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Antimicrob. Agents Chemother. doi:10.1128/AAC.00684-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells

Department of Biology, Northeastern University, 360 Huntington Ave, Boston, MA, 02115; Department of Molecular Biology and Microbiology, Tufts University, 136 Harrison Ave, Boston, MA, 02111

^* To whom correspondence should be addressed. Email: k.lewis{at}neu.edu.

	Abstract

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. Expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that are a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibit a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug tolerant subpopulation. Interestingly, surviving C. albicans persisters were only detected in biofilms, and not in planktonic exponentially growing or stationary populations. Reinoculation of cells surviving killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants, but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters only formed in biofilms, mutants defective in biofilm formation were examined for tolerance to amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug tolerant persister cells in both planktonic and biofilm populations and it appears that yeasts and bacteria have evolved an analogous strategy that assigns the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy which leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.

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