Antifungal Susceptibility Testing

According to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 method (Reyes and Ghannoum, 2000), a broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of samples used alone and in combination with FLC against C. albicans strains. Here MIC is defined as the drug concentration that reduces growth by 80% compared to the control group. Serial five-fold dilutions of the samples were prepared using Sabouraud-dextrose-broth (SDB) medium (Weerasekera et al., 2016), with a final concentration of 0.32–1000 μg/mL. These were used in a preliminary study on the antifungal activity of TA and SL. Serial two-fold dilutions of the samples were then prepared using SDB to further analyze the antifungal activity of TA combined with FLC. The final concentration of the samples was 32–1000 μg/mL when used alone and 0.064–200 μg/mL and 0.0128–40 μg/mL when used in combination (TA and FLC, respectively). Each well contained 100 μL of 1 × 10⁵ colony-forming units (CFU)/mL of yeast cell suspension. Then, we added 50 μL of TA and FLC to each well (200 μL). Subsequently, we incubated the flat bottom 96-well plate (701001, NEST, China) at 37°C for 24 h. The wells with only fungi (no drugs) were defined as the control and wells with only SDB as the blank. Further, we determined the growth rate in each well via visual observation and measuring the optical density at a wavelength of 625 nm using a multifunctional microplate reader (SpectraMax 340PC384, Molecular Devices Corporation, United States). We analyzed the antifungal effects using fractional inhibitory concentration index (FICI) to assess the in vitro interactions between TA and FLC. The FICI was calculated for each combination using the following formula: FICI = FICI TA + FICI FLC = (MIC of TA in combination/MIC of TA alone) + (MIC of FLC in combination/MIC of FLC alone). Here, synergy was defined as FICI ≤ 0.5. 0.5 < FICI < 1 indicated partial synergy, FICI = 1 indicated additive effect, 1 < FICI ≤ 4 indicated indifference, and antagonism was defined as FICI > 4 (Khodavandi et al., 2010).