Simone Duarte

Influence of cranberry proanthocyanidins on formation of biofilms by streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

Effect of Starch and Sucrose on Dental Biofilm Formation and on Root Dentine Demineralization

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch → sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.

Própolis do sudeste e nordeste do Brasil: influência da sazonalidade na atividade antibacteriana e composição fenólica

The composition and biological activities of propolis, a resinous hive product collected by honeybees from various plant sources, depends on various factors such as season and vegetation of the area. The aim of this study was to evaluate the influence of the seasonal effect on the ethanolic extracts of Brazilian propolis (EEP) type 6 and type 12, collected during 6 months in terms of antibacterial activity and phenolic composition. The antimicrobial properties were evaluated by MIC and MBC on S. mutans Ingbritt 1600 and the profile of chemical composition by UV-visible spectrophotometry, HPLC-RF and GC-MS. The results demonstrated that the season in which propolis is collected influences its chemical composition, resulting in modifications in its antibacterial activity.

Influences of starch and sucrose on Streptococcus mutans biofilms.

INTRODUCTION The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms. METHODS S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose. RESULTS Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05). CONCLUSION The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

Effect of Photodynamic Antimicrobial Chemotherapy on in vitro and in situ Biofilms

Aim: The effect of photodynamic antimicrobial chemotherapy (PACT) on oral biofilms was evaluated. Methods: Biofilms formed in vitro were treated with sensitizer (S) and/or light (L) in the test (S⁺L⁺) and control (S^–L^–, S⁺L^–, S^–L⁺) groups. Additionally, 21 volunteers wore intraoral devices and the biofilms formed on each side of the device were referred to as S⁺L⁺ or S^–L^– groups. Results: Significant decreases in the viability of in vitro biofilms were observed after PACT. PACT was ineffective in inhibiting multi-species biofilms formed in situ. Conclusion: PACT was effective on in vitro biofilms and was ineffective on in situ biofilms.

Effect of Atmospheric-Pressure Cold Plasma on Pathogenic Oral Biofilms and In Vitro Reconstituted Oral Epithelium

Considering the ability of atmospheric-pressure cold plasma (ACP) to disrupt the biofilm matrix and rupture cell structure, it can be an efficient tool against virulent oral biofilms. However, it is fundamental that ACP does not cause damage to oral tissue. So, this study evaluated (1) the antimicrobial effect of ACP on single- and dual-species biofilms of Candida albicans and Staphylococcus aureus as well as (2) the biological safety of ACP on in vitro reconstituted oral epithelium. Standardized cell suspensions of each microorganism were prepared for biofilm culture on acrylic resin discs at 37°C for 48 hours. The biofilms were submitted to ACP treatment at 10 mm of plasma tip-to-sample distance during 60 seconds. Positive controls were penicillin G and fluconazole for S. aureus and C. albicans, respectively. The biofilms were analyzed through counting of viable colonies, confocal laser scanning microscopy, scanning electron microscopy and fluorescence microscopy for detection of reactive oxygen species. The in vitro reconstituted oral epithelium was submitted to similar ACP treatment and analyzed through histology, cytotoxocity test (LDH release), viability test (MTT assay) and imunnohistochemistry (Ki67 expression). All plasma-treated biofilms presented significant log10 CFU/mL reduction, alteration in microorganism/biofilm morphology, and reduced viability in comparison to negative and positive controls. In addition, fluorescence microscopy revealed presence of reactive oxygen species in all plasma-treated biofilms. Low cytotoxicity and high viability were observed in oral epithelium of negative control and plasma group. Histology showed neither sign of necrosis nor significant alteration in plasma-treated epithelium. Ki67-positive cells revealed maintenance of cell proliferation in plasma-treated epithelium. Atmospheric-pressure cold plasma is a promissing approach to eliminate single- and dual-species biofilms of C. albicans and S. aureus without having toxic effects in oral epithelium.

Comparison of SEM and VPSEM imaging techniques with respect to Streptococcus mutans biofilm topography.

The study compared images of mature Streptococcus mutans biofilms captured at increasing magnification to determine which microscopy method is most acceptable for imaging the biofilm topography and the extracellular polymeric substance (EPS). In vitro S. mutans biofilms were imaged using (1) scanning electron microscopy (SEM), which requires a dehydration process; (2) SEM and ruthenium red (SEM-RR), which has been shown to support the EPS of biofilms during the SEM dehydration; and (3) variable pressure scanning electron microscopy (VPSEM), which does not require the intensive dehydration process of SEM. The dehydration process and high chamber vacuum of both SEM techniques devastated the biofilm EPS, removed supporting structures, and caused cracking on the biofilm surface. The VPSEM offered the most comprehensive representation of the S. mutans biofilm morphology. VPSEM provides similar contrast and focus as the SEM, but the procedure is far less time-consuming, and the use of hazardous chemicals associated with SEM dehydration protocol is avoided with the VPSEM. The inaccurate representations of the biofilm EPS in SEM experimentation is a possible source of inaccurate data and impediments in the study of S. mutans biofilms.

