Antibacterial Activity of Epigallocatechin-3-gallate (EGCG) Loaded Lipid-chitosan Hybrid Nanoparticle against Planktonic Microorganisms.

Epigallocatechin-3-gallate (EGCG), a polyphenol derived from Green Tea, is one of the sources of natural bioactive compounds which are currently being developed as medicinal ingredients. Besides other biological activities, this natural compound exhibits anti-cariogenic effects. However, EGCG has low physical-chemical stability and poor bioavailability. Thus, the purpose of this study was to develop and characterize lipid-chitosan hybrid nanoparticle with EGCG and to evaluate its in vitro activity against cariogenic planktonic microorganisms. Lipid-chitosan hybrid nanoparticle (LCHNP-EGCG) were prepared by emulsion and sonication method in one step and characterized according to diameter, polydispersity index (PdI), zeta potential (ZP), encapsulation efficiency (EE), mucoadhesion capacity and morphology. Strains of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei were treated with LCHNP- EGCG, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated. LCHNP-EGCG exhibited a size of 217.3 ± 5.1 nm with a low polydispersity index (0.17) and positive zeta potential indicating the presence of chitosan on the lipid nanoparticle surface (+33.7 mV). The LCHNP-EGCG showed a spherical morphology, high stability and a mucoadhesive property due to the presence of chitosan coating. In addition, the EGCG encapsulation efficiency was 96%. A reduction of almost 15-fold in the MIC and MBC against the strains was observed when EGCG was encapsulated in LCHNP, indicating the potential of EGCG encapsulation in lipid-polymer hybrid nanoparticles. Taking the results together, the LCHNP-EGCG could be an interesting system to use in dental care due to their nanometric size, mucoadhesive properties high antibacterial activity against relevant planktonic microorganisms.

An in vitro coculture approach to study the interplay between dental pulp cells and Streptococcus mutans.

AIM: To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. METHODOLOGY: Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-ß1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = .05). RESULTS: While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-ß1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. CONCLUSIONS: The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

Serotipos prevalentes de Streptococcus mutans en América Latina / Prevalent serotypes of Streptococcus mutans in Latin America

La microbiota oral está conformada por diversas especies bacterianas que en condiciones normales desempeñan una función protectora del huésped; sin embargo, cuando existe un desequilibrio en el ecosistema, estos microorganismos son capaces de producir diversas manifestaciones como lo es el caso de la caries dental, enfermedad infecciosa producida principalmente por Streptococcus mutans, patógeno capaz de desmineralizar los tejidos duros del diente mediante la fermentación de hidratos de carbono obtenidos de la dieta. Se ha identificado en la pared celular de este microorganismo ocho serotipos que intervienen en la adhesión, agregación y coagregación bacteriana. En los seres humanos S. mutans presenta los serotipos c, e y f, siendo el serotipo c el más prevalente a nivel mundial, el cual se conoce que está asociado a pacientes sanos, a diferencia del e y f que son capaces de invadir las células endoteliales de las arterias coronarias. No obstante, en los últimos años se ha logrado identificar el serotipo k que de igual manera presenta alta capacidad de invadir el endotelio humano, actuando en la patogénesis de las enfermedades cardiovasculares. El objetivo de la presente revisión bibliográfica es lograr cuantificar los serotipos prevalentes de S. mutans en América Latina (AU)

The oral microbiota is made up of various bacterial species that under normal conditions perform a protective function of the host, however, when there is an imbalance in the ecosystem, these microorganisms are capable of producing various manifestations such as caries, an infectious disease. produced mainly by Streptococcus mutans, a pathogen capable of demineralizing the hard tissues of the tooth through the fermentation of carbohydrates obtained from the diet. Eight serotypes involved in bacterial adhesion, aggregation and coaggregation have been identified in the cell wall of this microorganism. In humans, S. mutans presents serotypes c, e, and f, serotype c being the most prevalent worldwide, which is known to be associated with healthy patients, unlike e and f, which are capable of invading the endothelial cells of the coronary arteries. However, in recent years it has been possible to identify serotype k, which also has a high capacity to invade the human endothelium, acting in the pathogenesis of cardiovascular diseases. The objective of this literature review is to quantify the prevalent serotypes of S. mutans in Latin America (AU)

ZccE, a P-type ATPase contributing to biofilm formation and competitiveness in Streptococcus mutans.

