Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; Center for Health Technology and Services Research - CINTESIS@RISE, Faculty of Medicine, University of Porto, Porto, Portugal.
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland.
Clin Microbiol Infect. 2022 Dec;28(12):1655.e5-1655.e8. doi: 10.1016/j.cmi.2022.08.014. Epub 2022 Aug 24.
Hereby, we describe the molecular mechanisms underlying the acquisition of azole resistance by a Candida parapsilosis isolate following fluconazole treatment due to candiduria.
A set of three consecutive C. parapsilosis isolates were recovered from the urine samples of a patient with candiduria. Whole-genome sequencing and antifungal susceptibility assays were performed. The expression of MRR1, MDR1, ERG11 and CDR1B (CPAR2_304370) was quantified by RT-qPCR.
The initial isolate CPS-A was susceptible to all three azoles tested (fluconazole, voriconazole and posaconazole); isolate CPS-B, collected after the second cycle of treatment, exhibited a susceptible-dose-dependent phenotype to fluconazole and isolate CPS-C, recovered after the third cycle, exhibited a cross-resistance profile to fluconazole and voriconazole. Whole-genome sequencing revealed a putative resistance mechanism in isolate CPS-C, associated with a G1810A nucleotide substitution, leading to a G604R change in the Mrr1p transcription factor. Introducing this mutation into the susceptible CPS-A isolate (MRR1) resulted in resistance to fluconazole and voriconazole, as well as up-regulation of MRR1 and MDR1. Interestingly, the susceptible-dose-dependent phenotype exhibited by isolate CPS-B was associated with an increased copy number of the CDR1B gene. The expression of CDR1B was increased in both isolates CPS-B and CPS-C and in the MRR1 strain, harbouring the gain-of-function mutation.
Our results describe clinical azole cross-resistance acquisition in C. parapsilosis due to a G1810A (G604R) gain-of-function mutation, resulting in MRR1 hyperactivation and consequently, MDR1 efflux pump overexpression. We also associated amplification of the CDR1B gene with decreased fluconazole susceptibility and showed that it is a putative target of the MRR1 gain-of-function mutation.
本研究描述了由于尿念珠菌病而接受氟康唑治疗后，一株近平滑念珠菌分离株获得唑类药物耐药的分子机制。
从一名尿念珠菌病患者的尿液样本中连续分离出三株近平滑念珠菌分离株。进行全基因组测序和抗真菌药敏试验。通过 RT-qPCR 定量测定 MRR1、MDR1、ERG11 和 CDR1B（CPAR2_304370）的表达。
初始分离株 CPS-A 对所有三种唑类药物（氟康唑、伏立康唑和泊沙康唑）均敏感；在第二次治疗周期后采集的分离株 CPS-B 表现出对氟康唑的敏感剂量依赖性表型，而在第三次治疗周期后采集的分离株 CPS-C 对氟康唑和伏立康唑表现出交叉耐药表型。全基因组测序揭示了 CPS-C 分离株中存在一种潜在的耐药机制，与 G1810A 核苷酸取代相关，导致 Mrr1p 转录因子的 G604R 变化。将该突变引入敏感的 CPS-A 分离株（MRR1）中，导致对氟康唑和伏立康唑的耐药性，并上调了 MRR1 和 MDR1 的表达。有趣的是，分离株 CPS-B 表现出的敏感剂量依赖性表型与 CDR1B 基因的拷贝数增加有关。在分离株 CPS-B 和 CPS-C 以及携带功能获得性突变的 MRR1 菌株中，CDR1B 的表达均增加。
我们的研究结果描述了由于 G1810A（G604R）功能获得性突变导致的近平滑念珠菌对唑类药物的交叉耐药性获得，导致 MRR1 过度激活，进而导致 MDR1 外排泵过度表达。我们还将 CDR1B 基因的扩增与氟康唑敏感性降低相关联，并表明它是 MRR1 功能获得性突变的潜在靶点。
