Voice prosthetic biofilm formation and Candida morphogenic conversions in absence and presence of different bacterial strains and species on silicone-rubber

Abstract

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.

Figure 1. Restoration of voice after total laryngectomy using a tracheostoma valve.

Schematic drawing of voice restoration after laryngectomy using a tracheostoma (left) and SEM of a mixed species (yeast and bacteria) biofilm formation on the tracheostoma valve after insertion for 40 days into a patient (right, bar marker indicates 600 µm). Taken with permission from .

Figure 2. Flow diagram of the feast and famine cycle and resulting hyphal ingrowth.

A. Time sequence of biofilm formation in silicone rubber tubes from mixed cultures of Candida species and a bacterial strain during exposure to feast and famine. B. Scanning electron micrograph of C. tropicalis hyphae penetrating into silicone-rubber after 12 days growth under conditions of feast and famine. Bar markers represent 10 µm and 2.5 µm for the left and right panel, respectively. Taken with permission from .

Figure 3. Visualization of mixed species biofilms on silicone-rubber tubes obtained using OCT and CLSM.

A. In situ cross section of a mixed species biofilm of C. tropicalis combined with L. crispatus by OCT. Average biofilm thickness is 80±24 µm. In situ cross section of a mixed species biofilm of C. albicans combined with R. dentocariosa by OCT. Average biofilm thickness is 26±8 µm. B. A cross section of a mixed species biofilm of C. tropicalis combined with L. crispatus by CLSM after live/dead staining. Average biofilm thickness is 109±29 µm. C. A cross section of a mixed species biofilm of C. albicans combined with R. dentocariosa by CLSM after live/dead staining. Average biofilm thickness is 7± µm. The bars in A and C denote 500 µm, while in B and D bars represent 75 µm.

Figure 4. Association between yeast and lactobacilli in an in vivo formed biofilm on a voice prosthesis.

Overlay-images of a biofilm from an explanted voice prosthesis (life time 318 days) hybridized with the FITC-labelled EUK516 probe indicating all yeasts (with hyphae) and Cy3-labelled Lab158 probe illustrating presence of lactobacilli. The high magnification panel on the right clearly shows the association of lactobacilli (red) and yeasts (green). Bars equal 20 µm and 5 µm for the left and right panels, respectively. Taken with permission from .