Effects of efflux pumps on antifungal activity of chitosan against Candida albicans

Background: Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan. Materials and Methods: The minimal fungicidal concentration (MFC) of oligomer (7-9 kD) and polymer (900-1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD triangle), and AD triangle overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility. Results: While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD triangle) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD triangle barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype. Conclusion: Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.