Microbial drug resistance (Larchmont, N.Y.) · 2026
Itraconazole-Induced Apoptosis in Candida glabrata (Nakaseomyces glabratus) Biofilms: Role of ROS and CgPDR1 Mutations (K274N, D1082G, and S343F) in Resistant Clinical Isolates.
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Valizadeh Farnaz, Fattahi Mahsa, Lotfali Ensieh, Motamed Nasrin
Аннотация
BACKGROUND: Nakaseomyces glabratus exhibits intrinsic tolerance to azole antifungals, frequently mediated by gain-of-function (GOF) mutations in the transcription factor CgPDR1. While efflux-mediated resistance is well established, its potential interaction with oxidative stress-dependent cell death pathways in biofilms remains insufficiently characterized.
MATERIALS AND METHODS: Fifteen clinical isolates (susceptible, n = 5; resistant, n = 10) were analyzed under planktonic and biofilm conditions. Antifungal susceptibility to itraconazole (0.03-32 µg/mL) was determined, while apoptosis and intracellular reactive oxygen species (ROS) were quantified using Annexin V/PI staining and fluorescence-based assays, respectively. The role of oxidative stress was evaluated using ascorbic acid co-treatment. CgPDR1 mutations were identified by sequencing.
RESULTS: Resistant isolates exhibited significantly elevated minimum inhibitory concentration (MIC) values (up to 32 µg/mL) and a high prevalence of CgPDR1 mutations (K274N: 100%; D1082G: 30%; S343F: 10%). Itraconazole induced a marked increase in ROS production and apoptosis in susceptible biofilms (Annexin V+ ≈ 70%), whereas resistant isolates demonstrated attenuated ROS generation and reduced apoptosis (≈10%, p < 0.01). Although ascorbic acid significantly decreased ROS levels in susceptible isolates, it did not alter MIC values or restore antifungal susceptibility.
CONCLUSIONS: CgPDR1 GOF mutations are associated with a coordinated resistance phenotype in N. glabratus biofilms, characterized by enhanced drug tolerance and reduced ROS-mediated apoptosis. These findings support a multifactorial resistance model and identify K274N as a potential population-specific biomarker.
MATERIALS AND METHODS: Fifteen clinical isolates (susceptible, n = 5; resistant, n = 10) were analyzed under planktonic and biofilm conditions. Antifungal susceptibility to itraconazole (0.03-32 µg/mL) was determined, while apoptosis and intracellular reactive oxygen species (ROS) were quantified using Annexin V/PI staining and fluorescence-based assays, respectively. The role of oxidative stress was evaluated using ascorbic acid co-treatment. CgPDR1 mutations were identified by sequencing.
RESULTS: Resistant isolates exhibited significantly elevated minimum inhibitory concentration (MIC) values (up to 32 µg/mL) and a high prevalence of CgPDR1 mutations (K274N: 100%; D1082G: 30%; S343F: 10%). Itraconazole induced a marked increase in ROS production and apoptosis in susceptible biofilms (Annexin V+ ≈ 70%), whereas resistant isolates demonstrated attenuated ROS generation and reduced apoptosis (≈10%, p < 0.01). Although ascorbic acid significantly decreased ROS levels in susceptible isolates, it did not alter MIC values or restore antifungal susceptibility.
CONCLUSIONS: CgPDR1 GOF mutations are associated with a coordinated resistance phenotype in N. glabratus biofilms, characterized by enhanced drug tolerance and reduced ROS-mediated apoptosis. These findings support a multifactorial resistance model and identify K274N as a potential population-specific biomarker.