Sobrinus Serotype Research Articles

Differentiation of banding patterns between Streptococcus mutans and Streptococcus sobrinus isolates in rep-PCR using ERIC primer

BackgroundStreptococcus mutans and Streptococcus sobrinus are considered to be important bacterial species in the initiation of human dental caries. Therefore, the establishment of a reliable genotyping method to distinguish S. mutans from S. sobrinus is of central importance.ObjectiveWe assessed the usefulness of repetitive extragenic palindromic polymerase chain reaction (rep-PCR) using ERIC primer banding patterns in differentiating S. mutans and S. sobrinus.DesignFive S. mutans and two S. sobrinus prototype strains and 50 clinical isolates (38 S. mutans serotype c, 4 S. sobrinus serotype d, and 8 S. sobrinus serotype g) were examined. The banding patterns of amplicons generated were compared among the prototype strains and clinical isolates, to find common bands that distinguish S. mutans and S. sobrinus.ResultsMultiple banding patterns were seen with all strains tested. The representative strains of S. mutans tested revealed six unique, strong bands at 2,000 bp, 1,700 bp, 1,400 bp, 1,100 bp, 850 bp, and 250 bp, whereas S. sobrinus had seven strong bands at 2,000 bp, 1,800 bp, 1,100 bp, 900 bp, 800 bp, 600 bp, and 550 bp. The band at 1,100 bp was the only band that was observed in both S. mutans and S. sobrinus. Furthermore, most clinical S. mutans isolates revealed identical banding patterns. All S. mutans had amplicons at 1,700 bp, 850 bp, and 250 bp, whereas those of S. sobrinus were at 1,100 bp, 900 bp, and 800 bp.ConclusionsThese results indicate that using rep-PCR with the ERIC primers can distinguish between S. mutans and S. sobrinus.

Effect of removable orthodontic appliances on oral colonisation by mutans streptococci in children.

Little is known about the effect of removable orthodontic appliances on oral colonisation by mutans streptococci (MS). In the present study, the frequency of isolation and serotype distribution of MS were evaluated in two groups of children, one undergoing therapy with removable appliances and the other not subjected to any kind of orthodontic treatment, respectively. Streptococci isolated from dental plaque samples from both groups of children were identified as mutans streptococci on the basis of their morphological and biochemical properties and were then serotyped in an enzyme immuno-assay using monoclonal antibodies. The number of subjects harbouring MS in their dental plaque was statistically higher in the group of orthodontic children without caries experience (CF) in comparison with CF children of the control group (10/12, 83.3% vs. 15/44, 34%). No clear difference was observed in the distribution of the different MS serotypes between the experimental and control group: S. mutans c,f serotype was the most frequently isolated in both groups of children followed by S. mutans serotype e and S. sobrinus serotype g. Such results suggest that the use of removable appliances may lead to the creation of new retentive areas and surfaces, which favour the local adherence and growth of MS. The data obtained stress the importance of a careful monitoring of patients treated orthodontically for risk of caries development.

Identification of monoclonal antibody-binding domains within antigen P1 of Streptococcus mutans and cross-reactivity with related surface antigens of oral streptococci

Eleven monoclonal antibodies (MAbs) specific for P1, the major protein surface antigen of Streptococcus mutans serotype c, were characterized by Western blot (immunoblot) analysis and by radioimmunoassay using whole bacterial cells. The approximate binding domains of the MAbs were determined by using full-length and truncated P1 polypeptides. The accessibility of these binding sites on the surfaces of intact bacteria was determined by radioimmunoassay. The ability of each MAb to cross-react with related proteins from strains of S. mutans serotypes e and f, S. sanguis, and S. sobrinus serotype g is also reported.

Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan

The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtfS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtf gene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtfS gene.

