Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

Oil droplet formation on pellicle covered tooth surfaces studied with environmental scanning electron microscopy.

Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.

Protein Profile of the Acquired Enamel Pellicle after Rinsing with Whole Milk, Fat-Free Milk, and Water: An in vivo Study.

This study detected changes in the protein profile of the acquired enamel pellicle (AEP) formed in vivo after rinsing with whole milk, fat-free milk, or water. Nine subjects in good oral condition took part in the study. The acquired pellicle was formed in the morning, for 120 min, after prophylaxis with pumice. Following this, the volunteers rinsed with 10 mL of whole milk, fat-free milk, or deionized water for 30 s, following a blinded crossover protocol. After 60 min, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry. The obtained tandem mass spectrometry spectra were searched against a human protein database (Swiss-Prot). The proteomic data related to protein quantification were analysed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected from all groups. Forty-nine were common to all 3 groups, while 72, 62, and 49 were specific to the groups rinsing with whole milk, fat-free milk, and water, respectively. Some were typical components of the AEP, such as cystatin-B, cystatin-SN, isoforms of α-amylase, IgA and IgG, lysozyme C, protein S100 A78, histatin-1, proline-rich protein 27, statherin, and lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in defence of the organism against pathogens. Distinct proteomic profiles were found in the AEP after rinsing with whole or fat-free milk, which could have an impact on bacterial adhesion and tooth dissolution. The use of fat-free milk could favourably modulate the adhesion of bacteria to the AEP as well as biofilm formation when compared with whole milk.

Oral astringent stimuli alter the enamel pellicle's ultrastructure as revealed by electron microscopy.

OBJECTIVES: This electron microscopic study aimed at investigating effects of oral astringent stimuli on the enamel pellicle's morphology. METHODS: Pellicles were formed in situ within 30min on bovine enamel slabs, fixed to individuals' upper jaw splints. The pellicle-coated specimens were immersed in vitro in seven diverse astringent solutions and subsequently analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, as well as transmission electron microscopy (TEM). Four biocompatible astringents, namely the polyphenol epigallocatechin gallate, the metal salt iron(III) sulfate, the basic protein lysozyme, and the aminopolysaccharide chitosan, were additionally applied in situ. After rinsing the oral cavity with these compounds, the pellicle's ultrastructure was imaged by SEM and TEM, respectively. Untreated pellicle samples served as controls. RESULTS: Exposure to polyphenols and lysozyme induced particularly thicker and electron-denser pellicles in comparison to the control pellicle with similar characteristics in vitro and in situ. In contrast, acidic chitosan and metal salt solutions, respectively, revealed minor pellicle alterations. The incorporation of Fe and Al into the pellicles treated with the corresponding inorganic salts was verified by EDX analysis. CONCLUSIONS: Astringent-induced pellicle modifications were for the first time visualized by TEM. The ultrastructural alterations of the dental pellicle may partly explain the tooth-roughening effect caused by oral astringent stimuli. CLINICAL SIGNIFICANCE: Astringents might modify the pellicle's protective properties against dental erosion, attrition, as well as bacterial adhesion, and by this means may influence tooth health. The findings may thus be particularly relevant for preventive dentistry.

In vitro comparison of commercial oral rinses on bacterial adhesion and their detachment from biofilm formed on hydroxyapatite disks.

PURPOSE: This in vitro study was designed to assess the effectiveness of three oral rinses on bacterial adherence to epithelial cells and hydroxyapatite surfaces. The role of oral rinses on the detachment of bacteria from biofilm was also evaluated. MATERIALS AND METHODS: The efficacy of three oral rinses, Acclean, Noplak and Prevention were tested against a wide range of oral bacteria. Oral rinse antimicrobial activity was determined by an MTT assay for bacterial viability, by live/ dead staining and by measuring the bacterial metabolic activity using an XTT assay. RESULTS: The two oral rinses that contained 0.12% chlorhexidine had the greatest antibacterial activity on both planktonic and bio lm-grown organisms when compared to the Prevention oral rinse. CONCLUSION: Both Acclean and Noplak were extremely effective in lowering the number of bacteria attached to buccal epithelial cells and pelllicles. In addition, these two oral rinses were also effective against the biofilm bacteria.

