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Antimicrobial Agents and Chemotherapy, July 2007, p. 2454-2463, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01237-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role for Cell Density in Antifungal Drug Resistance in Candida albicans Biofilms

Palani Perumal, Satish Mekala, and W. LaJean Chaffin^*

Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Received 3 October 2006/ Returned for modification 30 November 2006/ Accepted 25 April 2007

Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 x 10³ cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 x 10⁸ cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 x 10³ cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.

Published ahead of print on 14 May 2007.

Present address: Center for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, India 600025.

Present address: 1209 Fox Run Dr., Plainsboro, NJ 08536.

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