Determination of Minimal Inhibitory Concentration (MIC)

The antifungal susceptibility test was performed using the broth microdilution assay based on protocol M27-A3 of the Clinical and Laboratory Standards Institute (CLSI, 2008), for defining the minimal inhibitory concentrations (MICs) of fluconazole, HECL, BF, and BDP. Fluconazole solution was prepared in line with M27-A3 guidelines; the solution was diluted in RPMI 1640 buffered with 3-(N-morpholino) propanesulfonic acid (MOPS) at pH 7.0. All the natural products tested were re-suspended in the same culture medium recommended by the CLSI, after solubilization in 0.5% dimethyl sufoxide (DMSO). To standardize the inoculum, Candida spp. cells were initially cultivated for 24 h using Sabouraud Dextrose Agar (SDA) at 35°C, and an initial suspension was adjusted spectrophotometrically to match the 0.5 McFarland scale standard (1–5 × 10⁶ cells) at 530 nm. Subsequently, two serial dilutions were made, the first in saline solution (1:100), and the second in RPMI 1640 (1:20), in order to obtain the final concentration of 10³ cells/mL. Afterward, aliquots of 100 μL of the final inoculum solution were added to each well with 100 μL of different concentrations (2,500–0.001 μg/mL) of HECL, BF, or BDP and (64–0.03 μg/mL) fluconazole. Finally, the microtiter plates were incubated at 37°C and the reading was done after 24 h of incubation for fluconazole, and between 24 and 48 h for the natural products. In addition, the reference strains C. parapsilosis ATCC22019 (susceptible) and C. krusei ATCC6258 (resistant) were included as control microorganisms for the testing of fluconazole. The MIC was defined as the lowest concentration which showed about 50% reduction in visual growth as compared to the positive control for fluconazole, HECL, BF, or BDP. The isolates were classified as resistant at the following cutoff point: MIC ≥ 8 μg/mL to fluconazole for all the Candida spp. tested, except for C. glabrata, where strains with MIC ≥ 64 μg/mL were considered resistant. Candida krusei is intrinsically resistant to fluconazole (CLSI, 2017).