Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine
Pusateri, Christopher R.
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Candida albicans cells in a biofilm are resistant to many antifungal agents, however their sensitivity to natural antifungal peptides is unknown. Salivary histatins (Hsts) and defensins (hBDs) are naturally-occurring peptides in human saliva with antifungal activity. Objectives. (1) To evaluate the effect of Hst 5 on C. albicans resuspended from a biofilm compared to planktonic cells; (2) To evaluate the potential for surface treatment of denture acrylic with Hst 5 and hBD-3 to inhibit adherence of C. albicans and subsequent biofilm development. Methods. Heat-cured denture acrylic disks (Ivocap and Lucitone 199) were placed in polystyrene wells, preconditioned with 300μl saliva for 30 minutes, and inoculated with C. albicans cells (10 6 cells/ml) for 1h, at 37°C. Non-adherent cells were removed by washing, and YPD growth medium was placed over the disks and incubated for 24, 48, and 72 h at 37°C. Biofilm cells were recovered from disks using a cell scraper, and quantified under phase-contrast microscopy. A candidacidal assay was performed on cells recovered from 48-hour-biofilms and on planktonically-grown cells using Hst 5 (15.5μM, 31.25μM, 62μM). For the cell adhesion assay, disks were pre-coated with either 0.12% chlorhexidine gluconate, 50μM Hst 5, or 0.6μM hBD-3 and cell numbers were quantified at 24, 48, and 72 hours. Results. No significant difference was found in biofilm cell numbers on Ivocap and Lucitone acrylic disks at any time interval. There was no significant difference in sensitivity to Hst 5 of C. albicans cells resuspended from Lucitone acrylic compared to planktonic cells ( p > 0.05). C. albicans cells resuspended from Ivocap acrylic demonstrated decreased sensitivity to Hst 5 compared to planktonic cells or cells grown on Lucitone acrylic at the lowest concentration of Hst 5 (15.5μM) ( p < 0.03). Acrylic pre-coated with 0.12% chlorhexidine gluconate significantly inhibited biofilm development of C. albicans at all time intervals ( p < 0.05). Hst 5 significantly inhibited biofilm development of C. albicans at later stages of growth (72 hours) ( p = 0.011). Acrylic pre-coated with hBD-3 resulted in a non-significant inhibition of biofilm development of C. albicans. Conclusion. C. albicans cells recovered from biofilms on Lucitone denture acrylic have equivalent sensitivity to Hst 5 as planktonic cells; however, sensitivity was reduced on biofilms grown on Ivocap. These data suggest that substrate materials may affect the sensitivity of biofilm cells to killing by Hst 5. Pre-coating acrylic with chlorhexidine limited biofilm development of C. albicans at all time intervals, while Hst 5 limited development at later stages of growth. hBD-3 was less effective at inhibiting biofilm growth.