Candida albicans biofilm on titanium: effect of peroxidase precoating

Abstract

The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil) was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2-21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 10(6) blastoconidia per gram of titanium powder (n = 30) and 0.50 ± 0.04 × 10(6) blastoconidia per cm(2) of titanium foil (n = 12). The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate), Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated) and liquid environment (containing peroxidase substrates) to limit C. albicans biofilm formation.

Keywords: adhesion; material; oral; yeast.

Figure 1

MTT procedure. Top: Rate of tetrazolium salt MTT reduction as a function of substrate concentration in the presence of a fixed blastoconidia number (Cartesian presentation of data with double reciprocal plot displayed in the insert). Bottom: Reduction of tetrazolium salt MTT as a function of Candida albicans blastoconidia numbers (data for the lowest values are presented in the insert with more appropriate coordinates).

Figure 2

Washing process evaluation for different materials (titanium powder and foil, resin foil) after a two-day incubation in a contaminated environment. S_i: first supernatant after a two-day incubation; W_1–3: supernatant after each of the three washing steps. Note: After incubation (two days), the immersed materials (titanium powder, titanium, and resin foil) were washed three times (W_1–3) with sterile broth. The turbidity of the subsequent supernatant after sedimentation was monitored at 600 nm. Full columns represent assays where titanium powder has been washed immediately after contamination by a yeast liquid culture. Data are expressed as the percentage of the absorbance (A 600 nm) measured in the supernatant after a two-day incubation.

Figure 3

A) Candida albicans blastoconidia adherent to titanium granule (magnification X400 in the absence of any stain procedure) after a one-week incubation and three washings. B) Titanium grains surrounded by filamentous structures (hyphae and pseudohyphae) after a three-week incubation. C) Titanium particles (after three weeks of incubation and four washings) with some remaining blastoconidia and trabecular structure.

Figure 4

Time course of yeast biomass in the planktonic phase (top) and in the anchored phase (bottom). Note: Development of yeast biomass was weekly monitored for three weeks in the planktonic phase (absorbance at 600 nm) and in the anchored phase (absorbance at 570 nm after MTT procedure).

Figure 5

Yeast recovery from deposits onto titanium foil. Note: Droplets (20 μL of sterile water containing from 10 to 200 blastoconidia) were deposited onto titanium foil and incubated at 37°C for 30 minutes. Candida albicans recovery was evaluated after rubbing the foil prepared in the following conditions: a) blastoconidia droplets were directly deposited onto foil; b) blastoconidia droplets were deposited onto lactoperoxidase-coated titanium; and c) blastoconidia droplets with added G/GOD were deposited onto lactoperoxidase-coated titanium, d) blastoconidia droplets with added G/GOD/KI were deposited onto lactoperoxidase-coated titanium (n = 5 for each condition). Data were analyzed by an ANOVA test (P = 0.0009) completed by a Dunnett’s multiple comparison test (^***P < 0.001). Abbreviations: G, glucose; GOD, glucose oxidase; LP, lactoperoxidase; I⁻, iodide; SCN⁻, thiocyanate.