Antifungal Activity of <i>Thymus vulgaris</i> L. Essential Oil Against <i>Candida</i> Species Causing Otomycosis: Anti‐Virulence Activity, Synergistic Potential, and Cell Wall Effect

Otomycosis is a fungal infection of the external auditory canal, predominantly caused by Candida spp. The increasing resistance of these pathogens to conventional antifungal agents requires the exploration of alternative therapeutic approaches. This study was designed to assess the antifungal, anti-virulence, synergistic potential, and mechanism of action of Thymus vulgaris essential oil (EO) against Candida spp., causative agents of otomycosis. Gas chromatography-mass spectrometry (GC-MS) and GC-flame ionization detection (GC-FID) analyses identified p-cymene (38.7%), thymol (30.3%), γ-terpinene (2.6%), carvacrol (3.8%), and linalool (5.2%) as major components. The antifungal activity was assessed using the microdilution method, revealing minimal inhibitory concentrations (MICs) ranging from 0.156 to 2.5 mg/mL. Although none of the strains exhibited phospholipase activity, the production of all formed biofilms was significantly inhibited by T. vulgaris EO (21.4%-92.8%). The checkerboard assay revealed additive or indifferent interactions with nystatin in most tested strains, suggesting that combined use may offer limited therapeutic benefit. Cholesterol and sorbitol assays showed the affinity of the EO for binding to cholesterol rather than targeting the fungal cell wall. These findings highlight the potential of thyme EO as an alternative antifungal agent for otomycosis treatment, though further research is needed to optimize its therapeutic application.