Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 ؋ 10 10 sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease. These organisms prevail in the complex microcommunity of the oral biofilm in the presence of sucrose, under the extremely low pHs responsible for tooth demineralization. This organism is also a possible causative agent of infective endocarditis (9). S. mutans has been classified into four serotypes (c, e, f, and k) based on the chemical composition of its cell surface rhamnose-glucose polymers. The genes involved in the synthesis of serotypespecific polymers have been cloned and sequenced. Four rml genes (rmlA-rmlD) are related to the synthesis of dTDP-Lrhamnose (37, 38), and gluA is involved in the production of the immediate precursor of the glucose side chain (42). The six-gene operon (rgpA-rgpF) and rgpG, which are required for the synthesis of rhamnose-glucose polymers, have also been cloned and sequenced (41, 43). Serotype-specific genes just downstream from the rgpA-rgpF operon have also been sequenced, and this region is highly diverse among these serotypes (33). Therefore, the strains of each serotype of S. mutans are thought to have evolved and spread independently.In a previous study, we showed that some blood isolates of S. mutans that cannot be classified into the c, e, or f serotype have negligible amounts of glucose side chains, despite the presence of the rhamnose backbone in serotype-specific polysaccharides (21). We designated the novel serotype as serotype "k" and showed that serotype k strains are less susceptible to phagocytosis by human polymorphonuclear leukocytes. Most oral isolates are ...

Streptococcus mutans is a known pathogen of dental caries and its major cell surface antigens have been widely investigated. Recently, an approximately 120 kDa Cnm protein with binding properties to type I collagen was identified, and its encoding gene (cnm) cloned and sequenced. In the present study, we sequenced cnm from 47 different clinical S. mutans strains and found that the nucleotide alignment of the collagen-binding domain was well conserved. We devised a PCR method for identifying the cnm gene, examined the prevalence of cnm-positive S. mutans strains in various mother-child groups, and assessed the significance of such strains for transmission and dental caries. The detection rate of cnm-positive strains was significantly lower in strains isolated from Japanese children in the 2000s (8.0 %) as compared to those isolated in the 1980s (15.8 %) (P,0.05). Furthermore, the presence of S. mutans possessing cnm in salivary specimens collected from 55 S. mutans-positive mother-child pairs was 40 and 32.7 % in the mothers and children, respectively. The frequency of cnm-positive children whose mothers were also positive was 72 %, which was significantly higher than that of cnm-positive children with negative mothers (P,0.0001, odds ratio 17.5). In addition, clinical parameters indicating dental caries were significantly increased in children with cnm-positive S. mutans in saliva (n513), as compared to those with cnm-negative S. mutans (n515) and S. mutans-negative children (n520) (P,0.01). These results indicate that cnm-positive S. mutans strains are closely correlated with dental caries, while vertical transmission in cnm-positive mother-child pairs was also demonstrated.

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.

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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.

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.

Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.

Streptococcus mutans is one of the oral pathogens associated with infective endocarditis (IE).With respect to bacterial binding ability to the extracellular matrix, the Cnm protein, a cell surface collagen-binding adhesin of S. mutans, is known as one of the possible virulence factors with regard to IE. In this study, we aimed to determine the distribution of the cnm gene, which encodes Cnm, in a large number of clinical isolates of S. mutans from Thai subjects. Then, the cnm-positive strains were classified using a multilocus sequence typing (MLST) scheme, which we constructed previously. In addition, the data were analysed together with our previous MLST data of cnmpositive strains from Japan and Finland in order to evaluate the clonal relationship among S. mutans strains harbouring the cnm gene. The cnm gene was detected in 12.4 % of all 750 Thai isolates, and serotype f showed the highest rate of detection (54.5 %). According to the MLST data, two clonal complex groups were revealed as the important clones related to cnm-positive S. mutans from various origins of isolation. Moreover, the collagen-binding properties of S. mutans strains with the cnm gene were significantly greater than those of strains without the gene, although four cnm-negative strains classified into two sequence types (STs), ST110 and ST136, showed extremely high collagen-binding rates suggesting the presence of additional genes involved with collagen binding in these STs. Taken together, these results provided information on both epidemiological as well as evolutional aspects of S. mutans possessing the cnm gene. INTRODUCTIONStreptococcus mutans is a well-known pathogen of dental caries (Hamada & Slade, 1980), which consists of four serotypes, c, e, f and k (Linzer et al., 1986;). The detection rate of serotype c is highest among the strains isolated from oral cavities, followed by serotype e, while serotypes f and k are regarded as being present in minor proportions . Various pieces of evidence show the association of S. mutans with some life-threatening diseases such as infective endocarditis (IE), although the major species related to IE belong to the mitis group of oral streptococci (Banas, 2004;Mylonakis & Calderwood, 2001). Moreover, continuous detections of this bacterium in specimens from patients suffering from such disease emphasize the importance of S. mutans in IE (Gauduchon et al., 2001;Nomura et al., 2006;Vose et al., 1987;Ullman et al., 1988). Indeed, studies to clarify specific components involved in IE in S. mutans have also been performed worldwide (Beg et al., 2002;Jung et al., 2009; MatsumotoNakano et al., 2009;Miller-Torbert et al., 2008).IE is initiated by the binding of bacterial surface molecules to host extracellular matrix (ECM) proteins exposed on damaged heart tissue (Moreillon & Que, 2004). In S. mutans, collagen-binding adhesin or Cnm protein, encoded by the cnm gene, was reported as a strain-specific collagen-binding molecule (Sato et al., 2004). The Cnm protein possesses a collagen-binding domain (CBD) in ...