Cariogenic Streptococcus Mutans Research Articles

Lupinifolin from Albizia myriophylla wood: A study on its antibacterial mechanisms against cariogenic Streptococcus mutans

ObjectiveTo determine the anti-Streptococcus mutans mechanisms of action of lupinifolin from Albizia myriophylla Benth. (Fabaceae) wood and provide scientific evidence to support the traditional use of the plant against dental caries. MethodsThe minimum inhibitory concentration (MIC) was evaluated using the broth micro-dilution method. The effects of lupinifolin on bactericidal activity, bacterial cell walls, and membranes were investigated by time-kill, lysis, and leakage assays, respectively. Electron microscopy was utilized to observe any cell morphological changes caused by the compound. Localization of lupinifolin in S. mutans was detected using the thin layer chromatography technique. ResultsThe MIC range of lupinifolin against S. mutans (n=6) was 2–4 μg/ml. This compound displayed bactericidal effects on S. mutans ATCC 25175 by 90–99.9% killing at 4MIC-16MIC after 8–24 hours. Lupinifolin-treated cells demonstrated no lysis. However, significant cytoplasmic leakage through the bacterial membrane was observed after treatment with lupinifolin at 4MIC-16MIC. As revealed by ultrastructural analysis, lupinifolin produced some changes in bacterial cell walls and membranes. Moreover, the compound was observed in the cytoplasmic fraction of the lupinifolin-treated cells. These results suggest that lupinifolin can enter the cell of bacteria but does not accumulate in the cell envelope and subsequently disrupts the integrity of the cytoplasmic membrane, leading to cell death. ConclusionThe scientific evidence from this study offers valuable insights into the potential role of lupinifolin in pharmaceutical and antibiotic applications and supports the therapeutic effects of A. myriophylla, which has traditionally been used as an alternative treatment for dental caries.

Factors Influencing the Competition between Streptococcus oligofermentans and Streptococcus mutans in Dual-Species Biofilms

Previous studies have shown that Streptococcus oligofermentans inhibits the growth of cariogenic Streptococcus mutans in biofilms in vitro and is considered a probiotic candidate for caries prevention. This study aimed to examine the effects of various environmental factors on the competition between S. oligofermentans and S. mutans in a dual-species biofilm model. Single or dual S. oligofermentans and S. mutans biofilms were grown in a 96-well active attachment model for 48 h. Several growth conditions were examined in the model, namely: S. oligofermentans was inoculated 24 h before S. mutans or vice versa; the growth medium was supplemented with 0.2% sucrose or 0.4% glucose; biofilms were grown under a constantly neutral pH or pH-cycling condition, which included 8 h of neutral pH and 16 h of pH 5.5. The 48-h biofilms were examined for viable cell counts and lactic acid and hydrogen peroxide production ability. When S. oligofermentans was inoculated first, it clearly inhibited the growth of S. mutans and reduced the biofilm lactic acid production by up to 8-fold through hydrogen peroxide production, independently of sugar supply and pH conditions. When S. mutans was inoculated first, the level of inhibition by S. oligofermentans varied depending on the sugar supply and pH conditions. Thus, the inhibition efficacy of S. oligofermentans against S. mutans in dual-species biofilms is influenced by environmental factors. This study provides practical information on how to maximize the efficacy of S. oligofermentans.

The synthetic human beta-defensin-3 C15 peptide exhibits antimicrobial activity against Streptococcus mutans, both alone and in combination with dental disinfectants.

