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Background: Streptococcus mutans and Streptococcus sobrinus are Gram-positive bacteria involved in the development of dental caries, as they are able to form biofilms on tooth enamel, ferment sugars into acids, and survive under acidic conditions. This ultimately leads to a local lowering of the pH value on the tooth surface, which causes enamel cavities., Hypothesis: One measure to reduce caries is to limit the growth of cariogenic bacteria by using two anti-bacterial agents with different mechanisms of action. The hypothesis of this study was that the anti-bacterial activity of ω-6 polyunsaturated arachidonic acid (AA) against S. mutans and S. sobrinus can be enhanced by the sesquiterpene alcohol trans, trans-farnesol (t,t-farnesol)., Methods: The anti-bacterial activity of single and combined treatment was determined by the checkerboard assay. Bacterial viability was assessed by live/dead SYTO 9/propidium iodide (PI) staining on flow cytometry. Anti-biofilm activity was determined by MTT metabolic assay, crystal violet staining of biofilm biomass, SYTO 9/PI staining by spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM)., Results: t,t-Farnesol lowered the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of AA at sub-MICs. AA reduced the metabolic activity of preformed mature biofilms, while t,t-farnesol had no significant effect. The enhanced anti-bacterial effect of the combined t,t-farnesol/AA treatment was further evidenced by increased PI uptake, indicating membrane perforation. The enhanced anti-biofilm effect was further verified by SDCM and HR-SEM. Gene expression studies showed reduced expression of some biofilm-related genes., Conclusions: Altogether, our study suggests a potential use of the two naturally occurring compounds arachidonic acid and t,t-farnesol for preventing biofilm formation by the cariogenic bacteria S. mutans and S. sobrinus . These findings have implications for caries prevention.

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.

Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.

The potent antimicrobial effects of antimicrobial photodynamic therapy (aPDT) with visible light plus water-filtered infrared-A irradiation and natural compounds as photosensitizers (PSs) have recently been demonstrated. The aim of this study was to obtain information on the antimicrobial effects of aPDT with mother juices against typical cariogenic oral Streptococcus pathogens in their planktonic form and determine its eradication potential on total human salivary bacteria from volunteers. Mother juices of pomegranate, bilberry, and chokeberry at different concentrations were used as PSs. The unweighted (absolute) irradiance was 200 mW cm -2 , applied five minutes. Planktonic cultures of Streptococcus mutans and Streptococcus sobrinus and total mixed bacteria from pooled saliva of volunteers were treated with aPDT. Up to more than 5 log 10 of S. mutans and S. sobrinus were killed by aPDT with 0.4% and 0.8% pomegranate juice, 3% and 50% chokeberry juice, and 12.5% bilberry juice (both strains). Concentrations of at least 25% (pomegranate) and >50% (chokeberry and bilberry) eradicated the mixed bacteria in saliva samples. This pilot study has shown that pomegranate mother juice is superior to the berry juices as a multicomponent PS for killing pathogenic oral bacteria with aPDT.

Non-cariogenic sweet substances, like sugar alcohols, are used to decrease the risk of caries by reducing the growth of dental plaque. The aim of our study was to reveal the impact of xylitol and erythritol on the growth and biofilm formation of cariogenic bacteria including as a novelty, set of clinical mutans streptococci and Scardovia wiggsiae and to assess the possible synergistic influence of these polyols. We found both xylitol and erythritol to express high growth inhibition effect on cariogenic bacteria. In synergistic effect experiments, 10% polyol combination with excess of erythritol was found to be more effective against growth of Streptococcus mutans and the combination with excess of xylitol more effective against growth of Streptococcus sobrinus and S. wiggsiae. In biofilm inhibition experiments, solutions of 10% polyols in different combinations and 15% single polyols were equally effective against mutans streptococci. At the same time, higher biofilm formation of S. wiggsiae compared to experiments without polyols was detected in different polyol concentrations for up to 34%. In conclusion, both erythritol and xylitol as well as their combinations inhibit the growth of different cariogenic bacteria. Biofilm formation of mutans streptococci is also strongly inhibited. When applying polyols in caries prophylaxis, it is relevant to consider that the profile of pathogens in a particular patient may influence the effect of polyols used.

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping ( p  < 0.05). Hot steeping significantly reduced bacterial growth ( p  < 0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin-resistant and -susceptible Staphylococcus aureus, antibiotic-resistant and -susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin-resistant S. aureus, methicillin-susceptible S. aureus, antibiotic-resistant S. pneumoniae isolates, antibiotic-susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3-1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9-22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9-22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells., (© 2019 The Societies and John Wiley & Sons Australia, Ltd.)

Aims: To investigate the effects of the ginsenoside Rh2 on monospecies and multispecies cariogenic biofilms and explore the mechanism of the antibiofilm effect of Rh2 in vitro., Methods and Results: Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis were chosen to form the monospecies or multispecies biofilms. Crystal violet staining and laser scanning confocal microscopy were used to observe the effect of Rh2 on biofilms in vitro. Cytotoxicity was examined by the Cell Counting Kit-8. The effects of Rh2 on bacterial membranes were observed via transmission electron microscopy (TEM). The isobaric tags for relative and absolute quantification (iTRAQ) method were used to profile the common differentially expressed proteins. Gene expression was analysed by reverse transcription quantitative polymerase chain reaction. In general, the treatment of cariogenic biofilms with Rh2 significantly decreased biomass accumulation by inhibiting bacterial growth and extracellular polysaccharide synthesis without any cytotoxic effects. TEM imaging showed that Rh2 could disrupt the cell membranes of these bacteria. The iTRAQ results indicated that the levels of mannose-specific IIC/D and acetaldehyde/alcohol dehydrogenase were substantially down-regulated, while the mRNA expression of the corresponding genes were significantly changed., Conclusions: Our data revealed a potential application for Rh2 in the protection against dental caries via the inhibition of cariogenic biofilms., Significance and Impact of the Study: This study describes the first application of a ginsenoside against multispecies cariogenic biofilms. Rh2 may serve as an alternative agent to prevent dental caries by effectively modulating the pathogenic potentials of oral biofilms., (© 2018 The Society for Applied Microbiology.)

