Overexpression of efflux pump and biofilm associated genes in itraconazole resistant Candida albicans isolates causing onychomycosis

Abstract

Candida onychomycosis is a common fungal nail infection where treatment efficacy can be compromised by antifungal resistance. This study investigates the role of efflux pump genes (CDR1, CDR2, and MDR1) and biofilm-associated genes (ALS1, ALS3) in Candida albicans isolates classified as resistant to itraconazole from patients with onychomycosis. Ten itraconazole-resistant and 10 sensitive isolates were collected for efflux pump and biofilm-associated gene expression analysis by Real-Time PCR methods. Itraconazole resistance was induced in sensitive isolates through pulse exposure. Biofilm formation was quantified both with and without itraconazole. Biofilm structures were visualized by scanning electron microscopy. Our findings indicate a statistically significant upregulation of CDR1 (P-value = 0.049), CDR2 (P-value = 0.023), and ALS3 (P-value = 0.010) in resistant isolates when compared to sensitive isolates. While MDR1 and ALS1 showed some variation, the differences were not statistically significant. Rhodamine 6G efflux assays demonstrated significantly higher efflux activity in resistant isolates (P-value = 0.001 at 60 min). Biofilm formation assays showed itraconazole’s impact: pre-treatment reduced biofilm formation, while it had a limited effect on pre-formed biofilms. Scanning electron microscopy indicated less dense biofilms when the formation began in the presence of itraconazole. A pulse exposure to itraconazole also further upregulated CDR1, CDR2, and MDR1 in resistant isolates. Our results implicated CDR1, CDR2, and ALS3 in itraconazole resistance, suggesting their potential as therapeutic targets for future investigation. These findings emphasize the primary role of efflux pumps and biofilm-associated genes in the resistance of clinical C. albicans onychomycosis isolates, although specific mutations (e.g., ERG11, TAC1) were not examined.