EUCAST broth microdilution method.

All cultures and preparation for MIC and checkerboard assays adhered to EUCAST guidelines, except that 100% FBS (Gibco) was necessary to assess the growth inhibition of C. albicans (see “XTT reduction assay” below). A single colony was grown in YPD medium at 30°C for ∼20 h and then suspended in liquid RPMI medium (pH 7), which contained 8.4 g RPMI 1640, 34.5 g MOPS (morpholinepropanesulfonic acid), and 20 g dextrose in 1 liter of distilled water. Cells were then added to solutions of FK506 or its analogs as specified above in 96-well microdilution plates to give final suspensions of 100 μl per well at cell densities of 0.01 optical density (OD) unit/ml (63). The inoculated plates were incubated at 35 and 37°C (for A. fumigatus, 35°C; for C. neoformans, 37°C) for 48 h. The optical density at 600 nm (OD₆₀₀) was determined with an iMark microplate absorbance reader and was used to determine cell growth. After subtracting the background values for the remaining wells, percent growth relative to the amount of control growth in the absence of inhibitors was calculated under each condition. MICs and fractional inhibitory concentrations (FIC) were calculated as described previously (63, 64).