Antibacterial Efficacy of Herbal Toothpaste Containing Bamboo Salt: A Randomized Controlled Clinical Trial

Background: The inclusion of herbal antibacterial agents in the composition of toothpastes is becoming increasingly popular, due to lower side effects. The present study intended to investigate the antibacterial efficacy of a herbal toothpaste containing Bamboo salt on cariogenic oral bacteria. Methods: The present double-blinded parallel randomized controlled clinical trial was conducted on 60 dental students (age range: 18-30). Following the baseline saliva sampling, the participants were randomly assigned into the case and control groups, to use the Bamboo salt herbal toothpaste and conventional non-herbal toothpaste, respectively. They were instructed to brush their teeth twice a day using the Bass technique. Saliva sampling was repeated after four weeks. The salivary counts of Streptococcus mutans and Lactobacillus at baseline and 4-week follow-up were determined and presented as the logarithm of colony-forming units per milliliter (log CFU/mL). statistical analysis was performed using independent samples t-test and paired sample t-test (P<0.05). Results: A significant decrease in salivary Streptococcus mutans and Lactobacillus was observed using both toothpastes (*P<0.001). The difference between the antibacterial efficacy of two toothpaste types on Streptococcus mutans and Lactobacillus was not statistically significant (P=0.530, and P=0.137, respectively). Conclusion: Due to the comparable efficacy of the investigated herbal toothpaste with conventional toothpaste, it potentially qualifies as a complementary agent for self-care oral hygiene procedures. Trial registration: This trial was registered in the “Iranian Registry of Clinical Trials” (IRCT20210414050964N1) on 21/06/2021.

Inhibitory Effect of Oral Thin Films (OTFs) Containing Xylitol Against Streptococcus mutans

Dental Caries is a chronic disease affecting half of the global population, causing pain and discomfort due to progressive damage to the teeth. Whilst xylitol has been studied for its effect on dental caries prevention, current practices present few limitations for its successful oral delivery, including short residence time in the mouth and poor patient compliance. Recently, oral thin films (OTFs) emerged as an alternative to delivering xylitol in the oral cavity. This research aims to develop novel OTFs containing xylitol with extended-release properties (as determined by the disintegration time) and to investigate its effect on a cariogenic bacterial strain, Streptococcus mutans. The minimum inhibitory concentration (MIC) of xylitol was determined. Employing the microdilution broth method, the antibacterial activity of the oral thin films containing xylitol for oral S. mutans was performed with simulated salivary fluid, incubated at 1, 4, and 10 h. The MIC of xylitol was found at 10%. Meanwhile, there was no significant difference in the inhibition of S. mutans (p > 0.05) between the control, OTFs (10 h), and xylitol-OTF (1 h), with the latter, demonstrated only 16.58% inhibition. Interestingly, when compared to xylitol-OTF (1 h), xylitol-OTF showed significant inhibition (p < 0.05) to S. mutans after four h (+53.24 %) and almost a complete inhibition after ten h (-92.58 %). These results suggest that the OTFs demonstrated a gradual release of xylitol and inhibited oral biofilm formation by decreasing the growth of S. mutans in a time-dependent manner. Most importantly, the study indicated the successful development of a novel xylitol-OTF with potential as an oral health biotherapeutic agent.

Photodynamic Therapy (PDT) in Prosthodontics: Disinfection of Human Teeth Exposed to Streptococcus mutans and the Effect on the Adhesion of Full Ceramic Veneers, Crowns, and Inlays: An In Vitro Study

The use of PDT in prosthodontics as a disinfection protocol can eradicate bacteria from tooth surfaces by causing the death of the microorganisms to which the photosensitizer binds, absorbing the energy of laser light during irradiation. The aim of the study was to investigate the capacity of PDT to increase the bond strength of full ceramic restorations. In this study, 45 extracted human teeth were prepared for veneers, crowns, and inlays and contaminated with Streptococcus mutans. Tooth surfaces decontamination was performed using a diode laser and methylene blue as a photosensitizer. The disinfection effect and the impact on tensile bond strength were evaluated by scanning electron microscopy (SEM) and pull-out tests of the cemented ceramic prosthesis. Results show that the number of bacteria was reduced from colonized prepared tooth surfaces, and the bond strength was increased when PDT was used. In conclusion, the present study indicates that using PDT as a protocol before the final adhesive cementation of ceramic restorations could be a promising approach, with outstanding advantages over conventional methods.

