3-Hydroxy coumarin demonstrates anti-biofilm and anti-hyphal efficacy against Candida albicans via inhibition of cell-adhesion, morphogenesis, and virulent genes regulation

Abstract

Candida albicans, a common fungus of human flora, can become an opportunistic pathogen and causes invasive candidiasis in immunocompromised individuals. Biofilm formation is the prime cause of antibiotic resistance during C. albicans infections and treating biofilm-forming cells is challenging due to their intractable and persistent nature. The study intends to explore the therapeutic potential of naturally produced compounds by competitive marine bacteria residing in marine biofilms against C. albicans biofilm. To this end, 3-hydroxy coumarin (3HC), a compound identified from the cell-free culture supernatant of the marine bacterium Brevundimonas abyssalis, was found to exhibit anti-biofilm and anti-hyphal activity against both reference and clinical isolates of C. albicans. The compound demonstrated significant inhibitory effects on biofilms and impaired the yeast-to-hyphal transition, wrinkle, and filament morphology at the minimal biofilm inhibitory concentration (MBIC) of 250 µg mL^-1. Intriguingly, quantitative PCR analysis of 3HC-treated C. albicans biofilm revealed significant downregulation of virulence genes (hst7, ume6, efg1, cph1, ras1, als1) associated with adhesion and morphogenesis. Moreover, 3HC displayed non-fungicidal and non-toxic characteristics against human erythrocytes and buccal cells. In conclusion, this study showed that marine biofilms are a hidden source of diverse therapeutic drugs, and 3HC could be a potent drug to treat C. albicans infections.

Figure 1

Screening of commercially available compounds identified through HR-LCMS analysis in cell-free culture supernatant (CFCS) of a marine bacterial isolate Brevundimonas abyssalis for their anti-biofilm potential against C. albicans. Bar graph depicts the biofilm inhibition (%) and the line graph shows planktonic cells absorbance at 600 nm. Two compounds namely 3-hydroxy coumarin (3HC) and 11-amino undeconoic acid displayed potent anti-biofilm activity against C. albicans. Error bars represent standard deviations from the mean of experimental triplicates.

Figure 2

Determination of minimal biofilm inhibitory concentration (MBIC) and non-fungicidal concentration of 3-hydroxy coumarin (3HC). MBIC denotes maximum biofilm inhibition with negligible loss in planktonic cells. (a) A dose-dependent reduction biofilm was observed upon treatment with 3HC. The concentration of 250 µg mL⁻¹ was chosen as MBIC as biofilm formation was hindered > 95% without any loss in the planktonic growth. (b) Measurement of C. albicans metabolic viability after 24 h growth in the presence and absence of 3HC, using Alamar blue. The image shows a significant reduction in cell viability at 1000 µg mL⁻¹ (MIC) and no reduction in viability at sub-MICs. Error bars indicate the mean values of three experimental triplicates. (c) Representative 96-well microtiter plate stained with Alamar blue displaying the true metabolic state of 3HC-treated C. albicans cells.

Figure 3

Anti-biofilm efficacy of varying 3-hydroxy coumarine (3HC) concentrations against reference and clinical isolates of C. albicans. (a) Effect of 3HC on biofilm inhibition of C. albicans strains. The biofilm formation of all the four tested C. albicans isolates were hindered > 90% at MBIC (blue bar) of 3HC. Error bars represent standard deviation from the mean (n = 9). (b) Effect of different concentrations of 3HC on metabolic viability of biofilms formed by various C. albicans strains (×200 magnification). A dose dependent reduction in biofilm viability and was observed in all the four isolates of C. albicans. Treatment at MBIC had significant reduction in biofilm biomass viability. (c) Representative FESEM images of untreated C. albicans 90028 and those exposed to 3HC at 2 MBIC, MBIC, ½ MBIC and ¼ MBIC showing complete dose-dependent biofilm reduction and complete inhibition of hyphal formation.

Figure 4

Inhibition of primary virulence factors (filament growth and wrinkle morphology) by the treatment of 3HC. Representative images of Spider medium agar plates show colony morphology exposed to 2 MBIC, MBIC, ½ MBIC and ¼ MBIC of 3HC. After the treatment the colony appeared to have (a) smooth surface and (b) reduced filament growth compared to that of their respective control. False colour has been applied to the images (b) to represent the results clear.

Figure 5

Efficacy of 3-Hydroxy coumarin (3HC) on the morphological transition and mature biofilm disintegration of C. albicans cells. Microscopic images showing (a) inhibition of hyphal formation and (b) reversion of preformed hyphae to yeast cells at 2 MBIC, MBIC, ½ MBIC and ¼ MBIC. (c) Disintegration of mature biofilm by 3HC at various concentrations. Maximum dislodging of the pre-formed biofilm was observed at 2 MBIC. Each experiment was repeated at least three times and representative images are presented here.

Figure 6

Non-toxic effect of 3HC on (a) human buccal epithelial cells (HBECs) and (b) human erythrocytes. Hydrogen peroxide and Triton X-100 were used as positive controls for HBECs and erythrocytes, respectively. Both the cells treated with different concentrations of 3HC (62.5 to 500 µg mL⁻¹) exhibited no toxic effect, except for the positive control. VC denotes vehicle control (ethanol).