Comparative Pan-Genome Analysis of Oral Veillonella Species.

The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies.

Complete Genome Sequence of Veillonella nakazawae JCM 33966T (=CCUG 74597T), Isolated from the Oral Cavity of Japanese Children.

We report the complete genome sequence of Veillonella nakazawae JCM 33966T (=CCUG 74597T). This bacterium is a member of the oral Veillonella and has the potential to be anticariogenic as an oral probiotic seed.

Effects of chemical treatment as an adjunctive of air-abrasive debridement on restoring the surface chemical properties and cytocompatibility of experimentally contaminated titanium surfaces.

The aim of this study was to evaluate the effects of three different chemotherapeutic agents, following air-abrasive debridement, on surface chemical properties and cytocompatibility. Disks contaminated with Streptococcus gordonii biofilm were treated with air-abrasion and immersion in either 0.9% NaCl (Air + NaCl), 0.05% alkaline electrolyzed water (AEW) (Air + AEW), or 3% H2 O2 (Air + H2 O2 ). Noncontaminated and untreated titanium disks served as a control (As-polished). The efficacy of biofilm removal, magnitude of initial cytocompatibility toward human bone marrow mesenchymal stem cells, and surface chemical properties were determined. In all treatment groups, biofilms containing microorganisms were observed to be completely removed. The data showed discrepancies for cell affinities among treatment groups, whereby: (1) the number of cells attached to the Air + AEW treated surfaces was approximately two times greater than that to the Air + NaCl treated surfaces; and (2) cell spreading was significantly enhanced on the Air + AEW treated surfaces compared with the Air + NaCl or Air + H2 O2 treated surfaces. X-ray photoelectron spectroscopy data showed that the mean relative concentrations of nitrogen to titanium on the As-polished, Air + NaCl, Air + AEW, and Air + H2 O2 surfaces were 0.0079, 0.0237, 0.0071, and 0.0210, respectively, which would provide a clear understanding that these discrepancies could be attributed to sufficient removals of organic-nitrogen deposits at the same magnitude as the As-polished following the Air + AEW treatment. This study clarifies that chemical surface treatment with AEW, as an adjunctive to air-abrasive debridement may be beneficial in restoring surface properties for tissue integration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:183-191, 2020.

Draft Genome Sequences of Two Veillonella tobetsuensis Clinical Isolates from Intraoperative Bronchial Fluids of Elderly Patients with Pulmonary Carcinoma.

To date, Veillonella tobetsuensis has been known as an oral anaerobe and a facilitator of early-stage oral biofilm development with streptococci. Here, we report the draft genome sequences of 2 strains of V. tobetsuensis first isolated from intraoperative bronchial fluids of elderly patients with pulmonary carcinoma.

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children.

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children. Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains. Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V.infantiumConclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Establishment of a species-specific primer pair for detecting Veillonella infantium based on the 70 kDa heat shock protein gene dnaK.

Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.

Identification of an early stage biofilm inhibitor from Veillonella tobetsuensis.

Oral biofilm, the cause of dental caries and periodontal diseases, consists of multiple bacterial species. Streptococcus spp. and Veillonella spp. have been reported as to be initial and early colonizers of oral biofilms. Our previous studies showed that Veillonella tobetsuensis may play an important role on the development of S. gordonii biofilms without coaggregation involving extracellular biomolecules. In this study, the effect of a cyclic dipeptide autoinducer from culture supernatants from V. tobetsuensis at late-exponential growth phase on S. gordonii biofilm was examined. The cyclic dipeptide, identified as cyclo (-L-Leu-L-Pro) by gas chromatography/mass spectrometry, inhibited the development of S. gordonii biofilm. Furthermore, cyclo (-L-Leu-L-Pro) appeared not to cause bactericidal effects on planktonic cells of S. gordonii. This is the first report that oral Veillonella produces cyclo (-L-Leu-L-Pro) in their culture supernatants. Moreover, the results of this study suggest that cyclo (-L-Leu-L-Pro) may have an application to inhibit early stage development of oral biofilms.

Draft Genome Sequences of Four Strains of Recently Established Novel Veillonella Species Isolated from Human Oral Cavities.

Veillonella species are known to contribute to the formation of early oral biofilms and tend to be prevalent in people with poor oral hygiene status. Here, we report the draft genome sequences of 4 oral Veillonella strains that were established recently as novel species.

Veillonella infantium sp. nov., an anaerobic, Gram-stain-negative coccus isolated from tongue biofilm of a Thai child.

