Fluconazole

C. albicans, C. glabrata and E. coli obtained from Mycology Reference Center and Bacteriology Collection, faculty of Veterinary Medicine, University of Tehran. The Candida yeast was cultured on Sabouraud dextrose agar (SDA; Merck, Germany) and the E. coli bacteria were cultured in Mueller–Hinton broth medium (MHB; pH: 7.2–7.4, Merck, Germany) at 37 °C for 16 to 18 h.
Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used to control the validation of fluconazole susceptibility test method, according to CLSI M60 standard.
The oleuropein (purity 98%) was acquired from the Department of Comparative Biosciences, Faculty of Veterinary Medicine at the University of Tehran. The fluconazole was acquired from Sigma-Aldrich (UK; 98%; CAS number 86386–73-4).

We tested 12 strains of C. gattii (from the culture collection of the Laboratório de Micologia/Universidade Federal de Minas Gerais, Brazil). Initially, the MIC for fluconazole (Sigma-Aldrich, St. Louis, Missouri, USA) was determined in drug-supplemented solid culture medium in Sabouraud’s dextrose agar (SDA). The MICs for fluconazole and amphotericin B (Sigma-Aldrich) were also determined by the microdilution method [8] (link), [11] –[13] (link). Drug susceptibility testing was performed in three independent experiments in duplicate.

The broth micro-dilution method (according to CLSI M38-A2 guidelines [33 ]) was used for testing the sensitivity of the dermatophyte isolates to the most commonly used antifungal drugs. Fluconazole was obtained from Pfizer International (New York, NY, USA), itraconazole, and miconazole were obtained from the Janssen Research Foundation (Beerse, Belgium), griseofulvin was purchased from Sigma Chemical Company (St. Louis, MO, USA), and terbinafine was purchased from Novartis (Basel, Switzerland). All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich), with the exception of Fluconazole, which was dissolved in RPMI1640 medium (Sigma Co. St. Louis, USA), buffered at pH 7.0 with 165 mM of 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma), and serially diluted two-fold to final concentrations of 0.125–64 μg/mL for Fluconazole and 0.03–16 μg/mL for the other antifungal agents. MIC values, MIC₅₀, and MIC₉₀ were determined.

Human bronchial and tracheal epithelial cells were obtained from residual tissue from lungs destined for transplantation in collaboration with the University of Wisconsin - Health Lung Transplant Program. The protocol was reviewed by the University of Wisconsin Institutional Review Board and was deemed “not human subjects research.” Cryopreserved aliquots of cells were thawed and expanded as monolayers in PneumaCult-Ex Plus Medium (StemCell Technologies) supplemented with 0.1% [vol/vol] gentamicin (Sigma) and 0.1% [vol/vol] fluconazole (Novaplus). Once the cells reached 80% confluence, they were transferred to 12-well plates with Transwell semi-permeable inserts (Corning 3460) and were allowed to differentiate in PneumaCult-ALI medium (StemCell Technologies) supplemented with 0.1% [vol/vol] gentamicin (Sigma) and 0.1% [vol/vol] fluconazole (Novaplus) at the air-liquid interface for at least 21 days when ciliary motion was observed. The culture medium was changed to an antibiotic-free medium containing 0.05% [vol/vol] hydrocortisone 48 hours prior to RV infection. All ALI cultures were used between 28 – 35 days of air lifting.

Standard powders of quetiapine (Sigma-Aldrich, South Africa), olanzapine (Sigma-Aldrich, South Africa), fluconazole (Sigma-Aldrich, South Africa) and amphotericin B (Sigma-Aldrich, South Africa) were used in this study. quetiapine and olanzapine were prepared in dimethyl sulfoxide (Merck, South Africa) to each yield a stock solution of 1,000 mg/ml. fluconazole was reconstituted in distilled water (final stock solution of 1,000 mg/ml) while amphotericin B was dissolved in dimethyl sulfoxide (DMSO) (Merck, South Africa) to yield a stock concentration of 1,000 mg/ml. The concentrations of drug diluents, in which the stock solutions were prepared, never exceeded 1%.

The spot test was performed to evaluate the ability of Pb14-3-3 to complement the functions of Bmh1p or Bmh2p. fluconazole was chosen after searching the Saccharomyces Genome Database, where a decrease in sensitivity to fluconazole was described for S. cerevisiae Δbmh1 and Δbmh2.
Each transformant was grown in SD-URA medium until a concentration of 1×10⁷ cells/mL (A_600nm = 0.6–0.9), and suspensions of 2.5 × 10⁸ cells/mL were prepared in glycerol 50 %. Then, 100 μL of each sample was transferred to a 96-well plate, six serial dilutions were prepared and 2.5 μL of each suspension was spotted in SD-URA supplemented with 2 % galactose and 35 μM fluconazole (Sigma-Aldrich, St. Louis, MO, USA); control plates without fluconazole were also made. The plates were incubated at 30 °C until growth in all dilutions was observed in control plates. The experiment was conducted in triplicate with three independent experiments for each transformant.