Escape from the competence state in Streptococcus mutans is governed by the bacterial population density

Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co‐culture experiments performed with oral and non‐oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra‐species and extra‐species quorum‐sensing systems nor the other 13 two‐component regulatory systems identified in S. mutans were involved in the cell‐density‐dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut‐off involving cell‐density‐dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability.     

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans

We report the whole genome sequence of the serotype e Cbm⁺ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra‐oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall‐anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram‐positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.     

Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans

Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram‐positive bacteria, including Bacillus and Lactococcus, encode functions involved in protein post‐export. In this study, we constructed a PrsA‐deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/membrane protein profile as well as cell‐surface‐related phenotypes, including auto‐aggregation, increased surface hydrophobicity and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface‐display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to those identified in Bacillus, and so is likely to be involved in protein post‐export.     

Fermentative 2‐carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans

Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by‐product of the pyruvate bypass that converts pyruvate into acetyl‐Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate‐bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate‐bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl‐CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down‐stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.     

Salivary antimicrobial proteins associate with age‐related changes in streptococcal composition in dental plaque

Secretion of antimicrobial proteins (AMPs) and salivary antibodies can modify biofilm formation at host body surfaces. In adolescents, associations have been reported between dental caries and salivary AMPs. AMPs demonstrate direct antimicrobial effects at high concentrations, and at lower more physiological concentrations they mediate changes in host cell defenses, which may alter the local environment and indirectly shape local biofilm formation. The expression of salivary AMPs in preschool children, at an age when the oral bacteria are known to change, has not been investigated. We sought to investigate salivary AMP expression in the context of previously well‐documented changes in the oral cavities of this age group including salivary immunoglobulin A (IgA), oral bacteria and dental caries. Dental plaque and saliva were collected from 57 children aged 12–24 months at baseline, of whom 23 children were followed‐up at 3 years of age. At each time, saliva was assessed for LL37, human neutrophil peptides 1–3, calprotectin, lactoferrin, salivary IgA, total plaque bacteria and Streptococcus mutans. Over time, concentrations of AMPs, S. mutans and bacteria‐specific salivary IgA increased. Caries experience was also recorded when children were 3 years old. Concentrations of AMPs were highest in the saliva of 3‐year‐old children with the greatest burden of S. mutans. These data suggest that salivary AMPs are variable over time and between individuals, and are linked with bacterial colonization. At follow up, the majority of children remained caries free. Larger longitudinal studies are required to confirm whether salivary AMP levels are predictive of caries and whether their modulation offers therapeutic benefit.     

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum

Adhesin‐mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species‐specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non‐mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram‐positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD‐SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species‐ and subspecies‐dependent adhesin interactions.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Sublingual immunization with the phosphate‐binding‐protein (PstS) reduces oral colonization by Streptococcus mutans

Bacterial ATP‐binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate‐binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat‐labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A‐secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries.     

Comparison of immunological and microbiological characteristics in children and the elderly with or without dental caries

The purpose of this study was to determine the relationships among early childhood caries (ECC), root caries (RC), the quantity of Streptococcus mutans in saliva, and the concentrations of total and specific secretory IgA (sIgA). Saliva samples were collected from 70 children, 3–4 yr of age, with and without ECC, and from 43 adults, ≥60 yr of age, with and without RC. The decayed, missing, and filled teeth (dmft) and decayed, missing, and filled surfaces (dmfs) scores of each child, and the root decayed and filled teeth (RDFT) and root decayed and filled surfaces (RDFS) scores of each elderly subject, were determined. The S. mutans levels, total sIgA, and specific sIgA against two virulence antigens of S. mutans in saliva were analysed using quantitative real‐time PCR (qPCR) and ELISAs. The quantity of S. mutans was significantly higher in caries‐positive subjects within the two populations than in the caries‐free subjects; and a positive correlation was found between the quantity of S. mutans and the dmft, dmfs, RDFT, and RDFS scores. In addition, the salivary total sIgA was significantly higher in children with severe early childhood caries (SECC) and in the elderly subjects with RC. Moreover, although the S. mutans level was significantly higher, the concentrations of specific sIgA against S. mutans antigens were significantly lower in samples from elderly subjects than in samples from children. These results support the concept that S. mutans is positively associated with ECC and RC. Furthermore, the levels of S. mutans‐specific antibodies in saliva are too low to prevent infection with cariogenic bacteria and to inhibit development of ECC and RC.     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