A strain of a novel anaerobic, Gram-stain-negative coccus was isolated from the tongue biofilm of a Thai child. This strain was shown, at the phenotypic level and based on 16S rRNA gene sequencing, to be a member of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.8, 95.1 and 95.9 % similarity to partial 16S rRNA, dnaK and rpoB gene sequences, respectively, to the type strains of the two most closely related species, Veillonelladispar ATCC 17748T and Veillonellatobetsuensis ATCC BAA-2400T. The novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK and rpoB gene sequencing and average nucleotide identity values. The major acid end-product produced by this strain was acetic acid under anaerobic conditions in trypticase-yeast extract-haemin with 1 % (w/v) glucose or fructose medium. Lactate was fermented to acetic acid and propionic acid. Based on these observations, this strain represents a novel species, for which the name Veillonella infantium sp. nov. is proposed. The type strain is T11011-4T (=JCM 31738T=TSD-88T).

Exploring the salivary microbiome of children stratified by the oral hygiene index.

Poor oral hygiene often leads to chronic diseases such as periodontitis and dental caries resulting in substantial economic costs and diminished quality of life in not only adults but also in children. In this study, the salivary microbiome was characterized in a group of children stratified by the Simplified Oral Hygiene Index (OHI-S). Illumina MiSeq high-throughput sequencing based on the 16S rRNA was utilized to analyze 90 salivary samples (24 Good, 31 Moderate and 35 Poor oral hygiene) from a cohort of Thai children. A total of 38,521 OTUs (Operational Taxonomic Units) with a 97% similarity were characterized in all of the salivary samples. Twenty taxonomic groups (Seventeen genera, two families and one class; Streptococcus, Veillonella, Gemellaceae, Prevotella, Rothia, Porphyromonas, Granulicatella, Actinomyces, TM-7-3, Leptotrichia, Haemophilus, Selenomonas, Neisseria, Megasphaera, Capnocytophaga, Oribacterium, Abiotrophia, Lachnospiraceae, Peptostreptococcus, and Atopobium) were found in all subjects and constituted 94.5-96.5% of the microbiome. Of these twenty genera, the proportion of Streptococcus decreased while Veillonella increased with poor oral hygiene status (P < 0.05). Furthermore, an unassigned species of Veillonella, Veillonella dispar and Veillonella parvula tended to be elevated in the Poor oral hygiene group. This is the first study demonstrating an important association between increase of Veillonella and poor oral hygiene status in children. However, further studies are required to identify the majority of Veillonella at species level in salivary microbiome of the Poor oral hygiene group.

Effects of chitosan fiber addition on the properties of polyurethane with thermo-responsive shape memory.

We investigated the effects of the addition of chitosan fiber (biomass nanofiber made by Sugino (BiNFi-s)) to polyether-based thermoplastic polyurethane (TPU) on material properties. BiNFi-s (2 and 5 wt %)/TPU composite materials were prepared via compression molding, and glass fiber (2 and 5 wt %)/TPU composite materials and plain TPU were also prepared for comparison. The glass transition temperature was analyzed using differential scanning calorimetry, and the crystal structure was investigated using X-ray diffraction. 20-mm-long test specimens with cross-sectional dimensions of 1 mm × 1 mm were cut from sheets of the composite materials, and three-point bending tests were carried out using a universal testing machine to investigate their mechanical properties and shape memory. The addition of BiNFi-s or glass fiber to TPU did not influence the glass transition temperature, although the crystal structure changed from semi-crystalline to amorphous. The elastic modulus increased 40% by the addition of 5 wt % BiNFi-s (2.31 MPa) compared with plain TPU (1.65 MPa), and these composites exhibited shape recovery with clinically relevant changes in temperature. The addition of 5 wt % BiNFi-s into TPU resulted in an improvement in the elastic modulus without any decrease in the shape memory effect. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1151-1156, 2017.

Monitoring the decontamination efficacy of the novel Poseidon-S disinfectant system in dental unit water lines.

BACKGROUND: Contaminated dental unit waterlines (DUWLs) are a known source of specific health care-acquired infections because of the difficulty in keeping them clean during routine dental practice. Recently, an electrolysis apparatus that uses only the chlorine normally present in municipal water, the Poseidon-S system, was developed as a novel additive-free disinfectant system to control microbial contamination in DUWLs. METHODS: The microbiological quality of water samples collected from DUWLs was assessed before and after installation of the Poseidon-S system in terms of the total viable counts (TVCs) of microorganisms. The microbicidal effects of the electrolyzed water against oral organisms and its cytotoxicity against human oral-derived cell lines were also examined. RESULTS: Water samples from the DUWLs initially had average microbial TVCs of 103-106 colony-forming units (CFU)/mL. After installation of the Poseidon-S system, the number of microorganisms in the water samples decreased to less than 1 × 102 CFU/mL. The electrolyzed water also exhibited remarkable microbicidal effects on the microorganisms present in the DUWLs as well as microorganisms commonly isolated from human oral cavities, but showed low cytotoxicity towards human oral-derived cells. CONCLUSION: This study demonstrated that routine use of the Poseidon-S system can effectively maintain low microbial levels in DUWLs.

