Consecutive Amphotericin B Treatments Fail to Eradicate Candida Biofilm Persisters

Abstract

Objective: Candida infection is a common and critical problem with high morbidity and mortality worldwide. This study attempted to identify the profile of Candida biofilm persisters and establish a reliable in vitro model for their molecular analysis. Method: A total of four well-defined strains from three representative Candida species were employed, including C. albicans SC5314, C. albicans BF-1, C. glabrata ATCC 90030 and C. tropicalis ATCC 13803. These Candida biofilms were established on 24-well polystyrene plates respectively. Their antifungal susceptibility and MICs were determined by XTT reduction assay after treatments with different concentrations of Amphotericin B (1-256 μg/ml) for 24 h. Following 24 h treatment with 256 μg/ml of Amphotericin B, the Candida biofilms were challenged consecutively for another 72 h. The level of persisters was quantified by spiral plating for CFU counting. The cellular viability of Candida biofilms was confirmed by Live/Dead staining and confocal laser scanning microscopy.

Result: Overall, Amphotericin B displayed potent antifungal effects on the four Candida biofilms tested (MICs: 1 to 32 μg/ml). The 24 h-treatment with Amphotericin B at 256 μg/ml effectively killed majority of these Candida biofilms, and a small fraction of persister cells (0.01% to 0.62%) survived. Interestingly, consecutive treatments further with 256 μg/ml of Amphotericin B at 48 h, 72 h and 96 h failed to control these biofilms. The viable persister cells were confirmed by confocal imaging.

Conclusion: The pioneering study shows that consecutive Amphotericin B treatments were unable to eradicate the biofilm persisters of C. albicans SC5314, C. albicans BF-1, C. glabrata ATCC 90030 and C. tropicalis ATCC 13803. These in vitro models of persisters may facilitate further investigation on the pathogenesis of Candida infection and antifungal strategies.

This abstract is based on research that was funded entirely or partially by an outside source: Supported by Hong Kong HMRF Grant, No.12110752, and the Modern Dental Laboratory/HKU Endowment Fund to LJJ