Inhibitory effect of a synthetic human β-defensin-3-C15 peptide on Candida albicans biofilm

Abstract

Objectives. The purpose of this study was to compare the inhibitory effect of a synthetic peptide comprising 15 amino acids of human β-defensin 3 (HBD3-C15) with chlorhexidine (CHX) or calcium hydroxide (CH) against C. albicans biofilm.

Methods. To determine the minimal antifungal concentration (MAC), C. albicans was grown on cover glass bottom dishes or human dentin disks for 48 h, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200 and 300 μg/ml), CH (100 μg/ml) or Nys (20 μg/ml) for 7 days at 37℃. Using confocal laser scanning microscopy (CLSM) and field-emission scanning electron microscopy (FE-SEM), MAC was determined. To compare the inhibitory effect of HBD3-C15 peptide against C. albicans biofilms with conventional intracanal medicaments, C. albicans was also grown on cover glass bottom dishes or human dentin disks for 7 days, and then treated with HBD3-C15 (100 μg/ml, MAC), non-functional peptide (NP, 100 μg/ml), CH (100 μg/ml), 1% CHX, or saline for 7 days at 37 ℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer™ Biofilm viability assay, and observed by CLSM. On dentin disk, normal, diminished, or ruptured cells were observed by FE-SEM. The results were subjected to two-tailed t-test, one-way analysis variance and post hoc test at a significance level of P=0.05.

Results. C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥100 μg/ml). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥100 μg/ml) showed significant fungicidal activity compared to CH group (P < .05). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. FE-SEM demonstrated that the C. albicans aggregated in HBD3-C15 (25 μg/ml) treated group and was disrupted in more than 100 g/ml concentration of HBD3-C15 (MAC). Live/Dead Biofilm viability assay and CLSM demonstrated that HBD3-C15 treated biofilms had a significantly less biovolume than CH, NP, and saline (P < .05), but had no significant difference from the CHX-treated group (P > .05). FE-SEM demonstrated that there was a marked decrease in aggregations of cells and biofilm and wrinkled or ruptured cells were frequently observed in the groups of CHX and HBD3-C15.

Conclusions. Synthetic HBD3-C15 peptide (100 μg/ml) exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm growth, but had no significant difference compared to CHX.