Phenolic compounds in fruits such as cranberries have been shown to promote a number of biological activities. The purpose of this study was to investigate the effects of polyphenolic compound-containing lingonberry extract on oral streptococci and compare them with the known anti-cariogenic activity of cranberries. Water-soluble and polyphenol-rich fractions (Fractions I and II, respectively) were isolated from cranberries and lingonberries. The effects of those fractions on the biofilm formation ability and bioactivity of Streptococcus mutans MT8148R, Streptococcus sobrinus 6715, and Streptococcus sanguinis ATCC 10556 were then evaluated. Cranberry or lingonberry Fraction II (at 0.5-1 mg/ml) significantly reduced biofilm formation by S. mutans, S. sobrinus, and S. sanguinis. In contrast, cranberry or lingonberry Fraction I (at 0.5-2 mg/ml) increased biofilm formation by S. mutans and S. sobrinus, but not by S. sanguinis. Fractions I and II (at 1-2 mg/ml) also reduced the bioactivity of S. mutans, while Fraction II (at 0.5 mg/ml) enhanced the bioactivity of all tested strains. The results revealed that lingonberries contained a larger amount of polyphenol than cranberries and that they showed almost the same level of activity against the biofilm formation ability and bioactivity of oral streptococci. This indicates that polyphenol-rich lingonberry fraction offers a promising natural food derivative for prevention of dental caries.

Dental plaque biofilms cause various dental diseases; therefore, inhibiting the growths of the dental plaque bacteria which produce biofilms can be a strategy for preventing dental disease. Certain sulfated polysaccharides from marine algae exert antimicrobial activities against human bacterial pathogens in addition to their physiological benefits. On the basis of these observations, the antimicrobial and antibiofilm activities of sulfated polysaccharides from different marine algae were evaluated against dental plaque bacteria. Among the sulfated polysaccharides, a fucoidan from Fucus vesiculosus showed notable antimicrobial activities against the selected dental plaque bacteria, including some foodborne pathogenic bacteria. The minimum inhibitory concentrations were of 125 to 1000 µg mL -1 . Regarding the antibiofilm activity, the fucoidan at the concentrations of above 250 µg mL -1 completely suppressed the biofilm formations and planktonic cell growths of Streptococcus mutans and S. sobrinus . However, no eliminative effect on the completed biofilm was observed. The fucoidan consisted of almost fucose base polysaccharide containing approximately 14.0% sulfate content. The average molecular weight of the fucoidan was changed by heat treatment (121 °C for 15 min) and it affected the antimicrobial activity.

The purpose of the present study was to develop an antibacterial mouthguard (MG) material using a masterbatch of silvernanoparticle-embedded ethylene-vinyl acetate (EVA) copolymers. In order to verify that the testing material was clinically applicable as an antibacterial MG material, we conducted an antibacterial test, a shock absorption test, and analysis of in vitro silver release. The colony-forming activity of Streptococcus sobrinus, Porphyromonas gingivalis, and Escherichia coli were significantly inhibited on the testing materials compared with the commercial EVA sheet (p<0.05). The shock absorption capability of the testing material was not significantly different from that of the commercial EVA sheet. Cumulative silver release (in pure water) from the testing materials were infinitesimal after soaking for 20 days, which implied that there could be no harm in wearing the MG during exercise. These results showed that this testing material could be clinically applicable as an antibacterial MG material.

Sealing can arrest caries lesions. We aimed to evaluate if sealing effects and kinetics are bacterial-strain and sealing-material specific. Human dentin discs were mounted in a dual-chamber device. Caries lesions were induced chemically and contaminated with either Lactobacillus rhamnosus (LR) or Streptococcus sobrinus (SS). For (1) kinetics assessment, the initial bacterial load and the sealing period were varied, and lesions sealed using a self-etch adhesive and composite. For (2) comparing materials, six sealing protocols (#1-#6) were evaluated: 1# Self-etch adhesive plus composite placed without a liner, or #2 calcium hydroxide, or #3 mineral trioxide aggregate, or #4 Biodentine liners; #5 antibacterial adhesive plus composite; #6 glass ionomer cement. Pulpal fluid flow was simulated during sealing. The outcome was the number of surviving bacteria (CFU) per g dentin. For LR, bacterial survival increased significantly with increasing initial bacterial load and decreased with longer sealing periods. The relative reduction followed a first-order kinetics. More LR survived under calcium hydroxide or MTA than other materials (p < 0.001). For SS, nearly no bacteria survived sealing regardless of sealing period, initial bacterial load or sealing material. In conclusion, sealing effects and kinetics were strain- and material-specific.

