In vitro activities of antifungal combinations against biofilms and planktonic forms of clinical Trichosporon asahii isolates.

Trichosporon species can cause biofilm-associated infections related to indwelling medical devices, especially intravenous catheters, and unacceptable mortality rates have been reported despite the administration of antifungal treatments (1). Trichosporon asahii can form biofilms with structured microbial communities in vitro, embedded within an extracellular matrix, with significantly increased resistance to antifungal compounds (2, 3), which might ultimately lead to clinical treatment failure. Antifungal combination may be an alternative therapy strategy for biofilm-related fungal infections (4). The synergistic effects of antifungal combinations against other fungal biofilms have been detected in vitro, such as amphotericin B-posaconazole for Candida albicans (5) and amphotericin B-caspofungin or voriconazole-caspofungin for Aspergillus spp. (6). The synergistic effects of antifungal combinations of voriconazole, amphotericin B, and caspofungin against planktonic T. asahii have been found in vitro (7). We evaluated the in vitro activity of the combinations of voriconazole-amphotericin B, voriconazole-caspofungin, and amphotericin B-caspofungin against 16 clinical isolates of T. asahii in biofilm and planktonic forms by a broth microdilution checkerboard method (5). Trichosporon biofilms were prepared according to the 96-well plate-based method (8). The effect of antifungal agents was determined by the 2,3-bis(2-methoxy4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based colorimetric assay for both biofilms and planktonic cells (5, 8). The MIC and sessile MIC (SMIC) were determined as the lowest antifungal concentration (alone or in combination) that caused a 50% reduction in optical density for both biofilms and planktonic cells compared with the growth control (5, 6). The interaction was defined on the basis of the fractional inhibitory concentration indexes (FICIs) as follows: 0.5, synergy; 0.5 to 4, indifference; and 4.0, antagonism. Under planktonic conditions, the amphotericin B-caspofungin combination showed the highest percentage of synergistic effects (81.25%; FICI, 0.125 to 0.5) (Table 1), as indicated by a previous in vitro study (7). Under biofilm conditions, the voriconazole-amphotericin B combination showed the highest percentage of synergistic effects (87.5%; FICI, 0.078 to 0.313), and the SMIC90/SMIC ranges for these two drugs obviously decreased from 1,024/512 to 1,024 g/ml to 64/4 to 128 g/ml for voriconazole and from 1,024/32 to 1,024 g/ml to 32/4 to 128 g/ml for amphotericin B, respectively. The combinations of amphotericin B-caspofungin (93.75%) and voriconazole-caspofungin (81.25%) mainly yielded indifferent interactions, and no antagonistic interaction was observed in any of the combinations of either the biofilms or the planktonic forms of T. asahii isolates (Table 1). Trichosporon now ranks as the second most common pathogen causing fungemia in patients with hematological malignant disease, mainly catheter-related bloodstream infections (CR-BSIs) (1, 9). For biofilm-related infections, catheter removal is recommended as an adjunctive strategy for the management of Candida CR-BSIs (4), which is also suggested for Trichosporon CR-BSIs