ERG11基因与热带念珠菌唑类药物耐药关系的研究

The study on the relationship between ERG11 gene and azole-resistant isolates of Candida tropicalis

目的探讨临床唑类药物耐药热带念珠菌菌株ERG11基因突变及表达情况。方法连续收集临床分离的热带念珠菌菌株,采用微量肉汤稀释法检测其对氟康唑、伏立康唑及伊曲康唑的药物敏感性,对唑类药物耐药菌株及部分敏感菌株进行ERG11基因测序,同时采用RT-PCR测定ERG11基因表达量。结果临床共分离92株热带念珠菌菌株,其中有29株为唑类药物耐药菌株,耐药率31.52%。40株热带念珠菌(29株耐药菌株和11株敏感菌株)ERG11基因序列共发现2个错义突变(S154F、Y132F)和5个同义突变,其中24株唑类药物耐药菌株同时出现上述两个错义突变位点。实时荧光定量PCR结果显示,在29株唑类药物耐药菌株中有19株其ERG11基因表达量较敏感菌株增高。分析16株对3种唑类药物全耐药菌株及其余13株仅对一种或两种药物耐药菌株,显示前者ERG11基因表达水平高于后者,差异有统计学意义。结论临床热带念珠菌唑类药物耐药与ERG11基因突变及过表达有关,有关热带念珠菌唑类药物的耐药机制还需进一步研究。

Objective To investigate the mutation and expression of ERG11 gene in clinical azole-resistant Candida tropicalis isolates. Methods The clinical Candida tropicalis isolates were collected and identified, then antifungal susceptibility tests to fluconazole, voriconazole and itraconazole were performed. ERG11 which was the gene coding antifungal drug target enzyme of cytochrome P450 lanosterol 14 a-demethylase, was quantified by real-time RT-PCR and sequenced. The sequencing results were compared with the known standard sequence(M23673) in GenBank. Results There were 92 clinical Candida tropicalis isolates were obtained, of which 29 were resistant to at least one of the above three azole antifungals. Two missense mutations(S154 F,Y132 F) and five synonymous mutations were found in 40 clinical isolates, including 29 azole-resistant isolates and 11 azole-susceptible isolates. It is worth noting that the two missense mutations were existed in 24 of 29 azole-resistant isolates, however, no missense mutations were found in azole-susceptible isolates. The results of real-time PCR showed that the expression of ERG11 gene in azole-resistant clinical isolates was significantly higher than that in azole-susceptible isolates, and 16 isolates resistant to all three of the azole antifungals had a higher level of ERG11 gene expression than 13 isolates that were only resistant to one or two azole antifungals.Conclusions Azole resistance of clinical Candida tropicalis isolates is associated with the mutations and overexpression ofERG11 gene,and the resistance mechanism of Can didatropicalisto azole antifungals needs further study.