Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Bacteriology

Development of a PCR Method for Rapid Identification of New Streptococcus mutans Serotype k Strains

Kazuhiko Nakano, Ryota Nomura, Noriko Shimizu, Ichiro Nakagawa, Shigeyuki Hamada, Takashi Ooshima
Kazuhiko Nakano
1Departments of Pediatric Dentistry
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ryota Nomura
1Departments of Pediatric Dentistry
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Noriko Shimizu
1Departments of Pediatric Dentistry
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ichiro Nakagawa
2Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shigeyuki Hamada
2Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Takashi Ooshima
1Departments of Pediatric Dentistry
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: ooshima@dent.osaka-u.ac.jp
DOI: 10.1128/JCM.42.11.4925-4930.2004
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5′ region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.

Streptococcus mutans, which is known to be a major cariogenic bacterium as well as one of the pathogens involved with bacteremia and infective endocarditis (IE), was previously classified into three serotypes, c, e, and f, based on the chemical composition of serotype-specific polysaccharides (5). The serotype-specific polysaccharide was shown to be composed of rhamnose-glucose polymers, with a backbone of rhamnose and side chains of α- or β-linked glucosidic residues (7). The genes involved in the synthesis of serotype-specific rhamnose-glucose polymers have also been cloned and sequenced. Four rml genes (rmlA through rmlD) are related to the synthesis of dTDP-l-rhamnose (18, 19), and the gluA gene encodes the enzyme that catalyzes the production of the immediate precursor of the glucose side chain donor (23). In addition, the six genes (rgpA through rgpF) required for synthesis of rhamnose-glucose polymers have been cloned and sequenced (15, 24). Further, the rgpG gene has been implicated in the initiation of synthesis of rhamnose-glucose polymers (25).

In our previous study, S. mutans strains with a low amount of the glucose side chain in serotype-specific polysaccharide were defined as a new serotype, serotype k, which was estimated to have a distribution in the oral cavity of approximately 2% (10). Recently, PCR methods were developed to identify serotypes c, e, and f by using DNA extracted from saliva samples with primers constructed on the basis of the differences in the sequences of the region downstream of rgpF among each of the serotypes (16). However, there is no information for serotype k strains regarding the genes involved in formation of the side chain of the rhamnose backbone. In the present study, we analyzed those genes from serotype k strains and developed a PCR method to identify subjects with S. mutans serotype k.

MATERIALS AND METHODS

S. mutans strains.Table 1 lists the S. mutans strains used in this study. Blood isolates TW295 (k) and TW871 (k) (3) and orally isolated strain MT8148 (c) were selected from the stock culture collection in our laboratory (8). One hundred strains of S. mutans isolated from 100 children, which included 78 serotype c, 17 serotype e, 3 serotype f, and 2 serotype k (strains FT1 and SU1) strains (NN2000 series of isolates) (10), were also analyzed, as was another serotype k strain, YK1 (10). Strains AT1 and YT1, isolated in the present study, were confirmed as S. mutans according to the results of a double immunodiffusion method that utilized autoclaved extracts and serotype k-specific antiserum, which has been described previously (10). The specificity of the primers used was tested against the following organisms: Streptococcus sanguinis ATCC 10556, Streptococcus oralis ATCC 10557, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC 903, Streptococcus milleri NCTC10703, and Streptococcus salivarius HHT.

View this table:
  • View inline
  • View popup
TABLE 1.