Influence of substratum position and acquired pellicle on Candida albicans biofilm

The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on Candida albicans biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 107 cells/mL of Candida albicans was added to each well for biofilm development. The plates were aerobically incubated at 35°C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (p< 0.05). Significant differences in cell viability and metabolic activity were observed between the adhesion and other time points (p< 0.05), but these variables were not affected by the presence of the pellicle (p > 0.05). It can be concluded that the substratum position influenced biofilm development.

Relationship between fluoride concentration and activity against virulence factors and viability of a cariogenic biofilm: in vitro study.

Despite widespread use of various concentrations of fluoride for the prevention of dental caries, the relationship between fluoride concentration and activity against cariogenic biofilms has not been much studied. Herein we investigated the relationship between fluoride concentration and activity against virulence factors and viability of Streptococcus mutans biofilms. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs. The 70-hour-old biofilms were exposed to 0, 1, 3, 10, 30, 100, 300, 1,000 or 2,000 ppm F(-). The changes of virulence factors and viability of the biofilms were analyzed using biochemical methods and laser scanning confocal fluorescence microscopy. At 1-2,000 ppm F(-), the activity of fluoride against acid production, acid tolerance, and extracellular polysaccharide formation of S. mutans biofilms accurately followed a sigmoidal pattern of concentration dependence (R(2) = 0.94-0.99), with EC50 values ranging from 3.07 to 24.7 ppm F(-). Generally, the activity of fluoride against the virulence factors was concentration-dependently augmented in 10-100 ppm F(-) and did not increase further at concentrations higher than 100 ppm F(-). However, fluoride did not alter glucosyltransferase activity and viability of S. mutans biofilm cells in all concentrations tested. These results can provide a basis for the selection of appropriate fluoride concentrations that reduce the physiological ability of cariogenic biofilms.

Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms.

OBJECTIVE: This study aimed to evaluate the influence of NaF (2, 10, 50 and 125 ppm F(-)) on the virulence factors and composition of Streptococcus mutans biofilms. METHODS: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite discs. To assess the influence of NaF on the virulence factors of S. mutans biofilm cells, glycolytic pH drop, proton-permeability and F-ATPase activity assay were performed using 74 h old S. mutans biofilms. Glucosyltransferase (GTF) activity assay in suspension was also performed. To examine the influence of NaF on S. mutans biofilm composition, the biofilms were treated twice daily (5 min exposure/treatment) a total of five times during biofilm formation. After a total of 5 treatments, the biomass, colony forming unit (CFU) and polysaccharide composition of the treated 74h old S. mutans biofilms were analysed by microbiological and biochemical methods, and scanning electron microscopy. RESULTS: NaF showed inhibitory effects on the acid production and acid tolerance of S. mutans biofilm cells at 10, 50 and 125 ppm F(-), compared to the vehicle control (P<0.05) and the treatments at these concentrations also affected the biomass, water-insoluble extracellular polysaccharides and intracellular iodophilic polysaccharides of the biofilms, compared to the vehicle control (P<0.05). CONCLUSIONS: These results indicate that NaF (10, 50 and 125 ppm F(-)) has inhibitory effects on the virulence factors and composition of S. mutans biofilms, suggesting the potential use of these concentrations as an effective measure for controlling dental biofilms.

Potential of shock waves to remove calculus and biofilm.

