The reference strain of C. albicans used in this study was ATCC 10261. The clinical isolate strains of C. albicans were D1, D2, and non albicans species including D9 (Candida tropicalis), and D46 (Candida krusei). All the strains of C. albicans were cultured in YEPD broth with the composition of yeast extract 1% (w/v), peptone 2% (w/v) and dextrose 2% (w/v). For agar plates 2% (w/v) agar (Himedia, Mumbai, India) was added to the media. For morphological switching the growth media used were serum (10% serum in YEPD), SLAD (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose, 50µM ammonium sulfate, 2% Bacto Agar) and spider media (1% mannitol, 0.4% K₂HPO₄, 1% Nutrient Broth). All Candida strains were stored in 30% (v/v) glycerol stock at -80˚C. The cells were freshly revived on YEPD broth and transferred to agar plate. The cells were grown at 30˚C on agar plate before each study to ensure the revival of the strains.

Drug susceptibility was tested using spot assay, minimal inhibitory concentration (MIC) and filter disc assay as described below:

Spot assays for the strains were determined using a method as described elsewhere [14, 15]. Briefly, for the spot assay 5μL of fivefold serial dilutions of each yeast culture (each with cells suspended in normal saline to an OD₆₀₀ nm of 0.1) was spotted onto YEPD plates in the absence (control) and presence of the drugs. Growth was not affected by the presence of solvent used in the examination (data not shown). Growth difference was measured after incubation at 30˚C for 48 hours. The concentrations used in this study are specified in figure legends.

Fig. (2). Drug susceptibility assays against non-albicans species of Candida in the presence of MB. (a) Spot assay of C. krusei and C. tropicalis; in the absence (control) and presence of MB. (b) Disc diffusion assay against C. krusei and C. tropicalis; and their respective zone of inhibitions in the absence (control) and presence of MB. For control, discs were spotted with the solvent of MB (water). (c) Broth microdilution assay to determine the MIC80 of C. krusei and C. tropicalis in presence of MB. Data is quantitatively displayed with colour (see colour bar), where each shade of colour represents relative optical densities of the cell. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control is indicated as MIC80 for each strain.

MIC was determined by broth dilution method as described in method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS (National Committee for Clinical Laboratory Standards) [16]. Briefly, 100μl of media was placed at each well of the 96 wells plate following with the addition of the drug with the remaining media and then was serially diluted. 100μl of cell suspension (in normal saline to an OD₆₀₀ 0.1) was added to each well of the plate and OD₆₀₀ was measured after 48 hours at 30˚C. The MIC₈₀ was defined as the concentration at which the 80% of the growth was inhibited.

The filter disc assay was performed as described elsewhere [17]. The drug was spotted in a volume of 5-10μL at the indicated amount in the figure legends and the diameters of the respective zones of inhibition were measured after incubation of the plates for 48 hours at 30˚C.

The efflux of R6G was determined by using protocol described elsewhere [18]. Briefly, approximately 1x10⁶ yeast cells from an overnight-grown culture in the absence (Control) and presence of MB at its sub inhibitory concentration (25µg/ml) were transferred to YEPD medium and allowed to grow for 5h. Cells were pelleted, washed twice with phosphate-buffered saline (PBS) (without glucose), and resuspended as a 2% cell suspension, which corresponds to 10⁸ cells (w/v) in PBS without glucose. The cells were then de-energized for 1h in 2-DOG (5 mM) and 2,4 DNP (5 mM) in PBS (without glucose). The de-energized cells were pelleted, washed, and then resuspended as a 2% cell suspension (w/v) in PBS without glucose, to which R6G was added at a final concentration of 10μM and incubated for 40 min at 30°C. The equilibrated cells with R6G were then washed and resuspended as a 2% cell suspension (w/v) in PBS without glucose. Samples with a volume of 1 ml were withdrawn at the indicated time and centrifuged at 10,000 x g for 1 min. The supernatant was collected, and absorption was measured at 527nm. Energy dependent efflux (at the indicated time) was measured after the addition of glucose (2%) to the cells resuspended in PBS (without glucose). Glucose-free controls were included in all the experiments.

Fig. (3). Drug efflux assay in presence of MB. Extracellular concentrations of R6G for C. albicans (ATCC 10261) cells grown in absence (control) and presence of MB (25μg/ml) calculated as described in material and methods. Negative control represents C. albicans de-energized cells without glucose. Mean of OD527 ± SD of three independent sets of experiments are depicted on Y-axis with respect to time (minutes) on X-axis. (P value > 0.05).

