Quantification of the Adhesion Strength of Candida albicans to Tooth Enamel

Caries is one of the most prevalent diseases worldwide, which is caused by the degradation of the tooth enamel surface. In earlier research the opportunistic pathogen Candida albicans has been associated with the formation of caries in children. Colonization of teeth by C. albicans starts with the initial adhesion of individual yeast cells to the tooth enamel surface. In this study, we visualized the initial colonization of C. albicans yeast cells on pellicle-covered enamel by scanning electron microscopy. To quantitatively unravel the initial adhesion strength, we applied fluidic force microscopy-based single-cell force spectroscopy to examine the key adhesion parameters adhesion force, rupture length and de-adhesion work. We analyzed single saliva-treated or untreated yeast cells on tooth enamel specimens with or without salivary pellicle. Under all tested conditions, adhesion forces in the lower nanonewton range were determined. Furthermore, we have found that all adhesion parameters were enhanced on the pellicle-covered compared to the uncovered enamel. Our data suggest that initial adhesion occurs through a strong interaction between yeast cell wall-associated adhesins and the salivary pellicle. Future SCFS studies may show whether specific management of the salivary pellicle reduces the adhesion of C. albicans on teeth and thus contributes to caries prophylaxis.

Analysing Complex Oral Protein Samples: Complete Workflow and Case Analysis of Salivary Pellicles

Studies on small quantity, highly complex protein samples, such as salivary pellicle, have been enabled by recent major technological and analytical breakthroughs. Advances in mass spectrometry-based computational proteomics such as Multidimensional Protein Identification Technology have allowed precise identification and quantification of complex protein samples on a proteome-wide scale, which has enabled the determination of corresponding genes and cellular functions at the protein level. The latter was achieved via protein-protein interaction mapping with Gene Ontology annotation. In recent years, the application of these technologies has broken various barriers in small-quantity-complex-protein research such as salivary pellicle. This review provides a concise summary of contemporary proteomic techniques contributing to (1) increased complex protein (up to hundreds) identification using minute sample sizes (µg level), (2) precise protein quantification by advanced stable isotope labelling or label-free approaches and (3) the emerging concepts and techniques regarding computational integration, such as the Gene Ontology Consortium and protein-protein interaction mapping. The latter integrates the structural, genomic, and biological context of proteins and genes to predict protein interactions and functional connections in a given biological context. The same technological breakthroughs and computational integration concepts can also be applied to other low-volume oral protein complexes such as gingival crevicular or peri-implant sulcular fluids.

Influence of two polyphenols on the structure and lubrication of salivary pellicle: An in vitro study on astringency mechanism

This study investigated the influence of two polyphenols on the structure and lubrication of the salivary pellicle, aiming to extend the understanding of astringency mechanisms. The salivary pellicle was prepared by the adsorption of human whole saliva on the enamel substrate. Low-astringency catechin and high-astringency tannic acid were used as astringents. The changes induced by the two polyphenols in the structure and lubrication of the salivary pellicle were examined using quartz crystal microbalance with dissipation (QCM-D) and nano-indentation/scratch technique. The salivary pellicle suffers from changes in structure and physical properties owing to protein dehydration and protein-polyphenol complexation when encountering polyphenolic molecules, causing increases in the roughness and contact angle but a decrease in the load-bearing capacity. Therefore, the lubrication performance of the salivary pellicle is impaired, leading to an increase and fluctuation of the friction coefficient. The intensity of astringency has a strong positive correlation with the water contact angle, surface roughness, and friction coefficient of the salivary pellicle. In summary, astringency is a tactile perception driven by the roughness and wettability of the salivary pellicle rather than oral lubrication, and increased intraoral friction is an inevitable consequence of astringency. The findings of this study will help promote and assist the objective evaluation of astringency.