Background: Alkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries., Objective: To investigate the effect of arginine on the growth and biofilm formation of oral bacteria., Methods and Results: Polymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2h and 8h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P=0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P<0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P=0.32, 0.06, 0.44 respectively)., Conclusions: Arg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: Characterization of a number of pulp capping materials and assessment of the leachate for elemental composition, antimicrobial activity and cell proliferation and expression., Methodology: Three experimental light curable pulp-capping materials, Theracal and Biodentine were characterized by scanning electron microscopy, energy dispersive spectroscopy and X-ray diffraction. The elemental composition of the leachate formed after 24h was assessed by inductively coupled plasma (ICP). The antimicrobial activity of the leachate was determined by the minimum inhibitory concentration (MIC) against multispecies suspensions of Streptococcus mutans ATCC 25175, Streptococcus gordonii ATCC 33478 and Streptococcus sobrinus ATCC 33399. Cell proliferation and cell metabolic function over the material leachate was assessed by an indirect contact test using 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay., Results: The hydration behavior of the test materials varied with Biodentine being the most reactive and releasing the highest amount of calcium ions in solution. All materials tested except the unfilled resin exhibited depletion of phosphate ions from the solution indicating interaction of the materials with the media. Regardless the different material characteristics, there was a similar antimicrobial activity and cellular activity. All the materials exhibited no antimicrobial activity and were initially cytotoxic with cell metabolic function improving after 3days., Conclusions: The development of light curable tricalcium silicate-based pulp capping materials is important to improve the bonding to the final resin restoration. Testing of both antimicrobial activity and biological behavior is critical for material development. The experimental light curable materials exhibited promising biological properties but require further development to enhance the antimicrobial characteristics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: In spite of contradicting results, the high susceptibility of composites for secondary caries is still often associated with the bacterial growth-stimulating effect of released methacrylate monomers. However, most studies that showed this effect were performed with techniques having inherent limitations (spectrophotometry)., Objectives: Therefore, our objective was to determine the effect of four methacrylate monomers (2-Hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA)) on the growth of two caries-associated bacteria, Streptococcus mutans and sobrinus, and one non-cariogenic species, Streptococcus sanguinis, using TaqMan quantitative polymerase chain reaction (qPCR) to quantify bacterial DNA., Materials and Methods: Cultures were exposed to monomer solutions selected after spectrophotometric growth measurements. At baseline and predetermined time intervals, bacterial DNA was extracted and quantified with TaqMan qPCR. Biofilms grown in the presence of monomers were analyzed with scanning electron microscopy (SEM)., Results: Spectrophotometry indeed showed increased growth rates of all three strains with 5 mM TEGDMA, EGDMA, and DEGDMA and increased total biomass of S. sanguinis with 5 mM TEGDMA. However, qPCR failed to show any growth-stimulating effect of these monomers on S. mutans and S. sobrinus. In contrast, some monomers exhibited a growth-inhibiting effect on S. sanguinis. SEM revealed extracellular matter in S. sobrinus and S. sanguinis biofilms, which might be attributed to polymer formation., Conclusions: Techniques which quantify bacterial DNA are more appropriate to evaluate bacterial growth in the presence of monomers than spectrophotometry., Clinical Relevance: Even though methacrylate monomers did not affect the growth of cariogenic species, growth inhibition of S. sanguinis, a non-cariogenic antagonistic species, may lead to ecological shifts towards higher cariogenicity.

Objectives: The accumulation of oral bacterial biofilm is the main etiological factor of oral diseases. Recently, electrolyzed hydrogen-rich water (H-water) has been shown to act as an effective antioxidant by reducing oxidative stress. In addition to this general health benefit, H-water has antibacterial activity for disease-associated oral bacteria. However, little is known about the effect of H-water on oral bacterial biofilm. The objective of this study was to confirm the effect of H-water on streptococcal biofilm formation., Methods: In vitro streptococcal biofilm was quantified using crystal violet staining after culture on a polystyrene plate. The effect of H-water on the expression of genes involved in insoluble glucan synthesis and glucan binding, which are critical steps for oral biofilm formation, was evaluated in MS. In addition, we compared the number of salivary streptococci after oral rinse with H-water and that with control tap water. Salivary streptococci were quantified by counting viable colonies on Mitis Salivarius agar-bacitracin., Results: Our data showed that H-water caused a significant decrease in in vitro streptococcal biofilm formation. The expression level of the mRNA of glucosyltransferases (gtfB, gtfc, and gtfI) and glucan-binding proteins (gbpC, dblB) were decreased remarkably in MS after H-water exposure for 60s. Furthermore, oral rinse with H-water for 1 week led to significantly fewer salivary streptococci than did that with control tap water., Conclusions: Our data suggest that oral rinse with H-water would be helpful in treating dental biofilm-dependent diseases with ease and efficiency., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cold-light bleaching treatment has grown to be a popular tooth whitening procedure in recent years, but its side effect of dental enamel demineralization is a widespread problem. The aim of this study was to synthesize zinc-substituted hydroxyapatite as an effective biomaterial to inhibit demineralization or increase remineralization. We synthesized zinc-substituted hydroxyapatite containing different zinc concentrations and analysed the product using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and energy dispersive spectrometer (EDS). The biological assessment of Zn-HA was conducted by CCK-8 assay and bacterial inhibition tests. pH cycling was performed to estimate the effect of Zn-HA on the enamel surface after cold-light bleaching treatment. The XRD, FTIR, and EDS results illustrated that zinc ions and hydroxyapatite combined in two forms: (1) Zn 2+ absorbed on the surface of HA crystal and (2) Zn 2+ incorporated into the lattice of HA. The results indicated that 2% Zn-HA, 4% Zn-HA, and 8% Zn-HA effectively inhibited the growth of bacteria yet showed poor biocompatibility, whereas 1% Zn-HA positively affected osteoblast proliferation. The XRD and scanning electron microscopy (SEM) results showed that the use of Zn-HA in pH cycling is obviously beneficial for enamel remineralization. Zinc-substituted hydroxyapatite could be a promising biomaterial for use in cold-light bleaching to prevent enamel demineralization.

Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F- channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species., Competing Interests: The authors have declared that no competing interests exist.

