Molecular biological characteristics of Candida albicans strains causing recurrent vulvovaginal candidiasis with different susceptibility to antifungal drugs in vitro

Objective. To determine molecular biological characteristics of Candida albicans strains from patients with recurrent vulvovaginal candidiasis (RVVC) associated with the formation of resistance to azole drugs. Materials and Methods. The study included 36 clinical isolates of C. albicans from patients with RVVC who sought outpatient care at the P.N. Kashkin Research Institute of Medical Mycology from July to November 2024. Species identification of Candida spp. was performed using MALDI-TOF mass-spectrometry and targeted sequencing. The susceptibility of Candida spp. to antifungal drugs (AFDs) was determined using the Fungus AST test system (Autobio Diagnostics Co, China) with the interpretation of MIC clinical breakpoint values in accordance with CLSI M27A, 2022. The nucleotide sequence of the ERG11 gene was studied using targeted sequencing on an ABI PRISM 3500 genetic analyzer. The expression level of genes encoding drug transporter proteins CDR1, CDR2, MDR1 and FLU1 was analyzed by real-time PCR using the fluorescent dye EVA Green using the relative 2-∆∆Сt measurement method. Biofilm formation of C. albicans strains was assessed by total cell mass (staining with an aqueous solution of crystal violet) and metabolic activity of cells (tetrazolium salt reduction method). Statistical processing of the results was carried out using nonparametric evaluation criteria of the SPSS v.22.0. Results. 11 (30.5%) strains were susceptible to fluconazole, 6 (14.0%) classified as “susceptible dosesensitive”, 20 (55.5%) were resistant to this AFD. All strains were susceptible to polyenes, echinocandins and 5-flucytosine. The work shows a correlation between the nucleotide polymorphism of the ERG11 gene and the sensitivity of the strain to fluconazole. Resistant strains of C. albicans (MIC > 8 mg/l) were characterized by the lowest variability and the presence in the genotype of a single missense mutation T394C (Y132H), associated with resistance to azoles. A possible role of A1343G (E448G) as a suppressor intragenic mutation that neutralizes the effect of T394C (Y132H) on conformational changes in the Erg11 protein has been suggested. Further research is need in this aspect. The ERG11 and FLU1 genes showed increased expression levels (р = 0.004 and р = 0.012, respectively) in fluconazole-resistant C. albicans strains compared to susceptible strains. No significant differences were found in the expression of genes encoding drug transporter proteins CDR1, CDR2 and MDR1. All studied C. albicans strains formed biofilms in vitro. Fluconazole-resistant strains had higher metabolic activity and biofilm mass (p = 0.011 and р = 0.012, respectively). Conclusions. Resistance of RVVC pathogens in the North-West region of Russia is associated with increased biofilm formation, amino acid substitution Y132H and increased expression of the ERG11 and FLU1 genes.