This population-based study determined the salivary microbiota composition of 2,343 adult residents of Hisayama town, Japan, using 16S rRNA gene next-generation high-throughput sequencing. Of 550 identified species-level operational taxonomic units (OTUs), 72 were common, in ≥75% of all individuals, as well as in ≥75% of the individuals in the lowest quintile of phylogenetic diversity (PD). These "core" OTUs constituted 90.9 ± 6.1% of each microbiome. The relative abundance profiles of 22 of the core OTUs with mean relative abundances ≥1% were stratified into community type I and community type II by partitioning around medoids clustering. Multiple regression analysis revealed that a lower PD was associated with better conditions for oral health, including a lower plaque index, absence of decayed teeth, less gingival bleeding, shallower periodontal pockets and not smoking, and was also associated with tooth loss. By contrast, multiple Poisson regression analysis demonstrated that community type II, as characterized by a higher ratio of the nine dominant core OTUs, including Neisseria flavescens, was implicated in younger age, lower body mass index, fewer teeth with caries experience, and not smoking. Our large-scale data analyses reveal variation in the salivary microbiome among Japanese adults and oral health-related conditions associated with the salivary microbiome.The human oral cavity is colonized by numerous and diverse microorganisms as commensals. These bacteria constitute complex microbial communities on intraoral surfaces, and dental plaque microbiota that form on the teeth are the cause of two major oral diseases, dental caries and periodontitis. Mutans streptococci are the major etiologic agent of dental caries 1 and Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are prime suspects in periodontitis 2 . Furthermore, recent studies that have used open-ended molecular approaches and the 16S rRNA gene have implicated other commensal members with the etiology of each disease, such as lactobacilli for dental caries 3 and as many as 17 species, including Filifactor alosis for periodontitis 4 .Saliva is a biological fluid secreted from the salivary glands into the oral cavity and contains bacteria shed from adhering microbial communities on various intraoral surfaces, including tooth surfaces, gingival crevices, tongue dorsum, and buccal mucosa. Oral bacteria in a planktonic state (as in saliva) are not generally regarded as direct causal agents of the oral diseases. However, intraoral transmission of pathogenic bacteria is likely to be mediated by bacteria dispersed via saliva 5,6 .The salivary microbiome, which is comprised of indigenous bacteria that are specific to each person, exhibits long-term stability (on the scale of years) [7][8][9][10] . On the other hand, oral disorders alter the structure of the teeth and their surroundings. Along with the loss of teeth, tooth decay and its treatment alter the structure of the tooth surfaces on which bacteria are attached. Gingival crevi...

We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. These genes were located downstream from the rgpA through rgpF locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA through rgpF homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotypespecific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes. S. mutans was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one S. mutans isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of S. mutans was significantly higher than that in the group with a monoinfection by a single serotype.Streptococcus mutans strains are classified into three serotypes (c, e, and f), and the serologic specificity is defined by rhamnose-glucose polysaccharide (RGP) on the cell wall (6). We have characterized the genes involved in RGP synthesis in S. mutans Xc (serotype c) in the course of our previous studies. Four rml genes (rmlA through rmlD) are directly related to the synthesis of dTDP-L-rhamnose (12, 13), and the gluA gene encodes the enzyme producing UDP-D-glucose (18). The rgpG gene is implicated in the initiation of RGP synthesis by transfer of N-acetylglucosamine-1-phosphate to a lipid carrier (16). Furthermore, six other genes (rgpA through rgpF) required for RGP synthesis were identified in the region downstream from rmlD, and these genes are likely to be involved in the transport and assembly of RGP (11,19).The RGPs are composed of ␣1,2-and ␣1,3-linked rhamnan backbones with glucose side chains linked to alternate rhamnoses. Each serotype-specific polysaccharide has unique linkages of its glucose side chains (serotype c, ␣1,2-linkage; serotype e, ␤1,2-linkage; and serotype f, ␣1,3-linkage) (5, 10). Recently, we identified and characterized the genes required for glucose side chain formation of the serotype c-specific RGP (9). However, the loci responsible for the determination of the other serotypes have not yet been elucidated.In this study, we identified the loci involved in the glucose side chain formation of RGP in serotypes e and f of S. mutans and confirmed that these regions determine serotype specificities. Furthermore, we designed three pairs of primers from specific DNA sequences within each serotype determinan...