Streptococcus mutans is a major etiologic agent of human dental caries that forms biofilms on hard tissues in the human oral cavity, such as tooth and dentinal surfaces. Human β-defensin-3 (HBD3) is a 45-amino-acid natural antimicrobial peptide that has broad spectrum antimicrobial activity against bacteria and fungi. A synthetic peptide consisting of the C-terminal 15 amino acids of HBD3 (HBD3-C15) was recently shown to be sufficient for its antimicrobial activity. Thus, clinical applications of this peptide have garnered attention. In this study, we investigated whether HBD3-C15 inhibits the growth of the representative cariogenic pathogen Streptococcus mutans and its biofilm formation. HBD3-C15 inhibited bacterial growth, exhibited bactericidal activity, and attenuated bacterial biofilm formation in a dose-dependent manner. HBD3-C15 potentiated the bactericidal and anti-biofilm activity of calcium hydroxide (CH) and chlorhexidine digluconate (CHX), which are representative disinfectants used in dental clinics, against S. mutans. Moreover, HBD3-C15 showed antimicrobial activity by inhibiting biofilm formation by S. mutans and other dentinophilic bacteria such as Enterococcus faecalis and Streptococcus gordonii, which are associated with dental caries and endodontic infection, on human dentin slices. These effects were observed for HBD3-C15 alone and for HBD3-C15 in combination with CH or CHX. Therefore, we suggest that HBD3-C15 is a potential alternative or additive disinfectant that can be used for the treatment of oral infectious diseases, including dental caries and endodontic infections.

Photo-cross-linked Antibacterial Zein Nanofibers Fabricated by Reactive Electrospinning and its Effects against Streptococcus mutans

Native zein electrospun nanofibers have shown poor solvent resistance and low mechanical strength. Compared to other toxic cross-linkers, a safer method of stabilizing zein based fibers while retaining or with improved mechanical strength is needed to convert these materials for biomedical applications where culture media or body fluids may be present. We report here a method of fabricating non-toxic zein nanofibers using reactive electrospinning coupled with in situ photo-cross-linking. The cross-linked zein nanofibers exhibited significantly improved mechanical strength and sustained morphology against water and aqueous ethanol solution. This process doesn't require additional conventional cross-linking agents to form cross-linking network, which is advantageous for biomedical applications. Antimicrobial monomer with photo-reactive moiety was coupled with methacrylate zein nanofibers and showed strong inhibitory activity against cariogenic Streptococcus mutans. Cytotoxicity test with human gingival fibroblasts revealed high biocompatibility.

Effects of Lectins on initial attachment of cariogenic Streptococcus mutans.

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159.

Despite continuous efforts to control cariogenic dental biofilms, very few effective antimicrobial treatments exist. In this study, we characterized the activity of the novel synthetic cyclic lipopeptide 4 (CLP-4), derived from fusaricidin, against the cariogenic pathogen Streptococcus mutans UA159. We determined CLP-4's MIC, minimum bactericidal concentration (MBC), and spontaneous resistance frequency, and we performed time-kill assays. Additionally, we assessed CLP-4's potential to inhibit biofilm formation and eradicate preformed biofilms. Our results demonstrate that CLP-4 has strong antibacterial activity in vitro and is a potent bactericidal agent with low spontaneous resistance frequency. At a low concentration of 5 μg/ml, CLP-4 completely inhibited S. mutans UA159 biofilm formation, and at 50 μg/ml, it reduced the viability of established biofilms by >99.99%. We also assessed CLP-4's cytotoxicity and stability against proteolytic digestion. CLP-4 withstood trypsin or chymotrypsin digestion even after treatment for 24 h, and our toxicity studies showed that CLP-4 effective concentrations had negligible effects on hemolysis and the viability of human oral fibroblasts. In summary, our findings showed that CLP-4 is a potent antibacterial and antibiofilm agent with remarkable stability and low nonspecific cytotoxicity. Hence, CLP-4 is a promising novel antimicrobial peptide with potential for clinical application in the prevention and treatment of dental caries.

