A standard strain of C. albicans, ATCC 90028, was obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. Culture was maintained on yeast peptone dextrose (YPD) agar slants at 4°C (all the media components were purchased from HiMedia laboratories Pvt. Ltd. Mumbai, India). Antifungal agents Fluconazole (FLC) (Forcan, Cipla Ltd., India), Voriconazole (VOR) (Vonaz, United Biotech Pvt. Ltd., India), Caspofungin (CSP) (Cancidas™, Merck & Co. Inc., USA), Amphotericin B (AmB) (Lyka Pharm. Pvt. Ltd., India), and Chloroquine (CQ) (Lariago®, Ipca Laboratories Ltd., India) were obtained from local market.

Activation of culture was done by inoculating a single colony from YPD agar plate into 50mL YPD broth (yeast extract 1%, peptone 2%, and dextrose 2%) in a 250mL conical flask. The flasks were incubated at 30°C at 100rpm on an orbital shaking incubator for 24h. Cells were harvested by centrifugation at 2000×g and washed thrice with 0.1M phosphate buffer saline (PBS), pH 7.4. Final cell number was adjusted to 1×107cells/mL and used for experiments.

The susceptibility study was carried out by the standard broth micro dilution method as per CLSI guidelines. Various concentrations of drugs were prepared; FLC was in the range 128–0.125μg/mL, while VOR was between 8μg/mL and 0.015μg/mL. Range of concentrations used for CSP and AmB were 4–0.007μg/mL. The concentrations of CQ were in the range 500–0μg/mL. All the drug concentrations were prepared in RPMI-1640 medium pH 7.0 (with l-glutamine, without sodium bicarbonate, buffered with 165mM MOPS; purchased from HiMedia laboratories Pvt. Ltd., Mumbai, India), in the wells of micro plates by double dilution. Wells without drug served as a control. Cells were added to each well to obtain density of 1×103cells/mL. Plates were incubated at 35°C for 48h and absorbance was read at 620nm using a microplate reader (Multiskan EX, Thermo Electron Corp., USA). The lowest concentration of the drugs which caused fifty percentage reduction in the absorbance compared to that of control was considered as minimum inhibitory concentration (MIC).7

Dilutions of individual drugs and their different combinations were prepared in a checkerboard format. A two dimensional array of serial concentrations of test compounds was used for preparation of dilutions of the drugs. 100μL of the cell suspension was added to each well and the plates were incubated at 35°C. Plates were read spectrophotometrically at 620nm, after 48h of incubation. The FIC Indices were calculated using following equation: ∑FIC=FICA+FICB, where FICA=(MIC of drug A in combination/MIC of drug A alone), FICB=(MIC of drug B in combination/MIC of drug B alone). When the value of ∑FIC≤0.5 it is the synergism, and when ∑FIC>4 it is known as the antagonism. A ∑FIC result of >0.5 but ≤4 is considered as indifference.20,21

Biofilm formation was carried using in vitro biofilm model.7 Briefly, 100μL of cell suspension (1×107cells/mL in PBS) was added to wells of tissue culture grade polystyrene micro plates. Plates were incubated at 37°C on an orbital shaker for 90min of adhesion phase. All the wells were washed with sterile PBS to remove non-adhered cells. Two hundred μl of RPMI-1640 medium, pH 7.0, containing various concentrations of the drugs was added to each well. The plates were incubated at 37°C at 100rpm in an orbital shaker for 48h. Quantitation of growth was done by using XTT-metabolic assay, where the colored end product was read spectrophotometrically.

Biofilm formation was quantitated using XTT [i.e. 2,3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (Sigma–Aldrich, India) reduction assay.7 Briefly, XTT solution was prepared by mixing 1mg/mL XTT salt in PBS and stored at −20°C. Prior to use, menadione solution prepared in acetone (Sigma–Aldrich, India) was added to XTT to a final concentration of 4μM. The wells containing biofilms were washed with PBS to remove non-adhered cells and 100μL of XTT-menadione solution was added to it. The plates were incubated for 5h in dark at 37°C at 100rpm. Intensity of the colored formazan end product was measured at 450nm using a micro plate reader. Wells without biofilms served as a blank.

