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ABSTRACT: Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains.
Molecular oral microbiology. 08/2012; 27(4):308-23.
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ABSTRACT: Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners.
Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis, Porphyromonas gulae, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens were performed.
P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them.
These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.
Archives of oral biology 03/2012; 57(9):1183-8. · 1.65 Impact Factor
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ABSTRACT: Streptococcus mutans is known to be a primary causative agent of dental caries and its surface proteins have been investigated to specify their association with its virulence. Amongst those, 4 glucan-binding proteins (Gbps) are considered to be important factors due to their glucan-binding properties, of which GbpB has been shown to participate in cell-wall construction and cell separation.
We examined clinical isolates of S. mutans collected from the oral cavities of Japanese and Finnish subjects, and focused on the association of their GbpB expression profiles and biological properties related to virulence.
Western blot analysis of GbpB expression by the isolates revealed a variety of patterns. Strains that showed single and multiple bands were used to designate S and M type strains, respectively, whilst those with no GbpB expression were classified as N type. The distribution of GbpB expression patterns was shown to be quite different between the Japanese and Finnish isolates. Furthermore, the chain length and doubling time of the N type in both populations were significantly longer than those of the other types.
Our results suggest variations in S. mutans GbpB expression patterns, which may have relationships with the virulence of S. mutans.
Archives of oral biology 10/2010; 56(3):258-63. · 1.65 Impact Factor
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K Nakano,
K Wada, R Nomura,
H Nemoto,
H Inaba,
A Kojima,
S Naka,
K Hokamura,
T Mukai,
A Nakajima,
K Umemura,
Y Kamisaki,
H Yoshioka,
K Taniguchi,
A Amano,
T Ooshima
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ABSTRACT: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis.
Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed.
The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups.
These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.
Oral Diseases 10/2010; 17(4):370-8. · 2.49 Impact Factor
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ABSTRACT: Streptococcus mutans is a major pathogen of dental caries and occasionally isolated from the blood of patients with infective endocarditis, though the association of its cell-surface glucosyltransferases (GTFB, GTFC, and GTFD) with pathogenicity for infective endocarditis remains to be elucidated. In this study, we investigated the contribution of S. mutans GTFs to platelet aggregation and analysed GTF expression profiles in a large number of clinical oral isolates.
The platelet aggregation properties of GTF-defective isogenic mutant strains constructed from S. mutans reference strain MT8148 were evaluated using whole blood and platelet-rich plasma (PRP) taken from mice, as well as human PRP. In addition, GTF expression profiles for 396 S. mutans strains isolated from the oral cavities of 396 subjects were analysed by western blotting using antisera specific for each GTF.
The platelet aggregation activities of the GTF-defective isogenic mutants were significantly lower than that of MT8148 when added to a large number of cells. Western blotting revealed no strains without GTF expression, though six strains had alterations of GTFB and GTFC as compared to MT8148. PCR analyses indicated that the gtfB-gtfC region length was approximately 4.5 kb shorter in those strains as compared to MT8148. These were designated as "GTFBC-fusion" strains and they demonstrated lower levels of platelet aggregation.
Our findings indicate that GTFs are associated with platelet aggregation. Although the clinical detection frequency of S. mutans strains with altered expressions is extremely low, GTFBC-fusion strains have activities similar to GTF-defective mutant strains.
Archives of oral biology 06/2010; 55(6):410-6. · 1.65 Impact Factor
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K Nakano, R Nomura,
N Taniguchi,
J Lapirattanakul,
A Kojima,
S Naka,
P Senawongse,
R Srisatjaluk,
L Grönroos,
S Alaluusua,
M Matsumoto,
T Ooshima
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ABSTRACT: Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm.
A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed.
The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A.
Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.
Archives of oral biology 12/2009; 55(1):34-9. · 1.65 Impact Factor
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ABSTRACT: Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present.
A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains.
Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan.
Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.
Oral Microbiology and Immunology 11/2009; 24(5):431-3. · 2.81 Impact Factor
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ABSTRACT: Streptococcus mutans is considered to be one of the pathogens that cause infective endocarditis. The purpose of the present study was to examine the properties of S. mutans with regard to platelet aggregation by focusing on its high molecular protein antigen c (PAc).
