Hisanori Fukushima

Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis

Periapical pathosis cases were classified and the effect of bacteria on clinical symptoms was examined. A positive correlation between bacterial growth and clinical symptoms was found. Peptococcus magnus and Bacteroides species were commonly found in clinically acute cases, while oral streptococci and enteric bacteria were frequently isolated from clinically asymptomatic cases.

Microbial Colonization Drives Lymphocyte Accumulation and Differentiation in the Follicle-Associated Epithelium of Peyer’s Patches

Peyer’s patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4+CD86−), whereas mature DCs (CD86high) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.

Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis

Twenty-one teeth with clinically asymptomatic periapical pathosis (class 3) were extracted and the isolation, identification, and localization of bacteria in the root apex were examined. Mixtures involving several bacteria were isolated from more than 60% of the cases. Scanning electron microscopy revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our results indicate that the bacteria in class 3 cases may be derived from organisms which colonized before or during endodontic treatment, but not from anachoresis. The bacteria-positive cases of asymptomatic periapical pathosis have the potential to progress to symptomatic periapical pathosis.

Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17.

BackgroundPrevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays.ResultsStrain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (σE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.ConclusionThese results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.

Purification and chemical analysis of a bacteriocin from the oral bacterium Streptococcus mutans RM-10

Bacteriocin from supernatant of broth-grown cultures of Streptococcus mutans Rm-10 was purified by ammonium sulphate fractionation, precipitation at pH 3.1, and gel filtration on Sephadex G-200 and Sepharose 2B. The purified preparation was shown to be homogeneous by gel filtration and immunoelectrophoresis. A molecular weight of 973,000 was determined by equilibrium sedimentation with UV scanning. Electron microscopy of this purified preparation revealed a fibrous structure with a homogeneous morphology. The bacteriocin was essentially proteinaceous in nature, containing 90 μg phosphorus and 34 μg hexose per mg of protein, and was found to be rich in aspartic acid, glycine, alanine, valine, leucine and lysine.

Complete Genome Sequence of Rothia mucilaginosa DY-18: A Clinical Isolate with Dense Meshwork-Like Structures from a Persistent Apical Periodontitis Lesion

Rothia mucilaginosa is an opportunistic pathogen in the human oral cavity and pharynx. We found that R. mucilaginosa DY-18, a clinical isolate from a persistent apical periodontitis lesion, had biofilm-like structures. Similar structures were also observed on R. mucilaginosa ATCC25296. To further study these structures, we determined the complete genome sequence of DY-18 and found it a 2.26-Mb chromosome. Regarding stress responsive systems known to affect biofilm formation in many bacteria, DY-18 genome possessed only two sigma factor genes. One of these encoded an additional sigma factor whose promoter-binding activity may be regulated in response to environmental stimuli. Additionally, several genes assigned to two-component signal transduction systems were presented in this genome. To the best of our knowledge, this is the first complete genome of R. mucilaginosa species and our data raise the possibility that this organism regulates the biofilm phenotype through these stress responsive systems.

Identification and characterization of clinically isolated biofilm-forming gram-positive rods from teeth associated with persistent apical periodontitis.

We isolated spore-forming gram-positive aerobic rods from three patients with persistent periapical periodontitis. These cells possessed unique phenotypic characteristics by exhibiting dense meshwork-like structures on their cell surfaces that could be found in a number of biofilm-forming bacteria. We identified these strains as Bacillus subtilis by the API system and 16S ribosomal RNA gene (rRNA) sequencing. Treatment of the meshwork-like structures with protease K and staining with calcofluor for polysaccharides indicated that these structures were polysaccharides in nature and could be essential for biofilm formation by these isolates. Our findings suggest that B. subtilis could form biofilms in periapical periodontitis lesions, and this might contribute to the resistance to treatment resulting in the development of persistent periapical periodontitis observed in these patients. The particular mechanisms for B. subtilis biofilms to develop periapical periodontitis are still unknown. Further studies are needed to clarify the role of biofilms in persistent infections.

Characterization and mode of action of a purified bacteriocin from the oral bacterium Streptococcus mutans Rm-10

The purified bacteriocin was sensitive to proteolytic enzymes such as trypsin, alpha-chymotrypsin and pronase, but resistant to papain and pepsin. Lowering the pH of the bacteriocin caused precipitation, and the Abs280 of the supernatant reached a minimum at pH 3.6, suggesting that this is the isoelectric point. Such a pH value coincides with minimum bacteriocin activity. The effect of the bacteriocin was bactericidal rather than bacteriolytic and concentration dependent. Treatment of Streptococcus faecalis ODU with Rm-10 bacteriocin led rapidly to the cessation of biosynthesis of macromolecules, DNA, RNA and protein. Electron microscopy showed bacteriocin fibres attached in aggregated bundles to target cells; after ultra-sonication or detergent treatment they were individually attached to the cell surface and, at the same time, the apparent bacteriocin activity had doubled. Bacteriocin activity was not altered by filter-sterilized saliva components. Oral rinsing with Rm-10 bacteriocin reduced the number of viable salivary bacteria after 20 min, suggesting a possible therapeutic use for Rm-10 bacteriocin.

Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms

BackgroundEvidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built.MethodsThis study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis.ResultsEPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes.ConclusionsThese results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the hosts innate defence response.