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

INTRODUCTION: 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.

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.

Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ.

OBJECTIVE: The present in situ - investigation aimed to specify the impact of pure hydroxyapatite microclusters on initial bioadhesion and bacterial colonization at the tooth surface. DESIGN: Pellicle formation was carried out in situ on bovine enamel slabs (9 subjects). After 1min of pellicle formation rinses with 8ml of hydroxyapatite (HA) microclusters (5%) in bidestilled water or chlorhexidine 0.2% were performed. As negative control no rinse was adopted. In situ biofilm formation was promoted by the intraoral slab exposure for 8h overnight. Afterwards initial bacterial adhesion was quantified by DAPI staining and bacterial viability was determined in vivo/in vitro by live/dead-staining (BacLight). SEM analysis evaluated the efficacy of the mouthrinse to accumulate hydroxyapatite microclusters at the specimens' surface and spit-out samples of the testsolution were investigated by TEM. RESULTS: Compared to the control (2.36×106±2.01×106bacteria/cm2), significantly reduced amounts of adherent bacteria were detected on specimens rinsed with chlorhexidine 0.2% (8.73×104±1.37×105bacteria/cm2) and likewise after rinses with the hydroxyapatite testsolution (2.08×105±2.85×105bacteria/cm2, p<0.001). No demonstrable effect of HA-particles on Streptococcus mutans viability could be shown. SEM analysis confirmed the temporary adsorption of hydroxyapatite microclusters at the tooth surface. Adhesive interactions of HA-particles with oral bacteria were shown by TEM. CONCLUSION: Hydroxyapatite microclusters reduced initial bacterial adhesion to enamel in situ considerably and could therefore sensibly supplement current approaches in dental prophylaxis.

Anti-plaque effect of a synergistic combination of green tea and Salvadora persica L. against primary colonizers of dental plaque.

OBJECTIVE: Green tea (Gt), leafs of Camellia sinensis var. assamica, is widely consumed as healthy beverage since thousands of years in Asian countries. Chewing sticks (miswak) of Salvadora persica L. (Sp) are traditionally used as natural brush to ensure oral health in developing countries. Both Gt and Sp extracts were reported to have anti-bacterial activity against many dental plaque bacteria. However, their combination has never been tested to have anti-bacterial and anti-adherence effect against primary dental plaque colonizers, playing an initial role in the dental plaque development, which was investigated in this study. METHODS: Two-fold serial micro-dilution method was used to measure minimal inhibitory concentration (MIC) of aqueous extracts of Gt, Sp and their combinations. Adsorption to hexadecane was used to determine the cell surface hydrophobicity (CSH) of bacterial cells. Glass beads were used to mimic the hard tissue surfaces, and were coated with saliva to develop experimental pellicles for the adhesion of the primary colonizing bacteria. RESULTS: Gt aqueous extracts exhibited better anti-plaque effect than Sp aqueous extracts. Their combination, equivalent to 1/4 and 1/2 of MIC values of Gt and Sp extracts respectively, showed synergistic anti-plaque properties with fractional inhibitory concentration (FIC) equal to 0.75. This combination was found to significantly reduce CSH (p<0.05) and lower the adherence ability (p<0.003) towards experimental pellicles. CONCLUSION: Combination between Gt and Sp aqueous extracts exhibited synergistic anti-plaque activity, and could be used as a useful active agent to produce oral health care products.

Efficacy of citronella and cinnamon essential oils on Candida albicans biofilms.

