Strzelczyk Joanna Katarzyna, Slemp-Migiel Anna, Rother Magdalena, Gołąbek Karolina, Wiczkowski Andrzej
Chair and Department of General Biology, Medical University of Silesia, Zabrze, Poland.
Laboratory of Microbiology in Nowy Targ Hospital, Nowy Targ, Poland.
Acta Biochim Pol. 2013;60(4):547-52. Epub 2013 Dec 16.
One of the mechanisms of Candida albicans resistance to azole drugs used in antifungal therapy relies on increased expression and presence of point mutations in the ERG11 gene that encodes sterol 14α demethylase (14DM), an enzyme which is the primary target for the azole class of antifungals. The aim of the study was to analyze nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates. The Candida albicans isolates represented a collection of 122 strains selected from 658 strains isolated from different biological materials. Samples were obtained from hospitalized patients. Fluconazole susceptibility was tested in vitro using a microdilution assay. Candida albicans strains used in this study consisted of two groups: 61 of the isolates were susceptible to azoles and the 61 were resistant to azoles. Four overlapping regions of the ERG11 gene of the isolates of Candida albicans strains were amplified and sequenced. The MSSCP (multitemperature single strand conformation polymorphism) method was performed to select Candida albicans samples presenting genetic differences in the ERG11 gene fragments for subsequent sequence analysis. Based on the sequencing results we managed to detect 19 substitutions of nucleotides in the ERG11 gene fragments. Sequencing revealed 4 different alterations: T495A, A530C, G622A and A945C leading to changes in the corresponding amino acid sequence: D116E, K128T, V159I and E266D. The single nucleotide changes in the ERG11 gene did not affect the sensitivity of Candida albicans strains, whereas multiple nucleotide substitutions in the ERG11 gene fragments indicated a possible relation with the increase in resistance to azole drugs.
白色念珠菌对抗真菌治疗中使用的唑类药物产生耐药性的机制之一，依赖于编码甾醇14α脱甲基酶（14DM）的ERG11基因表达增加以及出现点突变，该酶是唑类抗真菌药物的主要作用靶点。本研究的目的是分析唑类敏感和耐药临床分离株的白色念珠菌ERG11基因中的核苷酸替换情况。白色念珠菌分离株是从658株从不同生物材料中分离出的菌株中选取的122株菌株的集合。样本取自住院患者。使用微量稀释法在体外测试氟康唑敏感性。本研究中使用的白色念珠菌菌株分为两组：61株分离株对唑类敏感，61株对唑类耐药。对白色念珠菌菌株分离株的ERG11基因的四个重叠区域进行扩增和测序。采用多温度单链构象多态性（MSSCP）方法选择在ERG11基因片段中存在遗传差异的白色念珠菌样本进行后续序列分析。基于测序结果，我们在ERG11基因片段中检测到19个核苷酸替换。测序揭示了4种不同的改变：T495A、A530C、G622A和A945C，导致相应氨基酸序列发生变化：D116E、K128T、V159I和E266D。ERG11基因中的单核苷酸变化不影响白色念珠菌菌株的敏感性，而ERG11基因片段中的多个核苷酸替换表明可能与对唑类药物耐药性的增加有关。
