Antifungal susceptibility testing

The antifungal susceptibility testing of AmB alone and with ascorbic acid and α-tocopherol was performed according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [ 14 ]. The minimum inhibitory concentration (MIC) values were determined in Roswell Park Memorial Institute 1640 medium (RPMI 1640) (Sigma-Aldrich, Germany) buffered with 3-(N-morpholino)-propane-sulfonic acid (pH 7.0 with 0.165 M) obtained from Sigma-Aldrich Chemie GmbH, Germany.

Concentration of the C. albicans 10231 in the study was adjusted by measuring the absorbance in a spectrophotometer at a wavelength of 530 nm and adding sterile distilled water as required. A working suspension was prepared from a 1 in 10 dilution of the standardized suspension in sterile distilled water to yield 1-5×10⁵ colony-forming unit (CFU)/mL.The antifungal stock solution was two-fold diluted with RPMI of 16-0.03 µg/mL for AmB. A volume of 100 µL of inoculum suspension was added to each well except the sterility control. It must be mentioned that sterile water was added to the well instead. The microtitre plates were incubated for 24 h at 35 °C and the MIC was determined visually.

The MIC was defined as the lowest concentration of the antifungal agent that produced no visible fungal growth, compared to the medication-free control well. The control wells included the well without the antifungal (i.e. growth control), without microorganisms (i.e. sterility control), and with the solvent.

In order to determine the MIC of AmB and the concentrations of the combined vitamines, they were manipulated in several ways according to the EUCAST protocol with some modificationsto determine the effective concentrations against C. albicans strains. For this purpose, the range of concentrations of ascorbic acid (48×10² to 9.37 μg/mL) and α-tocopherol (1.28×10² to 0.25 µg/mL) was reduced while the concentrations of the antifungals were kept fixed [ 15 , 16 ]. At each manipulation, the concentration of AmB was reduced by a ratio of 1:2 until the achievement of the effective MIC of the antifungal which was compatible with one of the concentrations of the tested vitamins.