The platelet aggregation properties of six clinical strains and one isogenic mutant strain of S. mutans were analysed using an aggregometer and confocal microscopy, as well as with an inhibition assay of platelet aggregation using anti-PAc serum.
S. mutans strains with PAc expression induced platelet aggregation, while a PAc-deficient mutant and two clinical isolates with no PAc expression did not. When platelets were pretreated with higher amounts of anti-PAc serum, the platelet aggregation rate was reduced in a dose-dependent manner, indicating that PAc binds directly to platelets.
S. mutans PAc is involved in human platelet aggregation and may be one of the virulence factors in the pathogenesis of infective endocarditis.
Oral Microbiology and Immunology 11/2009; 24(5):427-30. · 2.81 Impact Factor
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ABSTRACT: Porphyromonas gingivalis infection is thought to be a significant etiological factor in the development of cardiovascular diseases. However, scant definitive evidence has been presented concerning the pathological molecular mechanisms of these disorders. In the present study, we performed a molecular analysis of the developmental mechanisms of aortic intimal hyperplasia induced by P. gingivalis.
The effects of P. gingivalis-induced bacteremia on intimal hyperplasia were evaluated using a mouse model of aortic hyperplasia created by photochemical-induced endothelial cell injury. Alterations of gene expression profiles in injured blood vessels of the mice were extensively analyzed using DNA microarray assays to identify the key molecules involved in P. gingivalis-induced hyperplasia. In addition, human aneurismal specimens from patients with or without P. gingivalis infection were analyzed histochemically.
Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model. Concomitantly, S100 calcium-binding protein A9 (S100A9) and embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis-infected patients.
We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.
Journal of Periodontal Research 11/2009; 45(3):337-44. · 1.69 Impact Factor
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ABSTRACT: Infective endocarditis (IE) is caused by a microbial infection of the endothelial surface of the heart. Although blood culture examinations are commonly used to determine the associated bacterial species, molecular techniques, which enable rapid identification of targeted bacterial species, have recently been applied in clinical cases.
Nine heart valve specimens from IE patients (six subacute cases and three acute cases) were extirpated and collected, then bacterial DNA was extracted. Bacterial species in the specimens were determined by two different molecular methods and the results were compared with those from a conventional blood culture technique. In addition, a comparison between the two molecular methods was carried out using known numbers of six streptococcal species.
The conventional blood culture method revealed the bacterial species in eight cases, while one was found to be negative. Multiple species were identified in most of the cases by both molecular methods; however, those specified by one method were not always consistent with those specified by the other. Furthermore, the species determined by the blood culture technique were not always identified by the molecular methods. We also found that the two molecular methods used in the present study were extremely sensitive to detect from 1 to 100 cells of individual oral streptococcal species.
Our results suggest that species specified by molecular methods may have disseminated incidentally into the bloodstream, so interpretation of such results should be carefully undertaken in clinical situations.
Oral Microbiology and Immunology 03/2009; 24(1):43-9. · 2.81 Impact Factor
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ABSTRACT: Oral bacteria, including cariogenic and periodontal pathogens, are thought to be etiological factors in the development of cardiovascular diseases. To define this relationship, we analyzed the distribution of oral bacterial species in cardiovascular specimens.
Following acceptance into the study, 203 consecutive patients were analyzed, from whom 82 aortic valve specimens, 35 mitral valve specimens, and 86 aortic aneurysmal wall specimens, of which 16 contained aneurysmal thrombus tissues, were obtained. In addition, a total of 58 dental plaque specimens were collected from the same group of patients who underwent heart valve replacement or removal of aortic aneurysms. Bacterial DNA was extracted from both cardiovascular tissues and dental plaque in those cases and then species-specific polymerase chain reaction assays were used to analyze the occurrences of six oral streptococcal and six periodontal bacterial species.
