Are the mutans streptococci still considered relevant to understanding the microbial etiology of dental caries?

The mutans streptococci were once the primary focus of research dedicated to understanding the etiology of dental caries. That focus has now shifted to an emphasis on the ecological balances and complexities within the entirety of the plaque microbiome. Within that framework there are considerable differences of opinion regarding the importance and relative contributions of the mutans streptococci. This article explores the basis for the various viewpoints, the limitations of current knowledge, and the confounders that make it difficult to arrive at a consensus.

Comparison of biofilm models for producing artificial active white spot lesions.

OBJECTIVE: This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models. METHODOLOGY: In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001). RESULTS: In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols. CONCLUSION: The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Choline and geranic acid (CAGE) ionic liquids inhibit both elastase activity and growth of oral bacteria.

Choline and geranic acid (CAGE) ionic liquids have recently been shown to have applications in the delivery of macromolecules and poorly soluble drugs across epithelial barriers and in bacterial growth inhibition. Ionic liquids are known to denature proteins by the disruption of forces that guide natural protein folding, and the inflammatory enzyme elastase was recently shown to be inhibited by a variety of ionic liquids other than CAGE. Inhibition of collagenolytic enzymes, including elastase, has been shown to improve outcomes in cases of periodontitis via amelioration of periodontal inflammation and alveolar bone resorption. In this study, we investigated whether CAGE prepared with varying stoichiometries was able to inhibit elastase at varying concentrations and whether these CAGE formulations could inhibit the growth of key pathogenic bacterial species associated with oral health conditions. We found that CAGE was capable of inhibiting both porcine elastase and human neutrophil elastase at concentrations as low as 5 mM, and that CAGE formulations were effective at inhibiting the growth of all tested pathogenic oral bacteria. The inhibition of elastase by CAGE may be a mechanism by which CAGE can improve outcomes in periodontitis independent from CAGE's known antibacterial properties.

Spent culture supernatant of Streptococcus gordonii mitigates inflammation of human periodontal cells and inhibits proliferation of pathogenic oral microbes.

BACKGROUND: The oral commensal bacterial species Streptococcus gordonii has been reported to regulate the inflammation of oral epithelial cells stimulated by the periodontal pathogen Porphyromonas gingivalis. This study investigated the activities of S. gordonii metabolites in S. gordonii spent culture supernatants (Sg-SCS) on periodontal-related bacterial growth and periodontitis-associated inflammatory cytokines. METHODS: Sg-SCS was collected from S. gordonii cultures grown in Dulbecco Modified Eagle Medium and added to the growth media of representative health- and disease-related oral species: S. gordonii, Streptococcus sanguinis, Streptococcus mitis, Streptococcus oralis, P. gingivalis, Tannerella forsythia, and Treponema denticola. The Sg-SCS was also tested for its ability to regulate the expression of proinflammatory cytokines by human macrophages, epithelial cells, and gingival fibroblasts upon stimulation with P. gingivalis-derived lipopolysaccharide (Pg-LPS). RESULTS: Sg-SCS significantly reduced transcript and protein levels of interleukin (IL)-1ß, 6, and 8 induced by Pg-LPS stimulation in multiple types of periodontal cells. mRNA sequencing and bioinformatics analyses indicated that Sg-SCS significantly affects 10 inflammatory pathways. Additionally, Sg-SCS exhibited suppression of the growth of periodontal disease-related bacteria, including T. denticola and P. gingivalis, along with the primary plaque-colonizing species S. oralis. At a low concentration, Sg-SCS also inhibits P. gingivalis adhesion. CONCLUSIONS: These results strongly suggest that S. gordonii-derived SCS contains metabolites that have anti-inflammatory properties and an ability to inhibit periodontitis-associated pathogenic bacteria. Further investigation will be needed to identify the individual metabolites within the Sg-SCS to develop a novel metabolite-based approach to treating and preventing periodontitis.

Role of mutans streptococci, acid tolerant bacteria and oral Candida species in predicting the onset of early childhood caries.

Aim: Early childhood caries is the most common chronic infectious disease in children in the United States. This study, which is part of a larger, longitudinal study exploring oral microbiological components of caries development in children, reports on the impact of total mutans streptococci (MS), total acid tolerant bacteria and Candida species on the development of dental caries in a subset of these children. Of particular interest was the relationship between caries development and co-colonization of mutans streptococci and Candida species. Methods: Children between the ages of 12 and 47 months displaying no evidence of dental caries were recruited for a longitudinal study (n = 130). Twelve age- and gender-matched pairs were selected. In each pair, one child developed caries during the study, and one did not. Whole mouth plaque samples were collected by swab at baseline and every 6 months thereafter for a duration of 18 months and spiral plated for microbial counts (CFU/ml). Cut-offs based on percent of total cultivable flora were designated for all microbial measures. A scoring system designated the Plaque Microbial Index (PMI) was developed for use in statistical analyses to assess potential predictive factors for caries risk assessment. Results: Children who developed caries were significantly more likely to harbor higher percentages of acid tolerant bacteria (p = 0.003), MS (p < 0.001) and have Candida species present (p < 0.001) at ≥1 visit leading up to caries onset. Mean PMI scores derived from the aforementioned microbial measures, were higher for caries active children than caries free children (p = 0.000147). Co-colonization of MS and Candida species was significantly associated with caries development (p < 0.001) and detection of both at the same visit had a 100% positive predictive value and 60% negative predictive value for caries development. Conclusion: In children who developed caries, there was a statistically significant association with the percent of total flora that was acid tolerant, the percent of MS, the presence of Candida and co-colonization of MS and Candida species. Combining these microbial measures into PMI scores further delineated children who developed caries from those who remained caries-free. These microbiological measures show potential as predictive factors and risk assessment tools for caries development.