Objectives: In vitro methods to study dental biofilms are useful in finding ways to support a healthy microbial balance in the oral cavity. The effects of sucrose, xylitol, and their combination on three strains of Streptococcus mutans and one strain of Streptococcus sobrinus were studied using a dental simulator., Methods: A simulator was used to mimic the oral cavity environment. It provided a continuous-flow system using artificial saliva (AS), constant temperature, mixing, and hydroxyapatite (HA) surface in which the influence of xylitol was studied. The quantities of planktonic and adhered bacteria were measured by real-time qPCR., Results: Compared against the untreated AS, adding 1% sucrose increased the bacterial colonization of HA (p<0.0001) whereas 2% xylitol decreased it (p<0.05), with the exception of clinical S. mutans isolate 117. The combination of xylitol and sucrose decreased the bacterial quantities within the AS and the colonization on the HA by clinical S. mutans isolate 2366 was reduced (p<0.05). Increasing the concentration (2%-5%) of xylitol caused a reduction in bacterial counts even in the presence of sucrose., Conclusions: The continuous-culture biofilm model showed that within a young biofilm, sucrose significantly promotes whereas xylitol reduces bacterial colonization and proliferation. The results indicate that xylitol affects the ability of certain S. mutans strains to adhere to the HA. Clinical studies have also shown that xylitol consumption decreases caries incidence and reduces the amount of plaque. This study contributes to the understanding of the mechanism behind these clinical observations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Context: One traditional medicines, Hypericum perforatum L. (Hypericaceae), possesses several beneficial effects against depression, ulcers, dyspepsia, abdominal pains, burns, bacterial infections, migraine headaches, and sciatica., Objective: The present study investigates the antimicrobial activity of the ethanol extract (HP-EtOH) of H. perforatum and its sub-extracts, namely n-hexane (HP-hexane), chloroform (HP-CHCl3), ethyl acetate (HP-EtOAc), n-butanol (HP-n-BuOH), and water (HP-H2O) extracts, against Streptococcus mutans, S. sobrinus, Lactobacillus plantarum, and Enterococcus faecalis., Materials and Methods: For the evaluation of the antimicrobial activity, flowering aerial parts of H. perforatum were extracted with EtOH and then this extract was fractionated to obtain five sub-extracts in different polarities. Antimicrobial activities of HP-EtOH and its sub-extracts against Streptococcus mutans, S. sobrinus, L. plantarum, and E. faecalis were assessed by using colorimetric micro-well dilution at concentration ranges of 64-0.5 μg/ml as well as resazurin microplate and modified microtiter-plate assays between the ranges of 100 and 0.78125 μg/ml., Results: According to the results of the present study, HP-H2O sub-extract displayed strong antibacterial activity (MIC values 8 μg/mL) against S. sobrinus and L. plantarum, and exerted moderate activity against S. mutans and E. faecalis at 32 and 16 μg/mL concentrations, respectively. Other sub-extracts also demonstrated antimicrobial activity against S. sobrinus at a concentration of 16 μg/mL. HP-EtOAc and HP-n-BuOH showed antimicrobial activity against L. plantarum and HP-EtOAc and HP-H2O were also active against E. faecalis at the same concentrations (16 μg/mL)., Conclusion: According to the results, we suggest that H. perforatum could be employed as a natural antibacterial agent in oral care products.

Objectives: To investigate the effect of Galla chinensis chemical compounds on enamel caries remineralization in rats., Methods: A total of 40 rats were inoculated with Streptococcus sobrinus 6715 and fed a cariogenic diet (Keyes 2000). The rats were randomly divided into 4 groups and treated topically twice a day with each of the following extracts (or control) for 5 weeks: distilled and deionized water (DDW, negative control); 1,000 ppm NaF (positive control); 4,000 ppm G. chinensis crude aqueous extract (GCE), or 4,000 ppm gallic acid (GA). After the experimental period, Keyes' caries diagnosis and scoring technique was applied as a preliminary evaluation on the molar teeth. For more accurate remineralization data, the residual enamel volume of the first molar and the mineral density (MD) of the enamel were further analyzed by micro-CT., Results: The severity of molar enamel caries decreased in the following order of treatment groups: DDW > GA > GCE > NaF (p < 0.05). In addition, the residual first molar enamel volume and MD values increased in the order of DDW, GA, GCE and NaF treatment groups (p < 0.05). In most molars, remineralization layers were observed on the surface of lesions., Conclusion: G. chinensis compounds remineralize enamel caries lesions in a rat model., (© 2016 S. Karger AG, Basel.)

