2.6. Minimum Inhibitory Concentration (MIC) of Antifungal Drugs Benomyl, Fluconazole, and Amphotericin B

Fungi known to be opportunistic in humans were subjected to minimum inhibitory concentration (MIC) determination for the antifungal drugs benomyl (Sigma, Steinheim, North Rhine-Westphalia, Germany) and fluconazole (Sigma, Saint Louis, MO, USA). Isolates with an MIC ≥ 64 µg/mL for fluconazole that also exhibited growth at 37 °C were subjected to MIC determination for the antifungal drug amphotericin B (Sigma, Saint Louis, MO, USA). The MIC protocol was performed using a modified version of the method used for yeast [42] and filamentous fungi [43] in a Roswell Park Memorial Institute (RPMI-1640) medium (INLAB, Diadema, SP, Brazil) using 96-well microtiter plates. The yeast cells and fungal spores were treated with benomyl concentrations ranging from 0.078125 to 40 µg mL⁻¹ [44], fluconazole from 0.062 to 64 µg mL⁻¹, and amphotericin B from 0.007 to 8 µg mL⁻¹. Microtiter plates containing spores and yeast cells were incubated at 25 °C for 48 h for benomyl and fluconazole and at 37 °C for 48 h for amphotericin B. All MIC assays were performed in duplicate.