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目的:研究光滑念珠菌和近平滑念珠菌对氟康唑耐药的易感性及其诱导耐药株的耐药机制。方法 :选取对氟康唑敏感的光滑念珠菌、近平滑念珠菌临床株各1株以及对氟康唑敏感的光滑念珠菌标准株ATCC2001、近平滑念珠菌标准株ATCC22019,利用浓度递增法,在体外用氟康唑诱导使其成为耐药株,采用实时定量PCR检测外排泵相关基因、其转录因子和靶酶编码基因表达,并对光滑念珠菌PDR1转录因子、近平滑念珠菌MRR1转录因子和ERG11基因进行PCR扩增和测序。结果 :光滑念珠菌较近平滑念珠菌更易被氟康唑诱导为耐药株,CDR1的过度表达为其主要的耐药机制,对PDR1转录因子测序后发现了新的突变位点Y932C、V847F。近平滑念珠菌诱导耐药株的MDR1表达显著增加,其MRR1转录因子中亦存在新的突变位点P295S、I799S。结论 :光滑念珠菌和近平滑念珠菌对氟康唑的耐药易感性不同,耐药机制也存在差异。新发现的转录因子突变位点有待验证其功能。
Objective: To study the susceptibility to fluconazole-resistant Candida and Candida parapsilosis and the drug resistance mechanism of the drug-resistant strains. Methods: One strain of Candida glabrata, one strain of Candida parapsilosis and one strain of Candida glabrata sensitive to fluconazole ATCC2001 and ATCC22019 of Candida parapsilosis were selected. The concentration of In vitro, fluconazole induced it to become drug-resistant strain. Real-time quantitative PCR was used to detect efflux pump-related genes, transcription factor and target enzyme-encoding gene expression. Candida glabrata PDR1 transcription factor, Candida parapsilosis MRR1 transcription factor And ERG11 gene for PCR amplification and sequencing. Results: Candida glabrata was more likely to be induced by fluconazole than Candida parapsilosis. Overexpression of CDR1 was the main mechanism of drug resistance. New mutations Y932C and V847F were found after PDR1 transcription factor sequencing. Candida parapsilosis induced MDR1 expression was significantly increased MRR1 transcription factor also exists new mutation sites P295S, I799S. Conclusion: Candida glabrata and Candida parapsilosis are susceptible to fluconazole different drug resistance mechanisms are also different. Newly discovered transcription factor mutations are pending validation of their function.