Most living organisms require zinc for survival; however, excessive amounts of this trace element can be toxic. Therefore, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent introduction of exogenous zinc ions, present a serious challenge for bacteria colonizing the oral cavity. Streptococcus mutans is considered one of the main bacterial pathobiont in dental caries. Here, we verified the role of a P-type ATPase ZccE as the main zinc-exporting transporter in S. mutans and delineated the effects of zinc toxification caused by zccE deletion in the physiology of this bacterium. The deletion of the gene zccE severely impaired the ability of S. mutans to grow under high zinc stress conditions. Intracellular metal quantification using inductively coupled plasma optical emission spectrometer revealed that the zccE mutant exhibited approximately two times higher zinc accumulation than the wild type when grown in the presence of a subinhibitory zinc concentration. Biofilm formation analysis revealed less single-strain biofilm formation and competitive weakness in the dual-species biofilm formed with Streptococcus sanguinis for zccE mutant under high zinc stress. The quantitive reverse transcription polymerase chain reaction test revealed decreased expressions of gtfB, gtfC, and nlmC in the mutant strain under excessive zinc treatment. Collectively, these findings suggest that ZccE plays an important role in the zinc detoxification of S. mutans and that zinc is a growth-limiting factor for S. mutans within the dental biofilm.

Salivary microbiome and metabolome analysis of severe early childhood caries.

BACKGROUND: Severe early childhood caries (SECC) is an inflammatory disease with complex pathology. Although changes in the oral microbiota and metabolic profile of patients with SECC have been identified, the salivary metabolites and the relationship between oral bacteria and biochemical metabolism remains unclear. We aimed to analyse alterations in the salivary microbiome and metabolome of children with SECC as well as their correlations. Accordingly, we aimed to explore potential salivary biomarkers in order to gain further insight into the pathophysiology of dental caries. METHODS: We collected 120 saliva samples from 30 children with SECC and 30 children without caries. The microbial community was identified through 16S ribosomal RNA (rRNA) gene high-throughput sequencing. Additionally, we conducted non-targeted metabolomic analysis through ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry to determine the relative metabolite levels and their correlation with the clinical caries status. RESULTS: There was a significant between-group difference in 8 phyla and 32 genera in the microbiome. Further, metabolomic and enrichment analyses revealed significantly altered 32 salivary metabolites in children with dental caries, which involved pathways such as amino acid metabolism, pyrimidine metabolism, purine metabolism, ATP-binding cassette transporters, and cyclic adenosine monophosphate signalling pathway. Moreover, four in vivo differential metabolites (2-benzylmalate, epinephrine, 2-formaminobenzoylacetate, and 3-Indoleacrylic acid) might be jointly applied as biomarkers (area under the curve = 0.734). Furthermore, the caries status was correlated with microorganisms and metabolites. Additionally, Spearman's correlation analysis of differential microorganisms and metabolites revealed that Veillonella, Staphylococcus, Neisseria, and Porphyromonas were closely associated with differential metabolites. CONCLUSION: This study identified different microbial communities and metabolic profiles in saliva, which may be closely related to caries status. Our findings could inform future strategies for personalized caries prevention, detection, and treatment.

Nicotine is a potent extracellular polysaccharide inducer in Fusobacterium nucleatum biofilms

The purpose of this in vitro study was to analyze the influence of nicotine on the extracellular polysaccharides in Fusobacterium nucleatum biofilm. Methods: F. nucleatum (ATCC 10953) biofilms supplemented with different concentrations of nicotine (0, 0.5, 1, 2, 4, and 8 mg/mL) were grown in two different BHI broth conditions [no sucrose and 1% sucrose]. Extracellular polysaccharides assay, pH measurements, and a spectrophotometric assay were performed. Data were submitted for ANOVA and Tukey honestly significant difference analyses (HSD) tests (α =.05). Results: Extracellular polysaccharides synthesis was influenced by an interaction between nicotine concentrations and growth medium solution containing sucrose (P<.05). The pH values declined in the sucrose-exposed biofilm were greater than in the group exposed only to nicotine (P<.05). The biofilm exposed to sucrose and nicotine had a higher total biofilm growth (P<.05) than the nicotine-treated biofilm without sucrose. Conclusions: Regardless of sucrose exposure, biofilms exposed to different nicotine concentrations influenced the amount of extracellular polysaccharides

Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 ß), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1ß correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1ß and IL6 and the chemokine CXCL8.

Effect of Lemon Essential Oil Microemulsion on the Cariogenic Virulence Factor of Streptococcus mutans via the Glycolytic Pathway.

