Streptococcus mutans Clonal Variation Revealed by Multilocus Sequence Typing

Abstract

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 ...

Multilocus sequence typing analysis of Streptococcus mutans strains with the cnm gene encoding collagen-binding adhesin

“…We found that the distribution of the cnm gene in S. mutans from Thai subjects was highest in serotype f (54.5 %). Such distribution was also observed in S. mutans strains from Japanese and Finnish people (Nakano et al, 2007;Nomura et al, 2009). These results thus confirmed the association between serotype f and the presence of the cnm gene in S. mutans.…”

“…Equal numbers of S. mutans strains without the cnm gene in each serotype were also subjected to MLST as the control strains for the Thai subjects. Briefly, internal fragments of eight housekeeping gene loci were amplified (Nakano et al, 2007), and their nucleotide sequences determined (Table 1). The sequences for each housekeeping gene fragment were compared with the previously described data (Lapirattanakul et al, 2008;Nakano et al, 2007) in order to assign et al, 2001) were used to produce a matrix of pairwise differences in the allelic profiles and a dendrogram was constructed from the matrix using UPGMA.…”

“…Our previous studies have indicated the predominant frequency of this cnm gene in minor serotypes f and k, which usually showed unique characteristics concerning the pathogenesis of IE (Nakano et al, 2004a(Nakano et al, , 2007(Nakano et al, , 2008). An unambiguous typing method based on the nucleotide sequences of eight housekeeping genes, multilocus sequence typing (MLST), was developed for S. mutans, and showed a close evolutional relationship among cnmpositive strains (Nakano et al, 2007). MLST analysis of cnm-positive strains from various origins will provide more insight into S. mutans clones with collagen-binding ability due to the possession of the cnm gene.…”

“…Briefly, internal fragments of eight housekeeping gene loci were amplified (Nakano et al, 2007), and their nucleotide sequences determined (Table 1). The sequences for each housekeeping gene fragment were compared with the previously described data (Lapirattanakul et al, 2008;Nakano et al, 2007) in order to assign et al, 2001) were used to produce a matrix of pairwise differences in the allelic profiles and a dendrogram was constructed from the matrix using UPGMA. Then, related STs were grouped into lineages or clonal complexes using the eBURST (enhanced based upon related sequence types) program, version 3 (http://eburst.mlst.net) (Feil et al, 2004).…”

Getting to the heart of the matter: Role ofStreptococcus mutansadhesin Cnm in systemic disease

“…75% of isolates, typically lack the cnm locus. [21][22][23][24] Intriguingly, the less abundant serotypes are highly overrepresented among isolates associated with S. mutans extra-oral infections. 25,26 This disparity in distribution provided some of the first evidence that such collagen-binding adhesins may make a critical contribution to the capacity for S. mutans to cause systemic disease.…”