S. mutans strains used in the present study

Sequence analysis of genes involved in biosynthesis of serotype-specific polysaccharide.The rgp genes (rgpA through open reading frame 11 [ORF11]; total of 15,890 bp) were divided into six fragments. Each gene fragment was amplified by PCR with primers constructed on the basis of the nucleotide alignment of strain Xc (GenBank accession no. AB010970 ) or strain UA159 (GenBank accession no. AE014133 ) with LATaq Polymerase (Takara Shuzo, Otsu, Japan). The PCR products were separated by electrophoresis on a 0.7% agar gel, and the amplified DNA was extracted with QIAEX (Qiagen, Düsseldorf, Germany). The DNA was directly cloned into a pGEM-T Easy vector (Promega, Madison, Wis.), for which the nucleotide sequence was determined by a dye-terminator reaction with a DNA sequencing system (373-18 DNA sequencer; Applied Biosystems, Foster City, Calif.) and an ABI PRISM cycle sequencing kit. Data analysis was performed with Gene Works software (IntelliGenetics, Mountain View, Calif.), and a multiple-alignment analysis was carried out with CLUSTAL W from the DDBJ (Mishima, Japan) (17). The nucleotide alignments of the rgp genes in TW295 (serotype k), TW871 (serotype k), FT1 (serotype k), and YT1 (serotype k) were compared with that of MT8148 (serotype c). The rgp genes of strain Xc (serotype c), for which the rgp genes were originally sequenced, and those of strain UA159 (serotype c) (1), of which the complete genome has been sequenced, were also compared with those of the serotype k strains.

Clinical specimens.Expectorated whole saliva (approximately 1 ml in each sample) was collected from 200 children and adolescents (2 to 18 years of age; mean age, 7.9 ± 3.6 years) who visited the Pedodontics Clinic of Osaka University Dental Hospital in January and February 2004. Collection of the clinical specimens was carried out in accordance with the Osaka University Health Guideline for Studies Involving Human Subjects. The saliva samples were processed for the PCR assay by the method reported by Hoshino et al. (6) with some modifications. Briefly, nonstimulated whole saliva was collected in a sterile tube and kept on ice. Bacterial cells were collected in a microcentrifuge tube from 500 μl of saliva at 16,000 × g for 5 min, then treated in a microwave oven at 500 W for 5 min, and digested in N-acetylmuramidase SG (Seikagaku Corp., Tokyo, Japan) at 50°C for 1 h. Then, 80 μl of nuclei lysis solution (Promega) was added and incubated at 80°C for 5 min, followed by the addition of 60 μl of protein precipitation solution (Promega). The proteins were removed by centrifugation at 16,000 × g for 3 min, and the DNA was purified by phenol-chloroform extraction and ethanol precipitation. The extracted DNA was then dissolved in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]).

Development of PCR method for identification of serotype k strains.Table 2 lists the PCR primers used in this study. The forward primer (CEFK-F) was common for all of the serotypes and designed within the 3′ end region of rgpE, whereas the serotype c-, e-, and f-specific (non-serotype k) and serotype k-specific reverse primers (CEF-R and K-R, respectively) were designed within the serotype k-specific 5′ region of the rgpF gene (350 bp from the initial sequence) (Fig. 1). The specificities of the prospective primers were tested by the program Amplify (2), based on the DNA sequence information stored in GenBank EMBL. All of the primers were commercially synthesized (Proligo Japan, Kyoto, Japan).

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Illustration of the serotype k-specific nucleotide region and location of the primers constructed for detection of serotype k strains by PCR. A base pair (BP) scale is shown above the map. The black box indicates the serotype k-specific nucleotide alignment region in the rgpF gene. The nucleotide number corresponds to the rgpF sequence in strain Xc obtained from GenBank (accession number AB010970 ).

View this table:
  • View inline
  • View popup
TABLE 2.

PCR primers used in the present study

PCR amplification was performed in a total volume of 20 μl with 1 μl of template solution and AmpliTaq Gold polymerase (Applied Biosystems) according to the manufacturer's instructions. The PCR amplification reaction was performed in a thermal cycler (iCycler; Bio-Rad, Hercules, Calif.) with the following cycling parameters: an initial denaturation at 95°C for 4 min and then 30 cycles consisting of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. The PCR products were subjected to electrophoresis in a 1.5% agarose gel-Tris-acetate-EDTA buffer. The gel was stained with 0.5 μg of ethidium bromide per ml and photographed under UV illumination. A 100-bp DNA ladder (New England BioLabs, Beverly, Mass.) was used as the molecular size standard.