ACTIVITY OF PERICARP EXTRACT OF MANGOSTEEN AGAINST ORAL STREPTOCOCCI

The increasing prevalence of dental caries is still as a major world health problem. Caries is the direct result of acid production by cariogenic oral pathogens, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria with minimal side effects on the oral tissues are much needed, especially natural agents derived directly from plants. Phytochemical studies have shown that the extracts from various parts of mangosteen or Garciniamangostana Linn tree contain varieties of secondary metabolites such as prenylated and oxygenated xanthones, many of which have been found in vitro to have antimicrobial properties against oral pathogens. Several studies which examined the eficacy of herbal for human health have shown that xanthones from mangosteen have remarkable biological activities such as antioxidant, antimicrobial, anticancer etc, and had no cytotoxic effects on human gingival fibroblasts. Their results showed that among these xanthone derivatives obtain from pericarp extract of mangosteen, α-mangostin has the most potent antimicrobial activity against cariogenic Streptococcus mutans. It can be concluded that the strong antimicrobial activity of the pericarp extract of mangosteen is a good drug of choice that might be helpful in preventing the dental caries.

The antibacterial activity of green coffee and Arabica coffee extracts on cariogenic Streptococcus mutans isolated from dental caries: An in vitro study.

The antibacterial activity of green coffee and Arabica coffee extracts on cariogenic Streptococcus mutans isolated from dental caries: An in vitro study.

Isolation of cariogenic Streptococcus mutans and the inhibitory effect of egg yolk antibody on caries

Objective: To isolate the cariogenic Streptococcus mutans (Sm) strains and study the therapeutical effect of egg yolk antibody (IgY) of the Sm on dental caries development. Methods: Sm strains were isolated from the children's dental plaque samples. Morphological, biochemical and molecular biological methods were applied to identify the serotype, acid producing and adhesion abilities of isolated Sm strains. After inactivation one of the Sm strains was used as antigen to immune laying hens to collect and extract the specific anti-Sm IgY. The rats were infected with Sm (serotype e). After 16 weeks of infection, all the rats were found developing dental caries. The rats were then randomly divided into two groups. The rats in experimental group were supplied with diet containing anti-Sm IgY while the rats in control group with normal IgY. All rats were sacrificed after another 8 weeks' observation. The degree of caries for each rat was assessed using Keyes' method. Results: We isolated 7 Sm strains from the children's dental plaque samples in the present study. The numbers of serotype c, e, f, k were 3, 2, 0 and 2, respectively. All strains showed similar morphological and biochemical characters as standard UA159 Sm strain, and possessed strong capabilities of acid production and adherence. Interestingly, even the same serotypec strains, such as No.3 and No.7 strains, demonstrated significant difference on acid producing and adherence capabilities. After 16 weeks infection with serotype e strain, the rats' mandibular teeth were apparently decayed, and treatment with specific anti-Sm IgY obviously attenuated the development of caries in the experiment group rats (16.4±2.0) compared with that in the control group rats (30.2±9.3) (P<0.05) determined by Keyes' method. Conclusions: Seven cariogenic Sm strains of different serotypes were isolated, which possesses similar morphology and biochemical characters. Although belonging to the same serotype strains they always show significant difference in acid-producing and adherencec apabilities. Further experiment provides evidences that the serotype e strain could obviously induce caries independently, and employment of specific anti-Sm IgY as passive immunotherapy additive might effectively inhibit the further development of dental caries.

Metabolic-Activity-Based Assessment of Antimicrobial Effects by D2O-Labeled Single-Cell Raman Microspectroscopy.

To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (D2O)-based single-cell Raman microspectroscopy (D2O-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm-1 as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, D2O-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.

Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome

Dental caries is a costly and prevalent disease characterized by the demineralization of the tooth’s enamel. Disease outcome is influenced by host factors, dietary intake, cariogenic bacteria, and other microbes. The cariogenic bacterial species Streptococcus mutans metabolizes sucrose to initiate biofilm formation on the tooth surface and consequently produces lactic acid to degrade the tooth’s enamel. Persistence of S. mutans biofilms in the oral cavity can lead to tooth decay. To date, no anticaries therapies that specifically target S. mutans biofilms but do not disturb the overall oral microbiome are available. We screened a library of 2-aminoimidazole antibiofilm compounds with a biofilm dispersion assay and identified a small molecule that specifically targets S. mutans biofilms. At 5 µM, the small molecule annotated 3F1 dispersed 50% of the established S. mutans biofilm but did not disperse biofilms formed by the commensal species Streptococcus sanguinis or Streptococcus gordonii. 3F1 dispersed S. mutans biofilms independently of biofilm-related factors such as antigen I/II and glucosyltransferases. 3F1 treatment effectively prevented dental caries by controlling S. mutans in a rat caries model without perturbing the oral microbiota. Our study demonstrates that selective targeting of S. mutans biofilms by 3F1 was able to effectively reduce dental caries in vivo without affecting the overall oral microbiota shaped by the intake of dietary sugars, suggesting that the pathogenic biofilm-specific treatment is a viable strategy for disease prevention.

