The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence

Candida species cultivation.Eleven resistant isolates of Candida albicans (n = 2), Candida glabrata (n = 3), Candida krusei (n = 3), and Candida tropicalis (n = 3) were used in this study. Their susceptibilities were determined according to Clinical and Laboratory Standards Institute document M27-A3 (15, 16) with Candida parapsilosis ATCC 22019 as the reference strain. Frozen stocks of isolates were maintained at −80°C until testing. After thawing, the yeast cells were subcultured on yeast-peptone-dextrose (YPD) solid medium (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) at least twice at 35°C before each experiment to ensure viability and purity. RPMI 1640 was used as the liquid medium for diluting drugs and strains.

Drugs.All 4 drugs (fluoxetine, fluconazole, itraconazole, and voriconazole) were purchased from Dalian Meilun Biotech Co. Ltd., China. The stock solution was prepared following the manufacturer's instructions. Fluoxetine and fluconazole were dissolved in sterile demineralized water at room temperature to achieve stock solutions of 2,560 μg/ml. Itraconazole and voriconazole were dissolved in dimethyl sulfoxide (DMSO) to form stock solutions of 256 μg/ml. All stock solutions were stored at −20°C. From the stock solutions, 2-fold serial dilutions were prepared.

Determination of MICs of planktonic cells.MICs for the azoles were determined according to the approved CLSI standard reference method (document M27-3) for antifungal susceptibility testing of yeasts by broth diffusion. For the checkerboard assays (17–19), 50 μl of RPMI 1640 medium containing azoles was added to the wells in the 2nd to 11th columns of the microtiter plate, and 50-μl aliquots of RPMI 1640 medium containing fluoxetine with concentrations ranging from 128 to 2 μg/ml were added to the wells in the A to G lines of the microtiter plate. The final concentration of fluconazole ranged from 1 to 512 μg/ml for C. albicans with an MIC of 512 μg/ml and from 0.5 to 256 μg/ml for the remaining isolates. The concentrations of itraconazole and voriconazole ranged from 0.08 to 4 μg/ml for all isolates. Next, 100-μl aliquots of Candida cell suspensions (1.0 × 10³ cells/ml) were added to each well mentioned above. All of the wells on the plate were filled with RPMI 1640 to a final volume of 200 μl. The plate was covered with its lid, sealed with Parafilm and incubated at 35°C for 24 h. Readings were performed with both visual examination and optical density (OD) by determining the absorbance at 492 nm on a microplate reader. MIC endpoints were defined as the MIC₅₀ values. All experiments were performed in triplicate. Drug interactions were interpreted by the fractional inhibitory concentration index (FICI) model and the percentage of growth difference (ΔE) model (20–22), respectively. An FICI of ≤ 0.5 represents synergy, an FICI of >4.0 represents antagonism, and a 0.5 < FICI ≤ 4.0 represents no interaction (23). In the ΔE model, the ΔE value was calculated by the data obtained directly from experiments. When the average value of ΔE was positive and the 95% confidence interval (CI) among the three replicates did not include 0, statistically significant (SS) synergy was claimed; when the average value of ΔE was negative and the 95% CI did not include 0, SS antagonism was claimed.

Determination of sessile MICs of biofilms.Biofilms were formed as described by Ramage et al. (24) on 96-well plates in modified cell suspensions of 1 × 10³ cells/ml. Results demonstrated that biofilms formed with this cellular density over at least 4 h of cultivation. The biofilms were formed over five time intervals (4, 8, 12, 24, and 48 h) at 37°C by pipetting 100 ml of the standardized cell suspension into selected wells of a 96-well plate. At each time point, the biofilms were washed three times gently with sterile phosphate-buffered saline (PBS) to remove the planktonic yeast. Fluconazole and fluoxetine were then added to the biofilms in serially double-diluted concentrations. The final concentration of fluconazole in wells ranged from 1 to 512 μg/ml for resistant isolate CA10. The final concentration of fluoxetine in wells ranged from 2 to 128 μg/ml. The control wells were filled with RPMI 1640 without antifungal agents. Then the whole system was incubated for a further 48 h at 35°C. A colorimetric reduction assay was carried out with XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] according to the protocol of Melo et al. (25). The absorbance was measured with a microtiter plate reader (Thermo Labsystems Multiskan MK3) at 492 nm. The drug concentration that brought about a reduction in absorption by 50% contrasted to that in the control well was reported as the sessile MIC (SMIC) endpoint. Each test was carried out in duplicate and repeated three times on different days.

