Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

Streptococcus mutans is a component of oral plaque biofilm that accumulates on the surface of teeth. The biofilm consists of extracellular components including extracellular DNA (eDNA). This study was conducted to investigate the factors that may affect the eDNA levels of S. mutans in biofilms. For the study, S. mutans UA159 biofilms were formed for 52 h on hydroxyapatite (HA) discs in 0% (w/v) sucrose +0% glucose, 0.5% sucrose, 1% sucrose, 0.5% glucose, 1% glucose, or 0.5% sucrose +0.5% glucose. Acidogenicity of S. mutans in the biofilms was measured after biofilm formation (22 h) up to 52 h. eDNA was collected after 52 h biofilm formation and measured using DNA binding fluorescent dye, SYBR Green I. Biofilms cultured in 0.5% sucrose or glucose had more eDNA and colony forming units (CFUs) and less exopolysaccharides (EPSs) than the biofilms cultured in 1% sucrose or glucose at 52 h, respectively. The biofilms formed in 0% sucrose +0% glucose maintained pH around 7, while the biofilms grown in 0.5% sucrose had more acidogenicity than those grown in 1% sucrose, and the same pattern was shown in glucose. In conclusion, the results of this study show that the number of S. mutans in biofilms affects the concentrations of eDNA as well as the acidogenicity of S. mutans in the biofilms. In addition, the thickness of EPS is irrelevant to eDNA aggregation within biofilms.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite Objective: This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis (E. faecalis) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology: E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy.The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results: CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05).Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions:Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Aim To apply an innovative three‐dimensionally printed tooth model to investigate the efficacy of three ultrasonically activated irrigation (UAI) systems in removing multispecies biofilms from dentine samples. Methodology Three‐dimensionally printed teeth with a curved root canal were fabricated with a standardized slot in the apical third of the root to achieve precision fit of human root dentine specimens. Multispecies biofilms including Enterococcus faecalis, Streptococcus mitis and Campylobacter rectus were developed in the root canal for 21 days. The canals were allocated to be irrigated with 1% sodium hypochlorite (NaOCl) using a syringe and needle or ultrasonically activated NaOCl with a stainless‐steel file (Irrisafe), a conventional nickel‐titanium (Ni‐Ti) file (CK) or a blue heat‐treated Ni‐Ti file (Endosonic Blue). Infected root canals irrigated with distilled water served as controls. Bacterial reduction was determined by colony‐forming unit (CFU) counting (n = 20), whilst biofilms were analysed using confocal laser scanning microscopy (n = 7) and field emission scanning electron microscopy. For CFU counting, the independent two‐sample t‐test (Welch's t‐test) was examined to compare overall bacterial reduction amongst groups. For CLSM analysis, the data were analysed using one‐way analysis of variance (ANOVA), followed by the Scheffé post hoc test. The p‐values <.05 were considered to indicate statistical significance. Results All groups in which NaOCl was ultrasonically activated had significantly lower CFU values than the syringe‐and‐needle irrigation and control groups (p < .05). Ultrasonic activation with the stainless‐steel file and blue heat‐treated Ni‐Ti file significantly reduced the biofilm volume compared with other groups (p < .05). Overall, UAI with the blue heat‐treated file resulted in the highest antibacterial and biofilm removal efficacy. Conclusions UAI with different inserts had differential antibiofilm effects. The blue heat‐treated Ni‐Ti ultrasonic insert resulted in the greatest antibacterial and biofilm removal from dentine in this standardized root canal model.

The objective of this study was to evaluate the distribution of dental caries-associated Streptococcus mutans in human saliva and plaque. The samples for this study were collected from 90 subjects (30 children, 30 adolescents, and 30 adults). The decayed, missing, and filled teeth (DMFT) and significant caries indices were evaluated. We applied polymerase chain reaction (PCR) analysis to detect S. mutans in each sample. Enumeration of S. mutans was conducted through culturing on Dentocult-SM and mitis salivarius agar medium. For saliva samples enzyme-linked immunosorbent assay (ELISA) analysis using monoclonal antibodies specific to antigen I/II and glucosyltransferase was done. We detected S. mutans in 79.7% and 56.8% of all saliva and plaque samples, respectively using PCR analyses. S. mutans were detected in 59.1%, 88.0%, and 88.9%, and in 86.4%, 56.0%, and 33.3% of saliva and plaque samples from children, adolescents, and adults, respectively. There were significantly higher levels of S. mutans in adolescents' saliva more than any groups. There was a positive correlation between DMFT and the level of S. mutans reactivity measured using ELISA. Our results suggest that there should be more emphasis on adolescents' oral hygiene more than in children or adults early prevention and research of dental caries.

