In vitro evaluation of the antimicrobial properties of terpinen-4-ol on apical periodontitis-associated bacteria.

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms.

Actin alpha 2, smooth muscle, a transforming growth factor-ß1-induced factor, regulates collagen production in human periodontal ligament cells via Smad2/3 pathway.

Background/purpose: Actin alpha 2, smooth muscle (ACTA2) is an actin isoform that forms the cytoskeleton. Actin plays a crucial role in numerous cellular functions. ACTA2 is a marker of functional periodontal ligament (PDL) fibroblasts and is upregulated by transforming growth factor-ß1 (TGF-ß1); however, the underlying function of ACTA2 in PDL tissue is unknown. We aimed to examine the localization and potential function of ACTA2 in PDL tissues and cells. Materials and methods: RNA expression was determined using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Protein expression was determined using immunofluorescence staining and Western blot analysis. Soluble and insoluble collagen production was examined using the Sircol collagen assay and picrosirius red staining, respectively. Small interfering RNA (siRNA) was used for knockdown assay to examine the effect of ACTA2 in human PDL cells. Results: ACTA2 expression was observed in human primary PDL cells and PDL cell line (2-23 cells). TGF-ß1 upregulated ACTA2, collagen type Ⅰ alpha1 chain (COL1A1), periostin (POSTN), and fibrillin-Ⅰ(FBN1) expression and soluble and insoluble collagen production in 2-23 cells. However, ACTA2 depletion by siRNA strongly suppressed PDL-related gene expression and collagen production compared with those of TGF-ß1-stimulated control cells. Furthermore, ACTA2 knockdown significantly suppressed the phosphorylation of Smad2 and Smad3. Conclusion: ACTA2 plays a crucial role in PDL-related marker expression and collagen production via Smad2/3 phosphorylation. Our findings might contribute to the development of novel and effective periodontal therapies.

Microbial Community Structures and Methanogenic Functions in Wetland Peat Soils.

Methane metabolism in wetlands involves diverse groups of bacteria and archaea, which are responsible for the biological decomposition of organic matter under certain anoxic conditions. Recent advances in environmental omics revealed the phylogenetic diversity of novel microbial lineages, which have not been previously placed in the traditional tree of life. The present study aimed to verify the key players in methane production, either well-known archaeal members or recently identified lineages, in peat soils collected from wetland areas in Japan. Based on an ana-lysis of microbial communities using 16S rRNA gene sequencing and the mole-cular cloning of the functional gene, mcrA, a marker gene for methanogenesis, methanogenic archaea belonging to Methanomicrobiales, Methanosarcinales, Methanobacteriales, and Methanomassiliicoccales were detected in anoxic peat soils, suggesting the potential of CH4 production in this natural wetland. "Candidatus Bathyarchaeia", archaea with vast metabolic capabilities that is widespread in anoxic environments, was abundant in subsurface peat soils (up to 96% of the archaeal community) based on microbial gene quantification by qPCR. These results emphasize the importance of discovering archaea members outside of traditional methanogenic lineages that may have significant functions in the wetland biogeochemical cycle.

Author Correction: Biologically driven DOC release from peatlands during recovery from acidification.

The original version of this Article contained an error in the Acknowledgements, which incorrectly omitted from the end the following: 'In particular, we thank the staff of the Centre for Ecology and Hydrology (including A. Burden, N. Ostle and C. Evans) in relation to a NERC grant involving CF & TJ (NE/E011748/1; 2007-2010), which established the sites from which the UK samples were subsequently collected.' This has been corrected in both the PDF and HTML versions of the Article.

Biologically driven DOC release from peatlands during recovery from acidification.