Time-kill curve study.Inocula of 5.0 × 10³ cells/ml of C. albicans (CA10) were used in this experiment. The final concentration was 16 μg/ml for fluconazole, 0.25 μg/ml for itraconazole, and 0.125 μg/ml for voriconazole when combined with fluoxetine (16 μg/ml). A drug-free sample served as a growth control. The XTT test was performed to detect the cell viability after different treatments according to the method described previously (26). Briefly, samples were incubated at 35°C on an orbital shaker and vortexed prior to removal of a sample for the determination of colony counts. At predetermined time points (0, 12, 24, 36, and 48 h after incubation), a 100-μl aliquot from each treatment mixture was transferred to a well of a new 96-well microtiter plate, and then a 100-μl aliquot of XTT-menadione solution was added (XTT was purchased from Sigma). Prior to each assay, XTT was dissolved in a saturated solution at 500 μg/ml in Ringer's lactate. The solution was filter sterilized through a 0.22-μm filter, and 100 mM menadione in acetone was added to a final concentration of 10 μM. The plate was then incubated in the dark for up to 2 h at 35°C. After that, the XTT reduction was assessed by determining the absorbance at 492 nm on a microplate reader (SpectraMax 190; Thermo Lab Systems, USA). All experiments were conducted in triplicate, and the results are reported as mean values (25). Thus, the growth- and metabolism-inhibitory effects of the drugs alone and in combination were observed based on the results of the spectrophotometric analyses.

Survival assay.Galleria mellonella survival assays were carried out according to a previously described methodology (27). Galleria mellonella larvae (0.25 ± 0.03 g) were placed in petri dishes and incubated at 37°C in the dark the night before the experiments. Larvae with dark spots or apparent melanization were excluded. Yeasts were grown overnight in liquid Sabouraud medium, washed with PBS, and suspended in the same buffer. For survival assays, larvae were inoculated with 1 × 10⁷, 5 × 10⁶, 1 × 10⁶, and 5 × 10⁵cells/larva of C. albicans. The inocula were prepared in PBS plus 20 μg/ml of ampicillin to prevent bacterial contamination. Yeast suspensions were injected in the hemocele through the last left proleg of the larvae using a 10-μl syringe (Gaoge, China). The infected larvae were incubated at 37°C, and death was monitored daily for 4 days. Larval death was monitored by visual inspection of their color (brown-dark brown) and by the lack of movement after touching them with forceps. A group of larvae inoculated with PBS-ampicillin were studied in parallel in every infection investigation as controls. For each condition, a total of 20 larvae were used, and each experiment was repeated at least three times.

Efficacy of fluconazole and fluoxetine in G. mellonella infected with C. albicans.Larval-killing assays were carried out at 37°C as described above, using a dose of 5 × 10⁶ yeast cells/larva. Infected larvae were treated with fluconazole (80, 160, 320, and 640 μg/ml) (Meilun, Dalian, China) and fluoxetine (128 μg/ml) (Meilun). A combination of fluconazole and fluoxetine was also used. In addition, parallel groups of uninfected larvae treated with the same concentrations of drugs were included to eliminate possible drug toxicity and other effects of the drugs as factors contributing to the observed results. Antifungals were administered 2 h postinfection. Survival was monitored every 24 h for 4 days. Each experiment used groups containing 20 larvae, and experiments were repeated twice using larvae from different batches.