Effective calculus and biofilm removal is essential to treat periodontitis. Sonic and ultrasonic technologies are used in several scaler applications. This was the first feasibility study to assess the potential of a shock wave device to remove calculus and biofilms and to kill bacteria. Ten extracted teeth with visible subgingival calculus were treated with either shock waves for 1 min at an energy output of 0.4 mJ/mm(2) at 3 Hz or a magnetostrictive ultrasonic scaler at medium power setting for 1 min, which served as a control. Calculus was determined before and after treatment planimetrically using a custom-made software using a grey scale threshold. In a second experiment, multispecies biofilms were formed on saliva-preconditioned bovine enamel discs during 64.5 h. They were subsequently treated with shock waves or the ultrasonic scaler (N = 6/group) using identical settings. Biofilm detachment and bactericidal effects were then assessed. Limited efficiency of the shock wave therapy in terms of calculus removal was observed: only 5% of the calculus was removed as compared to 100% when ultrasound was used (P ≤ 0.0001). However, shock waves were able to significantly reduce adherent bacteria by three orders of magnitude (P ≤ 0.0001). The extent of biofilm removal by the ultrasonic device was statistically similar. Only limited bactericidal effects were observed using both methods. Within the limitations of this preliminary study, the shock wave device was not able to reliably remove calculus but had the potential to remove biofilms by three log steps. To increase the efficacy, technical improvements are still required. This novel noninvasive intervention, however, merits further investigation.

Oral streptococci growth on aging and non-aging esthetic restorations after radiotherapy.

The aim of this study was to examine Streptococcus mutans biofilm growth on both aged and non-aged restorative dental resins, which were submitted to therapeutic irradiation. Sixty-four disks of an esthetic restorative material (Filtek Supreme) were divided into 2 groups: aged group (AG) and a non-aged group (NAG). Each group was subdivided into 4 subgroups: non-irradiated and irradiated with 10Gy, 35Gy, and 70Gy. The biofilms were produced by Streptococcus mutans UA159 growing on both AG and NAG surfaces. The colony-forming units per mL (CFU/mL) were evaluated by the ANOVA and the Tukey LSD tests (α=0.05). AG presented smaller amounts of CFU/mL than the NAG before irradiation and after 10Gy of irradiation (p<0.05). AG irradiated with 35 and 70Gy showed increased amount of bacterial biofilm when compared to non-irradiated and 10Gy-irradiated disks (p<0.05). The exposure to ionizing radiation at therapeutic doses promoted changes in bacterial adherence of aged dental restorative material.

Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface.

The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 µg/mL). However, S. mutans cells exposed to rCSP-1 (10 µg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

Inhibition of Streptococcus mutans adherence and biofilm formation using analogues of the SspB peptide.

OBJECTIVE: Streptococcus gordonii is a pioneer colonizer of the enamel salivary pellicle that forms biofilm on the tooth surfaces. Recent reports show the surface protein analogue peptide {400 (T) of SspB 390-402 is substituted to K forming SspB (390-T400K-402)} from S. gordonii interacts strongly with salivary receptors to cariogenic bacteria, Streptococcus mutans. To characterize the analogue peptide biological activities, we investigated its binding and inhibiting effects, and the role of its amino acid moities. METHODS: We measured binding activity of analogue peptides to salivary components using the BIAcore assay; assayed inhibition activities of peptides for bacterial binding and growth on saliva-coated hydroxyapatite beads (s-HA); and describe the peptides interfering with biofilm formation of S. mutans on polystyrene surfaces. RESULTS: The SspB (390-T400K-402 and -401) peptides significantly bound with salivary components and inhibited the binding of S. mutans and S. gordonii to s-HA without bactericidal activity; but did not inhibit binding of Streptococcus mitis, a beneficial commensal. Further, the lack of D and E-L at position 390 and 401-402 in the peptide, and substituted peptide SspB (D390H- or D390K-T400K-402) did not bind to salivary components or inhibit binding of S. mutans. The SspB (390-T400K-402) peptide inhibited biofilm formation on salivary components-coated polystyrene surfaces in absence of conditioned planktonic cells. CONCLUSIONS: We found constructing the peptide to include positions 390(D), 400(K) and 401(E), two surface positive and negative connective charges, and at least 12 amino acids are required to bind salivary components and inhibit the binding of S. mutans and S. gordonii.

The road to ruin: the formation of disease-associated oral biofilms.