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1ß responsible for the development of the disease. However, the mechanism of IL-1ß induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1ß and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1ß production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1ß. The inhibitors for ATP receptor reduced IL-1ß release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1ß through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity.

Identification of Veillonella Species in the Tongue Biofilm by Using a Novel One-Step Polymerase Chain Reaction Method.

Six Veillonella species have been frequently isolated from human oral cavities including infectious sites. Recently, it was reported that diet, smoking, and possibly socioeconomic status can influence the bacterial profile in oral cavities. In addition, oral hygiene habits may also influence oral microbiota in terms of both numbers and diversity of microorganisms. In this study, the identification of Veillonella species in tongue biofilms of Thai children, divided into three groups dependent on their status of oral hygiene. For this, we used a novel one-step PCR method with species-specific primer sets based on sequences of the rpoB gene. As shown in the results, the number of isolates of Veillonella species was 101 strains from only 10 of 89 subjects. However, the total number of bacteria was high for all subjects. Since it was reported in previous studies that Veillonella species were easy to isolate in human tongue biofilms at high numbers, the results obtained in this study may suggest country- or age-specific differences. Moreover, Veillonella species were detected predominantly in subjects who had poor oral hygiene compared to those with good or moderate oral hygiene. From these results, there is a possibility that Veillonella species may be an index of oral hygiene status. Furthermore, V. rogosae was a predominant species in tongue biofilms of Thai children, whereas V. parvula and V. denticariosi were not isolated at all. These characteristics of the distribution and frequency of Veillonella species are similar to those reported in previous studies. Although further studies are needed in other countries, in this study, a successful novel one-step PCR method was established to detect Veillonella species in human oral cavities easily and effectively. Furthermore, this is the first report investigating the distribution and frequency of Veillonella species in tongue biofilms of Thai children.

Effect of roselle calyx extract on in vitro viability and biofilm formation ability of oral pathogenic bacteria.

OBJECTIVE: To investigate the effect of the roselle calyx extract (RCE) (Hibiscus sabdariffa L.) on the in vitro viability and biofilm formation ability of oral pathogenic bacteria. METHODS: RCE was prepared by soaking roselle calyx powder with ethyl alcohol for 24 h at room temperature. After centrifugation, the extract was lyophilized. Then, the extract was dissolved in phosphate-buffered saline, the pH was adjusted, and the extract was aseptically filtered. We used Streptococcus mutans, Streptococcus sanguinis, Lactobacillus casei, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in this study. The antibacterial activity of the RCE was determined by treating the cells of these bacteria with the extract for 10 or 20 min at room temperature. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration was determined using the microdilution method, and the effect of the RCE on the ability to form biofilm was determined using a polystyrene micro plate assay. In addition, we used the WST-1 assay to determine the cytotoxicity of the RCE on HGF, Ca9-22 and KB cells. RESULTS: The RCE had antibacterial activity against oral bacteria used in this study. In particular, most significant antibacterial activity was observed against Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. The MIC and minimum bactericidal concentration were 7.2 mg/mL-28.8 mg/mL and 14.4 to >57.6 mg/mL. The RCE had an inhibitory effect on biofilm formation at the MIC and sub-MIC levels. In addition, the RCE had low cytotoxic effects on HGF, Ca9-22 and KB cells. CONCLUSIONS: Thus, our results indicate that the RCE may be used for preventing oral diseases.

Draft Genome Sequence of Veillonella tobetsuensis ATCC BAA-2400T Isolated from Human Tongue Biofilm.

Here, we report the draft genome sequence of Veillonella tobetsuensis ATCC-BAA 2400(T). This bacterium has the remarkable ability to form oral biofilms. The genome is predicted to encode the necessary enzymes involved in the pathway that facilitates the conversion of lactate to propionate.

The interaction between Streptococcus spp. and Veillonella tobetsuensis in the early stages of oral biofilm formation.

Dental plaque is a multispecies oral biofilm, the development of which is initiated by adherence of the pioneer Streptococcus spp. Oral Veillonella spp., including V. atypica, V. denticariosi, V. dispar, V. parvula, V. rogosae, and V. tobetsuensis, are known as early colonizers in oral biofilm formation. These species have been reported to co-aggregate with Streptococcus spp. in a metabolic cooperation-dependent manner to form biofilms in human oral cavities, especially in the early stages of biofilm formation. However, in our previous study, Streptococcus gordonii showed biofilm formation to the greatest extent in the presence of V. tobetsuensis, without co-aggregation between species. These results suggest that V. tobetsuensis produces signaling molecules that promote the proliferation of S. gordonii in biofilm formation. It is well known in many bacterial species that the quorum-sensing (QS) system regulates diverse functions such as biofilm formation. However, little is known about the QS system with autoinducers (AIs), between Veillonella and Streptococcus. Recently, AI-1 and AI-2 were detected and identified in the culture supernatants of V. tobetsuensis as strong signaling molecules in biofilm formation with S. gordonii. In particular, the supernatant from V. tobetsuensis showed the highest AI-2 activity among 6 oral Veillonella species, indicating that AIs, mainly AI-2, produced by V. tobetsuensis may be important factors and may facilitate biofilm formation of S. gordonii. Clarifying the mechanism that underlies the QS system between S. gordonii and V. tobetsuensis may lead to the development of novel methods for the prevention of oral infectious diseases caused by oral biofilms.

