Novel transcriptional regulator OxtR1 regulates potential ferrodoxin in response to oxygen stress in Treponema denticola.

OBJECTIVE: Treponema denticola has been strongly implicated in the pathogenesis of chronic periodontitis. Previously, we reported that the potential transcriptional regulator TDE_0259 (oxtR1) is upregulated in the bacteriocin ABC transporter gene-deficient mutant. OxtR1 may regulate genes to adapt to environmental conditions during colonization; however, the exact role of the gene in T. denticola has not been reported. Therefore, we investigated its function using an oxtR1-deficient mutant. METHODS: The growth rates of the wild-type and oxtR1 mutant were monitored under anaerobic conditions; their antibacterial agent susceptibility and gene expression were assessed using a liquid dilution assay and DNA microarray, respectively. An electrophoretic mobility shift assay was performed to investigate the binding of OxtR1 to promoter regions. RESULTS: The growth rate of the bacterium was accelerated by the inactivation of oxtR1, and the mutant exhibited an increased minimum inhibitory concentration against ofloxacin. We observed a relative increase in the expression of genes associated with potential ferrodoxin (TDE_0260), flavodoxin, ABC transporters, heat-shock proteins, DNA helicase, iron compounds, and lipoproteins in the mutant. OxtR1 expression increased upon oxygen exposure, and oxtR1 complementation suppressed the expression of potential ferrodoxin. Our findings also suggested that OxtR1 binds to a potential promoter region of the TDE_0259-260 operon. Moreover, the mutant showed a marginal yet significantly faster growth rate than the wild-type strain under H2O2 exposure. CONCLUSION: The oxygen-sensing regulator OxtR1 plays a role in regulating the expression of a potential ferrodoxin, which may contribute to the response of T. denticola to oxygen-induced stress.

Complete genome sequence of Fusobacterium vincentii strain TDC100 isolated from an apical periodontitis lesion.

This study reports the complete genome sequence of Fusobacterium vincentii strain TDC100. The complete circular chromosome of strain TDC100 was obtained and assembled using a combination of short- and long-read sequencing.

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Antibacterial activity of mulberry extracts and purified fractions against oral pathogenic bacteria.

OBJECTIVES: This study aimed to isolate antibacterial compounds active against periodontopathic bacteria from mulberry (Morus alba) leaves. METHODS: The acetone-soluble fraction of mulberry leaves was extracted from the oil layer by oil/water separation. The extract was purified using silica gel open-column chromatography. The minimum inhibitory concentration (MIC) of the crude extract or purified fractions against Porphyromonas gingivalis was measured at each step. RESULTS: The MIC of the crude extract against P. gingivalis was 62.5-125 µg/mL. The fractions showing activity against P. gingivalis were designated Cf K and Cf P. The MICs of Cf K against P. gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus mutans were 6.25 µg/mL, 25 µg/mL, 12.5 µg/mL, and 12.5 µg/mL, respectively. In contrast, the MICs of Cf P against P. gingivalis, F. nucleatum, P. intermedia, and S. mutans were 25.0 µg/mL, >50 µg/mL, 50 µg/mL, and 12.5-25.0 µg/mL, respectively. CONCLUSIONS: Mulberry leaves contain antibacterial components against periodontopathic bacteria such as P. gingivalis, F. nucleatum, and P. intermedia.

Porphyromonas gingivalis diffusible signaling molecules enhance Fusobacterium nucleatum biofilm formation via gene expression modulation.

Background: Periodontitis is caused by a dysbiotic shift in the dental plaque microbiome. Fusobacterium nucleatum is involved in the colonization of Porphyromonas gingivalis, which plays a key role in dysbiosis, via coaggregation and synergy with this microorganism. Aim: We investigated the effect of diffusible signaling molecules from P. gingivalis ATCC 33277 on F. nucleatum TDC 100 to elucidate the synergistic mechanisms involved in dysbiosis. Methods: The two species were cocultured separated with an 0.4-µm membrane in tryptic soy broth, and F. nucleatum gene expression profiles in coculture with P. gingivalis were compared with those in monoculture. Results: RNA sequencing revealed 139 genes differentially expressed between the coculture and monoculture. The expression of 52 genes was upregulated, including the coaggregation ligand-coding gene. Eighty-seven genes were downregulated. Gene Ontology analysis indicated enrichment for the glycogen synthesis pathway and a decrease in de novo synthesis of purine and pyrimidine. Conclusion: These results indicate that diffusible signaling molecules from P. gingivalis induce metabolic changes in F. nucleatum, including an increase in polysaccharide synthesis and reduction in de novo synthesis of purine and pyrimidine. The metabolic changes may accelerate biofilm formation by F. nucleatum with P. gingivalis. Further, the alterations may represent potential therapeutic targets for preventing dysbiosis.

