Antifungal tolerance and resistance were determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol as described [25] (link), [27] (link). Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the solvent for fenpropimorph (FN, Sigma Aldrich Co) and terbinafine (TB, Sigma Aldrich Co.); fluconazole (FL, Sequoia Research Products) and micafungin (MF, generously provided by Julia R. Köhler) were dissolved in sterile ddH2O. Geldanamycin (GdA, A.G. Scientific, Inc.) was used to inhibit Hsp90 at the indicated concentrations. Cyclosporin A (CsA, Calbiochem) was used to inhibit calcineurin at the indicated concentrations. Cercosporamide and staurosporine (STS, A.G. Scientific, Inc.) were used to inhibit protein kinase C at the indicated concentrations. DMSO was the solvent for GdA, CsA, STS, and cercosporamide.
Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL, FN, TB and cercosporamide. FL gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.04882813, 0.02441406. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.24414063. Cercosporamide gradients were from 100 µg/ml with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼103 cells were inoculated into each well. Plates were incubated in the dark at 30°C or 35°C for the period of time indicated in the figure legend, at which point plates were sealed with tape and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate on at least 3 occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.1 (http://jtreeview.sourceforge.net ).
Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of cyclosporin A across the x-axis of the plate and 2-fold dilutions of STS across the y-axis of the plate. STS gradients were from 0.5 µg/ml to 0 in the following concentrations steps in µg/ml: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125. CsA gradients were from 48 µg/ml down to 0 in the following concentration steps in µM: 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875. Plates were inoculated and growth was measured as with MIC tests. To test for synergy, the fractional inhibitory concentration (FIC) was calculated as follows: [(MIC80 of drug A in combination)/(MIC80 of drug A alone)] + [(MIC80 of drug B in combination)/(MIC80 of drug B alone)]. Values of ≤0.5 indicate synergy, those of >0.5 but <2 indicate no interaction and those ≥2 indicate antagonism.
Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL, FN, TB and cercosporamide. FL gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.04882813, 0.02441406. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.24414063. Cercosporamide gradients were from 100 µg/ml with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼103 cells were inoculated into each well. Plates were incubated in the dark at 30°C or 35°C for the period of time indicated in the figure legend, at which point plates were sealed with tape and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate on at least 3 occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.1 (
Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of cyclosporin A across the x-axis of the plate and 2-fold dilutions of STS across the y-axis of the plate. STS gradients were from 0.5 µg/ml to 0 in the following concentrations steps in µg/ml: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125. CsA gradients were from 48 µg/ml down to 0 in the following concentration steps in µM: 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875. Plates were inoculated and growth was measured as with MIC tests. To test for synergy, the fractional inhibitory concentration (FIC) was calculated as follows: [(MIC80 of drug A in combination)/(MIC80 of drug A alone)] + [(MIC80 of drug B in combination)/(MIC80 of drug B alone)]. Values of ≤0.5 indicate synergy, those of >0.5 but <2 indicate no interaction and those ≥2 indicate antagonism.
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