PURPOSE: To investigate the effects and mechanisms of lemon essential oil products on dental caries prevention. MATERIALS AND METHODS: Lemon essential oil microemulsions (LEOM) with concentrations of 1/8 minimum inhibitory concentration (MIC), 1/4 MIC, and 1/2 MIC were applied to S. mutans at concentrations of 0.2%, 1%, and 5% glucose, respectively. Changes in acid production capacity of S. mutans were measured based on changes in pH. The effect of the reductive coenzyme I oxidation method on LDH activity was examined. The effect of lemon essential oil microemulsion on the expression of the lactate dehydrogenase gene (ldh) was detected by a quantitative real-time polymerase chain reaction. RESULTS: Lemon essential oil microemulsion at 1/2 MIC concentration reduced the environmental pH value at different glucose concentrations, compared to those observed in the control group (p < 0.05). LDH activity of S. mutans was decreased at three subinhibitory concentrations of lemon essential oil microemulsions (p < 0.05). The effect of lemon essential oil microemulsions on S. mutans LDH activity and bacterial acid production were positively correlated (r = 0.825, p < 0.05). Lemon essential oil microemulsion at 1/2 MIC concentration downregulated the expression of the ldh gene of S. mutans at different glucose concentrations (p < 0.05). In different glucose environments, lemon essential oil microemulsions at subminimum inhibitory concentrations can inhibit the acid production of S. mutans by reducing ldh expression and LDH activity in the glycolytic pathway, proving its anti-caries potential. CONCLUSIONS: LEOM can effectively prevent dental caries and maintain the microecological balance of the oral environment.

Novel strategy for dental caries by physiologic dentin regeneration with CPNE7 peptide.

OBJECTIVE: CPNE7-derived functional peptide (CPNE7-DP) has been introduced as a bioactive therapeutics for dentin diseases. CPNE7-DP regenerates tubular dentin on the pulpal side and occlude dentinal tubules. CPNE7-DP was capable to treat dentin hypersensitivity typically associated with dentinal wear at the neck of the tooth. However, the role of CPNE7-DP in another common dentin disease, dental caries, remains uninvestigated. In this study, we evaluated the potential application of CPNE7-DP in dentin caries using an experimental dentin caries model in rats. DESIGN: The stability of CPNE7-DP in caries-like environments including pathologic bacteria of caries or low pH was tested. We established a nutrition-time/hyposalivation-based dental caries rat model by inoculating caries-inducing bacteria and diet for sufficient time. Glycopyrrolate has been treated to induce reversible hyposalivation for accelerating caries progression. Then the tubular dentin regeneration was investigated with histologic methods. Also, modulation of inflammation or autophagy by CPNE7-DP was investigated with marker gene expression in human dental pulp cells (hDPCs) and immunohistochemistry. RESULTS: CPNE7-DP was stable with caries-inducing bacteria and low pH. Establishment of dentin caries was confirmed with radiographic and histologic evaluation. CPNE7-DP regenerated a substantial amount of tubular tertiary dentin and alleviated the pulp inflammation of dentin caries. Under inflammatory conditions, CPNE7-DP reduced the expression of inflammatory cytokines. These phenomena could be the consequence of the modulation of autophagy by CPNE7-DP, which reactivates inflamed odontoblasts. CONCLUSIONS: Overall, CPNE7-DP, which repairs caries through physiological dentin regeneration, might help overcoming the limitations of current restorative caries treatments.

Anti-Biofilm Activities of Chinese Poplar Propolis Essential Oil against Streptococcus mutans.

Streptococcus mutans (S. mutans) is a common cariogenic bacterium that secretes glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs) and plays an important role in plaque formation. Propolis essential oil (PEO) is one of the main components of propolis, and its antibacterial activity has been proven. However, little is known about the potential effects of PEO against S. mutans. We found that PEO has antibacterial effects against S. mutans by decreasing bacterial viability within the biofilm, as demonstrated by the XTT assay, live/dead staining assay, LDH activity assay, and leakage of calcium ions. Furthermore, PEO also suppresses the total of biofilm biomasses and damages the biofilm structure. The underlying mechanisms involved may be related to inhibiting bacterial adhesion and GTFs activity, resulting in decreased production of EPSs. In addition, a CCK8 assay suggests that PEO has no cytotoxicity on normal oral epithelial cells. Overall, PEO has great potential for preventing and treating oral bacterial infections caused by S. mutans.

