Chemical compounds and synergistic antifungal properties of Thymus kotschanus essential oil plus ketoconazole against Candida spp

Background: Owing to the increased resistance of microorganisms to chemical drugs, their side effects and high costs, the tendency to use of medicinal plants has been enhanced. Herein, the chemical composition and anti fungal effects of Thymus kotschanus (Th. kotschanus) essential oil (EO) was assessed against Candida albicans (C. albicans), C. kruzei and C. glabrata species. Methods: The Th. kotschanus EO chemical composition was determined using gas chromatography (GC) and mass spectrometry GC (MS/GC). The antifungal activity against Candida species was determined using agar diffusion and broth micro-dilution methods. The expression of biofilm-associated als gene in exposure to 1 mu g/mL of ketoconazole and 1-2 mu g/mL of Th. kotschanus EO was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) technique. Results: Forty-five compounds were identified, of which carvacrol (55.4%), thymol (11.5%) and gamma-terpinene (12.1%) were the major fractions. The EO exerted no cell cytotoxicity against normal culture cells at 10 mu g/mL and its 50% infectious dose was 20 mu g/mL. The minimum inhibitory concentrations (MICs) and the minimum fungicidal concentrations (MFCs) of EO against standard strains ranged 0.5-1 and 1-2 mu g/mL, and against clinical isolates included 4-16 mu g/mL and 16-64 mu g/mL, respectively. The highest activity was against C. kruzei. The combination of 0.5 mu g/mL of EO and 1 mu g/mL of ketoconazole decreased MIC90 (< 0.5 vs 0.5-1 mu g/mL, p < 0.001) and MBC90 (0.25 vs 1-2 mu g/mL, p < 0.001) of strains significantly. The effect of 1 mu g/mL of ketoconazole had no significant effect on the als gene expression, while 1 and 2 mu g/mL of EO decreased the expression of the als gene 3.2 (p < 0.001) and 7.4 (p < 0.0001) fold, respectively. Conclusion: The Th. kotschanus EO exerted substantial antifungal effects against Candida spp., particularly by synergy plus ketoconazole. The synergistic effect with ketoconazole is promising for the eradication of fungal infections. The Th. kotschanus EO exhibited anti-biofilm activity by significant decrease of als gene expression.