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

Molecular and immunochemical characterization of recombinant Escherichia coli containing the spaA gene region of Streptococcus sobrinus

We identified and characterized a recombinant Escherichia coli containing the entire gene for surface protein antigen A (spaA) of Streptococcus sobrinus 6715. The recombinant E. coli was isolated from a cosmid gene bank of size-fractionated S. sobrinus DNA fragments, and recombinants expressing the SpaA protein were detected immunologically. Subcloning experiments showed that the DNA sequences encoding the SpaA protein could be isolated on two contiguous EcoRI fragments, 3.7 and 3.3 kilobases (kb) in size, both contained on a 16.2-kb BglII fragment. Southern blot hybridization experiments using the EcoRI fragments to probe genomic DNAs from various serotypes of the mutans group of streptococci revealed DNA sequence homology not only to S. sobrinus 6715 (serotype g) chromosomal DNA but also to S. sobrinus serotype d DNA. Weak hybridization signals to Streptococcus mutans serotypes c, e, and f and to Streptococcus cricetus serotype a were observed with the 3.3-kb EcoRI fragment. These results suggest that the coding sequence for the spaA gene is probably conserved in S. sobrinus strains. Plasmid-encoded polypeptides made in E. coli minicells revealed that transcription of the spaA gene was initiated on the 3.7-kb EcoRI fragment and that its product size was about 210 kilodaltons. The cloned SpaA protein was purified from the periplasmic protein of E. coli, and monospecific antiserum against the cloned product was prepared in rabbits. The data obtained from various physiochemical and immunological procedures allowed us to conclude that the coding sequence for the entire spaA gene of S. sobrinus 6715 had been successfully cloned in E. coli and that faithful expression of the cloned product could be obtained.

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans

The structural gene ( pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that ( pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross-reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.

Mutans streptococci のビルレンス因子の発現に及ぼす継代培養の影響

The fresh isolates of mutans streptococci (two strains of Streptococcus mutans serotype c and a strain of Streptococcus sobrinus serotype g) from the human oral cavity were subcultured one hundred times in brain heart infusion broth. Considerable changes were found in the virulence-related factors of mutans streptococci during repeated subculture. The results were summarized as follows. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoassay showed that the amount of a cell-surface protein antigen with a molecular weight of 190, 000 (PAc) of S. mutans serotype c, which is considered to be important in the sucrose-independent cellular adherence, exhibited considerable changes after repeated subculture. Moreover, there was a correlation between the cell-surface hydrophobicity and the amount of PAc on the cell surface. On the other hand, the amount of a cell-surface protein antigen with a molecular weight of 210, 000 (PAg) of S. sobrinus serotype g was not influenced during repeated subculture, and the cell-surface hydrophobicity was relatively constant. In addition, changes in the expression of several cell-surface proteins and extracellular proteins other than the protein antigens were noticed after repeated subculture in S. mutans and S. sobrinus. 2. Changes in the glucan-synthesizing ability, which is known to be important in the sucrose-dependent cellular adherence, was also found after subculture in S. mutans and S. sobrinus. There was a correlation between the cell-associated 1N NaOH-soluble glucan-synthesizing ability and the in vitro sucrose-dependent cellular adherence ability. 3. Almost no change was observed in the abilities for acid production and cell growth. 4. The cariogenicity of S. mutans in hamsters was reduced after repeated subculture. It was suggested that the reduced cariogenicity might be ascribed to the changes of the PAc expression and the cell-associated 1N NaOH-soluble glucan-synthesizing ability.

Electron Microscopy and Immunoperoxidase Staining of Streptococcus Mutans During Controlled Growth in two Different Media

Streptococcus mutans subspecies sobrinus serotype d (B13) was cultivated in a fermentor under controlled conditions in two different media: a complex proteose-peptone medium and a defined minimal medium (C4). Specimens from different growth phases were examined by electron-microscopy and tested electron-immunohistochemically. Irrespective of the growth medium used, no differences were observed in the immunohistochemical staining pattern of bacteria during the lag, the exponential and the early staionary phases. Specimens obtained several hours after exponential growth contained areas where the bacteria showed staining that ranged from a strong deposit of reaction product to no deposit. This appearance seems partly to explain the differences in the intensity of immunohistochemical staining of certain bacteria observed in dental plaques stained for identification of S. mutans subspecies sorbrinus.