Association between Bifidobacterium and Scardovia Wiggsiae and caries-related factors in severe early childhood caries and caries-free Thai children: a quantitative real-time PCR analysis and a questionnaire cross-sectional study.

AIMS: To quantitatively identify Bifidobacterium, S. wiggsiae and S. mutans in plaque samples obtained from children with severe-ECC and caries-free groups and to analyze their association with caries-related factors retrieved from the questionnaire in each group. STUDY DESIGN: To establish the 2 study groups, clinical examination in 122 Thai children, aged 2-5 years, recorded decayed, missing and filled teeth scores (dmft), in addition to plaque and gingival indices. Sixty one children in the caries-free group and 61 in the S-ECC group were identified. A questionnaire was used to assess the parent's attitudes and behavior regarding the child's oral hygiene care and diet. METHODS: Pooled overnight supra gingival plaque was collected from each child using a sterile toothpick, released in 1 ml of TE buffer, transported on ice to the Laboratory and stored at - 20 °C. DNA was extracted from the plaque based on enzymatic lysis and quantitative real-time PCR using fluorescent dye (SYBR green) in addition to Agarose gel electrophoresis were performed. All laboratory and retrieved from the questionnaire data per child were recorded and statistically analysed. RESULTS: S. wiggsiae (p < 0.005) and S. mutans (p < 0.001) were higher in the S-ECC group. Bifidobacterium, S. mutans, and S. wiggsiae were associated with the dmft score and gingival index (p < 0.001). The dmft scores of children who detected only S. mutans were significantly lower than the dmft scores of children who detected two bacteria; S. mutans + S. wiggsiae (p = 0.028), S. mutans + Bifidobacterium (p = 0.026), and three bacteria; S. mutans + Bifidobacterium + S. wiggsiae (p = 0.007). Children who found all three bacteria (Bi + Sm + Sw) had the highest dmft scores, followed by children who had two bacteria (Bi + Sw, or Bi + Sm, or Sw + Sm). The guardians' education levels, occupations, household income, prolonged bottle feeding, taking of water after bottle or breast feeding, eating sugar-coated crackers or bread with sweetened cream, and premature birth were the factors that related to S-ECC. CONCLUSION: Levels of S. wiggsiae and S. mutans, guardian's education, family economics, prolonged bottle feeding, eating high sugar-containing snacks and premature birth were associated with S-ECC.

Supragingival microbiome alternations as a consequence of smoking different tobacco types and its relation to dental caries.

This study aimed to assess the effect of smoking different tobacco types on the supragingival microbiome and its relation to dental caries. Forty supragingival plaque samples were collected from smokers of a single tobacco type and non-smokers seeking treatment at the University Dental Hospital Sharjah, UAE. DMFT (decayed, missing and filled teeth) was determined for all participants who were divided into two groups: no-low caries (NC-LC: DMFT = 0-4; n = 18) and moderate-high caries (MC-HC: DMFT = 5-20; n = 22). 16S rRNA gene was sequenced using third-generation sequencing with Nanopore technology. Microbiome composition and diversity were compared. Caries was most common among cigarette smokers. Supragingival microbiota were significantly altered among smokers of different tobacco types. In cigarette smokers, cariogenic bacteria from genus Streptococcus (including S. mutans) were significantly more among subjects with NC-LC, while Lactobacilli (including L. fermentum) were more among subjects with MC-HC. In medwakh smokers, several periodontopathogens were significantly elevated in subjects with NC-LC, while other pathogenic bacteria (as Klebsiella pneumoniae) were more in those with MC-HC. Cigarette and alternative tobacco smoking had a significant impact on the supragingival microbiome. Indeed, further studies are required to unravel the consequences of oral dysbiosis triggered by smoking. This could pave the way for microbiota-based interventional measures for restoring a healthy oral microbiome which could be a promising strategy to prevent dental caries.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Title IgA Nephropathy and Oral Bacterial Species Related to Dental Caries and Periodontitis.