Hydrogel Containing an Extract of Tormentillae Rhizoma for the Treatment of Biofilm-Related Oral Diseases

In the present study, hydrogels containing the dried extract of tormentil (Potentilla erecta (L.) Raeusch., Rosaceae) were designed and qualitatively evaluated regarding their viscosity, thixotropy, and texture properties. As mucoadhesiveness is a crucial factor determining drug retention within the oral cavity, mucoadhesive properties expressed as the work of adhesion and maximum detachment force under the presence of porcine buccal mucosa and two different models of mucoadhesive layers: mucin and gelatin discs were evaluated. The fingerprints of the analyzed tormentil extract were established by using a LC- -ESI-MS method. The dominating compounds of the tested extract are oligomeric proanthocyanidins and agrimoniin. This study reveals that designed hydrogels are promising semi-solid delivery systems for the dry extract of tormentil with beneficial mucoadhesive, thixotropic, and texture characteristics and may be utilized as platforms for tormentil delivery to the oral cavity in periodontal diseases. In vitro evaluation of the efficacy of the tormentil hydrogel against cariogenic Streptococcus mutans biofilms was also performed. The hydrogel significantly reduced artificial biofilm formation. For all Streptococci, complete inhibition was revealed at a final concentration of the extract of 2 mg/mL. These beneficial characteristics, as well as the anti-biofilm activity, enable its use for dental care, but further clinical studies are required.

Characterization of Streptococcus sanguis molecular receptors for Streptococcus mutans binding molecules

Background: Dental caries is a major problem in oral cavity. If dental caries causes cavity, the structure of dental hard tissue will not be reversible because of damage in the structure of the hard tissue. The early pathogenesis mechanism of dental caries is an adhesion interaction between cariogenic Streptococcus mutans microorganisms and tooth surface pellicles. The attachment involves a specific molecular component interaction between the bacterial complement molecules and the surface of the host. Streptococcus sanguis as a dominant ecology at the beginning of bacterial plaque aggregation will colonize the tooth surface earlier than S. mutans. The surface of bacterial cells can express some adesin. The bacteria also can express receptors for adhesins of other bacteria. Specific receptors for adhesions of S. Mutans bacteria are not only found in the pellicles, but also present in pioneer bacteria, such as S. sanguis. Adhesion between those bacteria is called as coagregation. Purpose: This study aimed to analyze the characterization of Streptococcus sanguis molecular receptors for Streptococcus mutans binding molecules. Method: This study used a sonication method for protein isolation of S. mutans and S. sanguis bacterial biofilms, as well as electrophoresis method using 12 % SDS-PAGE gel and Western Blot analysis. Result: Results of the protein profile analysis of S. mutans biofilms using 12% SDS-PAGE showed that there were 17 bands, each of which molecular weights was 212, 140, 81, 65, 61, 48, 45, 44, 40, 39, 33 , 25, 23, 19, 17, 12, and 11 kDa. On the other hand, results of the protein profile analysis of S. sanguis biofilms using 12% SDS-PAGE showed that there were 15 bands, each of which molecular weight was 130, 85, 65, 61, 48, 46, 40, 37, 29, 25, 23, 21, 17, 15, and 12 kDa. And, results of the analysis of S. sanguis receptor molecules using Western blot showed that there were three bands, each of which molecular weight was 130, 85, and 40 kDa. Conclusion: S. sanguis bacteria have specific receptor molecules for S. mutans bacteria with the molecular weight of 130, 85, and 40 kDa.

