Antifungal Susceptibility Tests

Susceptibility tests were made according to the CLSI – Clinical and Laboratory Standards Institute (former NCCLS – National Committee for Clinical Laboratory Standards) guideline M27-A2 (NCCLS, 2002) with some adjustments, using the broth microdilution method. Yeast cells were grown on GYP medium and the inoculum suspension was prepared by picking fresh colonies and resuspending them in 5 mL of sterile 0.9% (w/v) saline (NaCl). The resulting suspension was vortexed for 15 s and the cell density was adjusted with a spectrophotometer to give an inoculum concentration of 10⁶ cells per mL. The final inoculum suspension was prepared by a 1:50 dilution followed by a 1:20 dilution with double-strength broth medium, which resulted in a final concentration of 10³ cells per mL. One other final inoculum concentration was tested in C. albicans, 10⁵ cells/mL, achieved by a 1:10 dilution with double-strength broth medium, for allowing a sufficient number of cells to be visualized under the microscope and to enable the determination of a MFC based on a 99.9% killing (see Minimum Fungicidal Concentration section below). The inoculum size was verified by enumeration of CFUs obtained by subculturing on GYP plates. The solution of the BCO was prepared in ultrapure sterile water and 200 μL were added to the first line of the microplate. A serial two-fold dilution was made, twelve times, using ultrapure sterile water, in the 96-well microplates. The final concentration of the BCO, after addition of the inocula, ranged from 0.002 to 4.762 μM when using PDB medium and from 0.012 to 23.81 μM with RPMI medium. The serial twofold dilutions of AMB ranged from 0.03 to 17.31 μM. The yeast inoculum (100 μL) was added to each well of the microplate, containing 100 μL of the drug solution (twofold concentrated). The final volume in each well was 200 μL. The microplate was incubated at 35°C and examined after 72 h. Minimum inhibitory concentrations (MICs) are the lowest drug concentration showing absence of growth, as recorded visually. To evaluate the effect of sorbitol on the fungal susceptibility to the BCO, the growth medium (twofold concentrated) was supplemented with 2.4 M sorbitol (final concentration 1.2 M). All these tests were performed with three different batches of the BCO (triplicates).