Conventional chemical antiseptics used for treatment of oral infections often produce side-effects, which restrict their long-term use. Plants are considered as perspective sources of novel natural antiseptics. However, little is still known about their inhibitory properties against oral pathogens. The objective of this study was to test in vitro antimicrobial activities of generally recognized as safe (GRAS) species against planktonic cultures of cariogenic, periodontal and candidal microorganisms and identify active compounds of the most active extracts. Growth-inhibitory effects of ethanol extracts from 109 GRAS plant species, six Humulus lupulus cultivars and two hop supercritical CO2 extracts were evaluated using broth microdilution method. The chemical analysis was done through high-performance liquid chromatography. Best results were obtained for supercritical CO2 and ethanol extracts of H. lupulus with minimum inhibitory concentrations (MIC) ≥8 μg/mL and ≥16 μg/mL, respectively. The chemical analysis of supercritical CO2H. lupulus extracts revealed that α- and β-acids were their main constituents. Capsicum annuum and Capsicum frutescens showed antibacterial effect against Streptococcus sobrinus and Streptococcus salivarius (MIC=64-128 μg/mL). These strains were further inhibited by Zanthoxylum clava-herculis (MIC=64-128 μg/mL) and Myristica fragrans (both MIC≥128 μg/mL). The latter also exhibited antimicrobial activity against Fusobacterium nucleatum (MIC=64 μg/mL). Punica granatum possessed inhibitory effects against Candida albicans (MIC=128 μg/mL) and F. nucleatum (MIC=64 μg/mL). The results indicate that supercritical CO2H. lupulus extracts together with ethanol extracts of C. annuum, C. frutescens, M. fragrans, P. granatum and Z. clava-herculis are promising materials for further investigation on new antiseptic agents of oral care products., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to determine the antibacterial effect of different formulations containing Lysozyme and Lactoferrin and drug delivery system as well as poloxamer 407 with the trade name of Pluronic F-127 and/or freeze dried liposome containing DOTAP [freeze dried Liposomal DOTAP] on Streptococcus sobrinus, Streptococcus mutans and Lactobacillus acidophilus in comparison with 0.2% chlorhexidine., Materials and Methods: The antibacterial effect was assessed by determining the minimum inhibitory concentrations (MIC) for the study and control groups on Streptococcus sobrinus, Streptococcus mutans and Lactobacillus acidophilus. The amounts of biofilm formation accumulation of Mutans Streptococci for 24h on sterile hydroxyapatite discs after application of different formulations were evaluated. The different formulations studied were: (1) Sorensen's Buffer Solution, (2) a gel formulation containing only poloxamer 407, (3) Lysozyme and Lactoferrin dissolved in Sorensen's Buffer Solution, (4) poloxamer 407 combined with the third formulation, (5) Freeze dried Liposomal DOTAP dissolved in Sorensen's Buffer Solution, (6) Freeze dried Liposomal DOTAP combined with poloxamer 407 dispersed in Sorensen's Buffer Solution, (7) Freeze dried Liposomal DOTAP combined with the third formulation, and (8) Lysozyme and Lactoferrin dissolved in Sorensen's Buffer Solution, which was then incorporated into poloxamer 407 and combined with Freeze dried Liposomal DOTAP. The positive and negative control groups were 0.2% chlorhexidine gel and empty hydroxyapatite discs, respectively. Statistical evaluation was carried out with Kruskal-Wallis and Dunn's multiple comparison tests., Results: It was observed that the first, third and fifth groups did not have any antibacterial effects on the tested bacteria. The groups that contained poloxamer 407 had nearly identical antibacterial effects on Mutans Streptococci and L. acidophilus. These formulations also inhibited biofilm formation of the bacteria (p<0.05) more effectively. In the positive control group, there was no biofilm formation., Conclusions: Among the formulations containing poloxamer 407, the one containing Lysozyme and Lactoferrin exhibited the highest inhibitory effect on the tested bacteria. This novel formulation can be beneficial as an antibacterial agent for the prevention of dental caries and biofilm formation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aim: Polymethyl-methacrylate (PMMA) is commonly used primarily for baseplates of orthodontic appliances (BOA). The activities of cariogenic bacteria in biofilm on these surfaces may contribute to dental caries, gingival inflammation and periodontal disease. The PMMA incorporated with nanoparticles of silver (NanoAg-I-PMMA) and NanoAg in situ in PMMA (NanoAg-IS-PMMA) have been shown to control the growth of cariogenic bacteria, but clinical trial of anti-cariogenic application of these novel materials in orthodontics has not been evaluated. The main aim of the study is to compare the clinical effectiveness of using NanoAg-IS-PMMA and NanoAg-I-PMMA for construction of new BOA in inhibiting the planktonic growth and biofilm formation of the cariogenic bacteria., Materials and Methods: Twenty four patients with a median age of 12.6 years (7-15) harboring Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus as well as Lactobacillus casei participated in the randomized, double-blind, cross-over study. The experimental BOA, NanoAg-IS-BOA and NanoAg-I-BOA, contained 0.5% w/w NanoAg while the control BOA was standard PMMA. Antibacterial effect of NanoAg-IS-BOA and NanoAg-I-BOA was assessed against test cariogenic bacteria by planktonic and biofilm bacterial cells growth inhibition., Results: The average levels of test cariogenic bacteria in saliva decreased about 2 to 70 fold (30.9-98.4%) compared to baseline depending on the microorganism type and test BOA. Biofilm inhibition analysis demonstrated that NanoAg-I-BOA and NanoAg-IS-BOA inhibited the biofilm of all test bacteria by 20.1 to 79.9% compared to BOA. NanoAg-IS-BOA had a strong anti-biofilm effect against S. mutans, S. sobrinus and L. casei. However, NanoAg-I-BOA showed only slight anti-biofilm effects on test bacteria. Most notably, at all period of the clinical trial, NanoAg-IS-BOA showed a higher antibacterial activity than NanoAg-I-BOA., Conclusion: Based on the novel data that presented here, the NanoAg-IS-BOA had strong antimicrobial activity in the planktonic phase and subsequent biofilm formation of the cariogenic bacteria., Clinical Significance: Wearing of NanoAg-IS-BOA has the potential to minimize dental plaque formation and caries during orthodontic treatment.

Purpose: To determine the antibacterial effect of nano-structured, sol-gel processed bioactive glasses that may be used for implants, coatings, and as adjuncts to dental restorative materials., Methods: Six bioactive glasses (BAG), three made with differing amounts of silica (65, 75 and 85 mole%), and three with different amounts of silica (61, 71, and 81 mole%) and 3 mole% fluoride were prepared by a sol-gel synthesis method and tested against clinically important bacteria species, Streptococcus sobrinus (ATCC33478), Streptococcus mutans (ATCC25175) and Enterococcus faecalis (ATCC19433). Bacterial suspensions were independently incubated with bioactive glass in particulate form (< 3 µm) for 4 and 24 hours. Viability was determined by colony-forming units., Results: At 4 hours, all BAG produced an order of magnitude reduction in all three bacteria. After 24 hours, all BAG produced a significant reduction in S. sobrinus colonies, but no further reduction in S. mutans; all BAG, except BAG 61-F, significantly reduced E. faecalis compared to the control. At 4 hours, an increase in the pH of the BAG groups (to pH 9) could also have contributed to the bactericidal effect. In further experiments it was found that the viability of S. sobrinus was significantly reduced following exposure to an extract of BAG in media adjusted to a pH of 7.4. Additionally media with pH adjusted to 9 exerted a significant antibacterial effect against S. sobrinus after 4 hours. To determine the influence of the calcium ions released from the BAG in the absence of the pH effect, a typical dose response was demonstrated after 4 hours of exposure of S. sobrinus to media containing various levels of calcium. The results of this study clearly suggest that the effect of BAG extract on bacteria is not only related to a pH effect, but is also linked to an effect of liberated ions, such as calcium, extracted from the surface of the bioactive glasses.

Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α-1,3-glucanase) and dextranase (α-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE-SUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microscopy. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 μg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries., (© 2014 S. Karger AG, Basel.)