Peatlands store 1/3 of global soil carbon, destabilisation of which contributes much to the recent increase in DOC (dissolved organic carbon) in freshwater ecosystems. One suggested mechanism for the enhanced decomposition of peat and the releases of DOC is recovery from acidification. However, no biological role in the process has yet been identified. Here we report extracellular enzyme activities and microbial composition in peatlands of Korea, the UK, Japan and Indonesia, and find higher pH to promote phenol oxidase activities, greater abundances in Actinobacteria and fungi, and enhanced pore-water DOC concentrations. Our pH manipulation experiments also showed that increase in pH enhanced phenol oxidase activity and DOC production with greater Actinobacterial and fungal abundances. Finally, knockout or addition of phenol oxidase dramatically changed DOC and phenolic production, indicating the central role of phenol oxidase in DOC mobilisation. Our findings provide evidence to support a previously unrecognized biological mechanism through which pH increases activate phenol oxidase, accelerating the release of DOC and phenolics.

Antibiotic effects against periodontal bacteria in organ cultured tissue.

Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony-forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

Innate immune-stimulatory activity of Porphyromonas gingivalis fimbriae is eliminated by phase separation using Triton X-114.

Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3µglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100µg/mL. Unfractionated fimbriae induced serum-dependent production of IL-1ß by MNC. Triton X-114-fractionated fimbriae (10µg/mL)-induced production of IL-1ß, IL-8 or TNF-α was much lower than that induced by unfractionated fimbriae or 10ng/mL P. gingivalis-LPS preparation. Triton X-114-fractionated fimbriae immobilized on polystyrene tubes induced adhesion-stimulated superoxide release by LPS-primed neutrophils in a ß2 integrin-dependent manner. P. gingivalis cells caused priming of neutrophils; however, Toll-like receptor (TLR) 4 antagonists did not affect this response. Thus, P. gingivalis fimbriae were ineffective in inducing innate immune response in leukocytes; however, they induced ß2 integrin-mediated response by neutrophils. Immune-stimulatory components of P. gingivalis might be recognized by receptors other than TLR4.

Anti-CD14 Antibody-treated Neutrophils Respond to LPS: Possible Involvement of CD14 Upregulated by Anti-CD14 Antibody Binding.

CD14 and Toll-like receptor 4/MD2 (TLR4/MD2) mediate the action of LPS on neutrophils. The anti-CD14 antibody and the TLR4/MD2-antagonist, synthetic lipid IVa (LA-14-PP), are known to inhibit the response of neutrophils to LPS. We studied the role of CD14 in LPS-induced priming of neutrophils for enhanced release of the superoxide anion. The anti-CD14 antibody at much higher concentrations than required to saturate CD14 was required to inhibit priming by LPS. The inhibitory effect of the anti-CD14 antibody was overcome by LPS. After washing, anti-CD14-treated neutrophils showed upregulated CD14 upon incubation at 37°C and responded to LPS with a delayed time-course. Thus, CD14-blocked neutrophils gained responsiveness to LPS through newly upregulated CD14. These results suggested that the unbound/free anti-CD14 antibody was essential to inhibit LPS-induced priming by blocking CD14 that were newly expressed during incubation at 37°C. LA-14-PP inhibited the response of neutrophils to LPS in an anti-CD14 antibody sensitive manner. When neutrophils were treated with LA-14-PP followed by treatment with the anti-CD14 antibody, CD14 was upregulated upon warming, but priming was blocked, suggesting that TLR4/MD2 was not newly expressed by warming in association with CD14 molecules. Thus, in addition to blocking CD14, the anti-CD14 antibody was found to induce the expression of new CD14.

Serotype-dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains.

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two-phase separation system using the detergent Triton X-114, crude LPS extracts of the study strains were separated into detergent-phase LPS (DP-LPS) and aqueous-phase LPS (AP-LPS). Repeated treatment of the aqueous phase with the two-phase separation system produced only a slight decrease in AP-LPS, suggesting that AP-LPS was resistant to the detergent and thus distinguishable from DP-LPS. The presence of divalent cations increased the yield of AP-LPS. AP-LPS expression patterns were serotype-dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP-LPS, suggesting involvement of the LPS structure in the generation of AP-LPS. The two-phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP-LPS likely represents stabilized LPS aggregates, whereas DP-LPS might be derived from non-stabilized aggregates. Furthermore, time-dependent expression of stabilized LPS aggregates was found to be serotype-dependent in A. actinomycetemcomitans.