Itraconazole-Induced Apoptosis in Candida glabrata ( Nakaseomyces glabratus ) Biofilms: Role of ROS and CgPDR 1 Mutations (K274N, D1082G, and S343F) in Resistant Clinical Isolates

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.