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Adherence and Streptococcus mutans infections: in vitro study with saliva from noninfected and infected preschool children.

An in vitro adherence experiment was designed to mimic the transmission of Streptococcus mutans from mother to child to test the hypothesis that differences in initial adherence reflect differences in susceptibility to infection. The data show that the pretreatment of S. mutans cells with the saliva of the mother in a mother-child pair and the pretreatment of spheroidal hydroxyapatite with that of the child may result in combinations which counteract or foster the initial adherence to a varying extent. The findings indicate that such combinations may determine the risk of S. mutants infection.

Adhesiveness↗

Comparative studies on the effect of crude aqueous (CA) and solvent (CM) extracts of clove on the cariogenic properties of Streptococcus mutans.

A study was conducted to compare the efficiency of crude aqueous (CA) and solvent extracts (CM) of clove on the caries-inducing properties of Streptococcus mutans. The cariogenic properties investigated included the cell adhesion, cell-surface hydrophobicity and glucan synthesis activities of S. mutans. There was a significant difference between the effect of the CA and CM extracts on the adhesion of S. mutans (P < 0.05) within a concentration range of 5-15 mg/ml, the CM extract demonstrating a slightly higher inhibitory effect. However, the effect of the CM extract on the cell-surface hydrophobicity of S. mutans was weaker than that of the CA extract. The two extracts were found to reduce the synthesis of water-insoluble glucan (WIG) by almost 50% at a concentration as low as 0.5 mg/ml and the CM extract exhibited a significantly higher inhibitory effect than the CA extract (P < 0.05). The present findings indicate that both the CA and CM extracts exert inhibitory effects on the cariogenic properties of S. mutans and that the CA extract is as equally effective as the CM extract.

Bacterial Adhesion↗

Overestimation of Streptococcus mutans prevalence by nested PCR detection of the 16S rRNA gene.

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

DNA Primers↗

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.

Blotting, Southern↗

Nucleotide sequence of the asd gene of Streptococcus mutans. Identification of the promoter region and evidence for attenuator-like sequences preceding the structural gene.

The complete nucleotide sequence of the asd gene of Streptococcus mutans encoding aspartate beta-semialdehyde dehydrogenase (EC 1.2.1.11), an enzyme comprised of 357 amino acids, having an Mr of 38,897 and active in the biosynthetic pathway of lysine, threonine, methionine, diaminopimelic acid, and isoleucine, has been determined. In addition we report the 276 nucleotides upstream of the structural gene which contain a highly efficient promoter identified by both RNA polymerase binding and in vitro transcription analysis. A leader transcript which terminates at a fixed point immediately preceding the asd promoter region was identified in the DNA sequence and confirmed by in vitro transcription analysis as well. The close proximity of this transcript and its p-independent transcriptional terminator to the asd coding sequence suggests involvement in a mechanism of regulation. Message stability experiments indicate the half-life of asd specific messages to be comparable to that of Escherichia coli messages. Conditions of varying concentrations of lysine, threonine, and methionine exert no apparent control over expression of the S. mutans asd gene in Escherichia coli suggesting the requirement of an accessory regulatory element specific for the S. mutans asd gene.

Aspartate-Semialdehyde Dehydrogenase↗

[Streptococcus mutans glucosyltransferase: isolation and its ability to synthesize polysaccharides].

The extracellular glucosyltransferases (GTF, EC 2.4.1.5) were extracted from trypticase yeast L-cystine culture supernatant of streptococcus mutans strains SSMC 167(g), 6715(g), SSMC 100 (c) and Ingbritt (c) by 60% saturated ammonium sulphate precipitation. The yield was more than 50%. All the enzyme preparations could synthesize both water-soluble and insoluble glucans. The results of polyacrylamide gel electrophoresis of enzyme preparations illustrated the complexity and multicomponent nature of the GTF of S. mutans. The abilities of synthesizing polysaccharides were different between S. mutans serotype g and serotype c. The ability of synthesizing water-soluble glucan was almost the same as that of synthesizing water-insoluble glucan for GTFs from the two S. mutans strains of serotype g, but the ability of synthesizing water-soluble glucan was 10-fold to that of synthesizing water-insoluble glucan for GTFs from other two strains serotype c. No frucotosyltransferase activity was found.

Glucans↗

Accumulation of Streptococcus mutans on light-cured composites and amalgam: an in vitro study.