First, the sensitivity of the PCR assay was determined by using titrated cultures of S. mutans MT8148 (serotype c), TW295 (serotype k), and FT1 (serotype k). The CEFK-F and CEF-R sets of primers were used for amplification of the genomic DNA of MT8148, and the CEFK-F and K-R sets were used for amplification of that of TW295 and FT1, respectively. The detection limit for simultaneous PCR was determined by using the known numbers of bacterial cells diluted in sterile distilled water. Genomic DNA extracted from strains MT8148 (serotype c), NN2001 (serotype c), NN2002 (serotype e), NN2003 (serotype f), TW295 (serotype k), TW871(serotype k), FT1 (serotype k), and YT1 (serotype k) was analyzed to confirm the specificity of the primers designed to classify the non-serotype k or serotype k type strains. In addition, the specificity of the primers was confirmed with genomic DNA from 100 S. mutans strains (78 serotype c, 17 serotype e, 3 serotype f, and 2 serotype k strains) previously isolated (NN2000 series of isolates) (10). After analyses of the sensitivity and specificity of the present method, 200 DNA samples extracted from the saliva of the 200 subjects were analyzed.

Analysis of subjects with serotype k strains identified previously.Saliva samples were collected from two subjects from whom FT1 (serotype k) or YK1 (serotype k) had been isolated from plaque samples in our previous study. DNA from these samples was collected by the method described above and subjected to PCR with the serotype k-specific primers.

RESULTS

Nucleotide alignment of rgp genes from S. mutans serotype k strains.The nucleotide alignments of rgpA, rgpB, rgpC, rgpD, rgpE, rgpF, ORF7, rgpH, rgpI, ORF10, and ORF11 of TW295 (serotype k), TW871 (serotype k), FT1 (serotype k), and YT1 (serotype k) were compared with those of MT8148 (serotype c), Xc (serotype c), and UA159 (serotype c). There were no significant differences in the putative amino acid sequences of RgpA, RgpC, RgpE, RgpH, ORF10, and ORF11 among all of the test and reference strains. On the other hand, 59 amino acids in the C-terminal region were estimated to be deleted in RgpB of strains FT1 (serotype k), YT1 (serotype k), and MT8148 (serotype c). In addition, 23 amino acids were estimated to be deleted in RgpD of TW871 (serotype k). As for ORF7, strains FT1 (serotype k) and YT1 (serotype k) were estimated to be composed of 603 and 675 amino acids, respectively, which were fewer than any of the other tested strains (803 amino acids). Further, 26 amino acids were estimated to be deleted in one-third of the N-terminal region of RgpI in strain FT1 (serotype k). The most prominent difference among the genes in each of the tested serotype k strains commonly compared to strains MT8148 (serotype c), Xc (serotype c), and UA159 (serotype c) was identified in the 5′ region of the rgpF gene (350 bp from the initial sequence) (Fig. 2). Thus, primers specific for the detection of serotype k strains were constructed on the basis of those sequences.

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

Multiple-sequence alignment of the serotype k-specific 5′ region in the rgpF gene compared to that of the reference strains. Only nucleotides different from MT8148 are shown. Primers used for detection of serotype k and non-serotype k strains were constructed on the basis of the differences in nucleotide alignment (highlighted by the black box). Numbers above the nucleotides are those that appear in GenBank under accession number AB010970 (strain Xc). Serotypes are indicated in parentheses.