Objectives: The antibacterial activity of two different formulations of a chlorhexidine/thymol varnish should be elucidated in vitro and in vivo., Methods: The agar diffusion assay with Cervitec(®) and CervitecPlus(®) and three reference strains each of streptococci, lactobacilli, actinomyces and periodontal pathogens was performed. In a split-mouth study, 40 volunteers applied the test (CervitecPlus(®), solvent water and ethanol) and control (Cervitec(®), solvent ethyl acetate) varnish at buccal recessions of premolar teeth at baseline as well as after two, four and seven days. Supra- and subgingival plaques were collected 2 weeks before baseline and at the screening appointments. Supragingival plaque was analysed for mutans streptococci and lactobacilli and subgingival samples for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Porphyromonas intermedia. Friedman/Wilcoxon tests and U-test were used for statistical analysis (P < 0.05)., Results: Most reference strains were susceptible with inhibition zones (mm) as follows: Cervitec(®)/CervitecPlus(®) streptococci 27 ± 1.7/21.3 ± 2.5, lactobacilli 26 ± 9.2/23.7 ± 4.9, actinomyces 36.3 ± 6.6/27.3 ± 1.5, periodontal pathogens 18.7 ± 7.6/18 ± 1.7. Both varnishes reduced significantly the counts of mutans streptococci and lactobacilli in the patients. However, no significant differences were found between test and control sides at any time. The total counts of periodontal pathogens were low. A tendency to higher counts of A. actinomycetemcomitans at the control side could be shown; the test side did not harbour significantly higher counts., Conclusion: Both varnishes may influence the plaque formation and reduce mutans streptococci in supragingival plaque., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The bacteria grow in oral cavity and product acids, which could induce dental caries. In this study, in order to obtain the relationship between procyanidin dimers from sorghum episperm (sorghum procyanidins, SPC) and its anticaries effect. The extract of SPC purified by macroporous resin was divided into three parts by gel chromatography, marked as GPC-1, GPC-2, and GPC-3 in order. The ESI-MS and MS/MS analysis indicated that the main composition of GPC-2 was procyanidin dimers. In addition, the capacities of antigrowth and antiacid on Sreptococcus sobrinus 6715 were analysised to investigate the effect of SPC dimers in protecting against dental caries. The results indicated that the minimum inhibitory concentration (MIC) of SPC dimers was 16 mg/mL. Furthermore, the SPC dimers had notable preventive effect < against the acid production of Sreptococcus sobrinus 6715 compared with the control group, which suppressed in a dose-dependent manner by pH decline. These findings indicated that SPC dimers had potential to be used as anticaries preventive medicine due to its strong capacity of antigrowth and antiacid.

The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)-Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 μg/ml were obtained against Streptococcus sobrinus (90 μg/ml), Lactobacillus casei (30 μg/ml), S. mutans (20 μg/ml), and S. mitis (18 μg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 μg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens., (Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.)

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants--(1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens--using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease., (© 2014 S. Karger AG, Basel.)

Dental caries is an oral disease, which has a high worldwide prevalence despite the availability of various prophylactic means, including the daily use of fluoride toothpastes, water fluoridation, dental sealants, oral health educational programs and various antiseptic mouth-rinses. One important reason for this is uncontrolled increase in consumption of foods containing considerable sucrose concentration, especially among children. Sucrose is easily metabolized by oral bacteria (mostly streptococci) to acids and, subsequently, causing tooth decay or dental caries. In the oral ecosystem, streptococci principally reside on tooth surfaces forming biofilm. Important structural and binding materials of biofilm are glucan polymers synthesized by several isoforms of glucosyltransferase enzyme present in certain species of oral bacteria, including mutans group streptococci - Streptococcus mutans and Streptococcus sobrinus, which preferably colonize humans. Thus, there is a constant need to develop the methods and chemotherapeutics for improving oral health care and decreasing teeth decay through the suppression of cariogenic biofilm formation in the oral cavity. The aim of this paper was to review literature related to the pathogenesis of dental caries as well as currently existing and experimental pharmaceutical substances used for prevention of this process.

Objectives: To examine the ability of a removable thermoplastic appliance (RTA) to adsorb hygienic solutions and inhibit bacterial growth and to examine the efficacy of three hygiene protocols in reducing bacterial biofilm adherence to RTA., Materials and Methods: Solution adsorption and bacterial growth inhibition were examined in vitro using paper vs RTA discs. Subsequently, 11 patients treated with RTA (mean age, 29.1 ± 4.7 years) were assigned into a sequence of three hygiene protocols: regular RTA brushing (baseline), immersion RTAs in chlorhexidine mouthwash (CHX), and using a vibrating bath with cleaning solution (VBC). For each patient, 12 upper RTAs were examined (2 baseline RTAs, 5 CHX RTAs, and 5 VBC RTAs), for a total of 132 RTAs. All RTAs were stained with gentian violet, and biofilm presence was measured using a photodensitometer., Results: The RTA discs did not adsorb CHX or cleaning solution. The later agent did not show antibacterial features. Baseline RTAs showed significant biofilm adherence (P < .001) on the posterior palatal side of the aligner and on the anterior incisal edge. CHX and VBC hygienic protocols significantly (P < .001) reduced baseline biofilm adherence by 16% and 50%, respectively. Hygienic improvement was maintained over 140 days when CHX and VBC were used. However, VBC was three times more efficient than CHX., Conclusions: This study highly recommends the use of a VBC protocol. Biofilm deposits on the RTA, especially on incisal edges and attachment dimples, could lead to inadequate tooth/RTA and attachment/RTA overlap and consequently impair tooth alignment.

Propolis is a non-toxic natural substance with multiple pharmacological properties including anti-cancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 μg mL(-1)). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short-chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram-negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram-negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96-well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress-response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane-damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti-GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane-damaged or dead cells. Production of GroEL may physically play an important role in biofilm development., (2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd)

The objective of this study was to evaluate anti-inflammatory and antimicrobial activities of neovestitol and vestitol isolated from Brazilian red propolis (BRP). BRP ethanolic extract (EEP), neovestitol, and vestitol were evaluated by anti-inflammatory properties using a neutrophil migration assay. The antimicrobial activity was evaluated by minimal inhibitory and bactericidal concentrations (MIC and MBC) against Streptococcus mutans , Streptococcus sobrinus , Staphylococcus aureus , and Actinomyces naeslundii . Neovestitol, vestitol, and EEP inhibited neutrophil migration at a dose of 10 mg/kg. Regarding antimicrobial activity, neovestitol showed MICs ranging from <6.25 to 25-50 μg/mL and MBCs ranging from 25-50 to 50-100 μg/mL, while vestitol showed MICs ranging from 25-50 to 50-100 μg/mL and MBCs ranging from 25-50 to 50-100 μg/mL. Both isoflavonoids neovestitol and vestitol are consistent bioactive compounds displaying anti-inflammatory and antimicrobial activities that can strongly act in a low dose and concentration and have a promising potential to be applied in the pharmaceutical and food industries.

