通过下一代测序技术鉴定光滑念珠菌对棘白菌素类、唑类和 5-氟胞嘧啶耐药的遗传标记：一项可行性研究。

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.

机构信息

Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia; Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia.

出版信息

Clin Microbiol Infect. 2017 Sep;23(9):676.e7-676.e10. doi: 10.1016/j.cmi.2017.03.014. Epub 2017 Mar 23.

DOI:10.1016/j.cmi.2017.03.014

PMID:28344162

Abstract

OBJECTIVES

Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata.

METHODS

The antifungal susceptibility of 12 clinical isolates and one ATCC strain of C. glabrata was determined using the Sensititre YeastOne YO10 assay. These included three isolate pairs where the second isolate of each pair had developed a rise in drug MICs. Single nucleotide polymorphisms (SNPs) in genes known to be linked to echinocandin, azole and 5-fluorocytosine resistance were analysed in all isolates through NGS.

RESULTS

High-quality non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgCDR1, CgPDR1 and FCY2 were identified. For two of three isolate pairs, there was a >60-fold rise in MICs to all echinocandins in the second isolate from each pair; one echinocandin-resistant isolate harboured a mutation in FKS1 (S629P) and the other in FKS2 (S663P). Of the third pair, both the 5-fluorocytosine-susceptible, and resistant isolates had a mutation in FCY2 (A237T). SNPs in CgPDR1 were found in pan-azole-resistant isolates. SNPs in other genes linked to azole resistance (CgCDR1, ERG9 and CgFLR1) were present in both azole-susceptible and azole-resistant isolates. SNPs were also identified in Candida adhesin genes EPA1, EPA6, PWP2 and PWP5 but their presence was not associated with higher drug MICs.

CONCLUSIONS

Genome-wide analysis of antifungal resistance markers was feasible and simultaneously revealed mutation patterns of genes implicated in resistance to different antifungal drug classes.

摘要

目的

光滑念珠菌的多类抗真菌药物耐药性正在增加。我们研究了下一代测序（NGS）在检测光滑念珠菌抗真菌药物耐药标志物中的可行性。

方法

采用 Sensititre YeastOne YO10 检测法测定了 12 株临床分离株和 1 株 ATCC 株光滑念珠菌的抗真菌药敏性。其中三对分离株的第二个分离株药物 MIC 值升高。通过 NGS 对所有分离株中与棘白菌素、唑类和 5-氟胞嘧啶耐药相关的基因中的单核苷酸多态性（SNP）进行分析。

结果

鉴定出了与抗真菌耐药基因（如 FKS1、FKS2、CgCDR1、CgPDR1 和 FCY2）相关的高质量非同义 SNP。在三对分离株中的两对中，第二个分离株对所有棘白菌素的 MIC 值升高了>60 倍；一个棘白菌素耐药分离株携带 FKS1（S629P）突变，另一个携带 FKS2（S663P）突变。第三对中，5-氟胞嘧啶敏感和耐药的分离株均在 FCY2（A237T）中发生突变。CgPDR1 中的 SNP 存在于泛唑类耐药分离株中。唑类耐药相关基因（CgCDR1、ERG9 和 CgFLR1）中的 SNP 存在于唑类敏感和唑类耐药的分离株中。还在 Candida 黏附素基因 EPA1、EPA6、PWP2 和 PWP5 中鉴定出了 SNP，但它们的存在与较高的药物 MIC 值无关。