A relationship between IgA nephropathy (IgAN) and bacterial infection has been suspected. As IgAN is a chronic disease, bacteria that could cause chronic infection in oral areas might be pathogenetic bacteria candidates. Oral bacterial species related to dental caries and periodontitis should be candidates because these bacteria are well known to be pathogenic in chronic dental disease. Recently, several reports have indicated that collagen-binding protein (cnm)-(+) Streptococcs mutans is relate to the incidence of IgAN and the progression of IgAN. Among periodontal bacteria, Treponema denticola, Porphyromonas gingivalis and Campylobacte rectus were found to be related to the incidence of IgAN. These bacteria can cause IgAN-like histological findings in animal models. While the connection between oral bacterial infection, such as infection with S. mutans and periodontal bacteria, and the incidence of IgAN remains unclear, these bacterial infections might cause aberrantly glycosylated IgA1 in nasopharynx-associated lymphoid tissue, which has been reported to cause IgA deposition in mesangial areas in glomeruli, probably through the alteration of microRNAs related to the expression of glycosylation enzymes. The roles of other factors related to the incidence and progression of IgA, such as genes and cigarette smoking, can also be explained from the perspective of the relationship between these factors and oral bacteria. This review summarizes the relationship between IgAN and oral bacteria, such as cnm-(+) S. mutans and periodontal bacteria.

Streptococcus mutans in atherosclerotic plaque: Molecular and immunohistochemical evaluations.

OBJECTIVES: To verify the presence of Streptococcus mutans (S. mutans) in atherosclerotic plaque (AP) using techniques with different sensitivities, correlating with histological changes in plaque and immunoexpression of inflammatory markers. MATERIALS AND METHODS: Thirteen AP samples were subjected to real-time polymerase chain reaction (qRT-PCR), histopathological analyses, histochemical analysis by Giemsa staining (GS), and immunohistochemical analysis for S. mutans, IL-1ß, and TNF-α (streptavidin-biotin-peroxidase method). Ten necropsy samples of healthy vessels were used as controls. RESULTS: All AP samples showed histopathological characteristics of severe atherosclerosis and were positive for S. mutans (100.0%) in qRT-PCR and immunohistochemical analyses. GS showed that Streptococcus sp. colonized the lipid-rich core regions and fibrous tissue, while the control group was negative for Streptococcus sp. IL-1ß and TNF-α were expressed in 100% and 92.3% of the AP tested, respectively. The control samples were positive for S. mutans in qRT-PCR analysis, but negative for S. mutans, IL-1ß, and TNF-α in immunohistochemical analyses. CONCLUSION: The detection of S. mutans in AP and the visualization of Streptococcus sp. suggested a possible association between S. mutans and atherosclerosis. The results obtained from the control samples suggested the presence of DNA fragments or innocuous bacteria that were not associated with tissue alteration. However, future studies are necessary to provide more information.

Deletion of the yqeK gene leads to the accumulation of Ap4A and reduced biofilm formation in Streptococcus mutans.

Diadenosine-5',5'''-P1, P4-tetraphosphate (Ap4A) is a second messenger playing a crucial role in various life activities of bacteria. The increase of Ap4A expression is pleiotropic, resulting in an impairment in the formation of biofilm and other physiological functions in some bacteria. However, Ap4A function in Streptococcus mutans, an important pathogen related to dental caries, remains unknown. In this work, the Ap4A hydrolase, YqeK, was identified and characterized in S. mutans. Then, the effects of yqeK deletion on the growth, biofilm formation, and exopolysaccharide (EPS) quantification in S. mutans were determined by the assessment of the growth curve, crystal violet, and anthrone-sulfuric acid, respectively, and visualized by microscopy. The results showed that the in-frame deletion of the yqeK gene in S. mutans UA159 led to an increase in Ap4A levels, lag phase in the early growth, as well as decrease in biofilm formation and water-insoluble exopolysaccharide production. Global gene expression profile showed that the expression of 88 genes was changed in the yqeK mutant, and among these, 42 were upregulated and 46 were downregulated when compared with the wild-type S. mutans UA159. Upregulated genes were mainly involved in post-translational modification, protein turnover, and chaperones, while downregulated genes were mainly involved in carbohydrate transport and metabolism. Important virulence genes related to biofilms, such as gtfB, gtfC, and gbpC, were also significantly downregulated. In conclusion, these results indicated that YqeK affected the formation of biofilms and the expression of biofilm-related genes in S. mutans.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 µg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

Bioinspired caries preventive strategy via customizable pellicles of saliva-derived protein/peptide constructs.

