克柔念珠菌临床分离株对伊曲康唑耐药的分子表达情况

Molecular expression of itraconazole resistance in clinical isolates of Candida krusei

目的初步探讨克柔念珠菌临床分离株耐伊曲康唑的分子表达情况。方法对16株克柔念珠菌唑类靶酶基因ERG11进行PCR扩增和测序，比较伊曲康唑敏感组（3株）、剂量依赖性敏感组（8株）和耐药组（5株）的基因突变情况。同时，利用实时荧光定量PCR检测3组靶酶基因ERG11、外排泵基因ABC1和ABC2的mRNA表达差异。将数据转换后服从正态分布且方差齐，两组间比较采用双侧t检验。结果16株克柔念珠菌ERG11基因共存在7个突变位点，其中6个同义突变，1个错义突变（C44T），3组均可出现该错义突变。克柔念珠菌临床分离株伊曲康唑耐药组ERG11mRNA表达量较剂量依赖性敏感组和敏感组高（P值分别为0．015、0．002）；耐药组ABC2mRNA表达量也较剂量依赖性敏感组和敏感组高（P值分别为0．016、〈0．01），且剂量依赖性敏感组mRNA表达量高于敏感组（P=0．007）；而耐药组ABC1mRNA表达量较低。1株同时耐伊曲康唑和伏立康唑的克柔念珠菌（cK10），其职G11、ABC1和ABC2mRNA表达水平在所有耐药株中均为最高。结论ERG11基因序列存在多态性，未发现与伊曲康唑耐药有关的突变位点。ERG11和ABC2基因表达上调可能是克柔念珠菌临床分离株耐伊曲康唑的主要分子机制。

Objective To investigate the main molecular expressions responsible for itraconazole resistance in clinical isolates of Candida krusei （C. krusei）. Methods The ERG11 gene in the 16 C. krusei clinical isolates was amplified by polymerase chain reaction （PCR）. Point mutations were determined by nucleotide sequence and compared among itraconazole-resistant （R, n = 5）, itraconazole- susceptible dose dependent （SDD, n = 8） and itraconazole-susceptible （S, n= 3 ） C. krusei. Meanwhile, ERG11 and efflux transporter genes （ABC1 and ABC2） were determined by quantitative real-time reverse transcription polymerase chain reaction （qRT-PCR） in 3 groups at the mRNA level. All variables were transformed to obey normal distribution and variance homogenity and compared by two sided t test between two groups. Results Seven point mutations in ERG11 gene of all the C. krusei clinical isolates were detected, including 6 synonymous mutations and 1 missense mutation （C44T）. However, the missense mutation was found in all three groups. The mRNA levels of ERG11 gene in itraconazole- resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones （P = 0. 015 and P = 0. 002, respectively）. ABC2 gene mRNA level in itraconazole-resistant group was significantly higher than the other two groups （P= 0. 016 and P〈0.01, respectively）, and the level of its expression in susceptible dose-dependent group was higher than dosesusceptible group （P= 0. 007）. ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in one C. krusei （CK10） strain resistant to both itraconazole and voriconazole were highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C. krusei. ERG11 gene mutations are not found to he involved in the development of itraconazole resistance in C. krusei. ERG11 and ABC2 upregulation might be responsible for the acquired itraconazole resistance of these clinical isolates.