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

The oral microbial composition and diversity affect the clinical course of palmoplantar pustulosis patients after dental focal infection treatment.

BACKGROUND: Palmoplantar pustulosis (PPP) is a chronic pustular dermatosis on the palms and soles. Dental focal infections are known as the major worsening factor for PPP. Recent our study of oral microbiome demonstrated dysbiosis in PPP patients. While almost half of the PPP patients improved after treatment of dental focal infections, a certain number of patients did not improve. OBJECTIVE: To investigate the oral microbial factors affecting the clinical course of PPP after treatment of dental focal infection. METHODS: The oral microbiota of healthy controls (n = 10), improved (n = 7) and not-improved (n = 6) patients were analyzed by sequencing of bacterial 16S ribosomal RNA gene. RESULTS: The UniFrac analysis suggested the differences of oral microbiota between improved and not-improved patients. The prevalence of the phylum Proteobacteria was lower in improved patients than in not-improved patients. When the alpha microbial diversity was assessed by Shannon index, Pielou's index and the average operational taxonomic units (OTUs), not-improved patients had a lower-diversity microbiota compared to improved patients. The degree of changes of oral microbiota after dental focal infection treatment was higher in improved patients than in not-improved patients. Six genera showed significant correlation with blood test data of PPP patients. CONCLUSION: Our findings suggested that oral microbial compositions and diversity could account for the distinct clinical course of PPP patients after treatment of dental focal infection. Oral microbiome analysis of PPP patients may provide a predictive factor for clinical responsiveness to dental focal infection treatment.

OxyR inactivation reduces the growth rate and oxidative stress defense in Capnocytophaga ochracea.

OBJECTIVE: The human oral cavity harbors several bacteria. Among them, Capnocytophaga ochracea, a facultative anaerobe, is responsible for the early phase of dental plaque formation. In this phase, the tooth surface or tissue is exposed to various oxidative stresses. For colonization in the dental plaque phase, a response by hydrogen peroxide (H2O2)-sensing transcriptional regulators, such as OxyR, may be necessary. However, to date, no study has elucidated the role of OxyR protein in C. ochracea. METHODS: Insertional mutagenesis was used to create an oxyR mutant, and gene expression was evaluated by reverse transcription-polymerase chain reaction and quantitative real-time reverse transcription-polymerase chain reaction. Bacterial growth curves were generated by turbidity measurement, and the sensitivity of the oxyR mutant to H2O2 was assessed using the disc diffusion assay. Finally, a two-compartment system was used to assess biofilm formation. RESULTS: The oxyR mutant grew slower than the wild-type under anaerobic conditions. The agar diffusion assay revealed that the oxyR mutant had increased sensitivity to H2O2. The transcript levels of oxidative stress defense genes, sod, ahpC, and trx, were lower in the oxyR mutant than in the wild-type strain. The turbidity of C. ochracea, simultaneously co-cultured with Streptococcus gordonii, was lower than that observed under conditions of homotypic growth. Moreover, the percentage decrease in growth of the oxyR mutant was significantly higher than that of the wild-type. CONCLUSIONS: These results show that OxyR in C. ochracea regulates adequate in vitro growth and escapes oxidative stress.

Crawling motility of Treponema denticola modulated by outer sheath protein.

Treponema denticola, a helically shaped motile microorganism, is a major pathogen of chronic periodontitis. Major surface protein (Msp) and dentilisin are virulence factors of T. denticola that are located on the outer sheath. The motility of T. denticola is deeply involved in colonization on and invasion into the host tissue. The outer sheath is located at the interface between the environment and T. denticola, and its components may also contribute to its motility via interaction with the materials outside the cells. The study aimed to clarify whether Msp or dentilisin contributes to the motility of T. denticola on solid surfaces, termed crawling, by investigating their effects using Msp-deficient and dentilisin-deficient T. denticola strains. Motility was analyzed by measuring the colony size in agar plates and velocity was analyzed using dark-field microscopy. The colony area of the mutant strains was smaller than that of the wild-type strain. The crawling velocity of the mutant strains was lower than that of the wild-type strain, with the lowest velocity observed in the dentilisin-deficient strain. Additionally, the ratio of the crawling distance by one revolution to the protoplasmic cylinder pitch (an indicator of the crawling efficiency) in the dentilisin mutant was significantly lower than that in the wild type strain and the Msp mutant. Together, these results indicate that dentilisin facilitates the crawling-dependent surface spreading of T. denticola.

Non-surgical treatment for periodontitis and peri-implantitis: longitudinal clinical and bacteriological findings-A case report with a 7-year follow-up evaluation.

