Fig 2.

Assessment of the drug interactions for the combinations of caspofungin and FK506, caspofungin and geldanamycin, and FK506 and geldanamycin against the AF293 (wild-type) strain and the resistant strains. The antifungal activity of both drugs was assessed visually according to the following criteria: no effect, morphological abnormalities (hyphal blunting and impaired branching) with <25% growth reduction, 25 to 50% growth reduction, 50 to 75% growth reduction, 75 to 90% growth reduction, and >90% growth reduction, as illustrated by the gradient of green colors from white (no activity) to dark green (>90% growth reduction). CSP, caspofungin; GDA, geldanamycin; FICI, fractional inhibitory concentration index; MEC, minimal effective concentration. (A) The greatest antifungal activity achieved with one single drug (FK506, caspofungin, or geldanamycin, as indicated) is shown for each strain and does not exceed 50 to 75% growth reduction (upper panel). When these drugs are combined at the concentrations associated with their maximal effect (caspofungin, 1 μg/ml; FK506, 0.1 μg/ml; geldanamycin, 4 μg/ml), only geldanamycin in combination with caspofungin or FK506 substantially increases activity, with near-complete growth inhibition (>90%) against the majority of the azole-resistant strains. The combination of caspofungin and FK506 is increasingly active against the wild-type strain AF293 but does not provide a substantial benefit against most of the resistant strains. (B) Results of the checkerboard dilution testing of all three drug combinations are shown for the representative multi-azole-resistant strain F16134. The same MEC cutoff is observed for each drug when used alone or in combination (0.5 μg/ml for caspofungin, 0.016 μg/ml for FK506, and 4 μg/ml for geldanamycin). On the basis of this cutoff (MEC), the interaction is described as indifferent for all three combinations (FICI [MEC] = 2). When a higher cutoff is considered (MIC), this level of activity is achieved only with the combination of geldanamycin with either caspofungin or FK506, defining these interactions as synergistic (in this case, the FICI is reported as <0.5, as it cannot be assessed mathematically in the absence of a defined MIC value for the single drugs).