Streptococcus mutans was the most frequently detected species in the cardiovascular specimens, followed by Aggregatibacter actinomycetemcomitans. As for dental plaque specimens from patients who underwent cardiovascular operations, most of the tested periodontitis-related species as well as oral streptococci were detected at high frequencies. Furthermore, the positive rate of S. mutans in cardiovascular specimens from patients whose dental plaque specimens were also positive for S. mutans was 78%, which was significantly higher than any other tested species when the same analysis was performed.
Our results suggest that specific oral bacterial species, such as S. mutans and A. actinomycetemcomitans, are related to bacteremia and may be etiologic factors for the development of cardiovascular diseases.
Oral Microbiology and Immunology 03/2009; 24(1):64-8. · 2.81 Impact Factor
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ABSTRACT: A new reliable genotyping method, multilocus sequence typing (MLST), was used to evaluate vertical transmission of the cariogenic pathogen Streptococcus mutans. A total of 136 S. mutans strains were isolated from saliva samples of 20 Japanese mother-child pairs, including 5 girls and 5 boys with primary dentition, and 5 girls and 5 boys with mixed dentition. The nucleotide sequences of 8 partial housekeeping genes, aroE, murI, gltA, glnA, glk, tkt, lepC, and gyrA, were analyzed and a similarity for all of those sequences between strains from a mother-child pair was regarded as indicating transmission, which was shown in 70% of the pairs. Interestingly, the rate of transmitted strains from mothers was significantly higher in the girls (90%) than in the boys (p = 0.001). Furthermore, the S. mutans sequence type (ST) with the highest distribution percentage in each maternal saliva sample was found to be transferred to their children. In addition, variations in two large conjugative-transfer associated regions, TnSmu1 and TnSmu2, were determined and compared with the STs defined by MLST. No variations in those two regions shown by PCR patterns were present in any of the strains isolated from the same families with the same STs, though isolates of some STs from different families showed distinct patterns for TnSmu2. Our results indicate that mothers are the main source for transmission of S. mutans to their children, while the present MLST method was also shown to be useful for investigating bacterial transmission.
Caries Research 12/2008; 42(6):466-74. · 2.33 Impact Factor
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ABSTRACT: Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.
Journal of dental research 11/2008; 87(10):964-8. · 3.46 Impact Factor
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ABSTRACT: There is scant information available regarding the distribution of periodontal bacterial species in children and adolescents over an extended period. The purpose of this study was to compare bacterial profiles in the same individuals over a period of 7 years.
Twenty-six children and adolescents from whom dental plaque and saliva specimens were obtained during both the first (1999-2000) and second (2006-2007) periods, were analyzed. Bacterial DNA was extracted from each specimen and the presence of 10 periodontal bacterial species was determined using a PCR method, with a focus on the red complex species of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.
Subjects with red complex species in saliva specimens obtained during the second collection possessed a significantly higher number of total bacterial species than those without. The detection rate of the red complex species in the second collection period samples was significantly greater in subjects who had two or more species detected in samples taken during the first collection compared with the other subjects.
Subjects possessing red complex species may be at possible risk for infection with a high number of periodontal bacterial species during adolescent and younger adult years.
Oral Diseases 07/2008; 14(7):658-64. · 2.49 Impact Factor
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ABSTRACT: Porphyromonas gingivalis, a major periodontal pathogen, is gaining increasing attention for its possible association with cardiovascular diseases. Its fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. In this study, fimA genotypic distribution was analyzed in P. gingivalis-infected cardiovascular specimens.
A total of 112 heart valves and 80 atheromatous plaque specimens were collected from patients undergoing cardiovascular surgery, as well as 56 dental plaque specimens. Bacterial DNA was extracted from each, and polymerase chain reaction analysis was carried out with a P. gingivalis-specific set of primers. P. gingivalis-positive specimens were further analyzed to discriminate the fimA genotype using polymerase chain reaction with fimA type-specific primer sets.
P. gingivalis was detected in 10.4% of the cardiovascular specimens and 50.0% of the dental plaque samples. In the latter, type II was most frequently detected (35.7%), followed by types I (28.6%) and IV (21.4%), while types IV and II were detected with considerable frequencies of 45.0% and 30.0%, respectively, in the cardiovascular specimens. In contrast, the occurrence of type I was limited (5.0%) in the cardiovascular specimens.