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.

The peptide LYS-[TRP(6)]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH2) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37°C under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500-1.9 μg mL(-1)). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Backgrounds: Silver diamine fluoride (SDF) has clinical success in arresting dentin caries, this study aimed to investigate its mechanism of action., Methods: Using a computer-controlled artificial mouth, we studied the effect of 38% SDF on cariogenic biofilms and dentin carious lesions. We used five common cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus rhamnosus and Actinomyces naeslundii) to form a cariogenic biofilm that generated carious lesions with a depth of approximately 70 um on human dentin blocks. We applied 38% SDF to the lesions in the test group and water to those in the control group. The blocks were incubated in the artificial mouth for 21 days before evaluation. Microbial kinetics, architecture, viability and distribution were evaluated every 7 days using colony forming unit (CFU), scanning electron microscopy and confocal laser scanning microscopy. The physical properties of the carious lesions were evaluated with microhardness testing, energy dispersive spectroscopy (EDS) and Fourier transform infra-red spectroscopy (FTIR)., Results: The CFU results revealed fewer colony forming units in the test group compared with the control group (p < 0.01). Scanning electron microscopy and confocal microscopy showed less bacterial growth in the test group, and confluent cariogenic biofilm in the control group (p < 0.01). The microhardness and weight percentages of calcium and phosphorus in the test group from the outermost 50mum were higher than in the control group (p < 0.05). EDS showed that calcium and phosphous were higher in outer 50 mum in test groups than in the control FTIR revealed less exposed collagen I in the test lesions compared with the control group (p < 0.01)., Conclusions: 38% SDF inhibits multi-species cariogenic biofilm formation on dentin carious lesions and reduces the demineralization process.

The aim of this study was to evaluate the effect of the methanol extract of Withania somnifera (MEW) on the growth and virulence properties of Streptococcus mutans and Streptococcus sobrinus at sub-minimum inhibitory concentration (MIC) levels and to identify the main components of MEW. First, antibacterial activity of MEW against oral bacteria was determined using a micro-dilution method. Then, the effect of MEW on the growth of S. mutans and S. sobrinus was investigated at sub-MIC levels. To test the effect of MEW on the virulence properties of S. mutans and S. sobrinus, assays for acid production, acid tolerance, and biofilm formation were performed at sub-MIC levels. A GC-MS analysis for the main components of MEW was also carried out. MEW showed a broad antibacterial range against oral bacteria (MIC: 0.125-2 mg/mL). At sub-MIC levels, MEW dose-dependently increased doubling times of S. mutans and S. sobrinus up to 258% and 400%, respectively. Furthermore, MEW inhibited acid production, acid tolerance, and biofilm formation of S. mutans and S. sobrinus at sub-MIC levels. The GC-MS analysis revealed the presence of mono- and disaccharides, sugar alcohols, and organic acids as main components. These data suggest that MEW might be useful for restraining physiological activities of cariogenic bacteria., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dental caries is a multifactorial infectious disease that leads to the destruction of dental hard tissue. The main goal of research into medicinal plants is to seek compounds with antimicrobial activity for subsequent use in prevention strategies and control of infectious diseases. The aim of this study was to evaluate the antimicrobial activity of fractions and subfractions obtained from Elaeagia utilis against Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus. The plant material was collected in the town of Alban (Cundinamarca, Colombia), which is located at an altitude of 2245 meters above sea level. Two extracts were obtained by cold maceration of E. utilis leaves in (a) petroleum ether extract and (b) ethanol extract. Fractions were obtained from the petroleum ether extract by column vacuum chromatography, and from the ethanol extract by continuous liquid/liquid partitioning. The antimicrobial activity of fractions and subfractions was evaluated by the well diffusion method. At a concentration of 10 mg/well, several fractions from both extracts showed antimicrobial activity against S. mutans, S. sobrinus and L. acidophilus. Among the ethanol extract fractions, the dichloromethane fraction had notably greater antimicrobial activity. It was sub-partitioned, yielding three subfractions with inhibitory activity, of which the most active was MeOH: H2O (Bp) with minimum inhibitory concentration 0.1 mg/well on the 3 study bacteria. Terpenes, sesquiterpenlactones and simple phenolic compounds were identified in it. In conclusion, this study shows the antimicrobial potential of fractions and subfractions obtained from extracts of E. utilis leaves against bacteria that are important in dental caries.

Objective: To investigate the mechanism of lemon peel essential oil (LPE) on the cariogenicity of Streptococcus sobrinus (Ss)., Methods: LPE was extracted by the authors, and the minimum inhibition concentration (MIC) was measured by disc diffusion method. The LPE was used as the experimental group with concentrations ranging from 2.250 g/L to 0.281 g/L prepared with trypticase peptone yeast (TPY) culture medium, and TPY culture medium was used as the control group. Ss at the concentration of 10(8) CFU/ml was added to each group, and cultured for 6, 18, 24, 48 hours. Neson-Somogyi method was used to measure the content of reducing sugar, and glucosyltransferase (GTF) activity. The activity of lactate dehydrogenase (LDH) was measured by lactic acid and pyruvic acid continuous monitoring method. The content of water insoluble glucan (WIG) was measured by anthrone method, and the pH value of the culture solution was detected. The value of pH before the experiment and the time difference was alculated as ΔpH., Results: At the same time point, the activity of GTF and LDH and the concentration of WIG and the value ΔpH decreased gradually with the increase of concentration of LPE. There were significant differences between each experimental group and control group (P < 0.01). The control group had the maximum value, GTF: (6.71 ± 0.61) mIU, LDH: (135.8 ± 1.7) U/L, WIG: (47.15 ± 5.12) mg/L, ΔpH: (2.67 ± 0.01). The highest drug concentration group had the minimum value: GTF: (0.39 ± 0.07) mIU, LDH: (95.0 ± 5.4) U/L, WIG: (2.44 ± 0.38) mg/L, ΔpH: (0.61 ± 0.01)., Conclusions: The LPE below the MIC could still inhibit the GTF, LDH activity and lead to the decrease of WIG and the acid production.