INTRODUCTION: The discovery of new antimicrobials derived from plants could aid in the management of biofilm-associated infections, including denture-induced stomatitis (DS). DS is an oral infection caused by Candida biofilms on the surfaces of poorly cleansed dentures. Effective treatment of DS requires the use of an appropriate denture cleanser and preferably one that exhibits antimicrobial properties. OBJECTIVE: This study aimed to evaluate the anti-Candida and anti-biofilm efficacy of two essential plant oils from Cymbopogon winterianus (citronella) and Cinnamon cassia (cinnamon). MATERIALS AND METHODS: Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs) were determined by broth microdilution, whilst anti-biofilm activity was measured against mature (cultured for 72 h) biofilms on acrylic surfaces. Candida cell viability was assessed immediately (0 h) after treatment (T0) and 48 h after biofilm re-growth (T48). Biofilm structure was determined using Scanning Electron Microscopy (SEM) at T0 and T48. RESULTS: The respective MICs of cinnamon and citronella oils were 65 and 250 µg/ml and these were also the MFC values. For anti-biofilm efficacy, both oils significantly (p < 0.05) reduced the number of viable micro-organisms and accumulation of biofilms at T0. However, at T48, there was no difference between treated and untreated biofilms. CONCLUSIONS: It is concluded that citronella and cinnamon essential oils have potential for daily anti-candidal denture cleansing.

Antibacterial Efficacy of a Propolis Toothpaste and Mouthrinse Against a Supragingival Multispecies Biofilm.

PURPOSE: To determine in vitro the antibacterial properties of propolis toothpaste and mouthrinse against an in vitro multispecies biofilm model. MATERIALS AND METHODS: Six-species biofilms grown anaerobically on pellicle-coated hydroxyapatite disks were fed with glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in 37°C saliva between feedings for up to 64.5 h. At each interval, biofilms were exposed to six different slurries and solutions, including: 1) toothpaste without propolis, 2) toothpaste with propolis, 3) toothpaste with chlorhexidine, 4) mouthrinse with propolis, 5) mouthrinse with chlorhexidine, 6) saline solution (control). Afterwards, biofilms were harvested and the number of colony forming units were determined (CFU). The results were analysed using ANOVA, followed by the Bonferroni test at a 5% significance level. RESULTS: The strongest CFU reduction was shown after treatment with 0.12% chlorhexidine (p<0.0004). When comparing the different toothpastes, there was no statistically significant difference (p<0.05) in CFU reduction. However, they all showed a significant reduction in CFU of more than one log-step vs the saline control group. Nevertheless, the propolis-containing mouthrinse showed no significant reduction in CFU. CONCLUSION: All toothpastes under investigation displayed some growth inhibition in this supragingival biofilm model, which accounted for an approximately 80%-88% linear reduction. However, the propolis mouthwash had no effect.

An in vitro study on the anti-adherence effect of Brucea javanica and Piper betle extracts towards oral Candida.

OBJECTIVE: The adherence of Candida to mucosal surfaces is the initial step for successful invasive process of the oral cavity. The study aimed to investigate the effect of two plant extracts on the non-specific and specific bindings of oral candida. METHODS: In the former, adsorption to hexadecane was used to measure the hydrophobic interaction of the candida cells. In the later, glass beads coated with saliva represented the experimental pellicles in specific adhesion of oral candida to hard tissue surface. RESULTS: Candida krusei, Candida dubliniensis and Candida tropicalis showed the highest adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others within the range of 7-10%. All candidal species were significantly affected by the extracts (P<0.05) with Brucea javanica exhibited more than 60% reduction of CSH than Piper betle. Candida parapsilosis showed the highest affinity in specific-bindings to pellicle with 18.72±0.71×10(5)CFU/ml. Exposing to P. betle-treated pellicle has drastically reduced the adherence of C. tropicalis, Candida albicans and C. krusei by 86.01%, 61.41% and 56.34%, respectively. B. javanica exhibited similar effect on C. tropicalis (89.86%), Candida lusitaniae (88.95%), C. albicans (79.74%), Candida glabrata (76.85%) and C. krusei (67.61%). CONCLUSION: The extracts demonstrated anti-adherence activities by modifying the CSH and the characteristics of the experimental pellicle.

Effect of microbubbled water on the removal of a biofilm attached to orthodontic appliances--an in vitro study.

