4.5. Antifungal Susceptibility Testing

Different colonies of 15 Candida albicans isolates and a control strain from overnight culture on SDA at 37 °C were suspended in 6 mL distilled water and homogenized with a gyratory vortex to the final density of the 0.5 McFarland standard. Working suspension was made by further 1:10 dilution of the standardized suspension resulting in 1–5 × 10⁵ CFU/mL. Cell counts and viability of yeasts were confirmed in triplicates using a hemocytometer chamber by adding trypan blue.

LAEO stock solution was prepared in pure ethanol (1:2) and then diluted in RPMI 1640 broth with L-glutamine (Capricorn Scientific, Ebsdorfergrund, Germany), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, Taufkirchen, Germany), supplemented with 2% glucose to obtain its final concentration of 8% (vol./vol.). Tween 80 in final concentration of 0.5% was added to enhance the EO solubility. Initially, 200 µL of freshly prepared LAEO stock solution was poured in the first rows of flat-bottomed 96-well microtiter plates. One hundred µL of RPMI 1640 with MOPS and 2% glucose were added in the remained wells. Nine serial two-fold dilutions were performed by the withdrawal of a 100 µL aliquot from the concentrated well into the succeeding one. Preparations of fluconazole and caspofungin work solutions, together with the susceptibility testing, were performed according to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) document E.DEF 7.3.2. After adding of 100 µL appropriate working yeast suspension (1–5 × 10⁵ CFU/mL), the final concentration of LAEO ranging between 4–0.008%, 64–0.125 µg/mL for fluconazole and 4–0.008 µg/mL for caspofungin were obtained. EO-free and antifungals-free grow control and sterility control wells were also included. The final solvent concentrations did not affect yeast growth as their concentration did not exceed 2% for ethanol and 1% for DMSO. All microtiter plates were incubated at 37 °C for 24 h in aerobic atmosphere.

After incubation, the plates were estimated to fungal growth. Minimal inhibitory concentration (MIC) of LAEO was defined as the lowest concentration, showing complete inhibition of visible growth. For fluconazole and caspofungin, MIC determinated the lowest concentration reducing ≥ 50% of the culture growth in comparison with the drug-free growth control reading by the microplate reader on 450 nm (Tecan Sunrise, Mannedorf, Switzerland). EUCAST clinical breakpoints are used to categorize the fluconazole susceptibility. Due to the significant inter-laboratory variation in caspofungin MIC ranges, a recommended epidemiological cut-off (ECV) value of 0.25 µg/mL was used to categorize the isolates into wild-type (WT) and non-WT [41]. MIC₅₀ and MIC₉₀ were defined as the lowest concentrations of each substance capable of inhibiting 50% and 90% of the tested isolates, respectively.

The minimal fungicidal concentration (MFC) of EO and tested drugs were estimated after MIC determination as the lowest concentration that kills 99.9% yeast cells after subculturing 10 μL of broth taken from all the wells without turbidity on SDA. MFC₅₀ and MFC₉₀ were defined as the lowest concentrations that kill 50% and 90% of tested isolates, respectively. Each experiment was performed in duplicates on three different dates. The results were reported as modal values.