2.4. Antifungal Susceptibility Testing

Susceptibility testing followed the recommendations proposed by the Clinical and Laboratory Standards Institute (CLSI-M27-A3) for yeasts with results interpreted according to CLSI-M27-S4 guidelines. Powders of amphotericin B (AMB, Sigma-Aldrich USA), voriconazole (VOR, Sigma-Aldrich USA), itraconazole (ITR, Sigma-Aldrich USA), caspofungin (CAS, Sigma-Aldrich USA), and fluconazole (FLU, Sigma-Aldrich USA) were obtained from the respective manufacturers. Roswell Park Memorial Institute1640 (RPMI-1640) (Sigma, St.Louis, Missouri) was made according to the manufacturer's protocol and buffered to pH 7.0 with 0.165 N-morpholino propanesulfonic acid (MOPS) buffer (Sigma, USA). Stock solutions with a 10-fold concentration for each antifungal were prepared in dimethyl sulfoxide (DMSO) except FLU which is soluble in water. The final concentrations of the working solutions were obtained by using RPMI medium. The final concentrations of the antifungal agents were 0.032 to 16 μg/mL for AMB, VOR, ITR, and CAS; and 0.12–64 μg/mL for FLU [17]. Briefly, testing was performed in flat-bottom microdilution plates with RPMI-1640 medium supplemented with 2% glucose. The inoculum suspensions (0.5 McFarland) were prepared by the spectrophotometric method (at 530 nm) (Pharmacia Biotech Cambridge, England, Ultrospec 3000 UV/visible spectrophotometer), and diluted to 0.5 × 10³ to 2.5 × 10³ cells/ml using RPMI-1640 medium. A 100 μl volume of C. kefyr inoculum and an equal volume of antifungal agents were added to each well. Antifungal drug-free and yeast-free wells were included as positive and negative controls [18]. The MIC values of azoles and echinocandins were determined following the CLSI guidelines using the method of prominent and complete inhibition (80% inhibition) of growth compared to that of the drug-free growth control well. The MIC of AMB was determined using the method of no visible growth compared to that of the drug-free growth control well. For the antifungal tested, the ranges of MIC, MIC50, MIC90, and geometric means (GM), were read and calculated for five antifungal drugs against C. Kefyr isolates. This study employed the CLSI broth microdilution method to investigate the antifungal susceptibility profiles of C. kefyr isolates. It is important to acknowledge that established clinical breakpoints are not available for C. kefyr and antifungal drugs. Therefore, categorizing the isolates as susceptible or resistant is not possible. However, the MIC data were interpreted using the reported epidemiological cutoff value (ECV) classified the isolate as Non–wild type (Non-WT) and Wild type (WT) [19, 20]. A Wild type organism is defined as a strain that does not harbor any acquired resistance to the particular antimicrobial agent being examined for which the MIC results are lower than the ECV. Organisms with acquired or mutational resistance mechanisms may be included among those for which the MIC results are higher than the ECV as a Non-WT (1, 7, 18).