Disruption of the adh (acetoin dehydrogenase) operon has wide-ranging effects on Streptococcus mutans growth and stress response

The agent largely responsible for initiating dental caries, Streptococcus mutans produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2-7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii , did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex , encoding an NAD + /NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans . IMPORTANCE Dental caries is often initiated by Streptococcus mutans , which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

Tetramic Acids Mutanocyclin and Reutericyclin A, Produced by Streptococcus mutans Strain B04Sm5 Modulate the Ecology of an in vitro Oral Biofilm

The human oral microbiome consists of diverse microbes actively communicating and interacting through a variety of biochemical mechanisms. Dental caries is a major public health issue caused by fermentable carbohydrate consumption that leads to dysbiosis of the oral microbiome. Streptococcus mutans is a known major contributor to caries pathogenesis, due to its exceptional ability to form biofilms in the presence of sucrose, as well as to its acidophilic lifestyle. S. mutans can also kill competing bacteria, which are typically health associated, through the production of bacteriocins and other small molecules. A subset of S. mutans strains encode the muc biosynthetic gene cluster (BGC), which was recently shown to produce the tetramic acids, mutanocyclin and reutericyclins A, B, and C. Reutericyclin A displayed strong antimicrobial activity and mutanocyclin appeared to be anti-inflammatory; however the effect of these compounds, and the carriage of muc by S. mutans, on the ecology of the oral microbiota is not known, and was examined here using a previously developed in vitro biofilm model derived from human saliva. While reutericyclin significantly inhibited in vitro biofilm formation and acid production at sub-nanomolar concentrations, mutanocyclin did not present any activity until the high micromolar range. 16S rRNA gene sequencing revealed that reutericyclin drastically altered the biofilm community composition, while mutanocyclin showed a more specific effect, reducing the relative abundance of cariogenic Limosilactobacillus fermentum. Mutanocyclin or reutericyclin produced by the S. mutans strains amended to the community did not appear to affect the community in the same way as the purified compounds, although the results were somewhat confounded by the differing growth rates of the S. mutans strains. Regardless of the strain added, the addition of S. mutans to the in vitro community significantly increased the abundance of S. mutans and Veillonella infantium, only. Overall, this study illustrates that reutericyclin A and mutanocyclin do impact the ecology of a complex in vitro oral biofilm; however, further research is needed to determine the extent to which the production of these compounds affects the virulence of S. mutans.

Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

Molecular Docking of Phytochemicals against Streptococcus mutans Virulence Targets: A Proteomic Insight into Drug Planning

Streptococcus mutans (S. mutans) is the most prevalent and most associated with dental caries. Here we aim to identify, through an in silico study, potential bioactive molecules against S. mutans. Twenty-four bioactive molecules with proven action against S. mutans were selected: 1-methoxyficifolinol; 5,7,2′,4′-tetrahydroxy-8-lavandulylflavanone (sophoraflavanone G); 6,8-diprenylgenistein; apigenin; artocarpesin; artocarpin; darbergioidin; dihydrobiochanin A; dihydrocajanin (5,2′,4′-trihydroxy-7-methoxyisoflavanone); erycristagallin; Erystagallin; ferreirin; fisetin; kaempferol; licoricidin; licorisoflavan A; licorisoflavan C; licorisoflavan E; luteolin (3′,4′,5,7-tetrahydroxyflavone); malvidin-3,5-diglucoside; myricetin; orientanol B; quercetin; and quercitrin. Moreover, we selected nine important target proteins for the virulence of this microorganism to perform as drug targets: antigen I/II (region V) (PDB: 1JMM); Antigen I/II (carbox-terminal region) (PDB: 3QE5); Spap (PDB: 3OPU); UA159sp signaling peptide (PDB: 2I2J); TCP3 signaling peptide (PDB: 2I2H); ATP-binding protein ComA (PDB: 3VX4); glucanosucrase (PDB: 3AIC); dextranase (PDB: 3VMO), and Hemolysin (PDB: 2RK5). Five molecules were revealed to be the best ligands for at least three target proteins, highlighting the following compounds: 11 (erystagallin), 10 (erycristagallin), 1 (methoxyficifonilol), 20 (malvidin-3,5-diglucoside), and 2 (sophoraflavanone G), which indicates a possible multi-target action of these compounds. Therefore, based on these findings, in vitro and in vivo tests should be performed to validate the effectiveness of these compounds in inhibiting S. mutans virulence factors. Furthermore, the promising results of these assays will allow the incorporation of these phytoconstituents in products for oral use for the control of tooth decay.