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Actinomyces oris strain K20 was isolated from oral apical lesions. Here, we report the complete circular genome sequence of this strain, obtained by means of hybrid assembly using two next-generation sequencing datasets. The strain has a 3.1-Mb genome with 2,636 coding sequences.

Background: Oral microbiota has been linked to both health and diseases. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested. Results: Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed amplicon sequence variants (254 ± 53 in saliva and 175 ± 37 in tongue; P = 8.9e-7, Kruskal–Wallis test) and Shannon index (6.0 ± 0.4 in saliva and 5.4 ± 0.3 in tongue; P = 2.0e-7, Kruskal–Wallis test). Fusobacterium periodonticum, Saccharibacteria sp. 352, Streptococcus oralis subsp. dentisani, Prevotella melaninogenica, Granulicatella adiacens, Campylobacter concisus, and Haemophilus parainfluenzae were the core operational taxonomic units (OTUs) common to both sites. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differed significantly in terms of bacterial composition, they showed inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.

The bacterial composition of oral samples has traditionally been determined by PCR amplicon sequencing of 16S rRNA genes. Recent amplicon sequence variant (ASV)-based analyses of 16S rRNA genes differ from that based on operational taxonomic unit (OTU) clustering in the way it deals with sequences having potential errors. However, little information is available on its application in oral microbiome studies. Here, we conducted ASV-based analysis of oral microbiome samples using QIIME 2. We investigated the optimal parameters for sequence denoising, using DADA2, and found the trimming of the first 20 nucleotides from 5′-end of both paired reads avoided excessive sequence loss during chimera removal. Truncating reads at positions 240–245 allowed the removal of low-quality sequences while maintaining sufficient length to merge matching paired ends. Taxonomic assignment, using the naïve Bayes classifier trained with the V3-V4 region of reference 16S rRNA sequences in the extended human oral microbiome database (eHOMD), resulted in bacterial compositions similar to those of OTU-based analyses. Contrary to OTU-based clustering, ASV-based analysis showed taxonomic abundance at the genus or species level to not differ significantly in tongue microbiomes, regardless of brushing. QIIME 2 can, therefore, be a standard pipeline for ASV-based analysis of oral microbiomes.

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that inThermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out ofThermoplasma acidophilumandPyrobaculum calidifontiscells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist inT. acidophilumandT. kodakarensisbut not inP. calidifontisorSulfolobus solfataricus. In vitro reconstitution showed that, inT. acidophilum,the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in theT. acidophilumchromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on theP. calidifontischromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, includingT. acidophilum,P. calidifontis, andT. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

Active participation of students is paramount not only for their learning experiences but also for their academic performance. Therefore, various methods have been developed and proven to help students achieve active learning. However, several shortcomings in these methods have been indicated as increasing students’ sense of burden and discomfort, eventually preventing them from benefiting sufficiently. This study aimed to determine the efficiency of a low-load web-based review quiz built by the researchers on Google Forms to enhance students’ reviewing habits and active class participation. Participants in this study were 53 first-year dental hygiene students in a 10-class microbiology course. After each class, all students were given the web-based quiz to prepare for a paper-based review test, which assessed the learning of the content covered in the previous classes. We analyzed the correlations between frequency of participation in the web-based quiz and the average scores of the weekly review tests or the final examination scores. Consequently, voluntary participation in the web-based quiz positively correlated with both short-term and long-term students’ learning outcomes. Through this web-based quiz during the first year of the dental hygiene program, students can develop the “self-learning attitude” needed to pass the national examination.

Honghao Zhang,1,2 Satoshi Komasa,1 Chiho Mashimo,3 Tohru Sekino,4 Joji Okazaki1 1Department of Removable Prosthodontics and Occlusion, Osaka Dental University, Hirakata, Osaka, Japan; 2Department of Stomatology, Nanfang Hospital and College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China; 3Department of Bacteriology, Osaka Dental University, Hirakata, 4The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, Japan 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. Keywords: implant, nanonetwork, postoperative infection, UV treatment, superhydrophilicity, osteointegration

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.

The deterioration of human oral microbiota is known to not only cause oral diseases but also to affect systemic health. Various environmental factors are thought to influence the disruption and restoration of the oral ecosystem. In this study, we focused on the effect of nitric oxide (NO) produced by denitrification and NO synthase enzymes on dental plaque microbiota. Interdental plaques collected from 10 subjects were exposed to NO donor sodium nitroprusside (SNP) and then cultured in a specialized growth medium. Depending on the concentration of exposed SNP, a decrease in &alpha, diversity and a continuous change in &beta, diversity in the dental plaque community were shown by sequencing bacterial 16S rRNA genes. We also identified eight operational taxonomic units that were significantly altered by NO exposure. Among them, the exposure of NO donors to Fusobacterium nucleatum cells showed a decrease in survival rate consistent with the results of microbiota analysis. Meanwhile, in addition to NO tolerance, an increase in the tetrazolium salt-reducing activity of Campylobacter concisus cells was confirmed by exposure to SNP. This study provides an overview of how oral plaque microbiota shifts with exposure to NO and may contribute to the development of a method for adjusting the balance of the oral microbiome.

Here, we present the complete genome sequence of Rothia 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.

Here, we present the complete genome sequence of Actinomyces naeslundii strain ATCC 27039, isolated from an abdominal wound abscess. This strain is genetically transformable and will thus provide valuable information related to its crucial role in oral multispecies biofilm development.

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.

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.

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.

Here, we present the complete genome sequence of Rothia mucilaginosa NUM-Rm6536, a strain isolated from the tongue plaque of a healthy human adult. This strain is amenable to genetic manipulation by transformation and so provides a useful foundation for more detailed investigation of this species.

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.

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.

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.

S. Treptococcus constellatus, a member of the S. Treptococcus anginosus Group (SAG), and known as part of indigenous oral microbiota, has been described to cause abscesses in various regions of the body, despite this organism appears to be innocuous, in general at its habitat. In this communication, we report a biofilm-forming capacity of a facultative anaerobic gram-positive coccus isolated as a dominant bacterium in an odontogenic subperiosteal abscess lesiony. The clinical isolate designated as strain H39 formed dense meshwork structures around the cells that are typical for biofilm forming bacteria, and produced viscous materials in its spent culture media. The 16S rRNA gene sequence of strain H39 was 99% homologous to that of S. Treptococcus constellatus ATCC 27823, a type strain for S. Treptococcus constellatus. Phylogenetic analysis using data sets of recN, groEL, tuf and 16S rRNA genes showed a sister relationship between strain H39 and S. Treptococcus constellatus ATCC 27823. Dense meshwork-like structures found on strain H39 were observed on S. Treptococcus constellatus ATCC 27823, but not on S. intermedius ATCC 27335 and S. anginosus ATCC 33397. Biofilm assay using 96-wells polystyrene microtiter plates revealed that S. Treptococcus constellatus strains H39 and ATCC 27823 can form dense biofilm on abiotic materials consistently. S. intermedius ATCC 27335 was able to form biofilm on microtiter plates at a lesser extent to those of S. Treptococcus constellatus strains. S. anginosus ATCC 33397 did not form biofilm on an abitotic material. As conclusions, dense meshwork structures around the cells of S. Treptococcus constellatus, and the capacity of S. Treptococcus constellatus and S. intermedius to form biofilm on abiotic materials as observed in this study might be related to the pathogenicity of these two organism and the tropism of organisms in SAG. As recently suggested, phylogenetic analysis could be a powerful tool for differentiating and identifying clinical isolates belong to SAG.