Characterization and eradication of persisters in Candida albicans biofilms

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. We report that biofilms formed by the major human pathogen Candida albicans exhibit biphasic killing in response to amphotericin B and chlorhexidine. Biphasic killing indicates the existence of a subpopulation of highly tolerant cells, termed persisters. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed when the same antimicrobials were added separately. Thus, surviving persisters form a multidrug tolerant subpopulation. Reinoculation of cells surviving killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests C. albicans persisters are not mutants, but phenotypic variants of the wild type. Persisters were detected and isolated from strains defective in late stages of biofilm formation, indicating that attachment initiates persister formation. Persisters may be largely responsible for the multidrug tolerance of fungal biofilms. Inhibition of multidrug resistance (MDR) efflux transporters and subsequent treatment with imidazole antifungals was found to kill C. albicans biofilms. When a panel of imidazole drugs was tested against wild type Candida biofilms, no killing was observed at any concentration. However, biofilms of a strain deleted in known MDR transporters were killed with concentrations as low as 50 μM clotrimazole and 200 μM miconazole. Fluconazole did not exhibit lethality, which was found to be dependent on the deletion of Cdr1p and Cdr2p transporters. The combination of disulfiram, a known Cdr1p inhibitor, and miconazole also produced potent killing of biofilms. The combination of imidazole antifungals and multidrug efflux inhibitors may be exploited to treat biofilm infection. We have also identified small molecules that kill C. albicans biofilms and persisters in the presence of miconazole. A screen of 68,950 compounds revealed 323 molecules that reduced metabolic activity of biofilms in the presence of miconazole. Two compounds had no direct activity, but produced over 1000-fold reduction of biofilm colony counts in combination with miconazole. Importantly, one of these compounds, a hydrazone, completely eradicated persisters. These small molecules could lead to the development of therapeutics that synergize with currently available antifungals and sterilize infection.