Organism
The reference strains of C. albicans ATCC 10231 strain 3147 [C. albicans Drug Resistance Panel (ATCC® MP­8™)] and C. albicans CBS 5982 were used in this study. Fluconazole susceptibility was studied on 100 colonized clinical isolates of C. albicans obtained from 100 pregnant women, diabetics, cancer and maintenance hemodialysis patients at Shahid Beheshti Hospital, Yasooj, Iran, between September 2016 and January 2017. Fluconazole susceptibility of clinical isolates of C. albicans was determined using CSLI M27-A3 document with slight modification (16).

Combination Testing of Fluconazole with Amphotericin B on Fluconazole-resistant Candida albicans
The effects of fluconazole alone and in combination with amphotericin B were tested against fluconazole-resistant C. albicans by broth microdilution method using CLSI document (M27-A3) with slight modification. Briefly, the inocula from 24 h C. albicans cultures on Sabouraud Dextrose Agar (SDA, Merck Research Laboratories) were prepared to a turbidity equivalent to a 0.5 McFarland standard with a spectrophotometer at 530 nm. The C. albicans suspensions were further dilutted to yield an inoculum concentration of approximately 5×10² – 2.5×10³ cells/ml in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma-Aldrich) with 0.2% glucose [buffered to pH 7.0 with 0.165 M morpholinophos- phonyl sulfate (MOPS)]. The minimal inhibitory concentration (MIC) assay was performed with 96-well plates. C. albicans inoculum (100μl) was exposed to 100μl of the two-fold dilutions of the fluconazole (ranging 0.0313-64μg/ml) and amphotericin B (ranging 0.0313-16μg/ml) alone and in combination. The fluconazole and amphotericin B in combination were mixed in 1:1 ratio. The microplates were kept at 4°C for 2 h and incubated at 35°C for 24 h to evaluate the MICs. The end points of ≥50% and ≥90% reduction in turbidity relative to the positive control were determined using a Stat Fax 303 Reader (Awareness Technology, Inc., USA) at 530 nm.
To evaluate the interaction between fluconazole and amphotericin B, the FIC index was calculated from the data obtained with relative MICs determination with the antifungal alone and in combination as described earlier (16, 17).

Combination Testing of Fluconazole with Amphotericin B on Fluconazole-resistant Candida albicans Biofilm Formation
The biofilm formation assay was performed exactly as described previously (18-21). Combination testing of fluconazole with amphotericin B on fluconazole-resistant C. albicans biofilm formation was determined by crystal violet (CV) staining, microscopic analysis of biofilm formation and the expression analysis of C. albicans hypha specific gene.
The yeast cell suspension (1×10⁶ cells/ml) was dispensed into the wells of 96-well plates (100μl/well). The wells were filled with 100μl of four different concentrations based on the MIC values (1/4 MIC, 1/2 MIC, 1 MIC and 2 MIC) of fluconazole alone or in combination with amphotericin B. After incubation at 35°C for 90 min, the plates were incubated again at 35°C for 24 h with gentle shaking. After fixation of biofilm with 99% methanol, they were stained by aqueous crystal violet for 20 min and de-stained using 33% acetic acid. The absorbance values were obtained at 590 nm using Stat Fax 303 Reader (18, 19).
The effect of fluconazole alone or in combination with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation was qualitatively analyzed by the light field microscope. The biofilms were formed on glass cover slips in 6-well cell culture plates by growing the cells (1×10⁶ cells/ml) in RPMI-1640 treated with four different concentrations based on the MIC values (1/4 MIC, 1/2 MIC, 1 MIC and 2 MIC) of fluconazole alone or in combination with amphotericin B. The microplates were incubated at 35°C for 90 min, and then maintained for 24 h with gentle shaking at 35°C. Thereafter, the cover slips were washed with PBS and visualized with the Leica light field microscope using 40X objective lens (19, 20).
Subsequently, the effect of fluconazole alone or in combination with amphotericin B was evaluated on the expression of hypha specific gene (HWP1) on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation by two-step quantitative Real-time-polymerase chain reaction (qRT-PCR). The biofilms were formed on 96-well plates by growing the cells (1×10⁶ cells/ml) in RPMI-1640 treated with four different concentrations based on the MIC values (1/4 MIC, 1/2 MIC, 1 MIC and 2 MIC) of fluconazole alone or in combination with amphotericin B and total RNA of the cells was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from DNase I-treated total RNA using Moloney-Murine Leukemia Virus reverse transcriptase and random hexamers (Fermentas, USA). The qRT-PCR reaction was conducted using the primers for the target HWP1 and housekeeping internal control Beta actin genes (Table 1). The qRT-PCR was performed in TPersonal thermocycler (Biometra- Germany) with an initial denaturation at 95°C (4 min), followed by 26 cycles of denaturation (94°C/40 sec), annealing (56°C/45 sec) and extension (72°C/45 sec), and final extension at 72°C for 10 min. The specificity of the primers was confirmed by the sequencing analysis (First BASE Laboratories Sdn. Bhd., Malaysia). The amplified products were analyzed by Quantity One 1-D Analysis (Bio-Rad, USA, version 4.6). The relative quantification of the target transcripts were determined by the volume-based analysis by comparing to DNA mass standard (Fermentas, USA) (19-21)