The aim of this report is to show that periodontitis and peri-implantitis with horizontal bone resorption in a 68-year-old male patient were successfully treated by non-surgical treatment. Scaling with an ultrasonic device was performed for moderate periodontitis around the mandibular left first premolar and moderate peri-implantitis around the maxillary right molar implants. Root planing with a metal curette was performed for the periodontal site, and debridement with a plastic curette was performed for the peri-implant site. A month after treatment, probing depth decreased from 5 to 2 mm at the periodontal site and 8 to 3 mm at the peri-implant site. The investigation of bacterial composition by sequencing the 16S rRNA gene amplicons showed that the composition similarly changed at both sites, 5 years after treatment; the change reflected the typical recovery of periodontitis. The clinical condition was maintained for 7 years after treatment at both sites. This was a successful case of non-surgical treatment for peri-implantitis with horizontal bone resorption, promoting recovery of the microbiota from dysbiotic shift.

Taxonomic and Gene Category Analyses of Subgingival Plaques from a Group of Japanese Individuals with and without Periodontitis.

Periodontitis is an inflammation of tooth-supporting tissues, which is caused by bacteria in the subgingival plaque (biofilm) and the host immune response. Traditionally, subgingival pathogens have been investigated using methods such as culturing, DNA probes, or PCR. The development of next-generation sequencing made it possible to investigate the whole microbiome in the subgingival plaque. Previous studies have implicated dysbiosis of the subgingival microbiome in the etiology of periodontitis. However, details are still lacking. In this study, we conducted a metagenomic analysis of subgingival plaque samples from a group of Japanese individuals with and without periodontitis. In the taxonomic composition analysis, genus Bacteroides and Mycobacterium demonstrated significantly different compositions between healthy sites and sites with periodontal pockets. The results from the relative abundance of functional gene categories, carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism, replication and repair showed significant differences between healthy sites and sites with periodontal pockets. These results provide important insights into the shift in the taxonomic and functional gene category abundance caused by dysbiosis, which occurs during the progression of periodontal disease.

Systemic administration of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-Ig abrogates alveolar bone resorption in induced periodontitis through inhibition of osteoclast differentiation and activation: An experimental investigation.

BACKGROUND/OBJECTIVES: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule expressed on T cells. CTLA-4 also binds to the surfaces of monocytes and macrophages, precursors of osteoclasts. Research on rheumatoid arthritis demonstrated that CTLA-4 suppresses inflammation and bone resorption. However, its effects on alveolar bone have yet to be understood. The purpose of this study was to investigate the role and potential mechanism of CTLA-4 in bone resorption in periodontitis. MATERIALS AND METHODS: In vivo, the effects of systemic administration of CTLA-4 immunoglobulin fusion protein (CTLA-4-Ig) on alveolar bone resorption were investigated using a periodontitis mouse model. A total of 20 C57BL/6J mice were randomly assigned to two groups according to the administration modes. Periodontitis was induced by placing a ligature around the left maxillary second molar. The contralateral tooth was left un-ligated. In the CTLA-4-Ig (+) group, CTLA-4-Ig was administered by intraperitoneal injection at 1 and 3 days after ligature placement. Animals in the CTLA-4-Ig (-) group were given only phosphate-buffered saline each time. At 5 days after ligature placement, bone resorption was assessed by micro-computed tomography and histological examination, and the prevalence of osteoclast-like cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining. In vitro, the effects of CTLA-4-Ig on osteoclasts were evaluated. Viability of RAW 264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL) and CTLA-4-Ig was tested by WST-1 assay. Osteoclast-like cells were enumerated by TRAP staining, and osteoclast activity was evaluated by resorption pit assay. Gene expression levels of osteoclast differentiation markers (macrophage-colony stimulating factor receptor, carbonic anhydrase II, cathepsin K, and Trap) and protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, were assessed by quantitative real-time polymerase chain reaction. The effect of CTLA-4-Ig on the nuclear factor-κB (NF-κB) activation was assessed by enzyme-linked immunosorbent assay. RESULTS: In vivo, ligature-induced bone resorption and the numbers of osteoclast-like cells were significantly decreased by the administration of CTLA-4-Ig. In vitro, treatment with RANKL and CTLA-4-Ig had no significant effect on cell viability. CTLA-4-Ig significantly reduced the prevalence and activation of osteoclast-like cells and decreased the expressions of osteoclast differentiation markers, compared with the RANKL-treated control. CTLA-4-Ig significantly suppressed RANKL-induced phosphorylation of NF-κB p65 but increased PP2A expression. CONCLUSION: These results suggest that CTLA-4-Ig abrogates bone resorption in induced periodontitis, possibly via inhibition of osteoclast differentiation and activation. The regulation of the NF-κB pathway and PP2A expression may be one mechanism by which CTLA-4-Ig suppresses osteoclast behavior.

Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering.

