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MATERIALS AND METHODS

Strains. Table 1 describes the C. albicans strains used in this study. Strains CAF2-1, DSY448, DSY465, DSY654, and DSY1050 were generous gifts from D. Sanglard (Lausanne, Switzerland), and strain GDH2346 was a gift from LJ Douglas (Glasgow, United Kingdom). The wild-type strain CAF2-1 was used in Northern blotting, Rh123 efffux, and sterol analyses. Strains were grown overnight at 37 C in yeast nitrogen base (YNB) medium with amino acids (Difco Laboratories, Detroit, Mich.; catalog no. 0392-15-9) supplemented with 50 mM glucose.

Biofilm formation and quantitation. Biofilms were formed on 1.5-cm2 denture acrylic (polymethylmethacrylate [PMA]) strips (Makki Dental Prosthetics, Inc., Middleburg Heights, Ohio) as described previously (9, 10). Brieffy, a standard inoculum of 107 cells/ml from an overnight culture of the fungal strains was applied to the surface of PMA strips placed in a 12-well tissue culture plate. The cells were allowed to adhere for 90 min at 37 C. Nonadherent cells were removed from the strips by gentle washing with 5 ml of phosphate-buffered saline (PBS). Strips were then submerged in 4 ml of YNB medium supplemented with 50 mM glucose and incubated for various durations at 37 C on a rocker. Strips with no Candida cells served as negative controls. Control and experimental strips were then incubated at 37 C for various time periods. Planktonic cultures were grown in the same way as biofilms, in 12-well tissue culture plates, except that denture acrylic strips were not added to the wells. Biofilm and planktonic cultures were grown for 6, 12, and 48 h, corresponding to early …