Statistical Analysis
All the experiments were performed in triplicate. Data were analyzed using the statistical software SPSS 23.0 for Windows (SPSS Inc., Chicago, IL, USA). All data were expressed as mean values with the corresponding standard deviations (SD). The comparisons were performed using one-way ANOVA, with Tukey’s HSD post hoc test. A P-value less than 0.05 was considered statistically significant.
 

Table 1. Oligonucleotide primers used for qRT-PCR

Hundred colonized C. albicans isolated from the clinical samples were identified and used for the fluconazole susceptibility testing. Ten fluconazole-resistant C. albicans were identified and used in the study. For fluconazole-resistant C. albicans, the relative MIC of fluconazole was ≥16 μg/ml when used alone. The 90% of fluconazole-resistant C. albicans isolates were resistant to amphotericin B with MIC ≥2.0μg/ml. The susceptibility assay showed that the combination of fluconazole and amphotericin B was synergistic (20%), partial synergistic (20%), additive (30%) and indifferent (30%) as antifungal effects against fluconazole-resistant C. albicans (Table 2). Combination of fluconazole and amphotericin B exhibited synergistic and partial synergistic effects against fluconazole-resistant C. albicans isolates with FIC index ranged from 0.50 to 0.75
Table 3 shows the corresponding results for the reduction of biomass determined by the CV staining of the inhibitory effect of fluconazole combined with amphotericin B on fluconazole-resistant C. albicans biofilm at different concentrations based on the MIC values. There was no significant effect of fluconazole alone on the reduction of biomass on fluconazole-resistant C. albicans biofilm. Furthermore, no significant effect was observed of amphotericin B alone on the reduction of biomass in amphotericin B-resistant isolates. The effect of fluconazole combined with amphotericin B significantly decreased the biomass on fluconazole-resistant C. albicans biofilm (P<0.05).
The combination testing of fluconazole with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation was determined by the microscopic analysis of biofilm. Figure 1 shows the corresponding results for the reduction of biofilm, which resulted in the reduction of biofilm formation, hyphae and planktonic cells on fluconazole-resistant C. albicans treated with fluconazole combined with amphotericin B. As shown in the Figure 1, the fluconazole alone could not decrease the biofilm formation, hyphae and planktonic cells of fluconazole-resistant C. albicans compared to the untreated control (P>0.05).
Our results indicate that the combination of fluconazole with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation exerted their antifungal effect through inhibition of the expression levels of HWP1 (P≤0.05). No significant difference was observed in the mean of expression levels of HWP1 on fluconazole-resistant C. albicans grown in the presence of fluconazole and amphotericin B alone (P>0.05) (Figure 2 and Figure 3).

Table 2. Baseline characteristics of all participants


Table 3. Results of CV staining (μg/ml) for the inhibitory effect of fluconazole combined with amphotericin B on fluconazole-resistant C. albicans biofilm



Figure 1. Light field microscopy of the inhibitory effect of fluconazole combined with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation at different concentrations. Magnification × 40, Bar = 50 µm. Flu: fluconazole, AmB: amphotericin B.



Figure 2. Expression analysis of HWP1 in combination of fluconazole with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation at different concentrations. M: 100 bp DNA Ladder, A1: Beta actin with 2 × MIC of antifungals, H1: HWP1 with 2 × MIC of antifungals, C1: Internal control (no reverse transcriptase was added), A2: Beta actin with 1 × MIC of antifungals, H2: HWP1 with 1 × MIC of antifungals, C2: Internal control, A3: Beta actin with ½ × MIC of antifungals, H3: HWP1 with ½ × MIC of antifungals, C3: Internal control, A4: Beta actin with ¼ × MIC of antifungals, H4: HWP1 with ¼ × MIC of antifungals, C4: Internal control, A5: Beta actin without antifungals (untreated control), H5: HWP1 without antifungals, C5: Internal control. Flu: fluconazole, AmB: amphotericin B.