These results suggest that specific fimA genotypic clones, which are reportedly associated with periodontitis, are also frequently harbored in cardiovascular specimens, indicating the possible involvement of type II and IV clones in the initiation and progression of cardiovascular diseases.
Oral Microbiology and Immunology 05/2008; 23(2):170-2. · 2.81 Impact Factor
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ABSTRACT: Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. Accumulated evidence suggests that P. gingivalis strains with type II fimbriae are more virulent as compared to those with other types. However, it is unknown if strong virulence is uniformly conserved among clones with type II fimbriae. In the present study, we compared infectious inflammatory changes in clinical isolates of P. gingivalis with type II fimbriae using a mouse abscess model to examine their pathogenic heterogeneity and heterogeneity-related factors.
Suspensions of nine different clinical isolates with type II fimbriae were subcutaneously injected into female BALB/c mice and inflammatory parameters, such as serum sialic acid concentration, were compared.
Many of the type II fimbrial isolates caused severe inflammation in the mice, though some were less causative, as was the control strain ATCC 33277 (type I fimbria strain). These results showed that pathogenic heterogeneity exists among P. gingivalis clones with type II fimbriae. Further, the heterogeneity-related factors of P. gingivalis strains were analyzed and the pathogenic potentials showed positive relationships to gingipain activities and invasive efficiency but not to hydrophobicity or autoaggregation. In addition, invasive efficiency was related to the activities of gingipains that were extracellularly secreted.
These results suggest that pathogenic heterogeneity has relationships with the invasive and proteolytic activities of P. gingivalis clones with type II fimbriae.
Oral Microbiology and Immunology 03/2008; 23(1):29-35. · 2.81 Impact Factor
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ABSTRACT: Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, has also been detected in cardiovascular tissues. Sixty heart valves were collected during valve replacement surgery from 60 patients (one from each), 10 were from patients with infective endocarditis (IE group) and 50 were from patients with other valvular diseases (non-IE group). In addition, 46 samples of aneurysmal tissue were taken from 46 patients with a thoracic or abdominal aneurysm (Aneurysm group, one from each). Dental plaque samples were taken from 54 of the patients, 31 in the IE and non-IE groups and 23 in the aneurysm group. First, the distribution of A. actinomycetemcomitans in all specimens was analysed using a polymerase chain reaction method, which resulted in a positive reaction in 33 (31.1%) of the cardiovascular specimens and 25 (46.3%) of the dental plaque samples. Next, using serotype-specific sets of primers, the serotype distribution of A. actinomycetemcomitans in the cardiovascular specimens and dental plaque samples was found to be significantly different compared to dental plaque samples from Japanese subjects reported previously.
Oral Microbiology and Immunology 05/2007; 22(2):136-9. · 2.81 Impact Factor
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ABSTRACT: We previously reported the new serotype k of Streptococcus mutans, which, compared to serotypes c, e, and f, features a drastic reduction in the length of the glucose side chain linked to the rhamnose backbone of the serotype specific polysaccharide. The 5' region of the rgpF gene of serotype k strains contains a distinctive nucleotide sequence, which suggests that an alteration of the rgpF gene in serotype k strains may explain the shortened glucose side chain. However, in the present study, expression of the rgpF gene of MT8148 (serotype c) in serotype k isolates was not found to lead to serotype conversion. Furthermore, mRNA expression of rgpE, known to be associated with glucose side chain formation, was not detected in any of the tested serotype k isolates with an RT-PCR method. The nucleotide alignment of all genes known to be involved in the biosynthesis of serotype specific polysaccharide in serotype k strains was shown to be quite similar to that of serotype c strains, as compared to serotype e and f strains, especially in the region downstream of rgpF. Our results indicate that the common characteristics of serotype k isolates may be caused by a lack of expression of the gene involved in glucose side chain formation.
Oral Microbiology and Immunology 11/2005; 20(5):303-9. · 2.81 Impact Factor