Damage to the cells treated with MB at its MIC₈₀ value was observed by using TEM (Zeiss EVOMA10). The cells (~10⁶ cells) were administered to the media with and without MB and were incubated for 24h at 30˚C. Sample preparation and analysis were performed by using the method as described elsewhere [19, 20]. Briefly, all cells were fixed with 2% glutaraldehyde in 0.1% phosphate buffer for 1 h at room temperature (20˚C). Washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed 1% OsO₄ in 0.1 M phosphate buffer for 1 h at 4˚C. Then the cells were dehydrated in acetone and dropped on round glass cover slip with hexamethyldisilizane (HMDS) and dried at room temperature then sputter coating with gold and observed under the TEM (Zeiss EVOMA10) at 30K magnification.

Sterols were extracted by the alcoholic KOH method and the percentage of ergosterol was calculated as described previously [21, 22]. Briefly, a single C. albicans colony from an overnight YPD agar plate culture was used to inoculate 50 ml of YEPD in the presence and absence of MB. The cultures were incubated for 16 h with shaking at 30°C. The stationary-phase cells were harvested by centrifugation at 2,700 rpm for 5 min and washed once with sterile distilled water. The net wet weight of the cell pellet was determined to which 3ml of freshly prepared 25% alcoholic potassium hydroxide solution (25 g of KOH and 35 ml of sterile distilled water, brought to 100 ml with 100% ethanol), was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated in an 85°C water bath for 1 h. Following incubation, tubes were allowed to cool to room temperature. Sterols were then extracted by the addition of a mixture of 1 ml of sterile distilled water and 3 ml of n-heptane followed by vigorous vortex mixing for 3 min. The heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at -20°C. Both ergosterol and 24(28)-DHE absorb at 281.5 nm, whereas only 24(28)-DHE absorbs at 230 nm. Ergosterol content is determined by subtracting the amount of 24(28)-DHE (calculated from the OD₂₃₀) from the total ergosterol plus 24 (28)-DHE content (calculated from the OD_281.5). Ergosterol content was calculated as a percentage of the wet weight of the cells with the following equations: % Ergosterol + % 24 (28)-DHE = [(A281.5/290) ( F] / pellet weight; % 24(28)-DHE = [(A230/518) ( F] / pellet weight and % Ergosterol = [% ergosterol + % 24(28) DHE] - % 24(28) DHE, where F is the factor for dilution in petroleum ether and 290 and 518 are the E values (in percent per centimeter) determined for crystalline ergosterol and 24(28)-DHE, respectively.

Studies of hyphal induction on C. albicans were carried out on hyphal induction media like Spider (1% nutrient broth, 1% mannitol, 0.2% K₂HPO₄), 10% (v/v) horse serum and SLAD (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose, 50µM ammonium sulfate, 2% Bacto Agar). The dimorphic switching was performed using the protocol described elsewhere [23]. Briefly, the culture was grown overnight at 30˚C in YEPD broth before each study. The revived cells were harvested by centrifugation at 5000g rpm for 3 minutes and washed twice and incubated at 37˚C for 6h with PBS to induce starvation. After incubation, the cells were transferred to the required media for hyphal growth and hyphae were observed under microscope at magnification 40X.

Fig. (4). Effect of MB on mitochondrial function and redox status. (a) Spot assay demonstrating depleted growth of C. albicans in presence of MB (25µg/ml) when grown with glycerol as non fermentable source in contrast with the growth in presence of glucose. (b) Reversion in the growth of C. albicans at MIC80 concentration of MB when grown along with antioxidant AA at 5mM.

To check the antifungal effects of MB against C. albicans, we first of all performed drug susceptibility testings. Three independent methods to check the drug susceptibility were performed viz. spot assay, filter disc assay and broth microdilution assay. From both spot assay and filter disc assay, it was confirmed that MB was efficiently acting as antifungal against C. albicans at 100µg/ml (Fig. 1a and 1b). Broth microdilution assay which determined the MIC₈₀ value also showed similar result when performed against C. albicans (Fig. 1c). The antifungal activity of MB was further assessed against two clinical isolates of C. albicans referred as D1 and D2 and we found that both the strains displayed enhanced susceptibility to MB at 100µg/ml (Fig. 1). Thus, all the drug susceptibility testing results indicate that MB is inhibitory against reference as well as clinical isolates of C. albicans.