Biofilms were observed under an inverted light microscope (Metzer, India). Photographs were taken by a Labomed microphotography system (Labomed, Mumbai, India). For scanning electron microscopy (SEM), samples were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2), for 24h at 4°C. Samples were post-fixed in 2% aqueous solution of osmium tetraoxide for 4h, then dehydrated in a series of graded alcohols and finally dried to a critical drying point with a Critical Point Dryer unit. The samples were mounted over stubs, and gold coating was performed using an automated gold coater (model JOEL JFC-1600) for 3min. Photographs were taken under a scanning electron microscope (model JOEL-JSM 5600).7

Planktonic growth of C. albicans was susceptible to different antifungal drugs at varying concentrations. MICs of FLC, VOR, AmB, and CSP were found at 0.5μg/mL, 0.25μg/mL, 0.125μg/mL and 0.25μg/mL, respectively (Table 1). Chloroquine did not show any significant effect on growth of C. albicans and MIC was observed at 1mg/mL. No significant decrease in MICs of two azoles, FLC and VOR could be observed when cells were treated in combination with various concentrations of CQ. FIC indices for combination of 250μg/mL concentration of CQ with azole drugs were found to be >0.5. Similar observations were noted for CQ-AmB interactions indicating no significant reduction in the MIC of AmB. Addition of CQ could not lower down the CSP concentration required to inhibit growth of C. albicans. It was evident with the FIC index of 0.74 for this combination (Table 1).

FLC and VOR alone were ineffective against biofilm formation. No significant inhibition of C. albicans biofilm development could be obtained even at high concentrations of FLC and VOR, i.e. 128 and 8μg/mL, respectively. This indicated resistance toward the azole drugs. After combination with CQ, concentrations of FLC and VOR required to inhibit biofilms were lowered down dramatically. Various CQ-FLC combinations were found to effectively inhibit biofilm development in C. albicans at 2–8μg/mL. For example, addition of 250μg/mL of CQ resulted in inhibition of biofilm development at 4μg/mL of FLC. Similarly, CQ-VOR was also effective and MIC of VOR for formation of C. albicans biofilms was achieved at 0.25μg/mL (Fig. 1A and B). FIC indices for CQ-FLC and CQ-VOR combinations against biofilm development were found to be 0.156 and 0.187, respectively, indicating synergistic activity. Results of biofilm inhibition analyzed by XTT metabolic assay were confirmed by light as well as electron microscopy. Control without drug exhibited typical biofilm structure consisting of dense network of filamentous forms and yeast cells attached to the solid surface (Fig. 2A). Although cell density was less, normal biofilms were observed in presence of CQ, FLC and VOR alone (Fig. 2B, C and D, respectively). No biofilm growth was observed in presence of FLC-CQ combination (Fig. 2E). Similarly, VOR-CQ treatment resulted in inhibition of biofilms and only few yeast cells were found adhered to the surface (Fig. 2F). The MIC of AmB for biofilm development was at 0.5μg/mL, which was fourfold more than that of planktonic MIC. When combined with 250μg/mL of CQ the effective AmB concentration was 0.5μg/mL. The FIC index of 1.126 for this combination indicated indifference activity in combination. Similarly, combination of CQ with CSP exhibited no change in the MIC for C. albicans biofilm development (Table 1). FIC index of 0.621 for CQ-CSP was indicative of additive effect.

Fig. 1.

Activity of FLC and VOR antifungal drugs alone and in combination with CQ, against biofilm development and mature biofilms of C. albicans (ATCC 90028). Percentage of growth was analyzed by comparing relative metabolic activity (RMA) obtained through XTT metabolic assay. (A) FLC-CQ against biofilm development; (B) VOR-CQ against biofilm development; (C) FLC-CQ against mature biofilms; (D) VOR-CQ against mature biofilms.

(0.33MB).

Fig. 2.

Scanning electron micrographs of FLC and VOR antifungal drugs alone and in combination with CQ, against biofilm development of C. albicans (ATCC 90028). (A) Control; (B) CQ alone 250μg/mL; (C) FLC alone (128μg/mL); (D) VOR alone (8μg/m); (E) 4μg/mL FLC+250μg/mL CQ; (F) 0.25μg/mL+250μg/mL CQ.

(0.57MB).

Mature biofilms of C. albicans were highly resistant toward activity of FLC and VOR. MICs were not achieved even at very high concentrations, i.e. 128μg/mL of FLC and 8μg/mL of VOR. When 250μg/mL of CQ was added to the mature biofilms, MIC of FLC was obtained at 4μg/mL. This indicated effective combination, with FIC index of 0.25. Combination of CQ with VOR resulted in inhibition of mature biofilms at concentrations as low as 0.25μg/mL (Fig. 1C and D). The FIC index for CQ-VOR combination was calculated to be 0.187. Although C. albicans biofilms were found sensitive to AmB and CSP, it required high concentrations. When combined with CQ, twofold decrease in the effective concentrations of AmB and CSP was observed (Table 1). The FIC indices for CQ-AmB and CQ-CSP drug combinations were 0.496 and 0.366, which are considered to exert synergistic effect.