The colonization of oral surfaces by micro-organisms occurs in a characteristic sequence of stages, each of which is potentially amenable to external intervention. The process begins with the adhesion of bacteria to host receptors on epithelial cells or in the salivary pellicle covering tooth surfaces. Interbacterial cell-cell binding interactions facilitate the attachment of new species and increase the diversity of the adherent microbial population. Microbial growth in oral biofilms is influenced by the exchange of chemical signals, metabolites and toxic products between neighbouring cells. Bacterial cells on tooth surfaces (dental plaque) produce extracellular polymers such as complex carbohydrates and nucleic acids. These large molecules form a protective matrix that contributes to the development of dental caries and, possibly, to periodontitis. The identification of key microbial factors underlying each step in the formation of oral biofilms will provide new opportunities for preventative or therapeutic measures aimed at controlling oral infectious diseases.

Oral streptococci growth on aging and non-aging esthetic restorations after radiotherapy

The aim of this study was to examine Streptococcus mutans biofilm growth on both aged and non-aged restorative dental resins, which were submitted to therapeutic irradiation. Sixty-four disks of an esthetic restorative material (Filtek Supreme) were divided into 2 groups: aged group (AG) and a non-aged group (NAG). Each group was subdivided into 4 subgroups: non-irradiated and irradiated with 10Gy, 35Gy, and 70Gy. The biofilms were produced by Streptococcus mutans UA159 growing on both AG and NAG surfaces. The colony-forming units per mL (CFU/mL) were evaluated by the ANOVA and the Tukey LSD tests (α=0.05). AG presented smaller amounts of CFU/mL than the NAG before irradiation and after 10Gy of irradiation (p<0.05). AG irradiated with 35 and 70Gy showed increased amount of bacterial biofilm when compared to non-irradiated and 10Gy-irradiated disks (p<0.05). The exposure to ionizing radiation at therapeutic doses promoted changes in bacterial adherence of aged dental restorative material.

O objetivo deste estudo foi avaliar a formação do biofilme de Streptococcus mutans crescido em resina restauradora envelhecida e não-envelhecida, submetidas à radiação terapêutica. Sessenta e quatro discos do material restaurador Filtek Supreme foram divididos em 2 grupos: grupo envelhecido (AG) e grupo não-envelhecido (NAG) e cada grupo foi dividido em 4 sub-grupos: não-irradiado e irradiado com 10Gy, 35Gy e 70Gy. O biofilme de S. mutans UA159 foi produzido na superfície de ambos os discos AG e NAG. As unidades formadoras de colônia/mL (UFC/mL) foram avaliadas por ANOVA e teste de Tukey (α=0,05). O grupo AG demonstrou menores quantidades de UFC/mL que o grupo NAG antes da radiação e após a radiação de 10Gy (p<0,05). Os sub-grupos AG irradiados com 35 e 70Gy demonstraram aumento na quantidade de biofilme quando comparado aos não irradiados e irradiados com 10Gy (p<0,05). A exposição à radiação ionizante nas doses terapêuticas promoveu mudanças na aderência bacteriana no material restaurador.

Polyphenolic beverages reduce initial bacterial adherence to enamel in situ.

OBJECTIVES: Polyphenols are antibacterial and anti-oxidative natural agents. The present in situ study aimed to investigate the effect of different polyphenolic beverages on initial bacterial adherence to enamel in the oral cavity. METHODS: Initial biofilm formation was performed on bovine enamel specimens mounted buccally on individual upper jaw splints and carried by six subjects. After 1 min of pellicle formation, oral rinses with black tea, green tea, grape juice, Cistus tea or red wine were performed for 10 min. Afterwards the slabs were carried for another 19 or 109 min, respectively. Samples exposed to the oral fluids for 30 and 120 min served as controls. Following intraoral exposure, the slabs were rinsed with saline solution. The amount of adherent bacteria was determined with DAPI-staining (4',6-diamidino-2-phenylindole) and with fluorescence-in situ hybridization (FISH) of eubacteria and streptococci. RESULTS: Rinses with all beverages reduced the amount of detectable bacteria. Lowest number of adherent bacteria was found following rinses with red wine, Cistus tea and black tea as measured with DAPI (up to 66% reduction of adherent bacteria vs. controls). Also FISH revealed significant impact of most tested beverages. CONCLUSIONS: Rinses with certain polyphenolic beverages as well as consumption of these foodstuffs may contribute to prevention of biofilm induced diseases in the oral cavity.