BACKGROUND AND OBJECTIVE: Dysbiosis, a loss of balance in the microbiota, is a potential factor of peri-implantitis. However, compositional change of the peri-implant microbiota soon after implant uncovering is still unknown. In this study, bacterial composition in the peri-implant sulcus was examined to understand the establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering. METHODS: Microbiota samples were collected at weeks 1, 2, 4, and 6 after stage-two surgery. Bacterial DNA was isolated from the samples, and a 16S rRNA gene library was constructed. Sequence reads were obtained using a high-throughput sequencing platform and were taxonomically assigned at the phylum and genus levels. RESULTS: Alpha diversity indices, which did not include taxonomic information, were at similar levels throughout the four time points. At 1 and 2 weeks, the bacterial composition was similar among patients with the predominance of Firmicutes and Proteobacteria. However, the composition was diverse at 4 and 6 weeks and significantly dissimilar to the composition at 1 week. CONCLUSIONS: At 1 week, the peri-implant microbiota was already formed with alpha diversity as high as that at the later time points. However, the bacterial composition was not highly dissimilar among patients at 1 week. The composition changed over the passage of several weeks and was specific for each patient.

Role of Hyalin-like Protein in Gliding and Biofilm Formation by Capnocytophaga Ochracea.

Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.

Inhibitory Effect of Resveratrol on Candida albicans Biofilm Formation.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 µg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 µg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.

Characterization of the Treponema denticola Virulence Factor Dentilisin.

Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas gingivalis and Tannerella forsythia. It has many virulence factors, yet there are only a few reports detailing these factors. Among them, dentilisin is a well-documented surface protease. Dentilisin is reported to be involved in nutrient uptake, bacterial coaggregation, complement activation, evasion of the host immune system, inhibition of the hemostasis system, and cell invasion as a result of its action, in addition to its original proteolysis function. Therefore, characterization of dentilisin, and clarifying the relationship between T. denticola and the onset of periodontal disease will be important to better understanding this disease. In this chapter, we explain the methods for analysis of dentilisin activity and pathogenicity.

Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in Treponema denticola.

Objective Treponema denticola is involved in 'chronic' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Investigation of the antimicrobial activity of Bilberry (Vaccinium myrtillus L.) extract against periodontopathic bacteria.

OBJECTIVE: The purpose of this study was to isolate the active antibacterial compounds from Bilberry (Vaccinium myrtillus L.) against periodontopathic bacteria. METHODS: The acetone soluble fraction of Bilberry was extracted from the oil layer by oil/water separation. The extract was then purified by a silica gel open column chromatography. The minimum inhibitory concentration (MIC) of the total extract or purified fractions against bacteria was measured at each step. RESULTS: The MIC of the total extract against Porphyromonas gingivalis was 500 µg/mL. The fraction exhibiting antibacterial activity against P. gingivalis was called NU4-TDC, and its MICs against P. gingivalis, Fusobacterium nucleatum, and Prevotella intermedia were 26.0 ± 7.8 µg/mL, 59.0 ± 10.4 µg/mL, and 45.1 ± 16.5 µg/mL, respectively. The MIC against Streptococcus mutans was >62.5 µg/mL. CONCLUSION: Bilberry contains antibacterial components against periodontopathic bacteria, such as P. gingivalis, F. nucleatum, and P. intermedia.

Synergistic biofilm formation by Parvimonas micra and Fusobacterium nucleatum.

Parvimonas micra is frequently isolated from lesions of apical periodontitis and is a major disease-related pathogen. One of the main causes of apical periodontitis is extraradicular biofilm. In this study, we investigated polymicrobial biofilm formation by P. micra and species associated with apical periodontitis. The coaggregation activity of P. micra with partner strains was investigated by visual assays. Synergistic biofilm formation was evaluated by cocultures of P. micra and partner strains. Growth of planktonic cells was measured by evaluating the absorbance at OD660, and biofilm formation was examined by staining with crystal violet. The effects of soluble components on synergistic biofilm formation and planktonic cell growth were examined after coculture of P. micra and other strains separated with a 0.4-µm pore-size porous membrane. P. micra coaggregated with Fusobacterium nucleatum, Porphyromonas gingivalis, or Capnoctyophaga ochracea. P. micra showed no coaggregation with Staphylococcus aureus, S. epidermidis, or Prevotella intermedia. In mixed cultures, biofilm formation by P. micra and F. nucleatum was greater than that by P. micra and P. gingivalis or C. ochracea. In separated cocultures, planktonic cell growth of P. micra was enhanced by each of the three species. Biofilm formation by P. micra was enhanced by F. nucleatum or C. ochracea; however, no significant enhancement was observed with P. gingivalis. These data indicated that P. micra and F. nucleatum had synergistic effects in biofilm formation and that these effects may be important for colonization by these two species in apical periodontitis lesions.