Specificity and sensitivity of the PCR assay.The PCR assay used in the present study showed positive bands with a non-serotype k-specific set of primers with MT8148 (serotype c) and with a serotype k-specific set of primers with TW295 (serotype k) and FT1 (serotype k), each of which produced single bands with the expected size of 294 bp, as assessed by electrophoresis (Fig. 3). The detection limit was determined in the presence of titrated bacterial cells, and the sensitivity of the PCR assay was found to be between 5 and 50 cells for the serotype k-specific set of primers with strains TW295 (serotype k) and TW871 (serotype k) and between 50 and 500 cells for the non-serotype k-specific set of primers with strain MT8148 (serotype c). Non-serotype k-specific PCR products were selectively identified from among the serotype c, e, and f strains (listed in Table 1), and serotype k-specific PCR products were selectively detected from among the serotype k strains (Fig. 4). Positive bands were identified among the S. mutans strains while the other species showed negative reactions. The specificity of the primers was also confirmed with 100 strains (NN2000 series of isolates), which indicated that the PCR method utilized was valid.

FIG. 3.
  • Open in new tab
  • Download powerpoint
FIG. 3.

Sensitivity of PCR assays for detection of non-serotype k and serotype k strains of S. mutans. The sensitivity of our PCR method was examined by using titrated cultures with 108 cells per ml from strains MT8148 (serotype c), TW295 (serotype k), and FT1 (serotype k). PCR was performed with the CEFK-F and CEF-R primer sets for MT8148 while the CEFK-F and K-R primer sets were used for TW295 and FT1. The detection limit for simultaneous PCR was determined by using the known numbers of bacterial cells diluted in sterile distilled water. The following numbers of cells were added: 5 × 104 (lanes 1), 5 × 103 (lanes 2), 5 × 102 (lanes 3), 5 × 10 (lanes 4), and 5 (lanes 5). M, molecular size marker (100-bp DNA ladder).

FIG. 4.
  • Open in new tab
  • Download powerpoint
FIG. 4.

PCR assay for the detection of S. mutans serotype k strains. (A) The primer sets CEFK-F and CEF-R were used for the detection of serotype c, e, and f strains. (B) The primer sets CEFK-F and K-R were used for the detection of serotype k strains. Lane 1, MT8148 (serotype c); lane 2, NN2001 (serotype c); lane 3, NN2002 (serotype e); lane 4, NN2003 (serotype f); lane 5, TW295 (serotype k); lane 6, TW871 (serotype k); lane 7, FT1 (serotype k); lane 8, YT1 (serotype k); lane 9, S. sanguinis ATCC 10556; lane 10, S. oralis ATCC 10557; lane 11, S. gordonii ATCC 10558; lane 12, S. mitis ATCC 903; lane 13, S. milleri NCTC10703; lane 14, S. salivarius HHT.

Samples from subjects with a positive reaction to serotype k-specific primers.Figure 5 shows the results from saliva samples of representative subjects. Of the 200 samples, 190 showed a positive reaction to the non-serotype k-specific set of primers (specific for serotypes c, e, and f) but not to the serotype k-specific set, whereas 10 samples showed positive reactions to both primer sets. These 10 subjects included 3 males and 7 females, none of whom had heart disorders. In addition, saliva samples previously taken from two subjects from whom the serotype k strains YK1 and FT1 were isolated also showed a positive reaction to the serotype k-specific primers. In addition, the use of a double immunodiffusion method for confirmation of serotype k strains AT1 and YT1, isolated from a subject whose saliva showed a positive reaction to serotype k-specific primers, also positively reacted to the serotype k-specific primers.

FIG. 5.
  • Open in new tab
  • Download powerpoint
FIG. 5.

Identification of subjects with S. mutans serotype k in saliva samples by PCR. (A) The primer sets CEFK-F and CEF-R were used for the detection of serotype c, e, and f strains. (B) The primer sets CEFK-F and K-R were used for the detection of serotype k strains. Lanes 1 through 5, samples from five representative subjects with a negative reaction to the serotype k-specific primers. Lanes 6 through 8, samples from three representative subjects with a positive reaction to the serotype k-specific primers. Samples in lanes 6 through 8 also reacted positively to the serotype c-, e-, and f-specific primers.