The aim of this study was to investigate how carbohydrates (glucose or sucrose) affect the characteristics of Enterococcus faecalis (E. faecalis) planktonic and biofilm in vitro. For this study, E. faecalis was cultured in tryptone-yeast extract broth with 0% glucose + 0% sucrose, 0.5% glucose, 1% glucose, 0.5% sucrose, or 1% sucrose. Viability of E. faecalis was examined by colony forming unit counting assays. Biofilm formation was assessed by measuring extracellular DNA (eDNA), a component of the biofilm matrix. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression of virulence-associated genes. Field emission scanning electron microscopy analysis, confocal laser scanning microscopy analysis, and crystal violet colorimetric assay were conducted to study E. faecalis biofilms. E. faecalis showed the highest viability and eDNA levels in 1% sucrose medium in biofilms. The result of qRT-PCR showed that the virulence-associated genes expressed highest in 1% sucrose-grown biofilms and in 1% glucose-grown planktonic cultures. E. faecalis showed highly aggregated biofilms and higher bacteria and exopolysaccharide (EPS) bio-volume in sucrose than in 0% glucose + 0% sucrose or glucose. The results indicate that the production of eDNA and EPS and expression of virulence-associated genes in E. faecalis are affected by the concentration of carbohydrates in biofilm or planktonic culture.

Glucosyltransferase-B (GTFB) of Streptococcus mutans is considered a virulence factor because of its activity in the production of insoluble glucan, which is key to the bacterial attachment onto dental surfaces, leading to the formation of dental caries. Local passive immunization with monoclonal antibodies against GTFB is considered to be an effective way to prevent dental caries. Here we amplified a 1.3 kb fragment of the N-terminal half of the gtfB gene (193-1530) of S. mutans by PCR and expressed the truncated protein (GTFBN). The expressed, purified protein was used as an immunogen in BALB/c mice. We selected and established one hybridoma (HBN8) that was capable of producing anti-GTFBN using ELISA, dot blot, and Western blot analyses. The monoclonal anti-GTFBN antibody was purified by affinity chromatography and its isotype was confirmed as IgG2a. The anti-GTFBN antibody inhibited the enzymatic activity of crude glucosyltransferase of S. mutans GS-5 in a dose-dependent manner. These data suggest that the anti-GTFBN antibody could be used as a vaccine to prevent the aggregation of S. mutans on tooth surfaces, and thus prevent the formation of dental caries.

Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm This study investigated the effect of a calcium hydroxide (CH) paste (CleaniCal ®) containing N-2-methyl pyrrolidone (NMP) as a vehicle on Enterococcus faecalis (E. faecalis) biofilms compared with other products containing saline (Calasept Plus™) or propylene glycol (PG) (Calcipex II ®). Methodology: Standardized bovine root canal specimens were used. The antibacterial effects were measured by colony-forming unit counting. The thickness of bacterial microcolonies and exopolysaccharides was assessed using confocal laser scanning microscopy. Morphological features of the biofilms were observed using field-emission scanning electron microscopy (FE-SEM). Bovine tooth blocks covered with nail polish were immersed into the vehicles and dispelling was observed. The data were analyzed using one-way analysis of variance and Tukey tests (p<0.05). Results: CleaniCal ® showed the highest antibacterial activity, followed by Calcipex II ® (p<0.05). Moreover, NMP showed a higher antibacterial effect compared with PG (p<0.05). The thickness of bacteria and EPS in the CleaniCal ® group was significantly lower than that of other materials tested (p<0.05). FE-SEM images showed the specimens treated with Calasept Plus™ were covered with biofilms, whereas the specimens treated with other medicaments were not. Notably, the specimen treated with CleaniCal ® was cleaner than the one treated with Calcipex II ®. Furthermore, the nail polish on the bovine tooth block immersed in NMP was completely dispelled. Conclusions: CleaniCal ® performed better than Calasept Plus™ and Calcipex II ® in the removal efficacy of E. faecalis biofilms. The results suggest the effect might be due to the potent dissolving effect of NMP on organic substances.