Inhibition of initial bacterial adhesion on titanium surfaces by lactoferrin coating.

Because dental implant abutments are located at transmucosal sites, their surface should inhibit bacterial accumulation to prevent peri-implantitis. The authors examined the effects of human lactoferrin (LF), an antibacterial protein present in saliva, as an antibacterial coating on the titanium surface and evaluated its effects before and after mucin-containing artificial saliva (AS) incubation. In the control group, titanium disks were soaked in distilled water, whereas in the LF group, titanium disks were soaked in LF solution to coat the disks. In the control-AS and LF-AS groups, half of the control and LF disks were incubated with AS. To confirm LF adsorption, the fluorescence intensity of fluorescein isothiocyanate-labeled LF was measured. The LF and LF-AS groups showed significantly higher intensity than the control and control-AS groups (P < 0.01). There was no significant difference between the LF and LF-AS groups (P > 0.05). The amount of adhered Streptococcus gordonii significantly increased by incubation with AS (P < 0.01) and significantly decreased by adsorption of LF (P < 0.01). There was no interaction between the two factors, LF adsorption and AS incubation (P = 0.561). These results suggest that the adsorbed LF inhibited bacterial adhesion following AS incubation. According to qualitative LIVE/DEAD analysis, viable bacteria appeared to be decreased in the presence of LF and SEM observation indicated that altered morphologies increased in LF and LF-AS groups. These results suggest that the adsorbed LF remained on the titanium surface after incubation with AS, and the remaining LF inhibited bacterial adhesion and exhibited bactericidal effects. Therefore, the adsorption of LF on the abutment material appears to be effective in preventing peri-implantitis.

The influence of oral Veillonella species on biofilms formed by Streptococcus species.

Oral Veillonella, Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, Veillonella rogosae, and Veillonella tobetsuensis are known as early colonizers in oral biofilm formation. To investigate the role of oral Veillonella, biofilms formed by the co-culture of Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, or Streptococcus sanguinis, with oral Veillonella were examined at the species level. The amount of biofilm formed by S. mutans, S. gordonii, and S. salivarius in the presence of the six Veillonella species was greater than that formed in the control experiments, with the exception of S. mutans with V. dispar. In contrast, in the case of biofilm formation by S. sanguinis, the presence of Veillonella species reduced the amount of the biofilm, with the exception of V. parvula and V. dispar. The time-dependent changes in the amount of biofilm and the number of planktonic cells were grouped into four patterns over the 24 combinations. Only that of S. gordonii with V. tobetsuensis showed a unique pattern. These results indicate that the mode of action of this combination differed from that of the other combinations with respect to biofilm formation. It is possible that there may be several factors involved in the interaction between Streptococcus and Veillonella species.

Lipopolysaccharide extracted from Porphyromonas gingivalis induces DNA hypermethylation of runt-related transcription factor 2 in human periodontal fibroblasts.

BACKGROUND/PURPOSE: Epigenetic alterations such as DNA methylation and histone acetylation are described as changes in the pattern of gene expression not involving the DNA sequence. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis has been shown to inhibit osteoblastic cell differentiation. We examined whether DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL). METHODS: The HPDL cells were incubated with LPS derived from P. gingivalis at a concentration of 10 µg/ml for 24 h. The cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza). Untreated cells were used as a control. Cell viability was determined using cell proliferation reagent. DNA methyltransferase (DNMT1) and runt-related transcription factor 2 (RUNX2) mRNAs were evaluated by quantitative polymerase chain reaction (RT-PCR). Analysis of RUNX2 DNA methylation was performed using quantitative methylation-specific PCR. RESULTS: The expression level of RUNX2 was significantly lower in the cells stimulated with LPS than the controls. The presence of 5Aza increased the expression of RUNX2 in cells stimulated with LPS. The expression levels of DNMT1 mRNA in the cells stimulated with LPS were significantly higher than in the control. The presence of 5Aza completely abolished the upregulated expression of DNMT1 in cells stimulated with LPS. The methylation of DNA at 0.1 kb and -1.9 kb in the cells stimulated with LPS was significantly higher than the control. CONCLUSION: The results indicate that DNA hypermethylation may be involved in the inhibitory effect of LPS on osteoblastic differentiation in HPDL.