DISCUSSION

S. mutans is known to be a major causative bacterium of dental caries in humans and is occasionally isolated from the blood of the patients with IE (20, 21); however, its mechanism of invasion and survival in blood remains to be clarified. In a previous study (10), S. mutans serotype k strains were shown to be less susceptible to phagocytosis, which indicated that this newly elucidated serotype may be one of the risk factors of IE caused by the bacterium. To prevent the occurrence of IE, host risk factors, such as congenital heart failure and acquired valve replacement, have been the focus of clinical practitioners. However, it is also important to elucidate which pathogenic bacteria have a potential to cause bacteremia and the ensuing IE. Microbiological diagnoses of bacteria in blood are generally based on conventional blood culturing, although molecular diagnoses have also been performed recently (4). PCR methods with primers constructed based on the 16S rRNA alignment are widely utilized for their rapid and sensitive detection of bacteremia (13). With a PCR assay, not only can the presence of bacteria in blood be identified but also the bacterial species itself can be specified, even in cases of culture-negative IE (22). To identify S. mutans by PCR, several methods have been proposed (6, 11, 14) and a PCR technique for differentiating between serotypes c, e, and f has been developed (16). In the present study, we utilized a PCR method to identify S. mutans serotype k strains.

The frequency of subjects with serotype k in the present study was 5%, which was higher than that (2%) found when analyzing a single representative strain from a single subject by an immunodiffusion method in a previous study (10). The detection limit of the present PCR method was shown to be from 5 to 50 cells (Fig. 3), indicating that subjects with a small number of the serotype k strain bacteria could be identified as positive. We observed positive reactions by the serotype k blood and oral isolates, as well as negative reactions by the 98 c, e, and f serotype strains by using the present serotype k-specific set of primers. Further, analyses of saliva samples taken from two subjects with previously isolated serotype k strains (FT1 and YK1) (10) revealed positive reactions to the primers, as did two other serotype k strains (AT1 and YT1). As noted above, since serotype k strains are known to be less susceptible to phagocytosis by polymorphonuclear leukocytes (10), it is important to identify patients with serotype k strains, especially those regarded as having a risk for IE and who are receiving medical or dental treatment. We propose that the PCR method elucidated here will be beneficial for screening subjects with S. mutans serotype k strains.

The rgpE, rgpH, and rgpI genes have been shown to be correlated to glucose side chain formation in the serotype-specific polysaccharide of S. mutans (12, 24), however, there were no significant differences in the nucleotide alignment of these genes found in the present serotype k strains. On the other hand, the rgpB, rgpD, ORF7, and rgpI genes were shown to be altered in one or two serotype k strains, though such alterations were not commonly detected in all of the serotype k strains tested. In contrast, the 5′ region of the rgpF gene (350 bp from the initial sequence) was shown to be specific for the serotype k strains compared to the reference strains MT8148, Xc, and UA159, and we used it to develop the present PCR method for identification of serotype k strains. It is reasonable to speculate with high probability that there is a high possibility that rgpF itself is associated with glucose side chain formation in the serotype-specific polysaccharide of S. mutans. In order of level of involvement, RgpA, RgpB, and RgpF, encoded by the rgpA, rgpB, and rgpF genes, respectively, were each shown to be involved in the biosynthesis of the rhamnose backbone (16, 24). Therefore, we also considered that a variation of the rgpF gene may result in a short rhamnose backbone, causing a shortened attachment location of the glucose side chain. On the other hand, expression of the rgpF gene of MT8148 in the serotype k strains did not produce the properties of serotypes c, e, and f (data not shown), implicating the presence of other genes involved in biosynthesis of the glucose side chain. The functions of those genes in the rhamnose backbone of the O polysaccharide of Escherichia coli have been studied; however, the mechanisms in S. mutans remain to be elucidated in further molecular biological studies.