Deficiency of BrpA in Streptococcus mutans reduces virulence in rat caries model

Our recent studies have shown that BrpA in Streptococcus mutans plays a critical role in cell envelope biogenesis, stress responses, and biofilm formation. In this study, a 10‐species consortium was used to assess how BrpA deficiency influences the establishment, persistence, and competitiveness of S. mutans during growth in a community under conditions typical of the oral cavity. Results showed that, like the wild‐type, the brpA mutant was able to colonize and establish on the surfaces tested. Relative to the wild‐type, however, the brpA mutant had a reduced ability to persist and grow in the 10‐species consortium (P < .001). A rat caries model was also used to examine the effect of BrpA, as well as Psr, a BrpA paralog, on S. mutans cariogenicity. The results showed no major differences in infectivity between the wild‐type and the brpA and psr mutants. Unlike the wild‐type, however, infection with the brpA mutant, but not the psr mutant, showed no significant differences in both total numbers of carious lesions and caries severity, compared with the control group that received bacterial growth medium (P > .05). Metagenomic and quantitative polymerase chain reaction analysis showed that S. mutans infection caused major alterations in the composition of the rats’ plaque microbiota and that significantly less S. mutans was identified in the rats infected with the brpA mutant compared with those infected with the wild‐type and the psr mutant. These results further suggest that BrpA plays a critical role in S. mutans pathophysiology and that BrpA has potential as a therapeutic target in the modulation of S. mutans virulence.     

PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries‐risk prediction

The microbial factor is an important determinant in caries risk assessment. This study aimed to use detection, by PCR, of Scardovia wiggsiae, in combination with Streptococcus mutans, for the accurate prediction of caries risk in children. Detection of Lactobacillus, as a caries‐specific species, was also performed. Dental plaque, as well as infected dentine when available, was collected from children who were caries‐free (n = 30) or diagnosed with early childhood caries (n = 30), and the prevalence and abundance of S. wiggsiae and S. mutans were estimated using quantitative PCR. Lactobacillus was amplified by Lactobacillus genus‐specific primers and then sequenced. Both S. wiggsiae and S. mutans were concurrently detected in 19 children diagnosed with early childhood caries, but in none of the caries‐free children. The positive predictive value was 1 in children with S. wiggsiae‐ and S. mutans‐positive test results, compared with 0.58 when only S. mutans was detected and 0.9 when only S. wiggsiae was detected. The abundance of S. wiggsiae and S. mutans in infected dentine was higher than that in dental plaque from children. Diverse Lactobacillus species were observed in dental plaque but none appeared to be caries‐specific. In conclusion, the detection of S. wiggsiae in combination with S. mutans improves the positive predictive value and the specificity of the test.     

Antibacterial effects of blackberry extract target periodontopathogens

Background and Objective: Antimicrobial agents provide valuable adjunctive therapy for the prevention and the control of oral diseases. Limitations in their prolonged use have stimulated the search for new, naturally occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the antibacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens. Material and Methods: The effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated using the colorimetric water‐soluble tetrazolium‐1 assay. The bactericidal effects of whole BBE against Fusobacterium nucleatum were determined by quantitating the numbers of colony‐forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells. Results: BBE at 350–1400 μg/mL reduced the metabolic activity of Porphyromonas gingivalis, F. nucleatum and Streptococcus mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in the numbers of CFUs following exposure to BBE for as little as 1 h, indicative of its bactericidal properties. An anthocyanin‐enriched fraction of BBE reduced the metabolic activity of F. nucleatum, but not of P. gingivalis or S. mutans, suggesting the contribution of species‐specific agents in the whole BBE. Oral epithelial cell viability was not reduced following exposure to whole BBE (2.24–1400 μg/mL) for ≤ 6 h. Conclusion: BBE alters the metabolic activity of oral periodontopathogens while demonstrating a minimal effect on commensals. The specific antibacterial properties of BBE shown in this study, along with its previously demonstrated anti‐inflammatory and antiviral properties, make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

Streptococcus mutans and Candida albicans are frequently co‐isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non‐glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two‐species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two‐species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co‐infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two‐species biofilm, representing new activities associated with this known S. mutans adhesin.     

