Japanese subgingival microbiota in health vs disease and their roles in predicted functions associated with periodontitis.

The present study aimed to identify and compare the microbial signatures between periodontally healthy and periodontitis subjects using 454 sequences of 16S rRNA genes. Subgingival plaque samples were collected from ten periodontally healthy subjects and ten matched chronic periodontitis patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. The microbial composition differed between healthy subjects and periodontitis patients at all phylogenetic levels. Particularly, 16 species, including Lautropia mirabilis and Neisseria subflava predominated in healthy subjects, whereas nine species, including Porphyromonas gingivalis and Filifactor alocis predominated in periodontitis. UniFrac, a principal coordinate and network analysis, confirmed distinct community profiles in healthy subjects and periodontitis patients. Using predicted function profiling, pathways involved in phenylpropanoid, GPI-anchor biosynthesis, and metabolism of alanine, arginine, aspartate, butanoate, cyanoamino acid, fatty acid, glutamate, methane, proline, and vitamin B6 were significantly over-represented in periodontitis patients. These results highlight the oral microbiota alterations in microbial composition in periodontitis and suggest the genes and metabolic pathways associated with health and periodontitis. Our findings help to further elucidate microbial composition and interactions in health and periodontitis.

Influence of Porphyromonas gingivalis in gut microbiota of streptozotocin-induced diabetic mice.

OBJECTIVES: Increasing evidence suggests that periodontitis can exacerbate diabetes, and gut bacterial dysbiosis appears to be linked with the diabetic condition. The present study examined the effects of oral administration of the periodontopathic bacterium, Porphyromonas gingivalis, on the gut microbiota and systemic conditions in streptozotocin-induced diabetic mice. MATERIALS AND METHODS: Diabetes was induced by streptozotocin injection in C57BL/6J male mice (STZ). STZ and wild-type (WT) mice were orally administered P. gingivalis (STZPg, WTPg) or saline (STZco, WTco). Feces were collected, and the gut microbiome was examined by 16S rRNA gene sequencing. The expression of genes related to inflammation, epithelial tight junctions, and glucose/fatty acid metabolism in the ileum or liver were examined by quantitative PCR. RESULTS: The relative abundance of several genera, including Brevibacterium, Corynebacterium, and Facklamia, was significantly increased in STZco mice compared to WTco mice. The relative abundances of Staphylococcus and Turicibacter in the gut microbiome were altered by oral administration of P. gingivalis in STZ mice. STZPg mice showed higher concentrations of fasting blood glucose and inflammatory genes levels in the ileum, compared to STZco mice. CONCLUSIONS: Oral administration of P. gingivalis altered the gut microbiota and aggravated glycemic control in streptozotocin-induced diabetic mice.

Deep sequencing reveals specific bacterial signatures in the subgingival microbiota of healthy subjects.

OBJECTIVES: This study aimed to define the comprehensive bacterial flora of the healthy oral cavity by identifying and comparing bacterial species in different subgingival sites using 454 sequencing of 16S rRNA genes. MATERIALS AND METHODS: Subgingival plaque samples were taken from six target teeth (central incisor, first premolar, and first molar in both the maxilla and mandible) of 10 periodontally healthy patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. RESULTS: Bacterial composition in phylum level was similar for all sites within the same individual irrespective of tooth location. Unweighted UniFrac distance values of microbiome also showed that average distance was significantly larger between subjects than between tooth locations of the same subjects. CONCLUSIONS: The present results clarify the lack of effect of tooth location in the healthy subgingival microbiota. Results may suggest that any subgingival site can demonstrate similar subject-specific microbiota. CLINICAL RELEVANCE: This investigation offers a better understanding of the uniqueness of the oral microbiome. The present study will facilitate sampling in future subgingival microbiological studies.

Confounding effects of microbiome on the susceptibility of TNFSF15 to Crohn's disease in the Ryukyu Islands.

