Chiho Mashimo

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 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.

Effect of ultraviolet treatment on bacterial attachment and osteogenic activity to alkali-treated titanium with nanonetwork structures

Purpose Alkali-treated titanium with nanonetwork structures (TNS) possesses good osteogenic activity; however, the resistance of this material to bacterial contamination remains inadequate. As such, TNS implants are prone to postoperative infection. In this work, we attempted to alter the biological properties of TNS by treatment with short-duration high-intensity ultraviolet (UV) irradiation. Methods TNS discs were treated with UV light (wavelength =254 nm, strength =100 mW/cm2) for 15 minutes using a UV-irradiation machine. We carried out a surface characterization and evaluated the discs for bacterial film formation, protein adsorption, and osteogenic features. Results The superhydrophilicity and surface hydrocarbon elimination exhibited by the treated material (UV-treated titanium with a nanonetwork structure [UV-TNS]) revealed that this treatment effectively changed the surface characteristics of TNS. Notably, UV-TNS also showed reduced colonization by Actinomyces oris during an initial attachment period and inhibition of biofilm formation for up to 6 hours. Moreover, compared to conventional TNS, UV-TNS showed superior osteogenic activity as indicated by increased levels of adhesion, proliferation, alkaline phosphatase activity, osteogenic factor production, and osteogenesis-related gene expression by rat bone marrow mesenchymal stem cells (rBMMSCs). This inverse relationship between bacterial attachment and cell adhesion could be due to the presence of electron–hole pairs induced by high-intensity UV treatment. Conclusion We suggest that simple UV treatment has great clinical potential for TNS implants, as it promotes the osseointegration of the TNS while reducing bacterial contamination, and can be conducted chair-side immediately prior to implantation.

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.

Isolation and identification methods of Rothia species in oral cavities

Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable bacteria number on BHI-Y agar in the oral cavities of all subjects, respectively. It was indicated that among oral Rothia species, R. mucilaginosa is most predominant in the oral cavity of humans. A novel selective medium, ORSM, was useful for isolating each oral Rothia species.

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.

Exopolysaccharide Productivity and Biofilm Phenotype on Oral Commensal Bacteria as Pathogenesis of Chronic Periodontitis

Abstract : Exopolysaccharide (EPS) productivities in many bacteria have been associated with pathogenicity in mammalian hosts as providing extracellular matrices to form biofilm (Costerton et al., 1995). Bacteria assuming biofilm-forming capacity have enormous advantages in establishing persistent infections (Costerton et al., 1999). Chronic periodontitis is caused by dental plaque known as a complex biofilm which consists of several hundred different species of bacteria (Chen, 2001; Socransky and Haffajee, 2002; Lovegrove, 2004). While sucrose-derived homopolysaccharides are well known substrates which mediate adhesion of bacteria to the tooth surface and co-aggregation interactions between species of oral bacteria in the dental plaque (Russell, 2009), recent studies suggest that each bacterium produces distinctive EPS components in a sucrose-independent manner and can form so called single species biofilm (Branda et al., 2005). In the oral cavity, several species of oral bacteria are known to produce their own EPS with this manner (Okuda et al., 1987; Dyer and Bolton, 1985; Kaplan et al., 2004; Yamane et al., 2005; Yamanaka et al., 2009; Yamanaka et al., 2010). In this chapter, we will describe a possibility that a single species biofilm in the oral cavity can cause persistent chronic periodontitis along with the importance of dental plaque formation and maturation with sucrose-derived polysaccharides.

Primer Design for the Identification of Ten Oral Actinomyces Species Using Multiplex PCR

Background: Actinomyces is one of the predominant genera in the oral cavity. Oral Actinomyces species play a central role in the initial stages of biofilm formation on teeth; however, limited information is currently available on the distribution of individual species in different sites or clinical conditions. Moreover, a suitable method has yet to be developed to identify oral Actinomyces species because of the phenotypic and genetic similarities between these microorganisms. Objective: Actinomyces naeslundii, A. odontolyticus, A. oris, A. georgiae, A. gerencseriae, A. graevenitzii, A. dentalis, A. johnsonii, A. israelii, and A. meyeri among the genus Actinomyces are regarded as normal human oral Actinomyces species. The purpose of the present study was to design primers to identify oral Actinomyces species using multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of the representative oral Actinomyces species. The 16S rDNA genes of the representative oral Actinomyces species were obtained from the DNA Data Bank of Japan, and a multiple sequence alignment analysis was performed with the CLUSTAL W program. Homology among the primers selected for oral Actinomyces species and their respective 16S rRNA sequences was confirmed by a BLAST search. Results: These primers were able to distinguish each oral Actinomyces species and did not display cross-reactivity with representative oral bacteria or other Actinomyces species. Moreover, we developed a multiplex PCR method with the ability to identify and differentiate oral Actinomyces species (i.e., A. naeslundii, A. johnsonii, A. oris, A. odontolyticus, A. israelii, A. georgiae, A. dentalis, A. graevenitzii, A. gerencseriae, and A. meyeri) using only two PCR tubes per sample. Conclusion: The present results indicate that our multiplex PCR method with these primers is useful for identifying the representative oral Actinomyces species. This method is easy because the use of Mighty Amp DNA Polymerase Ver.2 (Takara) means that DNA extraction may be avoided, and species identification using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of oral Actinomyces species and their involvement in oral infections to be fully clarified in future studies.

Complete Genome Sequence of Rothia aeria Type Strain JCM 11412, Isolated from Air in the Russian Space Laboratory Mir

ABSTRACT Here, we present the complete genome sequence ofRothia aeria type strain JCM 11412, isolated from air in the Russian space laboratory Mir. Recently, there has been an increasing number of reports on infections caused by R. aeria. The genomic information will enable researchers to identify the pathogenicity of this organism.