Kazuyoshi Yamane

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.

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.

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.

Biofilm‐like structures and pathogenicity of Escherichia hermannii YS‐11, a clinical isolate from a persistent apical periodontitis lesion

Escherichia hermannii, formerly classified as enteric group 11 of Escherichia coli, is considered to be nonpathogenic. In this report, we described some of the pathogenic properties of a viscous material-producing E. hermannii strain YS-11, which was clinically isolated from a persistent apical periodontitis lesion. YS-11 possessed cell surface-associated meshwork-like structures that are found in some biofilm-forming bacteria and its viscous materials contained mannose-rich exopolysaccharides. To further examine the biological effect of the extracellular viscous materials and the meshwork structures, we constructed a number of mutants using transposon mutagenesis. Strain 455, which has a transposon inserted into wzt, a gene that encodes an ATP-binding cassette transporter, lacked the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials. Complementation of the disrupted wzt in strain 455 with an intact wzt resulted in the restoration of these phenotypes. We also compared these strains in terms of their ability to induce abscess formation in mice as an indication of their pathogenicity. Strains with meshwork-like structures induced greater abscesses than those induced by strains that lacked such structures. These results suggest that the ability to produce mannose-rich exopolysaccharides and to form meshwork-like structures on E. hermannii might contribute to its pathogenicity.

Identification of the Genes Involved in the Biofilm-like Structures on Actinomyces oris K20, a Clinical Isolate from an Apical Lesion

INTRODUCTION Although the production of biofilm is thought to be crucial in the pathogenesis of abscess formations caused by oral resident microorganisms, the particular mechanisms are still unknown. The aim of this study was to identify gene(s) responsible for maintaining the cell surface-associated meshwork-like structures, which are found in some biofilm-producing bacteria, in a clinical isolate of Actinomyces oris K20. METHODS Random insertional mutagenesis by using transposon EZ-Tn5 was performed against the strain K20. Transposon insertion mutants were screened by scanning electron microscopy for the absence of cell surface-associated meshwork-like structures. The disrupted genes by the transposon insertion were determined by direct genome sequencing with the transposon-end primers. RESULTS Five mutants without the meshwork-like structures were identified from 175 mutants. Sequencing of flanking regions of transposon insertion revealed that 3 mutants had a gene encoded polysaccharide deacetylase, Spo0J containing ParB-like nuclease domain, and hypothetical protein, respectively. The other 2 mutants had an insertion in a noncoding region and an unidentified region, respectively. CONCLUSIONS Our findings indicated that these genes might be involved in the formation of meshwork-like structures on Actinomyces oris K20.

Pathogenicity of exopolysaccharide-producing Actinomyces oris isolated from an apical abscess lesion

Aim To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. Methodology The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. Results The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 108 cells mL−1, whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. Conclusions Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

Rabbit M1 and M2 macrophages can be induced by human recombinant GM-CSF and M-CSF.

Macrophages can change their phenotype in response to environmental cues. Polarized macrophages are broadly classified into two groups: classical activated M1 and alternative activated M2. Characterization of human macrophages has been widely studied, but polarized macrophages in rabbits have not been characterized. We characterized rabbit macrophages that were polarized using human recombinant GM‐CSF and M‐CSF. GM‐CSF‐treated macrophages had higher mRNA expression of proinflammatory cytokines (M1 phenotype) than did the M‐CSF‐treated counterpart. By contrast, high levels of TGF‐β and IL‐10 expression (M2 phenotype) were found in M‐CSF‐treated macrophages. The present study may be useful to understand roles of polarized macrophages in rabbit disease models.

Identification of disulphide stress–responsive extracytoplasmic function sigma factors in Rothia mucilaginosa

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.

Complete Genome Sequence of Prevotella intermedia Strain 17-2

ABSTRACT Prevotella intermedia, a Gram-negative black-pigmented anaerobic rod, is frequently isolated from not only periodontal pockets but also purulent infections. We report here the complete genome sequence of P. intermedia strain 17-2, which is a non-exopolysaccharide-producing variant obtained from exopolysaccharide (EPS)-producing P. intermedia strain 17 stock culture.