Inhibition of Candida albicans and Mixed Salivary Bacterial Biofilms on Antimicrobial Loaded Phosphated Poly(methyl methacrylate)

Biofilms play a crucial role in the development of Candida-associated denture stomatitis. Inhibition of microbial adhesion to poly(methyl methacrylate) (PMMA) and phosphate containing PMMA has been examined in this work. C. albicans and mixed salivary microbial biofilms were compared on naked and salivary pre-conditioned PMMA surfaces in the presence or absence of antimicrobials (Cetylpyridinium chloride [CPC], KSL-W, Histatin 5 [His 5]). Polymers with varying amounts of phosphate (0–25%) were tested using four C. albicans oral isolates as well as mixed salivary bacteria and 24 h biofilms were assessed for metabolic activity and confirmed using Live/Dead staining and confocal microscopy. Biofilm metabolism was reduced as phosphate density increased (15%: p = 0.004; 25%: p = 0.001). Loading of CPC on 15% phosphated disks showed a substantial decrease (p = 0.001) in biofilm metabolism in the presence or absence of a salivary pellicle. Salivary pellicle on uncharged PMMA enhanced the antimicrobial activity of CPC only. CPC also demonstrated remarkable antimicrobial activity on mixed salivary bacterial biofilms under different conditions displaying the potent efficacy of CPC (350 µg/mL) when combined with an artificial protein pellicle (Biotene half strength).

Acid tolerance in early colonizers of oral biofilms

Background In caries, low pH drives selection and enrichment of acidogenic and aciduric bacteria in oral biofilms, and development of acid tolerance in early colonizers is thought to play a key role in this shift. Since previous studies have focussed on planktonic cells, the effect of biofilm growth as well as the role of a salivary pellicle on this process is largely unknown. We explored acid tolerance and acid tolerance response (ATR) induction in biofilm cells of both clinical and laboratory strains of three oral streptococcal species (Streptococcus gordonii, Streptococcus oralis and Streptococcus mutans) as well as two oral species of Actinomyces (A. naeslundii and A. odontolyticus) and examined the role of salivary proteins in acid tolerance development. Methods Biofilms were formed on surfaces in Ibidi® mini flow cells with or without a coating of salivary proteins and acid tolerance assessed by exposing them to a challenge known to kill non-acid tolerant cells (pH 3.5 for 30 min) followed by staining with LIVE/DEAD BacLight and confocal scanning laser microscopy. The ability to induce an ATR was assessed by exposing the biofilms to an adaptation pH (pH 5.5) for 2 hours prior to the low pH challenge. Results Biofilm formation significantly increased acid tolerance in all the clinical streptococcal strains (P < 0.05) whereas the laboratory strains varied in their response. In biofilms, S. oralis was much more acid tolerant than S. gordonii or S. mutans. A. naeslundii showed a significant increase in acid tolerance in biofilms compared to planktonic cells (P < 0.001) which was not seen for A. odontolyticus. All strains except S. oralis induced an ATR after pre-exposure to pH 5.5 (P < 0.05). The presence of a salivary pellicle enhanced both acid tolerance development and ATR induction in S. gordonii biofilms (P < 0.05) but did not affect the other bacteria to the same extent. Conclusions These findings suggest that factors such as surface contact, the presence of a salivary pellicle and sensing of environmental pH can contribute to the development of high levels of acid tolerance amongst early colonizers in oral biofilms which may be important in the initiation of caries.

Inhibition of Candida albicans and Mixed Salivary Bacterial Biofilms on Antimicrobial Loaded Phosphated Poly(methyl methacrylate)

Biofilms play a crucial role in the development of Candida-associated denture stomatitis. Inhibition of microbial adhesion to PMMA and phosphate containing PMMA has been examined in this work. C. albicans and mixed salivary microbial biofilms were compared on naked and salivary pre-conditioned PMMA surfaces in the presence or absence of antimicrobials (cetyl pyridinium chloride [CPC], KSL-W, histatin 5 [his 5]). Polymers with varying amounts of phosphate (0-25%) were tested using four C. albicans oral isolates as well as mixed salivary bacteria and 24 h biofilms were assessed for metabolic activity and confirmed using Live/Dead staining and confocal microscopy. Biofilm metabolism was reduced as phosphate density increased (15%: P=0.004; 25%: P=0.001). Loading of CPC on 15% phosphated disks showed a substantial decrease (P=0.001) in biofilm metabolism in the presence or absence of a salivary pellicle. Salivary pellicle on uncharged PMMA enhanced the antimicrobial activity of CPC only. CPC also demonstrated remarkable antimicrobial activity on mixed salivary bacterial biofilms under different conditions displaying the potent efficacy of CPC (350 &micro;g/ml) when combined with an artificial protein pellicle (Biotene half strength).