To further elaborate our study, we test whether MB has similar effects against non-albicans species of Candida as well. By performing the spot assay and disc diffusion assay, it was clear that MB was efficiently inhibiting the Candida tropicalis and Candida krusei when grown at the concentration of 200 and 300µg/ml respectively (Fig. 2a and 2b). Broth microdilution assay was also performed for the verification where the MIC₈₀ values against non-albicans species were found to be in the range of 200-300μg/ml and correlates with spot and filter disc assays (Fig. 2c). However, Candida glabrata and Canddia parapsilosis remained resistant to MB even at concentration above 600μg/ml (data not shown).

Overexpression of Multidrug Efflux transporter is one of the predominant mechanisms that is used by Candida to avoid antifungal drug action [24]. To study the role of major drug efflux pumps, if any, in the antifungal mechanism of MB, we performed Rhodamine 6G (R6G) efflux assay. R6G is well known substrate of efflux pumps and can be efficiently used to study the efflux activities. Our data suggest that there was no significant difference (P value > 0.5) in the extracellular R6G concentration in the absence (control) or presence of MB (Fig. 3).

Fig. (5). Effect of MB on membrane composition. (a) Spot assay demonstrating the attenuated growth of C. albicans in the presence of MB with membrane perturbing agent SDS (0.02% w/v). (b) Transmission electron micrographic image showing the tampered membrane integrity of C. albicans in presence of MB as described in Materials and methods. (c) Left panel shows UV spectrophotometric sterol profiles of C. albicans scanned between 220 and 300 nm from a culture grown for 16 hours with and without MB (25µg/ml). Right panel shows relative percentages of ergosterol content in the absence (control) and presence of MB (25μg/ml). Mean of % ergosterol levels is calculated as described in materials and methods normalized by considering the untreated control as 100 (absolute value of 0.002) ± SD of three independent sets of experiments are depicted on Y-axis and * depicts P value <0.05.

MB has been previously shown to disrupt the mitochondrial integrity [25], therefore, we checked whether mitochondrial functioning of Candida cells is also affected in the presence of MB. For this, spot assays were performed in the media where glycerol is acting as sole non-fermentable carbon source in place of glucose (fermentable sugar) and it was observed that the growth of C. albicans was inhibited in the presence of MB in comparison to the cells grown in the presence of glucose (Fig. 4a). Next, we tested whether the observed disturbances in mitochondria is also linked with any alteration in redox status of the cell. For this, Candida cells were spotted in the presence of ascorbic acid (AA) which is a known antioxidant. We observed that the antifungal activity of MB could be rescued in the presence of AA which suggests that the antifungal activity of MB is also linked with the alteration in the redox status (Fig. 4b).

Since many of the current therapeutic drug regimes target the membrane integrity, hence we checked whether MB has any influence on the membrane integrity of C. albicans. Firstly, we performed the spot assay with SDS (membrane perturbing agent) and found that the C. albicans was unable to grow in the presence of MB when grown in SDS (Fig. 5a). This was further confirmed through TEM experiment which clearly depicts that MB disrupts the membrane integrity of C. albicans (Fig. 5b). Furthermore, we assessed whether the alteration in the membrane integrity due to MB could also have any influence on its membrane composition. We estimated the ergosterol levels and found that there was decrement in ergosterol content significantly by 66% (P value ≤ 0.05) in presence MB (Fig. 5c).

Yeast to hyphal switching is also one of the major factors which govern the virulence in C. albicans. Thus the effect of MB on this morphological switching was checked by providing various hyphae inducing conditions in liquid as well as solid media at 37˚C. We observed that filamentation was completely lacking in MB treated cells and appears only in yeast form in contrast to the untreated Candida cells suggesting that MB is an active inhibitor of morphogenetic switching for this dimorphic fungus (Fig. 6).

Fig. (6). Effect of MB on Dimorphic Switching of C. albicans. Left panel shows Hyphal morphogenesis in the liquid hyphal inducing media (YEPD with 10% Serum and Spider media) in the absence (control) and presence of MB (25µg/ml) in the C. albicans (ATCC 10261) at 4 hours. Right panel shows hyphal morphogenesis in the solid hyphal inducing medium (Spider agar medium, 10% Serum and SLAD medium) in the absence (control) and presence of MB (25µg/ml) in the C. albicans (ATCC 10261) at 4 hours.