Massively Increased Caries Susceptibility in an Irf6 Cleft Lip/Palate Model

Patients with cleft lip/palate (CLP) have been reported, in some studies, to exhibit an increased prevalence of caries, although the underlying cause for this increase is unknown. In genetically defined mouse models, studies of postnatal sequelae associated with CLP have been hampered by neonatal lethality. Using a conditional targeting approach, we ablated the major CLP gene Irf6 only in the late embryonic oral epithelium (Irf6 cKO), bypassing the role of the gene in lip and palate morphogenesis and thus ensuring survival to adulthood. We report that Irf6 cKO mice present with 1) dysplastic salivary glands due to disruptions of epithelial junctional complexes, likely secondary to elevated activation of RHO GTPases, and 2) increased salivary cell proliferation. These changes result in significantly reduced saliva flow rate and buffering capacity and increased mucus acidity. A marked decrease in expression of CCL27, one of the major mucosal and skin cytokines, was found that correlated with increased bacterial colonization of the oral cavity with the cariogenic pathogen Streptococcus mutans and other bacteria. When placed on a high-sugar diet, Irf6 cKO mice show a 35-fold increase in presentation and severity of dental caries as compared with wild-type control mice. Strikingly, within the 8-wk test period, many molars extensively dissolved, and there was progressive loss of the alveolar bone, likely as a result of increased colonization of periodontal pathogens. These data provide the first mechanistic insight into the heightened caries susceptibility associated with CLP and indicate a direct role for the major CLP gene Irf6 in salivary gland development and a significant role in regulating oral immunity. Our data suggest that careful evaluation of salivary gland function and the implementation of early oral health preventive strategies are warranted to reduce the burden of dental care in this at-risk population.

Characterization of a plasmid carrying cat, ermB and tetS genes in a foodborne Listeria monocytogenes strain and uptake of the plasmid by cariogenic Streptococcus mutans

A multi-drug resistant (MDR) Listeria monocytogenes isolate (serotype 1/2c) was recovered from a quick-frozen rice flour product collected from Langfang city in northern China. PCR screening identified the presence of cat, ermB and tetS genes. The plasmid profile of the strain showed the presence of an approximately 22.4-kb plasmid. Curing of this plasmid resulted in the loss of cat, ermB and tetS genes and increased susceptibility to several antibiotics, suggesting the involvement of the plasmid in multiple antibiotic resistances. Moreover, the plasmid was able to be uptaken by human oral pathogen Streptococcus mutans by natural transformation and resulted in the acquiring of multiple resistances in the transconjugants. This study contributes to our knowledge on acquired multi-drug resistance in foodborne pathogenic L.monocytogenes, which will add to a better understanding of effective clinical management of listeriosis.

Cationic Lipid Content in Liposome-Encapsulated Nisin Improves Sustainable Bactericidal Activity against Streptococcus mutans.

An oral infectious disease, dental caries, is caused by the cariogenic streptococci Streptococcus mutans. The expected preventive efficiency for prophylactics against dental caries is not yet completely observed. Nisin, a bacteriocin, has been demonstrated to be microbicidal against S. mutans, and liposome-encapsulated nisin improves preventive features that may be exploited for human oral health. Here we examined the bactericidal effect of charged lipids on nisin-loaded liposomes against S. mutans and inhibitory efficiency for insoluble glucan synthesis by the streptococci for prevention of dental caries. Cationic liposome, nisin-loaded dipalmitoylphosphatidylcholine/phytosphingosine, exhibited higher bactericidal activities than those of electroneutral liposome and anionic liposome. Bactericidal efficiency of the cationic liposome revealed that the vesicles exhibited sustained inhibition of glucan synthesis and the lowest rate of release of nisin from the vesicles. The optimizing ability of cationic liposome-encapsulated nisin that exploit the sustained preventive features of an anti-streptococcal strategy may improve prevention of dental caries.