Crohn's disease (CD) involves chronic inflammation in the gastrointestinal tract due to dysregulation of the host immune response to the gut microbiome. Even though the host-microbiome interactions are likely contributors to the development of CD, a few studies have detected genetic variants that change bacterial compositions and increase CD risk. We focus on one of the well-replicated susceptible genes, tumor necrosis factor superfamily member 15 (TNFSF15), and apply statistical analyses for personal profiles of genotypes and salivary microbiota collected from CD cases and controls in the Ryukyu Islands, southernmost islands of the Japanese archipelago. Our association test confirmed the susceptibility of TNFSF15 in the Ryukyu Islands. We found that the recessive model was supported to fit the observed genotype frequency of risk alleles slightly better than the additive model, defining the genetic effect on CD if a pair of the chromosomes in an individual consists of all risk alleles. The combined analysis of haplotypes and salivary microbiome from a small set of samples showed a significant association of the genetic effect with the increase of Prevotella, which led to a significant increase of CD risk. However, the genetic effect on CD disappeared if the abundance of Prevotella was low, suggesting the genetic contribution to CD is conditionally independent given a fixed amount of Prevotella. Although our statistical power is limited due to the small sample size, these results support an idea that the genetic susceptibility of TNFSF15 to CD may be confounded, in part, by the increase of Prevotella.

Complete genome sequence of the serotype k Streptococcus mutans strain LJ23.

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease.

Porphyromonas gingivalis is a black-pigmented asaccharolytic anaerobe and a major causative agent of periodontitis. Here, we report the complete genome sequence of P. gingivalis strain TDC60, which was recently isolated from a severe periodontal lesion in a Japanese patient.

Dysbiosis in the Salivary Microbiome Associated with IgA Nephropathy-|A| |Japanese Cohort Study.

IgA nephropathy is one of the leading causes of chronic kidney disease in Japan. Since the origin and mechanisms by which IgA nephropathy develops currently remain unclear, a confirmed disease diagnosis is currently only possible by highly invasive renal biopsy. With the background of the salivary microbiome as a rich source of biomarkers for systemic diseases, we herein primarily aimed to investigate the salivary microbiome as a tool for the non-invasive diagnosis of IgA nephropathy. In a comparison of salivary microbiome profiles using 16S rRNA amplicon sequencing, significant differences were observed in microbial diversity and richness between IgA nephropathy patients and healthy controls. Furthermore, recent studies reported that patients with IgA nephropathy are more likely to develop inflammatory bowel diseases and that chronic inflammation of the tonsils triggered the recurrence of IgA nephropathy. Therefore, we compared the salivary microbiome of IgA nephropathy patients with chronic tonsillitis and ulcerative colitis patients. By combining the genera selected by the random forest algorithm, we were able to distinguish IgA nephropathy from healthy controls with an area under the curve (AUC) of 0.90, from the ulcerative colitis group with AUC of 0.88, and from the chronic tonsillitis group with AUC of 0.70. Additionally, the genus Neisseria was common among the selected genera that facilitated the separation of the IgA nephropathy group from healthy controls and the chronic tonsillitis group. The present results indicate the potential of the salivary microbiome as a biomarker for the non-invasive diagnosis of IgA nephropathy.

Ancient DNA analysis of food remains in human dental calculus from the Edo period, Japan.

Although there are many methods for reconstructing diets of the past, detailed taxon identification is still challenging, and most plants hardly remain at a site. In this study, we applied DNA metabarcoding to dental calculus of premodern Japan for the taxonomic identification of food items. DNA was extracted from 13 human dental calculi from the Unko-in site (18th-19th century) of the Edo period, Japan. Polymerase chain reaction (PCR) and sequencing were performed using a primer set specific to the genus Oryza because rice (Oryza sativa) was a staple food and this was the only member of this genus present in Japan at that time. DNA metabarcoding targeting plants, animals (meat and fish), and fungi were also carried out to investigate dietary diversity. We detected amplified products of the genus Oryza from more than half of the samples using PCR and Sanger sequencing. DNA metabarcoding enabled us to identify taxa of plants and fungi, although taxa of animals were not detected, except human. Most of the plant taxonomic groups (family/genus level) are present in Japan and include candidate species consumed as food at that time, as confirmed by historical literature. The other groups featured in the lifestyle of Edo people, such as for medicinal purposes and tobacco. The results indicate that plant DNA analysis from calculus provides information about food diversity and lifestyle habits from the past and can complement other analytical methods such as microparticle analysis and stable isotope analysis.

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content.

BACKGROUND: Streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood. RESULTS: We have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome. CONCLUSION: These observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.

Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis.

