来自尿液、伤口分泌物和腹水标本,其中对氟康唑耐药11株,氟康唑剂量依赖性耐药10株,氟康唑敏感29株;ERG11基因扩增及测序分析发现15个突变位点,其中11个为同义突变,无氨基酸改变,4个为错义突变;白假丝酵母氟康唑耐药株、剂量依赖性敏感株和氟康唑敏感菌株ERG11基因的mRNA表达水平之间比较差异有统计学意义(F=78.30,P=0.002);随着白假丝酵母对氟康唑敏感性的降低,ERG11基因mRNA的表达量均有不同程度的上升。结论侵袭性感染白假丝酵母氟康唑耐药严重,耐药水平与ERG11基因的错义突变及表达量密切相关。

OBJECTIVE To investigate the molecular mechanisms of fluconazole resistance of Candida albicans causing invasive infection and its relationship with ERG11 mutation so as to provide guidance for clinical antifungal therapy.METHODS From Jan 2015 to Jun 2017,50 strains of C.albicans causing invasive infection were isolated from hospitalized patients,all the strains were detected and drug susceptibility testing was performed by using VITEK 2 COMPACT,the genome DNA was extracted,the ERG11 gene was detected by means of PCR and sequenced,the relative expression of the ERG11 mRNA was detected by using RT-PCR,and the relationship between the fluconazole resistance and ERG11 mutation and relative expression was analyzed with the use of SPSS19.0 software.RESULTS Among the 50 strains of C.albicans,most of them were isolated from urine,wound secretions and ascites specimens,and there were 11 fluconazole-resistant strains,10 fluconazole-susceptible and dose-dependent strains and 29 fluconazole-susceptible strains.There were 15 mutation sites in the ERG11,including 11 synonymous mutations without change of amino acid and 4 missense mutations.There was significant difference in the level of mRNA expression of ERG11 gene among the fluconazole-resistant,fluconazole-susceptible and dose-dependent and fluconazole-susceptible strains（F=78.30,P=0.002）.With the decrease of susceptibility to fluconazole,the level of mRNA expression of the ERG11 gene was elevated.CONCLUSION The C.albicans strains causing invasive infection are highly resistant to fluconazole,and the drug resistance is closely associated with the missense mutations of the ERG11 gene and the expression level.