Streptococcus mutans and oral streptococci in dental plaque

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.     

Nested PCR for detection of mutans streptococci in dental plaque

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.     

Synergistic degradation of bovine serum albumin by mutans streptococci and other dental plaque bacteria

Mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) exhibited low levels of proteolytic activity against the model protein substrate, FITC-labelled bovine serum albumin, when incubated alone. Inclusion of other members of the dental plaque microflora in the assay usually resulted in marked increases in the degree of proteolysis and a high level of synergy. Interactions between mutans streptococci and either Streptococcus oralis or Fusobacterium nucleatum gave rise to the greatest degree of synergistic proteolytic degradation.     

Identification of non-mutans streptococci organisms in dental plaques recovering on mitis-salivarius bacitracin agar medium

The objective of this study was to both isolate and identify non-mutans streptococci organisms (non-MSO) from dental plaques recovered on mitis-salivarius sucrose bacitracin agar (MSB) plates. The dental plaque samples, which had been collected from 63 human subjects, were diluted and plated on MSB. The bacteria growing on the MSB plates were then identified with biochemical tests, as well as with 16S rDNA cloning and sequencing techniques. Our data indicated that bacteria from 30 subjects had been recovered on the MSB plates. Among the 21 typical colonies selected from the 30 subjects, 12 colonies, derived from 10 subjects, were identified as non-MSO. These 12 colonies were determined to be Streptococcus anginosus (8 colonies), S. sanguinis (1 colony), and Pantoea agglomerans (3 colonies). These results strongly suggest that a new selective medium will be required for the reliable isolation of mutans streptococci.     

Isolation and biochemical characterization of a novel lantibiotic mutacin from Streptococcus mutans.

Certain members of the indigenous biota of humans produce antimicrobial substances called bacteriocins, which inhibit other bacteria, including members of their own species. One of these substances, mutacin, is made by Streptococcus mutans, a member of the oral biota. Mutacin inhibits other mutans streptococci as well as many gram-positive exogenous pathogens. Here, we report for the first time the purification and partial biochemical characterization of a lanthionine-containing mutacin peptide from S. mutants T8. The biologically active peptide was isolated from the broth cultures by ultrafiltration and differential precipitation. The final mutacin preparation was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. A molecular mass of the peptide was estimated by electrospray ionization mass spectroscopy to be 3,244.64 +/- 1.15 Da. Its amino acid composition indicates the presence of lanthionine and likely beta-methyllanthionine in a total of about 25 amino acids. Because alpha,beta-unsaturated amino acids, the precursors of lanthionine residues, are often found in lantibiotics, we carried out the addition reaction of the mutacin with N-(methyl)mercaptoacetamide. The subsequent electrospray ionization mass spectroscopy analysis indicated the presence of two reaction products with M(r)s of 3,350.45 and 3,456.0. These are interpreted as the mutacin molecule with the addition of one and two molecules of reagent to the unsaturated amino acids, respectively. Sequencing of the peptide revealed an N-terminal amino acid sequence of Asn-Arg-Trp-Trp-Gln-Gly-Val-Val.     

New serotype of mutans streptococci isolated from pig oral cavity

Gram-positive streptococcal mutans-like strains, but with clearly different colony formation than S. orisuis on Mitis Salivarius agar, were isolated from the pig oral cavity and identified by 16S rRNA sequencing, G+C content, DNA-DNA homology and extensive biochemical and serological testing. The phenotypic data showed that the strains were similar to S. orisuis except for susceptibility to bacitracin. DNA-DNA homology between the isolates and S. orisuis was 72∼81%. However, serological data showed that they have a different sero-specific antigen from S. orisuis and other mutans streptococci. A new serotype, designated p, strains are classified in a serovar of S. orisuis, one of mutans streptococci.     

Antimicrobial effect of Korean propolis against the mutans streptococci isolated from Korean

The aim of this study was to determine the optimal concentration of Korean propolis against clinical isolates of mutans streptococci (MS) from Koreans. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and time-kill curves against mutans streptococci. The MIC(90) values of propolis for MS were 35 μg/ml. Propolis had a bacteriostatic effect on Streptococcus mutans ATCC 25175(T) and bactericidal effects on Streptococcus sobrinus ATCC 33478(T) at > 2 × MIC (70 μg/ml). These results suggest that the propolis can be used in the development of oral hygiene products for the prevention of dental caries.     

Isolation,partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.     

Identification of mutans streptococci with monoclonal antibodies

Mutans streptococci have been correlated with dental caries. The identification of the species within this group is still a problem. The characterization of a monoclonal antibody (Mab) OMVU10 against S. sobrinus as well as the isolation and characterization of Mabs against S. mutans (OMVU30 and OMVU31), S. cricetus (OMVU40) and mutans streptococci (OMVU2) is described. The epitope specificity for OMVU10 and OMVU31 was cell-wall antigen B in both cases although both Mabs recognized different species-specific epitopes. OMVU40 was cross reactive with Steptococcus sanguis taxon 3. All other Mabs were specific for one species. Using these Mabs, a key to the identification of mutans streptococci is developed. This key was tested for 85 wild type isolates of mutans streptococci and proved to be highly reliable and easy to perform.     

Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci.

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.     

Amoxycillin-resistant streptococci in dental plaque

This investigation was undertaken to study the prevalence of amoxycillin-resistant oral streptococci in normal healthy patients and patients with a cardiac condition, susceptible to infective endocarditis. Samples of supragingival dental plaque were collected from two test groups, children with congenital heart disease and adults with a history of rheumatic fever, and two control groups comprising normal healthy children and normal healthy adults. Bacteria from these samples were grown on a medium selective for oral streptococci, as well as on the same medium containing known concentrations of amoxycillin. The results indicate that a high percentage of rheumatic heart patients and children with congenital heart disease harboured amoxycillin-resistant oral streptococci. The level of amoxycillin resistance in the plaque of adults with rheumatic heart disease was significantly greater than in that of normal adults. In view of the high percentage of patients at risk harbouring amoxycillin-resistant streptococci, it is important that the individual clinical situation be monitored. Perhaps antibiotic sensitivity tests should be performed to select an appropriate antibiotic for prophylaxis of infective endocarditis.     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

Factors influencing cell shape in the mutans group of streptococci.

Electron and light microscopic and growth studies of representatives of the diverse species of mutans streptococci revealed the cells to be either bacillary or coccoid in shape. Some strains changed from bacillary to coccoid if the HCO3-/K+ ratio of the media was increased and from coccoid to bacillary if the ratio was decreased. Doubling times of rods and cocci were the same despite an HCO3-/K+ ratio change between 0.008 and 2.84. For strain 10449S, no tested anions or cations substituted for HCO3- or K+ to produce this effect, except for B4O7(2-). Strain 10449S grown at a high B4O7(2-)/K+ ratio became ellipsoid, and this phenomenon was associated with slower doubling times. Up to three incomplete septa could be observed in one rod, but no more than one incomplete septum could be observed in either ellipsoid or spherical cells. Interseptal distances were greatest in rods, shorter in spheres, and shortest in ellipses. All of the above differences were statistically significant (P less than 0.001).     

First isolation of Streptococcus downei from human dental plaques

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.     

Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.     

Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.     

Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries

The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170773D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the proline-rich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.     

Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite − 1. On the other hand, novel structural features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.