Orthodontic appliances often cause oral diseases such as dental caries and gingivitis due to the attachment of an oral biofilm. However, there are few reliable methods to remove the biofilm from the orthodontic appliances. The aim of this study was to investigate the effects of microbubbled water on the removal of biofilms made with Streptococcus mutans or Candida albicans on orthodontic appliances. The orthodontic appliances with biofilm were immersed with microbubbled water and the remaining biofilm on the appliances was detected and measured using a micro-plate reader and an absorbance meter. The microbubbled water had a sufficient effect on the removal of biofilm from orthodontic appliances. The effects of microbubbled water were significantly higher than those of tap water (S. mutans: p<0.05, C. albicans: p<0.01). The results of this study suggest that microbubbled water is effective in the removal of biofilm from the mouth of orthodontic patients.

Denture disinfection by microwave energy: influence of Candida albicans biofilm.

OBJECTIVE: This study evaluated the influence of the area of Candida albicans biofilm on denture disinfection by microwave energy. MATERIALS AND METHODS: Candida albicans biofilm was allowed to form for 72 h on resin discs, and three small coverage or seven large coverage discs were placed onto the palatal surface of sterile maxillary dentures. Each denture was immersed in 200 ml distilled water and individually irradiated at a power of 450, 630 or 900 W for different time intervals (1, 2 or 3 min) (n = 6). The effectiveness of disinfection was evaluated by counting the residual cells. The data were analysed by anova and Tukey's HSD test (α = 0.05). Pearson's correlation test was performed to determine the correlation between effectiveness of sterilisation and temperature. RESULTS: Dentures with a larger area of biofilm demanded a longer irradiation exposure to achieve disinfection (p < 0.001), irrespective of power setting, and in this time no yeast growth was detected. Dentures with small areas of biofilm were disinfected after 1 min at 900 W and 2 min at 450 or 630 W. A positive correlation was found between water temperature and effectiveness of disinfection (r = 0.6170; p < 0.001). CONCLUSION: The C. albicans biofilm area influenced disinfection by microwave energy; therefore dentures with larger biofilm areas required longer irradiation exposure to be disinfected.

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.

The effect of Piper betle and Psidium guajava extracts on the cell-surface hydrophobicity of selected early settlers of dental plaque.

The adhesion of early settlers of dental plaque to the tooth surface has a role in the initiation of the development of dental plaque. The hydrophobic surface properties of the bacteria cell wall are indirectly responsible for the adhesion of the bacteria cell to the acquired pellicle on the tooth surfaces. In this study, the effect of aqueous extract of two plants (Psidium guajava and Piper betle) on the cell-surface hydro-phobicity of early settlers of dental plaque was determined in vitro. Hexadecane, a hydrocarbon was used to represent the hydrophobic surface of the teeth in the oral cavity. It was found that treatment of the early plaque settlers with 1 mg/ml extract of Psidium guajava reduced the cell-surface hydrophobicity of Strep. sanguinis, Strep. mitis and Actinomyces sp. by 54.1%, 49.9% and 40.6%, respectively. Treatment of these bacteria with the same concentration of Piper betle however, showed a comparatively lesser effect (< 10%). It was also observed that the anti-adhesive effect of the two extracts on the binding of the early plaque settlers to hexadecane is concentration dependent.

The anti-adherence effect of Piper betle and Psidium guajava extracts on the adhesion of early settlers in dental plaque to saliva-coated glass surfaces.

The aqueous extracts of Piper betle and Psidium guajava were prepared and tested for their anti-adherence effect on the adhesion of early plaque settlers (Strep. mitis, Strep. sanguinis and Actinomyces sp.). The saliva-coated glass surfaces were used to simulate the pellicle-coated enamel surface in the oral cavity. Our results showed that the anti-adherence activities of Piper betle and Psidium guajava extracts towards the bacteria were different between the bacterial species. Psidium guajava was shown to have a slightly greater anti-adherence effect on Strep. sanguinis by 5.5% and Actinomyces sp. by 10% and a significantly higher effect on Strep. mitis (70%) compared to Piper betle. The three bacterial species are known to be highly hydrophobic, and that hydrophobic bonding seemed to be an important factor in their adherence activities. It is therefore suggested that the plant extracts, in expressing their anti-adherence activities, could have altered the hydrophobic nature of the bonding between the bacteria and the saliva-coated glass surfaces.