Pyrogallol and Fluconazole Interact Synergistically In Vitro against Candida glabrata through an Efflux-Associated Mechanism

Twenty-two C. glabrata isolates were used to evaluate the anti-Candida activity of PG alone or together with FLC, ITC, or VRC. PG was active against all isolates, with MIC values of 16 to 64 mg/liter, and no difference was found between FLC-susceptible and FLC-resistant isolates. Combination of PG with FLC showed synergistic effects against all isolates (Table 1). The MICs in the PG+FLC group decreased by 4- to 8-fold for PG and 4- to 128-fold for FLC compared with those of each drug alone, with the fractional inhibitory concentration index (FICI) ranging from 0.188 to 0.375. PG+ITC and PG+VRC yielded similar synergistic effects. PG+ITC showed synergism against 68.2% (15/22) of isolates and no interaction against the other 31.8% (7/22). The MICs were reduced 2- to 8-fold for PG and 2- to 16-fold for ITC compared with those of each drug alone, with FICI ranging from 0.250 to 0.750. Similarly, PG+VRC showed synergism against 63.6% (14/22) of isolates and no interaction against the other 36.4% (8/22). The MICs were reduced by 4- to 8-fold for PG and 2- to 64-fold for VRC compared with those of each drug alone, with FICI ranging from 0.141 to 0.750.

The extracellular fluorescence of rhodamine 6G increased steadily over time in all isolates (Fig. 1). The fluorescence intensity of rhodamine 6G in the presence of FLC alone was higher than that in the other groups. In contrast, the fluorescence intensity of rhodamine 6G in the presence of PG alone was slightly lower than that in the control and was even lower in PG+FLC at synergistic concentrations.

The qRT-PCR assays showed that FLC alone significantly upregulated CgCDR1, CgCDR2, and CgPDR1 expression compared with that in the control group in eight, nine, and nine of the isolates, respectively, whereas the corresponding expression levels in the other isolates hardly changed. The results with PG alone were inconsistent among the isolates compared with those in the control group. CgCDR1 expression increased in four isolates, decreased in four other isolates, and was unchanged in the remaining isolates. CgCDR2 expression increased in four isolates but remained unchanged in all other isolates. CgPDR1 expression increased in two isolates, decreased in one isolate, and remained unchanged in all other isolates. PG+FLC significantly downregulated CgCDR1 and CgPDR1 expression in all isolates compared with the control group, whereas six isolates displayed CgCDR2 downregulation, and the remaining isolates showed no significant change. In addition, compared with PG or FLC alone, PG+FLC resulted in 2.22-fold (P < 0.01) and 3.00-fold (P < 0.01) decreases in CgCDR1 expression, respectively. Similarly, compared with PG and FLC alone, PG+FLC resulted in 1.56-fold (P < 0.05) and 2.10-fold (P < 0.01) decreases in CgCDR2 expression and 1.47-fold (P < 0.01) and 2.01-fold (P < 0.01) decreases in CgPDR1 expression, respectively, (Table 2, Fig. 2).

Disruption and replacement mutants were generated as described in the supplemental material. During drug-susceptibility testing, the MIC values of FLC alone were reduced in the CgPDR1-deficient strain C. glabrata 66/ura3Δpdr1 (Table 3). However, PG failed to enhance FLC activity against C. glabrata 66/ura3Δpdr1, with a FICI value of 1. When CgPDR1 was replaced, the MIC values of FLC alone recovered, and PG+FLC showed a strong synergistic effect. Similar results were obtained with PG+ITC and PG+VRC.

The fluorescence intensity of rhodamine 6G in the extracellular matrix of the CgPDR1-deficient strain C. glabrata 66/ura3Δpdr1 grown in the presence of FLC, alone or in combination with PG, was markedly higher than that in the other groups (Fig. 1). In contrast, the fluorescence intensity of rhodamine 6G in the extracellular matrix of the CgPDR1-replacement strain C. glabrata 66/ura3Δpdr1-PDR1 grown in the presence of PG+FLC at synergistic concentrations decreased to a level lower than that in the other groups.

Twenty-two clinical C. glabrata isolates (11 FLC-resistant and 11 FLC-susceptible isolates) and C. glabrata 66 CgPDR1-disruption and -replacement mutants were used. All strains were routinely stored at −80°C in yeast-peptone-dextrose liquid medium (1% yeast extract, 2% peptone, and 2% dextrose), supplemented with 30% (vol/vol) glycerol, and recultured at least twice on Sabouraud agar (Kehua Biotech Co., Shanghai, China) at 35°C before use in the experiments.