Our laboratory previously demonstrated that a serotype k blood isolate of TW871 showed a lower level of sucrose-independent adherence to saliva-coated hydroxyapatite than MT8148, as well as less cariogenicity in experiments with the caries-inducing rat model (9). We hypothesized that an alteration of glucan-binding protein C in TW871 caused the reduction of in vitro adhesion and the caries scores in vivo. However, the caries-inducing activities of TW871 have not been discussed in terms of alteration of the serotype-specific polysaccharide compared with that in MT8148. By utilizing a mutant strain without a glucose side chain in the serotype-specific polysaccharide as well as wild-type serotype k strains, the unknown function related to dental caries may be elucidated in further studies.

ACKNOWLEDGMENTS

This study was a part of a 21st Century COE entitled “Origination of Frontier Biodentistry” at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and was supported by a Grant-in-Aid for Scientific Research (B) 16390605 from the Japan Society for Promotion of Science.

FOOTNOTES

    • Received 19 March 2004.
    • Returned for modification 28 June 2004.
    • Accepted 6 July 2004.
  • Copyright © 2004 American Society for Microbiology

REFERENCES

  1. 1.↵
    Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B. Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H. Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA99:14434-14439.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    Engels, W. R. 1993. Contributing software to internet: the Amplify program. Trends Biochem. Sci.18:448-450.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    Fujiwara, T., K. Nakano, M. Kawaguchi, T. Ooshima, S. Sobue, S. Kawabata, I. Nakagawa, and S. Hamada. 2001. Biochemical and genetic characterization of serologically untypable Streptococcus mutans strains isolated from patients with bacteremia. Eur. J. Oral Sci.109:330-334.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Gauduchon, V., L. Chalabreysse, J. Etienne, M. Celard, Y. Benito, H. Lepidi, F. Thivolet-Bejui, and F. Vandenesch. 2003. Molecular diagnosis of infective endocarditis by PCR amplification and direct sequencing of DNA from valve tissue. J. Clin. Microbiol.41:763-766.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Hamada, S., and H. D. Slade. 1980. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev.44:331-384.
    OpenUrlFREE Full Text
  6. 6.↵
    Hoshino, T., M. Kawaguchi, N. Shimizu, N. Hoshino, T. Ooshima, and T. Fujiwara. 2004. PCR detection and identification of oral streptococci in saliva samples using gtf genes. Diagn. Microbiol. Infect. Dis.48:195-199.
    OpenUrlCrossRefPubMed
  7. 7.↵
    Linzer, R., M. S. Reddy, and M. J. Levine. 1986. Immunochemical aspects of serotype carbohydrate antigens of Streptococcus mutans, p. 29-38. In S. Hamada, S. M. Michalek, H. Kiyono, L. Manaker, and J. R. McGhee (ed.), Molecular microbiology and immunology of Streptococcus mutans. Elsevier Science Publishers, Amsterdam, The Netherlands.
  8. 8.↵
    Minami, T., T. Fujiwara, T. Ooshima, Y. Nakajima, and S. Hamada. 1990. Interaction of structural isomers of sucrose in the reaction between sucrose and glucosyltransferases from mutans streptococci. Oral Microbiol. Immunol.5:189-194.
    OpenUrlPubMed
  9. 9.↵
    Nakano, K., M. Matsumura, M. Kawaguchi, T. Fujiwara, S. Sobue, I. Nakagawa, S. Hamada, and T. Ooshima. 2002. Attenuation of glucan-binding protein C reduces the cariogenicity of Streptococcus mutans: analysis of strains isolated from human blood. J. Dent. Res.81:376-379.
    OpenUrlCrossRefPubMed
  10. 10.↵
