Antifungal susceptibility testing protocol

Antifungal susceptibility was determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol, as described [44] (link). Minimum inhibitor concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of caspofungin (CF, generously provided by Rochelle Bagatell). Echinocandin gradients were from 16 µg/ml down to 0 with the following concentration steps in µg/ml: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Geldanamycin (GdA, A.G. Scientific, Inc.) and radicicol (RAD, A.G. Scientific, Inc.) were used to inhibit Hsp90 at the indicated concentrations, and cyclosporin A (CsA, CalBiochem) and FK506 (A.G. Scientific, Inc.) were used to inhibit calcineurin at the indicated concentrations. Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the vehicle for GdA, RAD, CsA, and FK506. Sterile water was the vehicle for CF. Plates were incubated in the dark at 30°C for the time period indicated, at which point plates were sealed and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and was corrected for background from the corresponding medium. Each strain was tested in duplicate on at least two occasions. MIC data was quantitatively displayed with colour using the program Java TreeView 1.1.3 (http://jtreeview.sourceforge.net).
Clinical antifungal MICs were determined using broth microdilution with RPMI 1640 broth for amphotericin, fluconazole, ketoconazole, itraconazole, voriconazole, and caspofungin following Clinical and Laboratory Standards Institute document M27-A3 [56] . Visual MIC endpoints were read after 24 hours of incubation at 35°C for caspofungin and after 48 hours of incubation for all other drugs. Complete inhibition was used to determine amphotericin endpoints; 50% inhibition (compared to growth control) was used for caspofungin and 80% inhibition was used for the other drugs.