Evaluation of Anti-Biofilm Capability of Cordycepin Against Candida albicans

Abstract

Introduction: The opportunistic pathogen Candida albicans can form biofilms, resulting in drug resistance with great risk to medical treatment.

Methodology: We investigated the ability of C. albicans to form biofilms on different materials, as well as the inhibitory and eradicating effects of cordycepin on biofilm. The action mechanism of cordycepin against biofilm was studied by crystal violet staining, XTT [2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction method, phenol-sulfuric acid method, cellular superficial hydrophobicity (CSH) assay, and confocal laser scanning microscope observation. We also evaluated the acute toxicity of cordycepin in vivo.

Results: The results showed facile formation of biofilms by C. albicans on polypropylene. The 50% minimum inhibitory concentration (MIC₅₀) of cordycepin was 0.062 mg/mL. A concentration of 0.125 mg/mL significantly decreased biofilm formation, metabolic activity, secretion of extracellular polysaccharides, and relative CSH. Cordycepin could inhibit biofilm formation at low concentration without affecting fungal growth. In addition, cordycepin effectively eradicated 59.14% of mature biofilms of C. albicans at a concentration of 0.5 mg/mL. For acute toxicity, the LD₅₀ (50% of lethal dose) of cordycepin was determined as higher than 500 mg/kg for mice.

Conclusion: The results of this study show that cordycepin significantly inhibited and eradicated biofilms by decreasing metabolic activity, the ratio of living cells, the hydrophobicity, and damaging the extracellular polysaccharides of biofilm. These findings should facilitate more effective application of cordycepin and suggest a new direction for the treatment of fungal infections.

Keywords: Candida albicans; biofilm; cordycepin; eradication; inhibition.

Figure 1

Growth curves of C. albicans in the absence or presence cordycepin.

Figure 2

Effects of cordycepin on biofilms of C. albicans. (A) Inhibitory effects on biofilm formation; (B) Eradicating effects on mature biofilms. Error bars represent the standard deviations, and different letters represent statistical differences among bars (n = 3, P < 0.05).

Figure 3

CLSM images of C. albicans biofilm treated with different concentrations of cordycepin. (A), (C) and (E) show the effects of 0, 0.031, and 0.125 mg/mL of cordycepin on the formation of biofilms and live/dead cells ratio. (B), (D) and (F) show the fluorescence intensities of live (green)/dead (red) cells in biofilms treated with 0, 0.031, and 0.125 mg/mL of cordycepin, respectively. Viable cells appear green due to FDA staining, and cells with damaged membranes appear red due to PI staining.

Figure 4

Changes of mature biofilms and live/dead cells before and after cordycepin treatment. (A), (C) and (E) show the effects of 0, 0.125, and 0.5 mg/mL of cordycepin on the eradication of mature biofilms and live/dead cells. (B), (D) and (F) show the fluorescence intensities of live (green)/dead (red) cells in biofilms treated with 0, 0.125, and 0.5 mg/mL of cordycepin, respectively. Viable cells appear green due to FDA staining, and cells with damaged membranes appear red due to PI staining.

Figure 5

Effects of cordycepin on the metabolic activity of C. albicans biofilm. (A) Effects on biofilm formation; (B) Effects on mature biofilms. Error bars represent the standard deviations, and different letters represent statistical differences among bars (n = 3, P < 0.05).

Figure 6

Effects of cordycepin on EPS secretion of C. albicans biofilm. (A) Effects on EPS secretion in biofilm formation; (B) Effects on EPS secretion in mature biofilms. Error bars represent the standard deviations, and different letters represent statistical differences among bars (n =3, P < 0.05).

Figure 7

Distribution of polysaccharides and dead cells in cordycepin-treated biofilms. (A), (C) and (E) show the distribution of EPS (green) and dead cells (red) in biofilms treated with 0, 0.031, and 0.125 mg/mL of cordycepin. (B), (D) and (F) show the distribution of EPS (green) and dead cells (red) in mature biofilms treated with 0, 0.125, and 0.5 mg/mL of cordycepin.

Figure 8

Effects of cordycepin on CSH of C. albicans biofilm. (A) Addition of cordycepin before biofilm formation; (B) Addition of cordycepin after biofilm formation. Error bars represent the standard deviations, and different letters represent statistical differences among bars (n = 3, P < 0.05).