Ex vivo decontamination of yeast-colonized dentures by iodine-thiocyanate complexes

Abstract

Introduction: Under well-defined experimental conditions, and in the presence of hydrogen peroxide, lactoperoxidase produces stable iodine-thiocyanate complexes that have antimicrobial properties. A novel process was developed to short circuit the consumption of hydrogen peroxide by microbial catalases by producing iodine-thiocyanate complexes prior to contact with microorganisms, with the aim of being able to decontaminate the ex vivo dentures colonized by yeasts.

Materials and methods: Teabags containing lactoperoxidase adsorbed on inert clay beads were immersed for 1 minute in phosphate buffer solution (0.1 M pH 7.4) containing 5.2 mM potassium iodide, 1.2 mM potassium thiocyanate, and 5.5 mM hydrogen peroxide. After removing the adsorbed lactoperoxidase, the stability and efficacy of iodine-thiocyanate complexes for Candida-colonized denture decontamination were verified. Investigations were performed in vitro on Candida albicans ATCC 10231 and on clinical isolates from 46 dentures. A Candida plate count was performed after a 24-hour incubation at 37°C on Sabouraud-chloramphenicol or CHROMagar solid media; then, the yeast growth was evaluated in Sabouraud broth by turbidimetry and biofilm biomass by crystal violet staining.

Results: In vitro tests demonstrated the effectiveness of the oxidant solution in sterilizing a suspension of 10⁶Candida cells per milliliter after a 5-minute incubation. A single ex vivo immersion of contaminated dentures in a solution of iodine-thiocyanate complexes led to a decrease of at least 1 log unit in the number of colony-forming units in 58.3% of the tested dentures, while immersing in water alone had no effect on denture colonization (significant c2: p = 0.0006).

Conclusion: These data suggest a promising new strategy for decontamination of dentures.

Keywords: Candida; biofilm; hygiene; lactoperoxidase; oral cavity.

Figure 1

DTNB oxidation by iodine–thiocyanate complexes: relation between absorbance at 412 nm and oxidant concentration in the reaction medium (mean ± SEM). Inset: relation between absorbance and oxidant solution volume transferred in the reaction medium. The various volumes were made up to 0.5 mL with phosphate buffer and then added to the same reagent volume (2 mL) to obtain a 2.5 mL final volume. Abbreviations: DTNB, dithiobisnitrobenzoic acid; SEM, standard error of the mean.

Figure 2

Evaluation of iodine–thiocyanate complexes concentration in solutions stored at 4°C (average of 4 replicates for each of the solutions tested). Oxidant production was assayed by the DTNB method in 47 different oxidant solutions after variable storage times (from 1 day to 132 weeks). Initial concentrations were 1929 ± 46 μM (mean ± SEM, n = 8). Dotted line indicates the value corresponding to the mean − 3 SD of the initial concentrations. Abbreviations: DTNB, dithiobisnitrobenzoic acid; SEM, standard error of the mean.

Figure 3

Candida inhibition after a 5-minute incubation at room temperature by various combinations of reagents used to form iodine–thiocyanate complexes. Candida albicans ATCC 10231 survival rate was evaluated by counting colony-forming units (CFUs) after cultivation on Sabouraud agar. Lp was clay-adsorbed inside a teabag. Data are expressed as the percentage of CFUs counted after incubation in 0.1 M phosphate buffer (pH 7.4). Error bars indicate the standard error of the mean (N = 4). Abbreviation: Lp, lactoperoxidase; H₂O₂, hydrogen peroxide; KSCN, potassium thiocyanate; KI, potassium iodide.

Figure 4

Effect of different concentrations of iodine–thiocyanate complexes on Candida albicans growth (ATCC 10231 and 6 clinical isolates) after a 5-minute incubation at room temperature in the wells of a 96-well polystyrene plate. Yeast growth was then evaluated after addition of Sabouraud broth and incubation at 37°C for 24 hours. Data are expressed as the percentage of turbidity observed in the same conditions after incubation in 0.1 M phosphate buffer (pH 7.4). The different oxidant concentrations were compared with the paired control by ANOVA complemented by a Dunnett’s multiple comparison test (***p < 0.001). Error bars indicate the standard error of the mean (N = 6). Abbreviation: ANOVA, analysis of variance.

Figure 5

Effect of different concentrations of iodine–thiocyanate complexes on biofilm formation by Candida albicans strains (ATCC 10231 and 6 clinical isolates) after a 5-minute incubation at room temperature in the wells of 96-well polystyrene plate. Biofilm was then evaluated by staining the attached biomass after incubation at 37°C for 24 hours in Sabouraud broth. Data are expressed as the percentage of attached biomass observed in the same conditions after incubation in 0.1 M phosphate buffer (pH 7.4). The different oxidant concentrations were compared with the paired control by ANOVA complemented by a Dunnett’s multiple comparison test (***p < 0.001). Error bars indicate the standard error of the mean (N = 6). Abbreviation: ANOVA, analysis of variance.

Figure 6

Sabouraud agar plates inoculated by swabbing contaminated acrylic resin pieces and incubated for 48 hours at 37°C. The resin pieces were previously subjected to different processes. (A) Uncontaminated acrylic resin piece processed to attest the sterility of handling. (B) Contaminated (Candida albicans ATCC 10231) acrylic resin piece immersed for 30 minutes in phosphate buffer (0.1 M, pH 7.4) at room temperature. (C) Contaminated (C. albicans ATCC 10231) acrylic resin piece immersed for 30 minutes in the solution of iodine–thiocyanate complexes at room temperature. The data are representative of 4 independent experiments.