Air plasma effect on dental disinfection

A nonthermal low temperature air plasma jet is characterized and applied to study the plasma effects on oral pathogens and biofilms. Experiments were performed on samples of six defined microorganisms’ cultures, including those of gram-positive bacteria and fungi, and on a cultivating biofilm sample of Streptococcus mutans UA159. The results show that the plasma jet creates a zone of microbial growth inhibition in each treated sample; the zone increases with the plasma treatment time and expands beyond the entire region directly exposed to the plasma jet. With 30s plasma treatment twice daily during 5 days of biofilm cultivation, its formation was inhibited. The viability of S. mutans cells in the treatedbiofilms dropped to below the measurable level and the killed bacterial cells concentrated to local regions as manifested by the fluorescence microscopy via the environmental scanning electron microscope. The emission spectroscopy of the jet indicates that its plasma effluent carries an abundance of reactive atomic oxygen, providing catalyst for the observed plasma effect.

Effect of Twice-Daily Blue Light Treatment on Matrix-Rich Biofilm Development

Background The use of blue light has been proposed as a direct means of affecting local bacterial infections, however the use of blue light without a photosensitizer to prevent the biofilm development has not yet been explored. The aim of this study was to determine how the twice-daily treatment with blue light affects the development and composition of a matrix-rich biofilm. Methodology/Principal Findings Biofilms of Streptococcus mutans UA159 were formed on saliva-coated hydroxyapatite discs for 5 days. The biofilms were exposed twice-daily to non-coherent blue light (LumaCare; 420 nm) without a photosensitizer. The distance between the light and the sample was 1.0 cm; energy density of 72 J cm-2; and exposure time of 12 min 56 s. Positive and negative controls were twice-daily 0.12% chlorhexidine (CHX) and 0.89% NaCl, respectively. Biofilms were analyzed for bacterial viability, dry-weight, and extra (EPS-insoluble and soluble) and intracellular (IPS) polysaccharides. Variable pressure scanning electron microscopy and confocal scanning laser microscopy were used to check biofilm morphology and bacterial viability, respectively. When biofilms were exposed to twice-daily blue light, EPS-insoluble was reduced significantly more than in either control group (CHX and 0.89% NaCl). Bacterial viability and dry weight were also reduced relative to the negative control (0.89% NaCl) when the biofilms were treated with twice-daily blue light. Different morphology was also visible when the biofilms were treated with blue light. Conclusions Twice-daily treatment with blue light without a photosensitizer is a promising mechanism for the inhibition of matrix-rich biofilm development.

Streptococcus mutans photoinactivation by combination of short exposure of a broad-spectrum visible light and low concentrations of photosensitizers

OBJECTIVE Investigate the photodynamic antimicrobial effect by the combination of a novel noncoherent broad spectrum visible light and low concentrations of curcumin and toluidine blue over suspensions of Streptococcus mutans. BACKGROUND DATA Long illumination times to activate photosensitizers (PS) and the use of high concentrations of these drugs in photodynamic antimicrobial chemotherapy (PACT) are limitations of its application as an antimicrobial technology in dental practice. MATERIALS AND METHODS Planktonic suspensions of S. mutans were standardized and submitted to PACT treatment at low concentrations of curcumin (C) (0.075; 0.75 and 7.5 μM) and toluidine blue (T) (0.25; 2.5 and 25 μM) exposed to 42 J/cm2 (12.2 sec; set power: 3.930 mW) of a white light (WL) (output wavelength range: 400-700 nm; beam diameter: 12 mm) (C+WL+ and T+WL+, PACT groups; incubation time, C: 60 sec; T: 5 min); isolated effect of both C (C+WL-) and T concentrations (T+WL-); effect of light source (C-WL+ and T-WL+) and suspensions neither submitted to PS nor to light-emitting diode (LED) illumination (control groups, C-WL- and T-WL-). Aliquots of each group were diluted and cultured on blood agar plates and the number of colony-forming units (CFU)/mL was recorded, transformed into log10 and analyzed by ANOVA and Tukeys test at a cutoff value at 0.05. RESULTS The groups submitted to PACT presented a bacterial reduction value of>5-log10 to both tested PS in comparison with control groups (p<0.05). PS or light source used alone demonstrated no antimicrobial effect on the number of viable bacterial counts. CONCLUSIONS The combination of a novel noncoherent light at short illumination exposure time with low concentrations of studied PS achieved a lethal photoinactivation of S. mutans, and can be considered an effective antimicrobial in vitro approach for reducing the number of micro-organisms involved with the dental caries process.