Figure 3. Relative gene expression levels of HWP1 in combination of fluconazole with amphotericin B on fluconazole-resistant C. albicans (ATCC 10231) biofilm formation at different concentrations. Flu: fluconazole, AmB: amphotericin B.* Significant downregulation. Data are means of fold changes ± SD from three independent experiments amplified in triplicates.

Discussion

In this study, we evaluated the inhibitory effect of fluconazole combined with amphotericin B against C. albicans. Our data supported the influences of fluconazole combined with amphotericin B against C. albicans. Although theoretically the use of fluconazole combined with amphotericin B may lead to the antagonism based on the specific modes of action of the azoles and polyenes, different reports have shown the inhibitory effect of fluconazole combined with amphotericin B against C. albicans (13, 15, 22, 23).
The frequency of biofilm-related infections caused by C. albicans has been increased disproportionately in the last few decades and become incurable because of the increasing number of immunocompromised hosts (3, 4). The current therapeutic options for the Candida infections are extremely limited as compared to those for the treatment of bacterial infections. The new formulations development for the antifungal drugs is not an easy task; only one new formulation of antifungal drugs has been created over the past 30 years. Several lines of evidence demonstrated that drug combinations are strategies to meet some of these challenges (13, 24).
In the present study we investigated the inhibitory effect of fluconazole combined with amphotericin B on fluconazole-resistant C. albicans biofilm formation. Our findings, based on the biomass inhibitory effects, consistently demonstrated that fluconazole combined with amphotericin B exerted a significant reduction on fluconazole-resistant C. albicans biofilm formation. Moreover, the morphological alterations on fluconazole-resistant C. albicans biofilms correlated well with the inhibitory effect of the fluconazole combined with amphotericin B treatments. In addition, fluconazole combined with amphotericin B indicated the inhibitory effect on HWP1 gene that contributes to the biofilm formation. Since hyphal morphogenesis is a key virulence factor in C. albicans infection, the inhibition of HWP1 could decrease the ability of C. albicans to cause the infection. C. albicans biofilms are intrinsically resistant to the various antifungal agents (2, 25). Combination therapy is an option for the eradication of Candida biofilm (26). Haque et al (13) showed that combination of antifungal agents was the most effective against C. albicans biofilm.
However, the results of the present study demonstrated that fluconazole alone was not effective against fluconazole-resistant C. albicans biofilms in biomass reduction, morphological changes and the gene expression alterations. Although fluconazole has been proved as highly effective agent against Candida biofilms (27), there was no inhibitory effect observed on the fluconazole-resistant C. albicans biofilms. Several possible mechanisms of resistance to fluconazole have been identified in Candida species including genes involved in the ergosterol biosynthesis pathway, drug transporters, ploidy changes and loss of heterozygosity (28). In addition, the cells in a biofilm structure display a reduced susceptibility to azoles that target ergosterol biosynthesis pathway via possibly causing the ineffectiveness blockage of the enzyme lanosterol 14α-demethylase (26).
Our results demonstrate that the amphotericin B alone exhibited inhibitory effects against fluconazole-resistant and amphotericin B susceptible C. albicans (ATCC 10231) biofilm. The amphotericin B application alone resulted in the reduction in biomass of the biofilm and morphological alterations at different concentrations against fluconazole-resistant C. albicans (ATCC 10231). Kuhn et al (29) confirmed the resistance of mature C. albicans to the conventional antifungal agents. They demonstrated that lipid formulations of amphotericin B exhibited inhibitory effects against C. albicans biofilms. However, amphotericin B alone was not able to inhibit HWP1 gene on fluconazole-resistant C. albicans biofilms. This result suggests that the amphotericin B alone may inhibit other genes involved in the biofilm development on fluconazole-resistant C. albicans.

 

Conclusion

In conclusion, these findings demonstrated that fluconazole combined with amphotericin B exhibits potent inhibitory effects against fluconazole-resistant C. albicans biofilm formation. Evaluating the treatment efficacy of fluconazole combined with amphotericin B against fluconazole-resistant C. albicans biofilm with other measures is important in determining the antifungal effectiveness.

 

Acknowledgements

The authors wish to thank the Islamic Azad University of Yasooj for financial support. Results of the current study are part of the MSc thesis (1280679).

 

Conflicts of Interest

Authors declared no conflict of interests.