Nanoscale adhesion forces of glucosyltransferase B and C genes regulated Streptococcal mutans probed by AFM

Glucosyltransferases (Gtfs), represented by GtfB and GtfC, are important virulence factors of Streptococcus mutans and the major etiologic pathogens of tooth decay. However, the individual roles of gtfB and gtfC in the initial attachment of S. mutans are not known. We used atomic force microscopy to explore the contribution of gtfB and gtfC, as well as enamel‐surface roughness, on the initial attachment of S. mutans. Adhesion forces of four S. mutans strains (wild‐type, ΔgtfB, ΔgtfC, and ΔgtfBC), onto etched enamel surfaces, were determined. Force curves showed that, with increasing etching time from 0 to 10 s, the forces of all strains increased accordingly with acid‐exposure time, the adhesion forces of wild‐type strains were significantly greater than those of mutant strains (p < .05), and the forces of the three mutants were similar (p < .05). When the etching time was increased from 10 to 30 s, difference in force between 20 and 30 s was not observed, and adhesion forces among ΔgtfB, ΔgtfC, and wild‐type strains were not significantly different when the etching time was >20 s (p > .05). These data suggest that the roughness and morphology of enamel surfaces may have a significant influence upon the initial attachment of bacteria, and that gtfB and gtfC are essential for the adhesion activity of bacteria. Furthermore, gtfB seems to be more important than gtfC for bacterial‐biofilm formation, and gtfB inactivation is an effective strategy to inhibit the virulence of cariogenic biofilms.     

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.     

Human T‐cell responses to oral streptococci in human PBMC‐NOD/SCID mice

We investigated cellular and humoral immune responses to oral biofilm bacteria, including Streptococcus mutans, Streptococcus anginosus, Streptococcus sobrinus, and Streptococcus sanguinis, in NOD/SCID mice immunized with human peripheral blood mononuclear cells (hu‐PBMC‐NOD/SCID mice) to explore the pathogenicity of each of those organisms in dental and oral inflammatory diseases. hu‐PBMC‐NOD/SCID mice were immunized by intraperitoneal injections with the whole cells of the streptococci once a week for 3 weeks. FACS analyses were used to determine the percentages of various hu‐T cell types, as well as intracellular cytokine production of interleukin‐4 and interferon‐γ. Serum IgG and IgM antibody levels in response to the streptococci were also determined by enzyme‐linked immunosorbent assay. S. anginosus induced a significant amount of the proinflammatory cytokine interferon‐γ in CD4⁺ and CD8⁺ T cells in comparison with the other streptococci. However, there was no significant differences between the streptococci in interleukin‐4 production by CD4⁺ and CD8⁺ T cells after inoculation. Further, S. mutans significantly induced human anti‐S. mutans IgG, IgG₁, IgG₂, and IgM antibodies in comparison with the other organisms. In conclusion, S. anginosus up‐regulated Th1 and Tc1 cells, and S. mutans led to increasing levels of their antibodies, which was associated with the induction of Th2 cells. These results may contribute to a better understanding of human lymphocyte interactions to biofilm bacteria, along with their impact on dental and mucosal inflammatory diseases, as well as endocarditis.     

Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans

Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4‐aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.     

DMBT1 involvement in the human aortic endothelial cell response to Streptococcus mutans

Streptococcus mutans is a causative organism of dental caries and has been reported to be associated with the development of cardiovascular disease (CVD). Previous studies have demonstrated that S. mutans invades human aortic endothelial cells (HAECs) and HAECs invaded by S. mutans produce higher levels of CVD–related cytokines than non‐invaded HAECs. DMBT1 (deleted in malignant brain tumors 1), also known as salivary agglutinin or gp‐340, belongs to the scavenger receptor cysteine–rich superfamily. DMBT1 is expressed in epithelial and non‐epithelial tissues and has multiple functions. The interaction between S. mutans and DMBT1 has been demonstrated in cariogenesis, but DMBT1 involvement in CVD has not been examined. In this study, we investigated DMBT1 expression in HAECs stimulated with S. mutans and examined the role of DMBT1 in the interaction between S. mutans and HAECs. All of the tested S. mutans strains induced higher production levels of DMBT1 in HAECs than those in unstimulated HAECs. More S. mutans cells adhered to DMBT1 knock down HAECs than to DMBT1–producing HAECs. Invasion of DMBT1 knock down HAECs by S. mutans was stronger than that of DMBT1–producing HAECs, and externally added DMBT1 reduced bacterial invasion. Cytokine production by DMBT1 knock down HAECs by S. mutans stimulation was higher than that by DMBT1–producing HAECs. These phenomena seemed to be due to the effect of released DMBT1, namely, the inhibition of bacterial adherence to HAECs by DMBT1. These results suggest that DMBT1 plays a protective role against the S. mutans–induced CVD process in HAECs.