Fig. 1. Effect of MA-PA combinations against Candida spp. biofilm. The graphs represent the percentage biofilm inhibition of MA-PA combinations in (A) C. albicans ATCC 90028, (B) CI 4, (C) CI 5, (D) CI 6, (E) C. tropicalis MTCC 186, (F) CI 1, (G) CI 2, and (H) CI 3 with red, black and green colours representing high (60–80 %), moderate (30–50 %), and low (0–30 %) level of biofilm inhibition in Candida spp., respectively.

Fig. 2. Effect of MA-PA combination on the viability of reference and clinical isolates of Candida spp. such as (A) C. albicans ATCC 90028 (B) CI 4 (C) CI 5 (D) CI 6 (E) C. tropicalis MTCC 186 (F) CI 1 (G) CI 2 and (H) CI 3. When compared to untreated control, the viability was not affected in MA + PA treatment with ≤125 + 200 μg mL⁻¹.

Fig. 3. Inhibitory effect of MA-PA combination at 31.25 + 50 μg mL⁻¹ on virulence aspects of A) C. albicans and B) C. tropicalis, (i) Light micrographs representing antibiofilm effect of MA-PA combination against Candida spp. at the magnification of 400 × . (ii) CLSM images showing the effect of MA-PA combinatorial inhibition of biofilm construction and hyphae of Candida spp. Scale bar – 50 μm. (iii) Filamentous growth of Candida spp. in the absence and presence of MA-PA combination in spider agar. Reduced filamentous growth was observed in MA-PA combinations treated Candida spp. in both reference and clinical isolates. (iv) Effect of MA-PA on Candida spp. extracellular protease on BSA solid medium. Reduced zone of precipitation was observed in treated reference strains of Candida spp. and the inhibition was moderate in clinical isolates.

Fig. 4 Download : Download high-res image (195KB)Download : Download full-size image Fig. 4. A) Effect of MA-PA combinations on lipases production in reference and clinical isolates of C. albicans and C. tropicalis. Bar graph represents percentage inhibition of lipase in the treated Candida strains with error bars indicate standard deviation (n = 6). Single asterisk represents the statistical significance of lipase reduction in treated Candida strains with p < 0.05. B) Gene expression analysis of untreated and MA-PA combination treated C. albicans at 31.25 + 50 μg mL⁻¹. Expression of virulence genes was normalized with housekeeping genes and the relative fold change in differential expressions was calculated using 2-ΔΔCT method. The values are expressed as means and error bars indicate standard deviation (n = 3). The sign “*” indicates the statistical significance of gene expression regulation in treated C. albicans with p < 0.05.

Fig. 5. Dose dependent effects of A) MA (250, 125, 62.5 and 31.25 μg mL⁻¹) and B) PA (400, 200, 100 and 50 μg mL⁻¹) on the survival of Danio rerio (Zebrafish) for 7 days by Kaplan-Meier method. The significance of survival curves were calculated using chi-square test of Log rank and Gehan-Breslow-Wilcoxon method.

Fig. 6. Anti-infective efficacy of individual and combinatorial effect of MA and PA against systemic candidiasis in Zebrafish model system. A) Experimental setup for establishing C. albicans and C. tropicalis mediated systemic candidiasis for 12 h and post infection treatment with individual and combinatorial effect of MA and PA. The concentrations of individual compounds for the treatment were 125 μg mL⁻¹ of MA and 200 μg mL⁻¹ of PA and for combination, 31.25 + 50 (F1) and 62.5 + 100 μg mL^-1 (F2) of MA + PA were tested. B) Compared to normal Zebrafish, inflammatory responses were spotted at a) respiratory, b) abdominal, c) urinary tract and d) skin in both C. albicans and C. tropicalis infected animals. Recovery from the inflammation at various regions could be visualized in all treated animals. Anti-pathogenic potency of MA, PA, F1 and F2 against C) C. albicans and D) C. tropicalis infections in Zebrafish by survival assay. Compared to infected controls, the survival percentage increased in all treated animals.

Fig. 7. Quantitative fungal burden in A) C. albicans and B) C. tropicalis infected Zebrafish. Compared to the infection controls, MA, PA, F1 and F2 treatment revealing decreased level of fungal load in Zebrafish. The values are expressed as means and error bars indicate standard deviation (n = 3). The signs “**” and “***” indicates the statistical significance with p < 0.01 and p < 0.001, respectively.

Fig. 8. Histopathology of vital organs on 3rdday of post infections. A) Histopathology analysis of Gills, Normal control – regular structure of (a) primary lamella, (b) secondary lamella and (c) chloride cells. Infected control – abnormalities such as d) Merging of lamellae, e) disorganization and f) Thickening of secondary lamella. B) Histopathology analysis of Kidney, Normal Control: Black arrow indicates the normal histology of kidney cells with regular a) renal tubules, b) Glomeruli and c) hematopoietic tissue. C. albicans infected controls – (d) dilation of glomeruli, (e) degeneration of renal tubules. (g) Yellow arrows indicate the debris of necrotic epithelium cells in C. tropicalis infected kidney. (C) Histopathology analysis of Spleen, Normal control – regular assembling of (a) Red pulp cells and (b) ellipsoidal sheath. In C. albicans infected controls, (c) expansion of ellipsoidal sheath was observed. But in C. tropicalis infected controls, (d) leisions in ellipsoidal sheath was observed. (D) Histopathology analysis of Intestine, Normal control – normal structure of (a) goblet cells and (b) columnar shaped cells. In both Candida spp. infected intestine, (c) hyperplasia of goblet cells and (d) deformation of columnar cells was observed. In MA, PA and F2 treated animals, the recovery rate of spotted abnormalities in all organs was rapid but in case of F1 treated, the revival was moderate.

Fig. 9. SEM analysis of Zebrafish skin morphology with Magnification of 1000 × .A) In C. albicans and C. tropicalis infections, inflamamtions and colonization in the skin was observed, compared to normal control. Red and yellow arrows indicate the inflammatory sites and colonization of Candida spp., respectively. In Post treatment of B) C. albicans and C) C. tropicalis infection, inflammatory responses were reduced in all treated animals.

Fig. 10. Inhibitory effect of individual and combinations of MA and PA against A) haemolysin production of C. albicans and C. tropicalis with 10 % SDS as positive control. Compared to control, haemolysin was significantly reduced in all treated samples. B) Representative image showing anti-haemolysin ability of MA, PA, F1 and F2. C) Effect of individual and combinations of MA and PA on the survival of Candida spp. in human blood. The values are expressed as means and error bars indicate standard deviation (n = 3). The sign “*” indicates the statistical significance with p < 0.05.