Hevea brasiliensis Müll. Arg. is a tree that produces natural rubber, an industrially vital isoprenoid polymer. Biosynthesis of natural rubber is known to take place biochemically by a mevalonate (MVA) pathway, but molecular biological characterization of related genes has been insufficient. From H. brasiliensis, we obtained full-length cDNA of genes encoding all of the enzymes that catalyze the six steps of the MVA pathway. Alignment analysis and phylogenetic analysis revealed that in H. brasiliensis there are three acetyl-CoA acetyltransferase genes, two HMG-CoA synthase (HMGS) genes, and four HMG-CoA reductase (HMGR) genes. Gene expression analysis by type of tissue indicated that MVA pathway genes were highly expressed in latex, as compared to other types of tissue and that HMGS and HMGR, which exist in multiple copies, have different expression patterns. Moreover, these MVA pathway genes in H. brasiliensis were found to complement MVA pathway deletion mutations in yeast.

Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

Dysbiosis of the salivary microbiota in pediatric-onset primary sclerosing cholangitis and its potential as a biomarker.

Primary sclerosing cholangitis (PSC) is a liver disease known for its frequent concurrence with inflammatory bowel disease. Dysbiosis of the gut microbiota in PSC was reported in several studies, but the microbiological features of the salivary microbiota in PSC have not been established. Here we compared the salivary microbial communities of 24 pediatric-onset PSC patients, 16 age-matched ulcerative colitis (UC) patients, and 24 healthy controls (HCs) by analyzing the bacterial 16S rRNA gene sequence data. The species-richness (α-diversity) showed no significant between-group differences, whereas the overall salivary microbiota structure (ß-diversity) showed significant differences among the three groups. Taxonomic assignment revealed that the PSC salivary microbiota were characterized by significant decreases in the abundance of Rothia and Haemophilus compared to the HC group, and significantly decreased Haemophilus and increased Oribacterium compared to the UC group. By combining the genera selected by the random forest algorithm in machine learning, followed by confirmation with 10-fold cross-validation, we were able to distinguish the PSC group from the HC group with the area under the curve (AUC) of 0.7423, and from the UC group with the AUC of 0.8756. Our results indicate the potential of salivary microbiota as biomarkers for a noninvasive diagnosis of PSC.

Circadian oscillations of microbial and functional composition in the human salivary microbiome.

The human microbiomes across the body evidently interact with various signals in response to biogeographical physiological conditions. To understand such interactions in detail, we investigated how the salivary microbiome in the oral cavity would be regulated by host-related signals. Here, we show that the microbial abundance and gene participating in keeping the human salivary microbiome exhibit global circadian rhythm. Analysis of the 16S rRNA sequences of salivary microbial samples of six healthy adults collected at 4-h intervals for three days revealed that the microbial genera accounting for 68.4-89.6% of the total abundance were observed to significantly oscillate with the periodicity of ∼24 h. These oscillation patterns showed high variations amongst individuals, and the extent of circadian variations in individuals was generally lower than that of interindividual variations. Of the microbial categories oscillated, those classified by aerobic/anaerobic growth and Gram staining, Firmicutes including Streptococcus and Gemella, and Bacteroidetes including Prevotella showed high association with the circadian oscillation. The circadian oscillation was completely abolished by incubating the saliva in vitro, suggesting that host's physiological changes mostly contributed to the microbial oscillation. Further metagenomic analysis showed that circadian oscillation enriched the functions of environmental responses such as various transporters and two-component regulatory systems in the evening, and those of metabolisms such as the biosynthesis of vitamins and fatty acids in the morning.

Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation.

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (TH1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.

Complete Genome Sequence of Bifidobacterium dentium Strain JCM 1195T, Isolated from Human Dental Caries.

Bifidobacterium dentium strain JCM 1195(T) was isolated from human dental caries. Here, we report the complete genome sequence of this organism.

Complete Genome Sequence of Scardovia inopinata JCM 12537T, Isolated from Human Dental Caries.

Scardovia inopinata JCM 12537(T) was isolated from human dental caries. Here, we report the complete genome sequence of this organism. This paper is the first report to demonstrate the fully sequenced and completely annotated genome of an S. inopinata strain.

Complete Genome Sequence of Parascardovia denticolens JCM 12538T, Isolated from Human Dental Caries.

Parascardovia denticolens JCM 12538(T) was isolated from human dental caries. Here, we report the complete genome sequence of this organism. This paper is the first report demonstrating the completely sequenced and assembled genome of P. denticolens.

Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers.

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohn's disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1ß levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

Determination of the genome sequence of Porphyromonas gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed extensive genome rearrangements in P. gingivalis.

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).