OBJECTIVES:To clarify the effect of tooth loss on development of all-cause dementia and its subtypes in an elderly Japanese population. DESIGN: Prospective cohort study. SETTING: The Hisayama Study, Japan. PARTICIPANTS: Community-dwelling Japanese adults without dementia aged 60 and older (N = 1,566) were followed for 5 years (2007)(2008)(2009)(2010)(2011)(2012). MEASUREMENTS: Participants were classified into four categories according to baseline number of remaining teeth (≥20, 10-19, 1-9, 0). The risk estimates of the effect of tooth loss on the development of all-cause dementia, Alzheimer's disease (AD), and vascular dementia (VaD) were computed using a Cox proportional hazards model. RESULTS: During follow-up, 180 (11.5%) subjects developed all-cause dementia; 127 (8.1%) had AD, and 42 (2.7%) had VaD. After adjusting for potential confounders, there was a tendency for the multivariableadjusted hazard ratio of all-cause dementia to increase with decrease in number of remaining teeth (P for trend = .04). The risk of all-cause dementia was 1.62 times as great in subjects with 10 to 19 teeth, 1.81 times as great in those with one to nine teeth, and 1.63 times as great in those with no teeth as in those with 20 teeth or more. An inverse association was observed between number of remaining teeth and risk of AD (P for trend = .08), but no such association was observed with risk of VaD (P for trend = .20).CONCLUSION: Tooth loss is associated with an increased risk of all-cause dementia and AD in the Japanese population. J Am Geriatr Soc 65:e95-e100, 2017.Key words: Alzheimer's disease; epidemiology; oral health; prospective cohort study; vascular dementia T he increase in the incidence of dementia is a substantial public health concern in aging societies. Approximately 46.8 million people worldwide have dementia, and the incidence is 9.9 million per year.1 The number of people living with dementia will nearly double every 20 years, but the causes of dementia, especially Alzheimer's disease (AD), are unclear, and there is a lack of treatments and health service settings for this disorder.2,3 Therefore, intensified research studies are needed to identify factors that have the potential to decrease the risk of dementia and thereby decrease the burden of this disease on health systems.A growing number of research studies have focused on the link between oral health and cognitive status. In particular, many research studies have assessed the association between number of teeth and cognitive function, [4][5][6][7][8][9][10][11][12][13][14][15] but the results of observational longitudinal studies on the effect of tooth loss on cognitive function are inconsistent. A recent systematic review of the literature suggested that tooth loss was associated with greater risk of cognitive impairment and dementia, 16 whereas another reported that tooth loss was not consistently associated with those outcomes. 17 The inconsistency might be due to methodological deficiencies in this field, such as the lack of representativeness of the ...

