Short-term antifungal treatments of caprylic acid with carvacrol or thymol induce synergistic 6-log reduction of pathogenic candida albicans by cell membrane disruption and efflux pump inhibition

Abstract

Background/Aims: Although naturally-derived antifungals have been investigated for their ability to inactivate Candida albicans, which is a major cause of candidiasis, they have shown a less than 3 log reduction in C. albicans or required treatment times of longer than 3 h. Thus, the naturally-derived antifungals used in previous studies could not substantially eradicate C. albicans within a short period of time. Methods: To improve the fungicidal effects of naturally-derived antifungals against C. albicans within short time periods, we developed composites showing antifungal synergism using caprylic acid (CA), carvacrol (CAR) and thymol (THM) for 1-10 min at 22/37°C. Using flow cytometry, we examined the mode of action for the synergism of these compounds on membrane integrity and efflux pump activity. Results: Whereas the maximum reduction by individual treatments was 0.6 log CFU/ml, CA + CAR/THM (all 1.5 mM) eliminated all pathogens (> 6.8 log reduction) after 1 min at 37°C and after 10 min at 22°C. The flow cytometry results showed that exposure to CA damaged the membranes in 15.7-36.5% of cells and inhibited efflux pumps in 15.4-31.3% of cells. Treatments with CAR/THM slightly affected cell membranes (in 1.8-6.9% of cells) but damaged efflux pumps in 14.4-29.6% of cells. However, the combined treatments clearly disrupted membranes (> 83.1% of cells) and pumps (> 95.0% of cells). The mechanism of this synergism may involve membrane damage by CA, which facilitates the entry of antifungals into the cytoplasm, and the inhibition of efflux pumps by CA, CAR or THM, causing their accumulation within cells and, leading to cell death. Conclusion: Antifungal composites (CA + CAR/THM) showing synergism (i.e., an additional 6 log reduction) within minutes at room/body temperature can be used to treat candidiasis and improve the microbiological safety of facilities contaminated with fungi as a novel alternative to synthetic antifungals. © 2019 The Author(s).