Metabolic Profile of Supragingival Plaque Exposed to Arginine and Fluoride.

Caries lesions develop when acid production from bacterial metabolism of dietary carbohydrates outweighs the various mechanisms that promote pH homeostasis, including bacterial alkali production. Therapies that provide arginine as a substrate for alkali production in supragingival oral biofilms have strong anticaries potential. The objective of this study was to investigate the metabolic profile of site-specific supragingival plaque in response to the use of arginine (Arg: 1.5% arginine, fluoride-free) or fluoride (F: 1,100 ppm F/NaF) toothpastes. Eighty-three adults of different caries status were recruited and assigned to treatment with Arg or F for 12 wk. Caries lesions were diagnosed using International Caries Detection and Assessment System II, and plaque samples were collected from caries-free and carious tooth surfaces. Taxonomic profiles were obtained by HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing), and plaque metabolism was assessed by the levels of arginine catabolism via the arginine deiminase pathway (ADS), acidogenicity, and global metabolomics. Principal component analysis (PCA), partial least squares-discriminant analysis, analysis of variance, and random forest tests were used to distinguish metabolic profiles. Of the 509 active lesions diagnosed at baseline, 70 (14%) were inactive after 12 wk. Generalized linear model showed that enamel lesions were significantly more likely to become inactive compared to dentin lesions (P < 0.0001), but no difference was found when treatment with Arg was compared to F (P = 0.46). Arg significantly increased plaque ADS activity (P = 0.031) and plaque pH values after incubation with glucose (P = 0.001). F reduced plaque lactate production from endogenous sources (P = 0.02). PCA revealed differences between the metabolic profiles of plaque treated with Arg or F. Arg significantly affected the concentrations of 16 metabolites, including phenethylamine, agmatine, and glucosamine-6-phosphate (P < 0.05), while F affected the concentrations of 9 metabolites, including phenethylamine, N-methyl-glutamate, and agmatine (P < 0.05). The anticaries mechanisms of action of arginine and fluoride are distinct. Arginine metabolism promotes biofilm pH homeostasis, whereas fluoride is thought to enhance resistance of tooth minerals to low pH and reduce acid production by supragingival oral biofilms.

Novel Probiotic Mechanisms of the Oral Bacterium Streptococcus sp. A12 as Explored with Functional Genomics.

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of S. mutans In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to that of the parental strain. The A12 pcfO mutant lost the ability to inhibit comX -inducing peptide (XIP) signaling by S. mutans, while mutants with changes in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.IMPORTANCE Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of S. mutans, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species.

Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for Streptococcus mutans UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in S. mutans These promoter-sfgfp fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled S. mutans UA159, Streptococcus gordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in Streptococcus mutans and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions in situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

Arginine Metabolism in Supragingival Oral Biofilms as a Potential Predictor of Caries Risk.

INTRODUCTION: Ammonia production via the arginine deiminase system (ADS) of oral bacteria can function to reduce the cariogenicity of oral biofilms by neutralizing glycolytic acids that cause tooth demineralization. OBJECTIVES: This cohort study investigated the relationship between ADS activity and bacterial profile changes of supragingival biofilms with caries experience among children over time. METHODS: A total of 79 children aged 2 to 7 y at baseline were assessed every 6 mo for a period of 18 mo. Children were grouped as caries free (CF), caries active with enamel lesions (CAE), or caries active with dentin lesions (CA). Supragingival plaque samples were collected from caries-free surfaces (PF) and from enamel (PE) and dentin (PD) lesions. Plaque ADS activity was measured by monitoring citrulline production from arginine and compared with ribosomal 16S rRNA-derived taxonomic profiles for the same samples. RESULTS: At baseline, 37% of the children were CF, 34% CAE, and 29% CA. At 18 mo, 26% were CF, 41% CAE, 23% CA, and 10% were caries experienced (new restorations but no caries activity). Throughout the study period, ADS activity was significantly higher in the CF group than the CA group (P < 0.0001), and ADS activity in the PF samples was significantly higher than in the PE and PD samples (P < 0.0001). Distance-based redundancy analysis showed that the bacterial communities could be differentiated when plaque samples are grouped into levels of high and low ADS activity. CONCLUSIONS: There is a positive correlation between caries activity and low arginolytic capacity of the supragingival oral biofilms of children and tooth surfaces over time. Measurements of arginine metabolism via ADS may be useful to differentiate the caries risk of individuals and tooth surfaces. KNOWLEDGE TRANSFER STATEMENT: Findings from this study support the development of new strategies for caries risk assessment and prevention based on modulation of the virulence of the oral microbiome through arginine metabolism in supragingival biofilms.

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence.

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore, MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

Getting to Know "The Known Unknowns": Heterogeneity in the Oral Microbiome.