The aim of the in vitro study was to examine the accumulation of Streptococcus mutans on light-cured composite materials and amalgam. Bacteria cultures were grown in a brain heart infusion medium, and their growth rate was determined through turbidity measurements. The data, so obtained, were evaluated statistically by analysis of variance (ANOVA) followed by Scheffe test. Experiments on amalgam showed better results compared to those on composite materials. There were no statistically significant differences in plaque accumulation on different composite materials after finishing and polishing procedures, compared to plaque accumulation on composite materials against a Mylar strip.

Analysis of Variance↗

Purification and properties of extracellular glucosyltransferase synthesizing 1,3-alpha-D-glucan from Streptococcus mutans serotype a.

Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.

Chromatography, Ion Exchange↗

Quasi-irreversible inhibition of enolase of Streptococcus mutans by fluoride.

Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al3+ or Be2+ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.

Fluorides↗

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme.

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.

Amino Acids↗

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a.

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.

Extracellular Space↗

Influence of bacterial cell concentration and inorganic anions on lysis of Streptococcus mutans BHT by salivary lysozyme.

The aim of this work was to study the influence of bacterial cell concentrations and inorganic anions on lysis of Streptococcus mutans BHT by human salivary lysozyme (HSL). HSL was partly purified from saliva by ion exchange chromatography. The bacteria were grown in a synthetic medium containing 3H-thymidine to monitor DNA release. The experiments demonstrated that release of 3H-thymidine was dependent on the bacterial cell concentration and an apparent Km-value corresponding to approximately 2.9 X 10(8) cells/ml was calculated. The influence of I-, Br-, Cl-, F-, HCO3- and SCN- on bacteriolysis was studied. All anions tested were slightly inhibitory on the action of HSL. The inhibition varied from 7 to 76% depending on the ion and ionic strength. The order of addition of HSL and sodium chloride caused different lytic responses. This was reflected by the amount of HSL adsorbed by the bacteria.

Animals↗

The LicT protein acts as both a positive and a negative regulator of loci within the bgl regulon of Streptococcus mutans.

An open reading frame (ORF) that would encode a putative antiterminator protein (LicT) of the BglG family was identified in the genomic DNA sequence of Streptococcus mutans. A DNA sequence that would encode a potential ribonucleic antiterminator (RAT) site in the mRNA at which the putative antitermination protein LicT would bind was located immediately downstream from this ORF. These putative antitermination components are upstream of a glucose-independent beta-glucoside-utilization system that is responsible for aesculin utilization by S. mutans NG8 in the presence of glucose. It was hypothesized that these putative regulatory components were an important mechanism that was involved with the controlled expression of the S. mutans bglP locus. A strain of S. mutans containing a licT : : Omega-Kan2 insertional mutation was created. This strain could not hydrolyse aesculin in the presence of glucose. The transcriptional activity associated with other genes from the bgl regulon was determined in the licT : : Omega-Kan2 genetic background using lacZ transcriptional fusions and beta-galactosidase assays to determine the effect of LicT on these loci. The LicT protein had no significant effect on the expression of the bglC promoter, a regulator of the bglA locus. However, it is essential for the optimal expression of bglP. These data correlate with the phenotype observed on aesculin plates for the S. mutans wild-type strain NG8 and the licT : : Omega-Kan2 strain. Thus, the glucose-independent beta-glucoside-specific phosphotransferase system (PTS) regulon in S. mutans relies on LicT for BglP expression and, in turn, aesculin transport in the presence of glucose. Interestingly, LicT also seems to negatively regulate the expression of the bglA promoter region. In addition, the presence of the S. mutans licT gene has been shown to be able to activate a cryptic beta-glucoside-specific operon found in Escherichia coli.

Bacterial Proteins↗

Inhibitory effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid production in dental plaque in vivo.

This study was conducted to find out whether sorbitol inhibits the sugar metabolism of Streptococcus mutans in vitro and the acid production in dental plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol-containing medium. The rate of acid production from sugars was estimated with a pH stat. The rate of acid production from glucose or sucrose was not changed at various concentrations of oxygen. By the addition of sorbitol to sugar, however, the acid production was decreased with increasing levels of oxygen. Intracellular NADH/NAD+ ratio and (dihydroxyacetone-phosphate+glyceraldehyde-phosphate)/3-phosphoglycerate ratio were high whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited the acid production in dental plaque in vivo. These results suggest that the increased NADH/NAD+ ratio during sorbitol metabolism through the inactivation of pyruvate formate-lyase by oxygen inhibited glyceraldehyde-phosphate dehydrogenase and then the acid production of S. mutans and the one in dental plaque.