Dental caries has been the most widespread chronic disease globally associated with significant health and financial burdens. Caries typically starts in the enamel, which is a unique tissue that cannot be healed or regrown; nonetheless, new preventive approaches have limitations and no effective care has developed yet. Since enamel is a non-renewable tissue, we believe that the intimate overlaying layer, the acquired enamel pellicle (AEP), plays a crucial lifetime protective role and could be employed to control bacterial adhesion and dental plaque succession. Based on our identified AEP whole proteome/peptidome, we investigated the bioinhibitory capacities of the native abundant proteins/peptides adsorbed in pellicle-mimicking conditions. Further, we designed novel hybrid constructs comprising antifouling and antimicrobial functional domains derived from statherin and histatin families, respectively, to attain synergistic preventive effects. Three novel constructs demonstrated significant multifaceted bio-inhibition compared to either the whole saliva and/or its native proteins/peptides via reducing biomass fouling and inducing biofilm dispersion beside triggering bacterial cell death. These data are valuable to bioengineer precision-guided enamel pellicles as an efficient and versatile prevention remedy. In conclusion, integrating complementary acting functional domains of salivary proteins/peptides is a novel translational approach to design multifunctional customizable enamel pellicles for caries prevention.

Effect of exopolysaccharides from cariogenic bacteria on human gingival fibroblasts.

Bacterial biofilm (dental plaque) plays a key role in caries etiopathogenesis and chronic periodontitis in humans. Dental plaque formation is determined by exopolysaccharides (EPSs) produced by cariogenic and periopathogenic bacteria. The most frequent cariogenic bacteria include oral streptococci (in particular S. mutans) and lactobacilli (most frequently L. acidophilus). In turn, the dominant periopathogen in periodontitis is Porphyromonas gingivalis. Development of dental caries is often accompanied with gingivitis constituting the mildest form of periodontal disease. Basic cellular components of the gingiva tissue are fibroblasts the damage of which determines the progression of chronic periodontitis. Due to insufficient knowledge of the direct effect of dental plaque on metabolic activity of the fibroblasts, this work analyses the effect of EPSs produced by S. mutans and L. acidophilus strains (H2O2-producing and H2O2-not producing) on ATP levels in human gingival fibroblasts (HGF-1) and their viability. EPSs produced in 48-hours bacterial cultures were isolated by precipitation method and quantitatively determined by phenol - sulphuric acid assay. ATP levels in HGF-1 were evaluated using a luminescence test, and cell viability was estimated using fluorescence test. The tests have proven that EPS from S. mutans did not affect the levels of ATP in HGF-1. Whereas EPS derived from L. acidophilus strains, irrespective of the tested strain, significantly increased ATP levels in HGF-1. The analysed EPSs did not affect the viability of cells. The tests presented in this work show that EPSs from cariogenic bacteria have no cytotoxic effect on HGF-1. At the same time, the results provide new data indicating that EPSs from selected oral lactobacilli may have stimulating effect on the synthesis of ATP in gingival fibroblasts which increases their energetic potential and takes a protective effect.

Fabrication of oral nanovesicle in-situ gel based on Epigallocatechin gallate phospholipid complex: Application in dental anti-caries.

The conventional anti-caries agents exhibit many shortcomings such as poor stability, low efficacy or short residence time in the oral environment, it is urgent to develop efficacy treatments to prevent dental caries. As the most active polyphenols from tea, Epigallocatechin gallate (EGCG) shows remarkable anti-cariogenic bioactivity. However, the poor stability and low bioavailability of EGCG limit its potential application. This study aimed to fabricate nanovesicles in-situ gel based on EGCG phospholipid complex in order to increase its stability and efficacy. The formation of EGCG phospholipid complex was characterized by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The ethanol injection method was used to prepare the EGCG-loaded nanovesicles, an optimal ratio of Poloxamer407 (P407) and Poloxamer188 (P188) as in-situ gel matrix was selected to fabricate oral nanovesicles in-situ gel. EGCG-loaded nanovesicle in-situ gel based on the phospholipid complex had uniform spherical shape without any agglomeration. The discrete nanoparticle with a size (131.44 ± 4.24 nm) and a negative zeta potential value at -30.7 ± 0.5 mV possessed good physical stability and high entrapment efficiency (83.66 ± 3.2%). The formulation exhibited a strong antibacterial activity on S. mutans, which could reduce acid production and tooth surface adhesion. In addition, EGCG formulation could inhibit the formation of glucan and biofilm from S. mutans by suppressing the activity of glycosyltransferase enzymes (GTF). In conclusion, the EGCG-loaded nanovesicle in-situ gel holds great promise as an efficient anti-cariogenic formulation for topical oral delivery.