Technological advances in DNA sequencing have provided unprecedented insights into the composition of the oral microbiome in health and disease, and RNA-sequencing and metabolomics-related technologies are beginning to yield information on the activities of these organisms. Importantly, progress in this area has brought the scientific community closer to an understanding of what constitutes a health-associated microbiome and is supporting the notion that the microbiota in healthy sites assumes an active role in promoting health and suppressing the acquisition, persistence, and activities of overt and opportunistic pathogens. It is also becoming clear that a significant impediment to developing a conclusive body of evidence that defines a healthy microbiome and the mechanisms by which beneficial bacteria promote health is that an inherent characteristic of the most abundant members of the oral flora, those that potentially play the greatest roles in health and disease, is intraspecies genomic diversity. In particular, individual isolates of abundant commensal and pathogenic streptococci show tremendous variability in gene content, and this variability manifests in tremendous phenotypic heterogeneity. Analysis of the consequences of this diversity has been complicated by the exquisite sensitivity these bacteria have evolved to environmental inputs, inducing rapid and substantial fluctuations in behaviors, and often only within subpopulations of the organisms. Thus, the conditions under which the oral microbiota is studied can produce widely different results within and between species. Fortunately, continually diminishing costs and ongoing refinements in sequencing and metabolomics are making it practical to study the oral microbiome at a level that will create a sufficiently robust understanding of the functions of individual organisms and reveal the complex interrelationships of these microbes ("the known unknowns") in a way that researchers will be able to engage in the rational design of reliable and economical risk assessments and preventive therapies.

The effect of arginine on oral biofilm communities.

Alkali production by oral bacteria via the arginine deiminase system (ADS) increases the pH of oral biofilms and reduces the risk for development of carious lesions. This study tested the hypothesis that increased availability of arginine in the oral environment through an exogenous source enhances the ADS activity levels in saliva and dental plaque. Saliva and supra-gingival plaque samples were collected from 19 caries-free (CF) individuals (DMFT = 0) and 19 caries-active (CA) individuals (DMFT ≥ 2) before and after treatment, which comprised the use of a fluoride-free toothpaste containing 1.5% arginine, or a regular fluoride-containing toothpaste twice daily for 4 weeks. ADS activity was measured by quantification of ammonia produced from arginine by oral samples at baseline, after washout period, 4 weeks of treatment, and 2 weeks post-treatment. Higher ADS activity levels were observed in plaque samples from CF compared to those of CA individuals (P = 0.048) at baseline. The use of the arginine toothpaste significantly increased ADS activity in plaque of CA individuals (P = 0.026). The plaque microbial profiles of CA treated with the arginine toothpaste showed a shift in bacterial composition to a healthier community, more similar to that of CF individuals. Thus, an anti-caries effect may be expected from arginine-containing formulations due in large part to the enhancement of ADS activity levels and potential favorable modification to the composition of the oral microbiome.

Oral arginine metabolism may decrease the risk for dental caries in children.

Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children's caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p < .001) and PE (194.8; p = .005). There was no significant difference in the salivary arginolytic activity among children with different caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children's age, caries status, and dentition (p < .001), with healthy plaque predicting higher ADS activity compared with diseased plaque. Plaque arginine metabolism varies greatly among children and tooth sites, which may affect their susceptibility to caries.

A galactose-specific sugar: phosphotransferase permease is prevalent in the non-core genome of Streptococcus mutans.

Three genes predicted to encode the A, B and C domains of a sugar : phosphotransferase system (PTS) permease specific for galactose\(EII(Gal) ) were identified in the genomes of 35 of 57 recently sequenced isolates of Streptococcus mutans, the primary etiological agent of human dental caries. Mutants defective in the EII(Gal) complex were constructed in six of the isolates and showed markedly reduced growth rates on galactose-based medium relative to the parental strains. An EII(Gal) -deficient strain constructed using the invasive serotype f strain OMZ175 (OMZ/IIGal) expressed significantly lower PTS activity when galactose was present as the substrate. Galactose was shown to be an effective inducer of catabolite repression in OMZ175, but not in the EII(Gal) -deficient strain. In a mixed-species competition assay with galactose as the sole carbohydrate source, OMZ/IIGal was less effective than the parental strain at competing with the oral commensal bacterium Streptococcus gordonii, which has a high-affinity galactose transporter. Hence, a significant proportion of S. mutans strains encode a galactose PTS permease that could enhance the ability of these isolates to compete more effectively with commensal streptococci for galactose in salivary constituents and the diet.

Progress dissecting the oral microbiome in caries and health.

Recent rapid advances in "-omics" technologies have yielded new insights into the interaction of the oral microbiome with its host. Associations of species that are usually considered to be acid-tolerant with caries have been confirmed, while some recognized as health-associated are often present in greater proportions in the absence of caries. In addition, some newly identified bacteria have been suggested as potential contributors to the caries process. In spite of this progress, two major challenges remain. The first is that there is a great deal of heterogeneity in the phenotypic capabilities of individual species of oral bacteria. The second is that the most abundant taxa in oral biofilms display remarkable phenotypic plasticity, i.e., the bacteria associated most strongly with health or with caries can morph rapidly in response to alterations in environmental pH, carbohydrate availability and source, and oxygen tension and redox environment. However, new technologic advances coupled with "old-fashioned microbiology" are starting to erode the barriers to a more complete understanding of oral biofilm physiology and ecology, and in doing so are beginning to provide insights for the creation of novel cost-effective caries control therapies.

BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans.

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.

Urease activity as a risk factor for caries development in children during a three-year study period: a survival analysis approach.

UNLABELLED: Recent cross-sectional studies suggest that reduced ability to generate alkali via the urease pathway in dental plaque may be an important caries risk factor, but it has not been assessed prospectively. OBJECTIVE: To evaluate the effect of plaque and saliva urease activity on the risk for developing new caries over a three-year period in children. METHODS: A panel of 80 children, three to six years of age at recruitment, was followed prospectively for three years. Plaque urease activity, saliva urease activity and dental caries were measured every six months. Survival analysis methodology was used to evaluate the effect of urease on caries development during the study period adjusted for gender, age, baseline caries levels, sugar consumption, amount of plaque, and mutans streptococci levels. RESULTS: The risk for developing new caries increased in a dose-responsive manner with increasing levels of urease activity in saliva (adjusted HR(Q4 vs. Q1): 4.98; 95% CI: 1.33, 18.69) and with decreasing urease activity in plaque (adjusted HR(Q4 vs. Q1): 0.29; 95% CI: 0.11, 0.76). Multiple measurements of urease activity were conducted to overcome the variability of urease activity in this study. Baseline caries and mutans streptococci in saliva were also important predictors of caries risk. CONCLUSIONS: Increased urease activity in saliva can be an indicator of increased caries risk in children, whilst increased urease activity in plaque may be associated with reduced caries risk. The reproducibility of urease measurements must be improved before these findings can be further tested and clinically applied.

Urease activity in dental plaque and saliva of children during a three-year study period and its relationship with other caries risk factors.

UNLABELLED: Bacterial urease activity in dental plaque and in saliva generates ammonia, which can increase the plaque pH and can protect acid-sensitive oral bacteria. Recent cross-sectional studies suggest that reduced ability to generate ammonia from urea in dental plaque can be an important caries risk factor. In spite of this proposed important clinical role, there is currently no information available regarding important clinical aspects of oral ureolysis in children. OBJECTIVE: The objective of this study was to evaluate the distribution and pattern of urease activity in the dental plaque and in the saliva of children during a three-year period, and to examine the relationship of urease with some important caries risk factors. METHODS: A longitudinal study was conducted with repeated measures over a three-year period on a panel of 80 children, aged 3-6 years at recruitment. The dynamics of change in urease activity were described and associated with clinical, biological, and behavioural caries risk factors. RESULTS: Urease activity in plaque showed a trend to remain stable during the study period and was negatively associated with sugar consumption (P<0.05). Urease activity in unstimulated saliva increased with age, and it was positively associated with the levels of mutans streptococci in saliva and with the educational level of the parents (P<0.05). CONCLUSIONS: The results of this study reveal interesting and complex interactions between oral urease activity and some important caries risk factors. Urease activity in saliva could be an indicator of mutans infection in children.

Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms.

We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.

The effect of sucrose on plaque and saliva urease levels in vivo.

OBJECTIVE: Dietary sugar exposures induce an immediate drop of the plaque pH. Based on in vitro observations, it was hypothesized that oral bacteria may rapidly respond to this environmental change by increasing the activity or expression of alkali-generating pathways, such as the urease pathway. The objective of this exploratory in vivo study was to determine the short-term effect of a brief sucrose exposure on plaque and saliva urease activity and expression, and to relate this effect to caries experience. METHODS: Urease activity levels were measured in plaque and saliva samples collected from 20 children during fasting conditions and 30 min after rinsing with a sucrose solution. Streptococcus salivarius ureC-specific mRNA in saliva was quantified using real-time RT-PCR. The impact of host-related factors, such as age, gender, sugar consumption, salivary mutans streptococci levels and caries status on urease activity was evaluated. RESULTS: Plaque urease activity under fasting conditions was higher in subjects with low caries and mutans streptococci levels. This difference was not observed after the sucrose exposure. The response of urease to sucrose in vivo did not depend on caries experience or salivary mutans levels. Significant increase in urease activity of plaque and saliva after exposure to sucrose was observed only in the subjects who had low urease levels at baseline. CONCLUSIONS: The findings of this exploratory study suggest that plaque urease activity may have an important long-term influence in caries development but not during a cariogenic challenge.

Inactivation of VicK affects acid production and acid survival of Streptococcus mutans.

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

Correlations of oral bacterial arginine and urea catabolism with caries experience.

BACKGROUND/AIM: Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects. METHODS: The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth >or= 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples. RESULTS: The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001). CONCLUSION: This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175.

INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

Distribution, regulation and role of the agmatine deiminase system in mutans streptococci.

The agmatine deiminase system (AgDS) was identified in seven strains of mutans streptococci. Genes encoding the AgDS of Streptococcus rattus FA-1 were sequenced and found to share homology with the agu genes of Streptococcus mutans UA159. With the exception of Streptococcus sobrinus, the AgDS of mutans streptococci appear to be sensitive to carbohydrate catabolite repression. Agmatine inhibited bacterial growth, suggesting that the AgDS degrades a deleterious substance into useful compounds.