Effects of Cistus-tea on bacterial colonization and enzyme activities of the in situ pellicle.

OBJECTIVES: Polyphenols are expected to have antibacterial properties. Cistus is a tea rich in polyphenols. The aim of the present in situ study was to investigate the effect of Cistus-tea on the pellicle and on the initial oral biofilm. METHODS: For in situ pellicle formation and initial biofilm formation, bovine enamel slabs were fixed on maxillary splints and carried by four subjects at buccal sites for up to 2 h. Bacteria present in 120-min pellicles were determined with DAPI-staining and fluorescence in situ hybridization with and without a 10 min rinse with Cistus-tea performed 1 min after incorporation of the slabs. In addition, amylase, lysozyme, glucosyltransferase and peroxidase activities immobilised in the pellicle layer were measured before and after rinsing for 10 min with Cistus-tea. RESULTS: The amount of bacteria detected in the 120-min biofilm was reduced significantly, if a 10 min rinse with Cistus-tea was performed one min after insertion of the enamel slabs. DAPI-staining yielded 13.2+/-3.5 for controls and 6.5+/-1.1 x 10(4) bacteria/cm(2), if a rinse with Cistus-tea was applied. Lysozyme, amylase and glucosyltransferase activities immobilised in the pellicle were not affected following a rinse with Cistus-tea. However, peroxidase activity was reduced significantly. CONCLUSIONS: Cistus-tea may be used to reduce the initial bacterial adhesion in the oral cavity.

Effects of Nidus Vespae extract and chemical fractions on glucosyltransferases, adherence and biofilm formation of Streptococcus mutans.

Nidus Vespae (the honeycomb of Polistes olivaceous, P. japonicus Saussure and Parapolybiavaria fabricius) have been extensively used in traditional Chinese medicine, given their multiple pharmacological activities, including antimicrobial, anti-inflammatory, anti-virus, anti-tumor and anesthetic properties. The present study evaluated the anti-glucosyltransferases (GTFs) activity, anti-adherence and anti-biofilm properties of 95% ethanol/water extract, cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol fractions of Nidus Vespae. Chloroform/methanol fraction showed a remarkable capacity for inhibiting the adherence of Streptococcus mutans ATCC 25175 to saliva-coated hydroxyapatite disc (S-HA) at sub-MC concentrations. In addition, the Nidus Vespae extract and chemical fractions significantly inhibited the activity of cell-associated and extracellular GTFs at sub-MIC concentrations, and the chloroform/methanol fraction was the most effective one. For the anti-biofilm activity assays, minimum biofilm inhibition concentrations (MBIC50) and minimum biofilm reduction concentrations (MBRC50) were determined using the microdilution method. The chloroform/methanol fraction showed the highest anti-biofilm activities with a MBIC50 of 8mg/ml and a MBRC(50) of 16mg/ml against Streptococcus mutans ATCC 25175. The significant inhibition of GTFs activity and biofilm formation demonstrated by Nidus Vespae shows it to be a promising natural product for the prevention of dental caries.

Película adquirida salival: revisión de la literatura / Acquired salivary pellicle: a review of the literature

En este artículo de revisión se presenta y analiza la información actualizada disponible sobre la composición química, mecanismo de formación y factores que afectan la producción de la película adquirida salival. Asimismo se discuten aspectos vinculados con la función que cumple dicho integumento, en especial la relacionada con su desempeño como antecesor de la placa bacteriana de la cual dependen las afecciones de mayor prevalencia e incidencia en odontología, como son la caries dental y la enfermedad periodontal.

In this article is presented and analyzes the information brought up to date on the chemical composition, mechanism of formation and factors that affect the production of salivary acquired pellicle. Likewise aspects related to the function are discussed that complies said integument, especially it related to its performance as the ancestor of the bacterial plaque of which the affections of grater prevalence and incidence in dentistry they depend, like are the dental decay and the periodontal illness.