    Nakano, K., R. Nomura, I. Nakagawa, S. Hamada, and T. Ooshima. 2004. Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype, k, in the human oral cavity. J. Clin. Microbiol.42:198-202.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Oho, T., Y. Yamashita, Y. Shimazaki, M. Kushiyama, and T. Koga. 2000. Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction. Oral Microbiol. Immunol.15:258-262.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Ozaki, K., Y. Shibata, Y. Yamashita, Y. Nakano, H. Tsuda, and T. Koga. 2002. A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis. FEBS Lett.532:159-163.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    Rothman, R. E., M. D. Majmudar, G. D. Kelen, G. Madico, C. A. Gaydos, T. Walker, and T. C. Quinn. 2002. Detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay. J. Infect. Dis.186:1677-1681.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    Sato, T., J. P. Hu, K. Ohki, M. Yamaura, J. Washio, J. Matsuyama, and N. Takahashi. 2003. Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal RNA genes. Oral Microbiol. Immunol.18:323-326.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Shibata, Y., Y. Yamashita, K. Ozaki, Y. Nakano, and T. Koga. 2002. Expression and characterization of streptococcal rgp genes required for rhamnan synthesis in Escherichia coli. Infect. Immun.70:2891-2898.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    Shibata, Y., K. Ozaki, M. Seki, T. Kawato, H. Tanaka, Y. Nakano, and Y. Yamashita. 2003. Analysis of loci required for determination of serotype antigenicity in Streptococcus mutans and its clinical utilization. J. Clin. Microbiol.41:4107-4112.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.22:4673-4680.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    Tsukioka, Y., Y. Yamashita, Y. Nakano, T. Oho, and T. Koga. 1997. Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans. J. Bacteriol.179:4411-4414.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Tsukioka, Y., Y. Yamashita, T. Oho, Y. Nakano, and T. Koga. 1997. Biological function of dTDP-rhamnose synthesis pathway in Streptococcus mutans. J. Bacteriol.179:1126-1134.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Ullman, R. F., S. J. Miller, M. J. Strampfer, and B. A. Cunha. 1988. Streptococcus mutans endocarditis: report of three cases and review of the literature. Heart Lung17:209-212.
    OpenUrlPubMed
  21. 21.↵
    Vose, J. M., P. W. Smith, M. Henry, and D. Colan. 1987. Recurrent Streptococcus mutans endocarditis. Am. J. Med.82:630-632.
    OpenUrlCrossRefPubMed
  22. 22.↵
    Wilck, M. B., Y. Wu, J. G. Howe, J. Y. Crouch, and S. C. Edberg. 2001. Endocarditis caused by culture-negative organisms visible by Brown and Brenn staining: utility of PCR and DNA sequencing for diagnosis. J. Clin. Microbiol.39:2025-2027.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Yamashita, Y., Y. Tsukioka, Y. Nakano, K. Tomihisa, T. Oho, and T. Koga. 1998. Biological functions of UDP-glucose synthesis in Streptococcus mutans. Microbiology144:1235-1245.
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    Yamashita, Y., Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga. 1998. Genes involved in cell wall localization and side chain formation of rhamnose-glucose polysaccharide in Streptococcus mutans. J. Bacteriol.180:5803-5807.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    Yamashita, Y., Y. Shibata, Y. Nakano, Y. Tsuda, N. Kido, M. Ohta, and T. Koga. 1999. A novel gene required for rhamnose-glucose polysaccharide synthesis in Streptococcus mutans. J. Bacteriol.181:6556-6559.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Development of a PCR Method for Rapid Identification of New Streptococcus mutans Serotype k Strains
Kazuhiko Nakano, Ryota Nomura, Noriko Shimizu, Ichiro Nakagawa, Shigeyuki Hamada, Takashi Ooshima
Journal of Clinical Microbiology Nov 2004, 42 (11) 4925-4930; DOI: 10.1128/JCM.42.11.4925-4930.2004

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Development of a PCR Method for Rapid Identification of New Streptococcus mutans Serotype k Strains
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Development of a PCR Method for Rapid Identification of New Streptococcus mutans Serotype k Strains
Kazuhiko Nakano, Ryota Nomura, Noriko Shimizu, Ichiro Nakagawa, Shigeyuki Hamada, Takashi Ooshima
Journal of Clinical Microbiology Nov 2004, 42 (11) 4925-4930; DOI: 10.1128/JCM.42.11.4925-4930.2004
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

blood
Mouth
polymerase chain reaction
Streptococcal Infections
Streptococcus mutans

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X