Acetic Acid↗

Effect of lactoperoxidase system on glucosyltransferase D of Streptococcus mutans.

OBJECTIVE: To investigate the effects of the peroxidase system (LP, H2O2, SCN) and its components on the activity of glucosyltransferase D (GtfD) originating from Streptococcus mutans (S. mutans). METHODS: GtfD was incubated in buffered-KCl assay mixture that contained 20 microM dextran and 100 mM sucrose including 14C-glucose. The activity of GtfD was measured with a scintillation counter. The effects of lactoperoxidase system and its components on GtfD activity were examined by incubating different components separately and together with GtfD. RESULTS: The lactoperoxidase system-generated hypothiocyanite (OSCN) had no effect on the activity of streptococcal GtfD, while the lactoperoxidase enzyme inhibited GtfD. The ratio between the 2 enzymes, LP and GtfD, was important for the inhibition. However, if LP was combined with its substrates, SCN and/or a high concentration of H2O2, it enhanced the activity of GtfD. CONCLUSION: Peroxidase-generated antimicrobial agent HOSCN/OSCN is not inhibitory against GtfD, whereas low concentrations of the LP-enzyme inhibit GtfD.

Anti-Bacterial Agents↗

Evidence for an immunological relationship between Streptococcus mutans and human cardiac tissue.

Two-dimensional immunoelectrophoresis, indirect immunofluorescence, and radioimmunoassay were used to demonstrate that antisera from rabbits immunized with some strains of Streptococcus mutans contain antibodies that cross-react with human cardiac tissue. These rabbits were sensitized to a shocking dose of human heart antigen, and anaphylactic deaths were sometimes produced. Myocarditis was also a result of the immunization procedure. Data obtained with all five techniques were comparable. Cross-reactivity could be associated with three antigens designated ID, IF, and HL. Antigens ID and IF were major immunogens of S. mutans Ingbritt, but HL antibodies were produced only after hyperimmunization. Corss-reactivity was of an immunological nature and not the result of nonspecific factors such as bacterial Fc reactive components or antibody elicited to growth medium constituents. These findings support the hypothesis that immunization with S. mutans can induce autoimmune reactions and indicate that antigens must be selected with caution before formulating any dental caries vaccine.

Anaphylaxis↗

Characterization of the conformational epitope of Guy's 13, a monoclonal antibody that prevents Streptococcus mutans colonization in humans.

Guy's 13 is a mouse monoclonal antibody which recognizes streptococcal antigen I/II (SA I/II), a major cell surface glycoprotein of Streptococcus mutans. In a number of clinical trials, this antibody has been shown to prevent colonization in the human oral cavity. The aim of this study was to identify the SA I/II epitope recognized by Guy's 13. The data suggest that the epitope is conformational, delimited by two noncontiguous regions of the antigen: residues 45 to 457, within the N-terminal half of SA I/II, and residues 816 to 983, within the C-terminal half. In fluid-phase immunoassays a strict requirement for the simultaneous presence of both regions was demonstrated for antibody binding. Furthermore, these two regions of SA I/II were shown to have the ability to interact with each other in the absence of Guy's 13 antibody, suggesting that the normal conformation of SA I/II might be determined by the interaction of these two regions.

Animals↗

Effect of xylitol chewing gum on salivary Streptococcus mutans in preschool children.

Dental caries remains a significant problem for poor children in the United States. One strategy for treating dental caries is to suppress streptococcus mutans, the chief pathogen responsible for the disease. The purpose of this study was to evaluate the effect of xylitol gum in salivary S. mutans levels in preschool children. Sixty-one children were randomly assigned into the xylitol group and the control group. The xylitol group chewed gum sweetened only with xylitol (XyliFresh100%, Hershey Food Corporation, U.S.A.) three times a day for three weeks. S. mutans counts were tested using the Dentocult-SM Strip Mutans-test (Orion Diagnostica, Finland) at baseline and after three weeks. The shift from higher S. mutans scores to lower was greater in the xylitol group than in the control group (p;lt0.05). This study supports the suggestion that chewing xylitol gum may reduce salivary S. mutans levels. Xylitol chewing gum may provide a feasible caries prevention method for preschool children.

Cariostatic Agents↗