Streptococcus mutans is resistant to bacitracin, which is a peptide antibiotic produced by certain species of Bacillus. The purpose of this study was to clarify the bacitracin resistance mechanism of S. mutans. We cloned and sequenced two S. mutans loci that are involved in bacitracin resistance. The rgp locus, which is located downstream from rmlD, contains six rgp genes (rgpA to rgpF) that are involved in rhamnose-glucose polysaccharide (RGP) synthesis in S. mutans. The inactivation of RGP synthesis in S. mutans resulted in an approximately fivefold-higher sensitivity to bacitracin relative to that observed for the wild-type strain Xc. The second bacitracin resistance locus comprised four mbr genes (mbrA, mbrB, mbrC, and mbrD) and was located immediately downstream from gtfC, which encodes the water-insoluble glucan-synthesizing enzyme. Although the bacitracin sensitivities of mutants that had defects in flanking genes were similar to that of the parental strain Xc, mutants that were defective in mbrA, mbrB, mbrC, or mbrD were about 100 to 120 times more sensitive to bacitracin than strain Xc. In addition, a mutant that was defective in all of the mbrABCD genes and rgpA was more sensitive to bacitracin than either the RGP or Mbr mutants. We conclude that RGP synthesis is related to bacitracin resistance in S. mutans and that the mbr genes modulate resistance to bacitracin via an unknown mechanism that is independent of RGP synthesis.Cariogenic Streptococcus mutans is known to be resistant to bacitracin. This property is often exploited in the isolation of this bacterium from the highly heterogeneous oral microflora (6). Bacitracin is a cyclic polypeptide antibiotic that is produced by certain species of Bacillus. The primary mechanism of action of this antibiotic is thought to be the inhibition of peptidoglycan synthesis (28). During peptidoglycan synthesis, C 55 -isoprenyl phosphate (IP) serves as a lipid carrier (24). After the translocation of sugar-peptide units to the ends of the linear peptidoglycan strands, the C 55 -isoprenyl pyrophosphate (IPP) is detached and dephosphorylated to IP by a membrane-bound pyrophosphatase, thus recycling IP for subsequent peptidoglycan synthesis (24, 28). Bacitracin binds tightly to IPP and prevents pyrophosphatase from interacting with IPP, thus reducing the amount of IP that is available for carrying sugarpeptide units.In Escherichia coli, increased phosphorylation of IP, due to elevated intracellular levels of the lipid kinase encoded by bacA, appeared to confer resistance to bacitracin (1). Alternatively, E. coli mutants lacking membrane-derived oligosaccharides had reduced sensitivity to bacitracin because of reduced IP utilization (5). Pollock et al. (20) reported that certain gram-negative bacteria that synthesized exopolysaccharides acquired resistance to bacitracin by shutting down the synthesis of exopolysaccharides. On the other hand, Podlesek et al. (19) suggested that an ABC-type efflux system, which consisted of the BcrA, BcrB, and BcrC proteins, might b...

Supragingival plaque is permanently in contact with saliva. However, the extent to which the microbiota contributes to the salivary bacterial population remains unclear. We compared the compositional shift in the salivary bacterial population with that in supragingival plaque following periodontal therapy. Samples were collected from 19 patients with periodontitis before and after periodontal therapy (mean sample collection interval, 25.8±2.6 months), and their bacterial composition was investigated using barcoded pyrosequencing analysis of the 16S rRNA gene. Phylogenetic community analysis using the UniFrac distance metric revealed that the overall bacterial community composition of saliva is distinct from that of supragingival plaque, both pre- and post-therapy. Temporal variation following therapy in the salivary bacterial population was significantly smaller than in the plaque microbiota, and the post-therapy saliva sample was significantly more similar to that pre-therapy from the same individual than to those from other subjects. Following periodontal therapy, microbial richness and biodiversity were significantly decreased in the plaque microbiota, but not in the salivary bacterial population. The operational taxonomic units whose relative abundances changed significantly after therapy were not common to the two microbiotae. These results reveal the compositional stability of salivary bacterial populations against shifts in the supragingival microbiota, suggesting that the effect of the supragingival plaque microbiota on salivary bacterial population composition is limited.

Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.Polysaccharides are the major constituents of streptococcal cell walls and are useful for the serological classification and identification of streptococci. The group-specific polysaccharide antigens of Lancefield group A, C, and E streptococci (3, 26), the serotype-specific antigen of Streptococcus mutans (18,27), and the rhamnose-glucose polysaccharide (RGP) antigen of Streptococcus sobrinus (19) share a common structural relationship. The backbones of these polysaccharides are polymers of ␣1,2-and ␣1,3-linked rhamnose units. Although the rhamnan backbone has been identified in many streptococci, little is known about the mechanism of its synthesis. Rhamnan is also present in O polysaccharides of phytopathogenic bacteria (Xanthomonas, Pseudomonas, and Stenotrophomonas), Yersinia enterocolitica, and Pseudomonas aeruginosa, and these O polysaccharides are regarded as pathogenic factors (1,6,25,28,32,40). However, the only report dealing with the assembly of rhamnan is that describing the synthesis of the A band, Drhamnan polysaccharide, of P. aeruginosa ...

We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved ␤-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the ␤-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.

The oral microbiota of orally healthy subjects comprises considerable amounts of bacteria possessing the ability to produce ACH, an oral carcinogen. Consumption of sugar alcohols may regulate ACH production by oral microbes.