Purpose: Acrylates for bonding of joint prostheses and stainless-steel brackets in orthopedics and orthodontics are prone to bacterial adhesion and biofilm formation, respectively, leading to serious infectious complications. Here we describe the preparation of a contact-killing acrylic resin by incorporation of [3-(methacryloylamino)propyl] trimethylammonium chloride (MAPTAC)., Methods: Physicochemical properties of the acrylates with and without MAPTAC incorporated were determined with X-ray photoelectron spectroscopy and water contact angles. The bond-strength of the acrylate with different percentages of MAPTAC was determined in a shear mode. The efficacy in contact-killing of the acrylate with MAPTAC incorporated with and without an adsorbed salivary coating was evaluated for various oral streptococcal strains. Cytotoxicity was tested against human skin fibroblasts., Results: Acrylates with 16 wt% and 20 wt% incorporated MAPTAC showed strong contact-killing of various oral streptococcal strains up to challenge concentrations of 109 mL-1 within 15 min, with no elution of antimicrobial polymers. Contact-killing reduced after coating with a salivary conditioning film, but still remained significant up to a challenge concentration of 105 mL-1. No cytotoxicity of acrylate with incorporated MAPTAC was observed toward human skin fibroblasts. The bond strengths of stainless-steel brackets fixed to etched enamel through the resin (12 ± 3 MPa) decreased with increasing amounts MAPTAC to half of the original value when 20 wt% of MAPTAC was incorporated, which remained within a clinically acceptable range., Conclusions: These results suggest that MAPTAC can be effectively incorporated in orthodontic resin to provide long-term bactericidal activity against oral bacteria, with potential application in orthopedics.

Linalool and α-terpineol exhibited strong antimicrobial activity against periodontopathic and cariogenic bacteria. However, their concentration should be kept below 0.4 mg/ml if they are to be used as components of toothpaste or gargling solution. Moreover, other compounds with antimicrobial activity against periodontopathic and cariogenic bacteria should be used in combination., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Background: Mexican-American children have a higher caries prevalence than the U.S. average. The Mothers and Youth Access (MAYA) study was a randomized clinical trial initiated to address this problem., Aim: Comparison of the efficacy of two prevention interventions in reducing early childhood caries (ECC)., Design: All 361 randomized mother-child dyads received oral health counselling. Beginning at 4 months postpartum, intervention mothers received chlorhexidine (CHX) mouthrinse for 3 months beginning 4 months postpartum and children received fluoride varnish (FV) every 6 months from age 12-36 months. Control group children received FV if precavitated lesions developed. Salivary mutans streptococci (MS) and lactobacilli were assessed., Results: No significant difference in children's 36-month caries incidence between groups; 34% in each group developed caries [(d(2+) fs) > 0]. About half of control group developed precavitated lesions and received therapeutic FV. Maternal MS levels declined during CHX use, but increased when discontinued., Conclusions: Maternal postpartum CHX regimen, oral health counselling and preventive child FV applications were not more efficacious than maternal counselling with child therapeutic FV for precavitated lesions for ECC prevention. FV for young children with brief maternal CHX use and oral health counselling may need to be combined with additional or longer-term therapies to significantly reduce ECC in high-risk populations., (© 2011 The Authors. International Journal of Paediatric Dentistry © 2011 BSPD, IAPD and Blackwell Publishing Ltd.)

Aims: The aim of this study was to evaluate and to compare the effect of two fluoride varnishes and one fluoride/chlorhexidine varnish on Streptococcus mutans and Streptococcus sobrinus biofilm formation, in vitro., Study Design: Standard acrylic discs were prepared and divided into groups based on the varnish applied to the disc surface: Fluor Protector, Bifluoride 12, and Fluor Protector + Cervitec (1:1). Untreated discs served as controls. In the study groups, biofilms of S. mutans and S. sobrinus were formed over 24 h, 48 h, and 5 days. The fluoride concentrations in the monospecies biofilms and viable counts of S. mutans and S. sobrinus were investigated., Results: In all study groups, a statistically significant increase in the viable number of S. mutans and S. sobrinus cells was observed between 24 h and 5 days. In both monospecies biofilms, the greatest antibacterial efficacy was detected in the Fluor Protector and Fluor Protector + Cervitec groups at 24 h. For all groups, the amount of fluoride released was highest during the first 24 h, followed by a significant decrease over the next 4 days. A negative correlation was detected between fluoride concentration and antibacterial effect in those groups with biofilms containing both species. Despite the release of high levels of fluoride, the greatest number of viable S. mutans and S. sobrinus cells was detected in the Bifluoride 12 group., Statistics: The data were analyzed using GraphPad Prism software (ver. 3)., Conclusions: The Fluor Protector + Cervitec varnish exerted prolonged antibacterial effects on S. mutans and S. sobrinus biofilms compared to the other varnishes tested.

Phytochemical investigation of a supercritical fluid extract of Glycyrrhiza uralensis has led to the isolation of 20 known isoflavonoids and coumarins, and glycycarpan (7), a new pterocarpan. The presence of two isoflavan-quinones, licoriquinone A (8) and licoriquinone B (9), in a fraction subjected to gel filtration on Sephadex LH-20 is due to suspected metal-catalyzed oxidative degradation of licoricidin (1) and licorisoflavan A (2). The major compounds in the extract, as well as 8, were evaluated for their ability to inhibit the growth of several major oral pathogens. Compounds 1 and 2 showed the most potent antibacterial activities, causing a marked growth inhibition of the cariogenic species Streptococcus mutans and Streptococcus sobrinus at 10 μg/mL and the periodontopathogenic species Porphyromonas gingivalis (at 5 μg/mL) and Prevotella intermedia (at 5 μg/mL for 1 and 2.5 μg/mL for 2). Only 1 moderately inhibited growth of Fusobacterium nucleatum at the highest concentration tested (10 μg/mL).