A Social Media Intervention for Promoting Oral Health Behaviors in Adolescents: A Non-Randomized Pilot Clinical Trial.

Poor oral hygiene and excessive consumption of soda are among the main drivers of systemic health issues in adolescents in the United States. This non-randomized pilot clinical trial focused on the effects of a health text message system and smartphone-based intervention on adolescent tooth-brushing behavior and dietary choices, with a convenience sample of 94 participants aged 12 to 14 years old. A group of 75 participants agreed to use a tooth-brushing app and received a health text message; the other group of 15 agreed to use the tooth-brushing app, but did not receive a health text message. Saliva specimens were collected directly before and at the end of each experiment; changes in the salivary presence of cariogenic bacteria over the duration of the study were evaluated and compared with the demographics and behavioral variables. Within the text message group, 5% of participants increased the frequency of daily tooth brushing. Within the non-intervention group, 29% of participants increased the frequency of their daily tooth brushing. There were reductions in the total salivary bacteria and total streptococci in both groups (p < 0.001), but no change in the presence of cariogenic Mutans streptococci. Raising adolescents' consciousness of oral health behavior resulted in marginal to moderate improvements to oral hygiene and dietary choices, as well as reductions in total salivary bacteria.

Rubesanolides C-E: abietane diterpenoids isolated from Isodon rubescens and evaluation of their anti-biofilm activity.

Phytochemical study of the leaves of the medicinal plant Isodon rubescens led to the isolation of three novel abietane diterpenoids, rubesanolides C-E (1-3). These diterpenes contain a unique γ-lactone subgroup formed between C-8 and C-20. Their structures were determined from analysis of spectroscopic data, and were further confirmed by X-ray crystallographic data. The compounds were evaluated for their antibacterial activity, and rubesanolide D (2) demonstrated inhibition activity against biofilm formation of the dental bacterium Streptococcus mutans.

Effects of DNA methylation on expression of virulence genes in Streptococcus mutans.

Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5'-GATC-3' appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential.

Vanillin-bioglass cross-linked 3D porous chitosan scaffolds with strong osteopromotive and antibacterial abilities for bone tissue engineering.

Chitosan scaffolds crosslinked by current methods insufficiently meet the demands of bone tissue engineering applications. We developed a novel effective crosslinking technique by using the natural and safe vanillin together with bioglass microparticles to generate an antibacterial, osteoconductive, and mechanically robust 3D porous chitosan-vanillin-bioglass (CVB) scaffold. In addition to the significantly improved mechanical properties, the CVB scaffolds had high porosity (>90%) and interconnected macroporous structures. Our data suggested that the crosslinking mainly resulted from the Schiff base reactions between the aldehydes of vanillin and amines of chitosan, together with the hydrogen and ionic bonds formed within them. Importantly, the CVB scaffolds not only showed good biocompatibility, bioactivity, and strong antibacterial ability but also significantly promoted osteoblastic differentiation, mineralization in vitro, and ectopic bone formation in vivo. Thus, the CVB scaffolds hold great promise for bone tissue engineering applications based on their robust mechanical properties, osteoconductivity, and antibacterial abilities.

Solubilized ubiquinol for preserving corneal function.

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol - the reduced and active form of coenzyme Q10 and a powerful antioxidant - significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or ß-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

Evaluating the relationship between acidogenicity and acid tolerance for oral streptococci from children with or without a history of caries.

Background: Dental caries etiology is attributed to a dysbiotic imbalance within the plaque microbiome leading to a dominance of strong acidogens. Some studies that investigate the link between acidogens and caries quantify the recovery of acid tolerant strains on acid agar as a measure of acidogenic potential. This methodology assumes that acidogenic potential and acid tolerance are directly related. Aim: The validity of that assumption was investigated by statistically evaluating that relationship using streptococci recovered from children with or without a history of dental caries. Methods: Thirty streptococcal isolates were isolated from each of 13 subjects. Acidogenicity was quantified by measuring the terminal pH after overnight growth in Brain Heart Infusion (BHI) and Chemically Defined Medium (CDM). Acid tolerance was quantified by measuring the lowest pH acid agar displaying growth. Results: A significant difference in acidogenicity in CDM between levels of acid tolerance was found, but no significant difference in acidogenicity in BHI was noted. Moreover, there were no significant interactions between acid tolerance and caries history on acidogenicity measures in either medium. Conclusion: An ability to grow on acid agar below pH 5.0 is best aligned with strong acidogenicity and best able to distinguish between subjects with differing caries histories.