English

Piper aduncum has been widely used for medicinal purposes, and it has also been known to possess antimicrobial activity. Dental plaque is a complex ecosystem that harbors benign and pathogenic bacteria. It is desirable that compounds targeted to treat dental plaque-related diseases should be selective in their action, preserving the benign bacteria and inactivating the pathogenic ones. Thus, the study evaluated the antibacterial activity of P. aduncum leaf extracts against cariogenic (Streptococcus mutans) and health-associated (Streptococcus sanguinis) bacterium. For this, ethanol extracts were obtained by decoction, maceration, Soxhlet or turbo-extraction. The minimum inhibitory concentration (MIC) of the extracts was determined using the broth microdilution method. The influence of extracts on virulence traits of S. mutans was evaluated by the adherence assay to glass surface and by the glycolytic pH drop assay. S. mutans was more susceptible to crude extracts of P. aduncum than S. sanguinis and the highest activity was obtained with the maceration extract (MAC). Thus, MAC was further fractionated by gel permeation chromatography and the most active fraction against S. mutans (MIC of 0.08 mg/mL) had a MIC of 0.62 mg/mL for S. sanguinis. In addition, this fraction inhibited sucrose-dependent adherence of S. mutans and also reduced the level of acid production by this bacterium. The preferential activity of P. aduncum extracts towards S. mutans compared with S. sanguinis, in addition to their ability to inhibit sucrose-dependent adherence and reduce the level of acid production by S. mutans, suggest that this plant may have a potential to prevent dental caries. Key words: Plant extracts, Piper aduncum, antibacterial activity, Streptococcus mutans, caries prevention.

L-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms.

l-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous l-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous l-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical l-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, l-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H2O2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H2O2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of l-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. Dental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria. l-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by which l-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms using l-arginine.

Lack of Buffering by Composites Promotes Shift to More Cariogenic Bacteria

Secondary caries (SC) remains a very important problem with composite restorations. The objectives of this study were to test the acid-buffering ability of several restorative materials and to evaluate whether buffering of the restorative material has an impact on the microbial composition of the biofilm. Disk-shaped specimens of conventional composite, composite with surface prereacted glass-ionomer filler particles (so-called giomer), glass-ionomer cement (GIC), amalgam, and hydroxyapatite (HAp) (control) were exposed to aqueous solutions with pH 4, 5, 6, and 7 and to the medium containing bacteria-produced acids, and pH changes were recorded over several days. Next, material specimens were immersed in bacterial growth medium with pH adjusted to 5. After a 24-h incubation, the extracts were collected and inoculated with a cariogenic (Streptococcus mutans) and a noncariogenic (Streptococcus sanguinis) species. The bacterial growth was monitored both in a single-species model by spectrophotometry and in a dual-species model by viability quantitative polymerase chain reaction. Amalgam and HAp showed the strongest acid-buffering ability, followed by the GIC and the giomer, while the conventional composite did not exhibit any buffering capacity. Furthermore, due to the lack of acid-buffering abilities, composite was not able to increase the pH of the medium (pH 5), which, in the absence of antibacterial properties, allowed the growth of S. mutans, while the growth of S. sanguinis, a less aciduric species, was completely inhibited. A similar effect was observed when bacteria were cultured together: there was a higher percentage of S. mutans and lower percentage of S. sanguinis with the conventional composite than with other materials and HAp. In conclusion, conventional composites lack the ability to increase the local pH, which leads to the outgrowth of more acidogenic/aciduric bacteria and higher cariogenicity of the biofilm. Together with lack of antibacterial properties, lack of buffering may account for the higher susceptibility of composites to SC.

Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.

Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenicStreptococcus mutansbacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome ofS. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance.We constructed SMU_1290c- and SMU_1289c-knockoutS. mutansUA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disruptedEscherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated.All three gene-knockoutS. mutansstrains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disruptedE.colicell from fluoride toxicity.We conclude that the chloride channel permeases contribute to fluoride resistance inS. mutans.