Modulation of antifungal drug resistance and combination activity of eugenol tosylate congeners with antifungal drugs in candida albicans

Abstract

Introduction The increased incidence of antifungal drug resistance in Candida albicans (C. albicans) species has been reported world-wide. Semi synthetic products have been investigated as potential treatment to treat Candida infections. To alleviate the burden of disease, new treatment options have been looked at such as combination therapy using natural products and synthetic drugs and also to develop new drug targets in C. albicans species. The aim of this study was to evaluate the modulation of antifungal drug resistance and combination activity of eugenol tosylate congeners with antifungal drugs in Candida albicans. Materials and methods Fifty C. albicans isolates collected from HIV and cancer patients who attended Charlotte Maxeke Academic Hospital were included in the study. Drug susceptibility testing was performed using fluconazole (FLC), nystatin (NYS) and caspofungin (CAS) antifungal drugs against C. albicans strains. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of six eugenol tosylate congeners (ETCs; C1-C6) and eugenol (EUG) were determined against drug resistant and susceptible C. albicans strains. The antifungal activity of the combination of ETCs with antifungal drugs (FLC, NYS and CAS) in a 1:1 combination ratio was investigated by determining the fractional inhibitory concentration index. The effect of ETCs on the activity of the proton ATPase (H+ ATPase) was evaluated by determining the rate of proton efflux in C. albicans cells treated with ETCs. The effect of ETCs on intracellular pH of C. albicans cells was investigated to evaluate the effect of a dysfunctional H+ ATPase enzyme on the intracellular pH of C. albicans in cells treated with ETCs and in untreated cells. In addition, the effect of ETCs on activity of drug efflux pumps was evaluated. The haemolytic effect of ETCs on horse red blood cells was investigated. Analysis of variance was used to investigate the significant differences between independent groups using graph pad prism 5. Results Of the 50 isolates tested for drug susceptibility, 78% were susceptible to FLC, 14% were resistant and 8% were susceptible dose dependent. Ten isolates (20%) were susceptible to CAS, 5 (10%) were resistant and 35 (70%) were categorized as intermediate. Forty four (88%) isolates were susceptible to NYS and 6 (12%) were resistant. Cross resistance was observed in one isolate which was resistant to both NYS and FLC. Multidrug resistance was also observed in one isolate which was resistant to both CAS and FLC. The MICs obtained for ETCs (C1-C6) ranged from 0.13 to 2 mg/ml. For EUG the MICs ranged from 0.33 and 0.5 mg/ml. Congener 1 was the most active compound with decreased MICs ranging between 0.13 and 0.250 mg/ml against both drug susceptible and resistant C. albicans strains. The combination activity of NYS with ETCs exhibited the most synergistic interactions (43%) compared to the combinations of ETCs with CAS (14%) and FLC (0%). All ETCs successfully reduced the rate of H+ efflux in both FLC susceptible and resistant strains at previously determined MICs, however congener 6 showed the greatest effect on reducing the rate of H+ efflux by 75% in glucose starved cells and by 38% in cells supplied with glucose in both FLC resistant and susceptible strains. Intracellular pH of FLC resistant and susceptible C. albicans cells treated with ETCs was less acidic (pH 6.6-6.8) than intracellular pH of untreated C. albicans cells with functional H+ ATPase (pH 6.40). ETCs showed no effect on activity of drug efflux pumps in FLC susceptible strains. However in FLC resistant strains C2, C4 and EUG exhibited inhibitory effects on activity of drug efflux pumps. ETCs induced hemolysis of RBC in a concentration dependent manner however the percentage hemolysis was below 15% for all ETCs in all tested concentrations. Conclusions Eugenol tosylate congeners exhibited antifungal activity against both drug resistant and susceptible C. albicans strains at acceptable concentrations for modified compounds. The synergistic effect exhibited by ETCs in combination with NYS is a promising therapeutic strategy to combat C. albicans infections. Congener 2, C4 and EUG significantly inhibited activity of drug efflux in FLC resistant strains indicating that the compounds can be successfully used for the reversal of FLC drug resistance in C. albicans strains. In addition the hemolysis assay indicated that ETCs have great potential as antifungal drugs and may be used as safe and effective treatment in clinical applications.