In vitro interaction of fluconazole and berberine chloride was investigated against 40 fluconazole-resistant clinical isolates of Candida albicans. Synergism in fungistatic activity was found with the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed the synergistic interaction, but no antagonistic action was observed.Candida albicans is the most common candidal pathogen, causing mucosal and invasive infections (7,9,23,29). With the increasing clinical use of fluconazole, a choice for the treatment of C. albicans infections (3, 5, 12), fluconazole-resistant isolates are occurring more frequently (2,19,28). Attempts have been made to cope with treatment failures by using combination therapy (14). However, contradictory results of either synergic or antagonistic actions in various antifungal combinations have been reported (8,11,16,27). As for fluconazoleresistant C. albicans, few data are available about the synergism of fluconazole with other antifungal agents (1,4,25).Berberine, a bioactive herbal ingredient, was demonstrated to have weak activity against C. albicans and C. glabrata (17,21,26). Recently, berberine was combined with amphotericin B to treat disseminated candidiasis in mice (10). To seek a novel combination therapy, we investigated the in vitro interaction of fluconazole and berberine chloride (BBR) against fluconazoleresistant clinical isolates of C. albicans.Strains and agents. Forty clinical isolates of fluconazoleresistant C. albicans were used in this study, and C. albicans ATCC 90028 was used as a quality control. Drugs prepared in dimethyl sulfoxide (DMSO) included fluconazole (PfizerRoerig Pharmaceuticals, New York, NY) and BBR (SigmaAldrich, St. Louis, MO).Checkerboard microdilution assay. Assays were performed on all 40 isolates according to methods of the CLSI (formerly NCCLS) (M27-A) (6, 18). The initial concentration of fungal suspension in RPMI 1640 medium was 10 3 CFU/ml, and the final concentrations ranged from 0.125 to 64 g/ml for fluconazole and 1 to 32 g/ml for BBR. Plates were incubated at 35°C for 24 h. Optical density was measured at 630 nm, and background optical densities were subtracted from that of each well. Each isolate was tested in triplicate. MIC 80 and MIC 50 were determined as the lowest concentration of the drugs (alone or in combination) that inhibited growth by 80% or 50%, respectively, compared with that of drug-free wells. The fractional inhibitory concentration (FIC) index is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FIC indices of Յ0.5 and Ͼ4, respectively. An FIC index result of Ͼ0.5 but Յ4 was considered indifferent (20).Agar diffusion test. C. albicans 0304103 (one fluconazole-resistant isolate with a MIC of Ͼ32 g/ml for BBR) was tested by agar diffusion assay. A 100-l aliquot of 10 6 -CFU/ml suspension was spread uniformly onto the yeast extract-peptone-dextrose agar plate with or without 64 g/ml ...