临床分离光滑假丝酵母菌唑类药物耐药机制研究

Study on mechanisms of zole-resistance of clinical Candida glabrata isolates

目的了解光滑假丝酵母菌临床分离株中唑类耐药株的耐药机制。方法收集2009年和2010年5家医院28例患者的38株光滑假丝酵母菌,根据CLSI M27-A3指南采用微量肉汤稀释法测定5种常用抗真菌药物(两性霉素B、5-氟胞嘧啶、氟康唑、伊曲康唑和伏立康唑)的最小抑菌浓度。采用Real-Time PCR法检测CDR1、CDR2及SNQ2等外排泵基因以及ERG11和转录因子PDR1的表达;流式细胞仪分析罗丹明6G外排能力,考察细胞膜外排泵功能;同时,PCR扩增ERG11基因全长以及PDR1基因主要功能区并测序比对。结果 38株光滑假丝酵母菌对两性霉素B和5-氟胞嘧啶均敏感,其中17株为唑类耐药菌。唑类耐药株CDR1基因表达明显高于敏感株(P=0.001),并且对罗丹明6G外排能力也显著强于敏感菌(P=0.001)。光滑假丝酵母菌敏感和耐药菌株ERG11表达差异无统计学意义(P=0.283),且ERG11基因未发现突变。此外,17株光滑耐药菌株PDR1基因表达量与敏感菌株比较差异无统计学意义(P=0.086),但每株耐药菌株PDR1基因均分别发现1处有义突变。结论转录因子PDR1突变、外排泵基因CDR1表达上调以及外排泵功能增强是目前临床上光滑假丝酵母菌唑类药物耐药的主要机制。

Objective To investigate the mechanism of azole resistance of clinical Candida glabrata isolates. Methods Thirty-eight clinical Candida glabrata isolates were collected from 28 patients of five hospitals between 2009 and 2010. The minimum acteriostatic concentrations of five commonly used antifungal agents （ araphotericin B, 5-flucytosine, fluconazole, itraconazole, and voriconazole） were determined by the broth microdilution antifungal susceptibility test. The expressions of efflux-related genes （CDR1, CDR2, and SNQ2）, ERG11, and transcription factor PDR1 were detected by the Real-Time PCR. The efflux ability of rhodamine 6G was analyzed by the flow cytometry. The function of the efflux pump of cell membrane was investigated. The whole sequence of ERG11 gene and the major functional domains of PDR1 were amplified by the PCR, sequenced, and compared to standard sequences. Results All 38 Candida glabrata isolates were sensitive to amphotericin B and 5-flucytosine and 17 of which were azole resistant. The expression of CDR1 and the efflux ability of rhodamine 6G of azole resistant isolates were significantly higher than those of sensitive isolates （P=0. 001）. The difference of the expression of ERG11 of sensitive isolates and azole resistant isolates was not statistically significant （P=0. 283）. No mutations of ERG11 were found in azole resistant isolates. The difference of the expression of PDR1 of 17 azole-resistant isolates and sensitive isolates was not statistically significant （P=0. 086）. But one sense mutation of PDR1 was found in each azole resistant isolate. Conclusion Mutations oftranscription factor PDR1, up-regulation of efflux pump related gene CDR1, and the increase of the function of efflux pumps are the main mechanisms of clinical zole resistance of Candida glabrata.