Surface thermodynamic homeostasis of salivary conditioning films through polar–apolar layering

Salivary conditioning films (SCFs) form on all surfaces exposed to the oral cavity and control diverse oral surface phenomena. Oral chemotherapeutics and dietary components present perturbations to SCFs. Here we determine the surface energetics of SCFs through contact angle measurements with various liquids on SCFs following perturbations with a variety of chemotherapeutics as well as after renewed SCF formation. Sixteen-hour SCFs on polished enamel surfaces were treated with a variety of chemotherapeutics, including toothpastes and mouthrinses. After treatment with chemotherapeutics, a SCF was applied again for 3 h. Contact angles with four different liquids on untreated and treated SCF-coated enamel surfaces were measured and surface free energies were calculated. Perturbations either caused the SCF to become more polar or more apolar, but in all cases, renewed SCF formation compensated these changes. Thus, a polar SCF attracts different salivary proteins or adsorbs proteins in a different conformation to create a more apolar SCF surface after renewed SCF formation and vice versa for apolar SCFs. This polar–apolar layering in SCF formation presents a powerful mechanism in the oral cavity to maintain surface thermodynamic homeostasis—defining oral surface properties within a narrow, biological range and influencing chemotherapeutic strategies. Surface chemical changes brought about by dietary or chemotherapeutic perturbations to SCFs make it more polar or apolar, but new SCFs are rapidly formed compensating for changes in surface energetics

Bacterial Density and Biofilm Structure Determined by Optical Coherence Tomography

Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104508#pone.0104508-Buijssen2" target="_blank">[14]</a>.</p

Restoration of voice after total laryngectomy using a tracheostoma valve.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104508#pone.0104508-Busscher1" target="_blank">[7]</a>.</p

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

<p>A. In situ cross section of a mixed species biofilm of <i>C. tropicalis</i> combined with <i>L. crispatus</i> by OCT. Average biofilm thickness is 80±24 µm. In situ cross section of a mixed species biofilm of <i>C. albicans</i> combined with <i>R. dentocariosa</i> by OCT. Average biofilm thickness is 26±8 µm. B. A cross section of a mixed species biofilm of <i>C. tropicalis</i> combined with <i>L. crispatus</i> by CLSM after live/dead staining. Average biofilm thickness is 109±29 µm. C. A cross section of a mixed species biofilm of <i>C. albicans</i> combined with <i>R. dentocariosa</i> 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.</p

Total number of CFU/cm² (<i>Candida</i> and bacteria) grown on silicone-rubber in an eight day time period after inoculation with a combination of <i>C. albicans</i> or <i>C. tropicalis</i> and a commensal bacterial strain or a <i>lactobacillus</i> strain, together with the percentage prevalence of the yeast in the final biofilm.

<p>During the growth period, biofilms were exposed to nutritional feast and famine. All results are from triplicate experiments with separate microbial cultures and are presented ± SD. a ≠ b ≠ c ≠ d ≠ e ≠ f at p<0.05 analyzed per column and separately for <i>C. albicans</i> and <i>C. tropicalis</i>.</p

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

<p>A. Time sequence of biofilm formation in silicone rubber tubes from mixed cultures of <i>Candida</i> species and a bacterial strain during exposure to feast and famine. B. Scanning electron micrograph of <i>C. tropicalis</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104508#pone.0104508-Busscher1" target="_blank">[7]</a>.</p

Percentage hyphae occurring in <i>Candida</i> biofilms grown in the absence or presence of selected bacterial strains on silicone-rubber in an eight day time period.

<p>During the growth period, biofilms were exposed to nutritional feast and famine. All results are from triplicate experiments with separate microbial cultures and are presented ± SD. Data are normalized with respect to the average percentage of hyphae in <i>Candida</i> biofilms grown in absence of bacteria. a≠b at p<0.05.</p