FLC (National Institutes for Food and Drug Control [NIFDC], Beijing, China), ITC (NIFDC), VRC (Haisi Co., Jincheng, Shanxi, China), and PG (U-sea Biotech, Shanghai, China) were obtained commercially. The purity of PG (>99.90%) was confirmed via high-performance liquid chromatography. FLC was prepared in sterile distilled water at 5,000 mg/liter. ITC was dissolved in dimethyl sulfoxide (DMSO) at 5,000 mg/liter. VRC was prepared in a dedicated solvent (ethanol and propylene glycol, 1:1) at 2,000 mg/liter. PG was prepared in DMSO at 10,000 mg/liter. All stock solutions were stored at −20°C.

The MICs of PG+FLC, PG+VRC, and PG+ITC against C. glabrata strains were tested using broth microdilution checkerboard assays based on Clinical and Laboratory Standards Institute standard M27-A3 (36). The MICs alone and in combination were defined as 50% of inhibition compared with the growth control. MICs were read visually. The drugs tested were serially diluted 2-fold in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) as previously described (37). The final concentrations were 4 to 256 mg/liter for PG, 2 mg/liter to 1.024 g/liter for FLC, 0.125 to 64 mg/liter for ITC, and 0.125 to 64 mg/liter for VRC. A 50-μl aliquot of each PG dilution and 50 μl of RPMI 1640 medium were added to individual wells in 96-well plates (Corning, Inc., Corning, NY, USA) in the first columns, and a 50-μl aliquot of each azole drug dilution and 50 μl of RPMI 1640 medium were added to row H. The well at the intersection of column 1 and row H was drug free and served as a control. Then, 50-μl aliquots of a PG-dilution series or an azole drug-dilution series were added to columns 2 to 11 and lines A to G, respectively. Next, 100 μl of cells was added to each well at a final concentration of 0.5 to 2.5 × 10³ cells/ml, except for column 12, to which 200 μl of RPMI 1640 medium was added as a negative control. The plates were incubated at 35°C for 24 or 48 h. Drug interactions were analyzed based on the FICI, calculated as MIC(A) combined/MIC(A) alone plus MIC(B) combined/MIC(B) alone. Synergism was defined as a FICI of ≤0.5, no interaction was defined as 0.5 < FICI ≤ 4.0, and antagonism was defined as a FICI of >4.0 (38). The experiments were performed in duplicate.

The rhodamine 6G efflux assay was performed as previously described (21), with a few modifications. Isolates were incubated at 37°C overnight without any drug or with PG alone, FLC alone, or PG+FLC at synergistic concentrations. Isolates were cultured overnight, then adjusted to a cell density of 5 × 10⁷ cells/ml in phosphate-buffered saline (PBS) and incubated at 37°C for 4 h in an orbital shaker (180 rpm; Yiheng Biotech, Shanghai, China). Rhodamine 6G was added at a final concentration of 10 mM, and the cultures were incubated at 37°C for 2 h. After the cells were washed twice with sterile PBS, glucose was added at a final concentration of 4 mM, and the cultures were shaken at 30°C for 1 h. During this period, the suspension was centrifuged at 3,000 × g every 10 min, and 100 μl of the supernatant from each group was transferred to individual wells of 96-well plates. The rhodamine 6G fluorescence in each sample was measured using a BioTek Synergy H4 microplate reader (BioTek Instruments, Winooski, VT, USA). The excitation and emission wavelengths were 515 and 555 nm, respectively.

The qRT-PCR analysis was performed as described previously (21), with minor modifications. Isolates were incubated without any drug or with PG alone, FLC alone, or PG+FLC at synergistic concentrations at 37°C overnight. The suspensions were adjusted to 5 × 10⁷ cells/ml in PBS, and the supernatants were collected after centrifugation at 3,000 × g. Total RNA was isolated using a yeast RNAiso reagent kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. RT-PCR was performed using RevertAid first-strand cDNA synthesis kits (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCRs for CgCDR1, CgCDR2, and CgPDR1 were run in triplicate using SYBR green real-time PCR master mix kits (Toyobo, Osaka, Japan) in an ABI 7500 real-time fluorescent quantitative PCR system (Applied Biosystems, Foster City, CA, USA). The primers used in this study are listed in Table 4. Each qRT-PCR mixture (25 μl) contained 12.5 μl SYBR green real-time PCR master mix, 9.5 μl double-distilled water, 2 μl each primer, and 1 μl cDNA. PCR conditions were as follows: initial denaturation at 95°C for 1 min, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 45 s at 72°C. Target gene expression was quantified using the 2^–ΔΔCT method, with ACT1 as a control (39).

Results are reported as the mean ± standard deviation (n = 3) and were calculated using IBM SPSS Statistics, version 24.0 (IBM Corp., Armonk, NY, USA). Differences among groups were analyzed using one-way analysis of variance, with the least-significant difference method. A P value of <0.05 was considered to reflect a statistically significant difference.

GenBank accession numbers of the molecular identification of the strains are MW709447 to MW709456 (https://www.ncbi.nlm.nih.gov/nuccore/?term=MW709447:MW709456[accn]) and MW729709 to MW729720 (https://www.ncbi.nlm.nih.gov/nuccore/?term=MW729709:MW729720[accn]).