Antimicrobial efficacy of platinum-doped silver nanoparticles.

Silver nanoparticles (AgNPs) have been proposed to combat oral infection due to their efficient ionic silver (Ag+ ) release. However, concentrations required for antimicrobial efficacy may not be therapeutically viable. In this work, platinum-doped silver nanoparticles (Pt-AgNPs) were explored to evaluate their potential for enhanced Ag+ release, which could lead to enhanced antimicrobial efficacy against S. aureus, P. aeruginosa, and E. coli. AgNPs doped with 0.5, 1, and 2 mol% platinum (Pt0.5 -AgNPs, Pt1 -AgNPs, and Pt2 -AgNPs) were synthesized by a chemical reduction method. Transmission electron microscopy revealed mixed morphologies of spherical, oval, and ribbon-like nanostructures. Surface-enhanced Raman scattering revealed that the surface of Pt-AgNPs was covered with up to 93% Pt. The amount of Ag+ released increased 16.3-fold for Pt2 -AgNPs, compared to AgNPs. The initial lag phase in bacterial growth curve was prolonged for Pt-AgNPs. This is consistent with a Ag+ release profile that exhibited an initial burst followed by sustained release. Doping AgNPs with platinum significantly increased the antimicrobial efficacy against all species. Pt2 -AgNPs exhibited the lowest minimum inhibitory concentrations, followed by Pt1 -AgNPs, Pt0.5 -AgNPs, and AgNPs, respectively. Doping AgNPs with a small amount of platinum promoted the release of Ag+ , based on the sacrificial anodic effect, and subsequently enhanced their antimicrobial efficacy.

Dental adhesive microtensile bond strength following a biofilm-based in vitro aging model.

OBJECTIVE: Laboratory tests are routinely used to test bonding properties of dental adhesives. Various aging methods that simulate the oral environment are used to complement these tests for assessment of adhesive bond durability. However, most of these methods challenge hydrolytic and mechanical stability of the adhesive- enamel/dentin interface, and not the biostability of dental adhesives. To compare resin-dentin microtensile bond strength (µTBS) after a 15-day Streptococcus mutans (SM) or Streptococcus sobrinus (SS) bacterial exposure to the 6-month water storage (WS) ISO 11405 type 3 test. METHODOLOGY: A total of 31 molars were flattened and their exposed dentin was restored with Optibond-FL adhesive system and Z-100 dental composite. Each restored molar was sectioned and trimmed into four dumbbell-shaped specimens, and randomly distributed based on the following aging conditions: A) 6 months of WS (n=31), B) 5.5 months of WS + 15 days of a SM-biofilm challenge (n=31), C) 15 days of a SM-biofilm challenge (n=31) and D) 15 days of a SS-biofilm challenge (n=31). µTBS were determined and the failure modes were classified using light microscopy. RESULTS: Statistical analyses showed that each type of aging condition affected µTBS (p<0.0001). For Group A (49.7±15.5MPa), the mean µTBS was significantly greater than in Groups B (19.3±6.3MPa), C (19.9±5.9MPa) and D (23.6±7.9MPa). For Group D, the mean µTBS was also significantly greater than for Groups B and C, but no difference was observed between Groups B and C. CONCLUSION: A Streptococcus mutans- or Streptococcus sobrinus-based biofilm challenge for 15 days resulted in a significantly lower µTBS than did the ISO 11405 recommended 6 months of water storage. This type of biofilm-based aging model seems to be a practical method for testing biostability of resin-dentin bonding.

Role of the Streptococcus mutans irvA gene in GbpC-independent, dextran-dependent aggregation and biofilm formation.

Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

Comparison of biofilm models for producing artificial active white spot lesions

Abstract Creating artificial caries-like lesions that mimic the complex changes observed in natural caries is essential for properly evaluating new strategies, dental materials, and devices designed to arrest their progression and avoid more costly and invasive treatments. Objective This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models. Methodology In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001). Results In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols. Conclusion The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture.

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture.

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.

PCR-Based Identification of Oral Streptococcal Species.

The microbial etiology of dental caries is still debated. Among the hypothesized contributors are the "low pH streptococci," a designation given to unusually acid proficient strains among the primary plaque colonizers S. oralis, S. mitis, S. gordonii, and S. anginosus. However, accurate assignment of species is difficult among the oral streptococci. Our objective was to develop a streamlined method for identifying strains of S. oralis and S. mitis from plaque samples so that they could be analyzed in a separate study devoted to low pH streptococci and caries. Two independent PCR amplifications of a locus highly conserved among streptococci were used for presumptive species identification. Multilocus sequence analysis (MLSA) was used to measure accuracy. Sensitivity was 100% for selecting S. oralis and S. mitis among the clones sampled. Specificity was good except for the most closely related species that could not be reliably distinguished even by MLSA. The results with S. oralis and S. mitis were used to identify new primer sets that expanded the utility of the approach to other oral streptococcal species. These novel primer sets offer a convenient means of presumptive identification that will have utility in many studies where large scale, in-depth genomic analyses are not practical.