Fungal burden determination.The fungal burden was determined (28) by CFU counts at different times after inoculation. For this purpose, four groups of 80 larvae were selected, all of which received 2.5 × 10⁶ cells/larva of the C. albicans strain. While one group remained untreated, the others were treated with fluconazole (320 μg/ml), fluoxetine (128 μg/ml), and the combination of fluconazole and fluoxetine. Every 24 h, 5 larvae were taken from each group, washed with ethanol, and cut into small pieces with a scalpel. No discrimination was made between live or dead larvae. The material was suspended in 1 ml of PBS-ampicillin and homogenized gently with a vortex for a few seconds. The homogenate was 10-fold diluted with same buffer, and 5-μl aliquots of the resulting dilutions were inoculated onto Sabouraud agar plates. The plates were incubated for 24 h at 37°C, and CFU were enumerated, with the results expressed as averages and standard deviations.

Histological study of larval tissue.To evaluate the presence of C. albicans inside tissues of G. mellonella, three larvae from different groups (uninfected, infected, and treated with the fluconazole-fluoxetine combination) were collected on the third day after infection. Larvae were fixed for 24 h in 4% buffered formalin and dehydrated with increasing concentrations of ethanol (70%, 80%, 90%, 96%, and 100%). The samples were then treated with xylene and paraffin embedded. Tissue sections of 8 μm were stained with periodic acid-Schiff (PAS) and observed under an Olympus FSX100 fluorescence microscope with 4.2× and 10× objectives. Samples from noninfected larvae were included as controls.

Statistics.Graphs were created and statistical analyses were performed with GraphPad Prism 5 (GraphPad, La Jolla, CA, USA). Survival curves were analyzed by the Kaplan-Meier method, and fungal burdens were analyzed using a t test.

Real-time quantitative PCR.For detecting the expression levels of aspartyl proteinase-related genes (SAP1, SAP2, SAP3, and SAP4), C. albicans (CA10) cells were grown to mid-log phase in RPMI 1640 medium at 35°C after treatment with drugs alone or in combination at the following final concentrations: fluconazole at 4 μg/ml and fluoxetine at 8 μg/ml. Cultures without drugs served as the controls. Cells were then harvested for RNA extraction. Cell total RNA was isolated using an RNApure yeast kit (DNase I) (CWBiotech, Beijing, China). Then, diluted RNA was treated with a first-strand cDNA synthesis SuperMix kit (CWBiotech) and was reverse transcribed at 42°C for 30 min and 85°C for 5 min according to the manufacturer's instructions. RT-PCR preparations were mixed with cDNA, ultra SYBR mixture (with ROX) (CWBiotech), and gene primers in triplicate. The ACT1 gene was used as the endogenous control (29). RT-PCRs were carried out with an ABI ViiA 7 (Applied Biosystems) sequence detection system using SYBR green I (CWBiotech) in duplicate for three separate experiments. An aliquot of 25 ml of PCR mix was used for each gene and the cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Results were analyzed using SPSS statistical software, and significance was defined as a P value of <0.05.

Phospholipase activity of C. albicans treated with fluoxetine.The isolates were screened for phospholipase activity by measuring the size of the zone of precipitation after growth on egg yolk agar as described by Price et al. (30) with some modifications. The test medium consisted of 15 g/liter peptone, 3 g/liter beef extract ointment, 5 g/liter NaCl, 15 g/liter agar, 10 g/liter glucose, and 10% sterile egg yolk emulsion. Then 10-μl aliquots of a suspension of 10⁷ CFU/ml were inoculated onto the surface of the test medium in duplicates. The plates were incubated at 37°C for 72 h, after which the diameter of the precipitation zone around the colony was measured. Phospholipase activity (P_z) was expressed as the ratio of the diameter of the colony to the diameter of the colony plus the precipitation zone. The phospholipase activity was classified as negative (P_z = 1), very low (P_z = 0.90 to 0.99), low (P_z = 0.80 to 0.89), high (P_z = 0.70 to 0.79), and very high (P_z = 0.69), as previously reported (31). Each experiment was performed twice.