Gingivalis Growth Research Articles

Synergistic Effect of Metronidazole and Chlorhexidine against Porphyromonas gingivalis Growth: An In Vitro Study.

Aim: To evaluate the potential synergistic activity of metronidazole (MTZ) and chlorhexidine (CHX) against Porphyromonas. gingivalis (P. gingivalis) growth. Methods: Antimicrobial susceptibility tests of P. gingivalis to MTZ and CHX were performed on in vitro serial 2-fold dilutions of MTZ (from 1 mg/mL to 0.015 mg/mL) and CHX (from 1 mg/mL to 0.03 mg/mL) in thioglycollate medium broth in a 96-well plate. The turbidity of each sample was analyzed by absorbance spectrophotometry at 450 nm wavelengths by using an enzyme-linked immunosorbent assay (ELISA) reader. The MIC50 (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were assessed. To investigate the potential synergism between MTZ and CHX, bacterial cells were treated with MTZ or CHX, as described above, either alone or in combination. Results: The MIC50 of MTZ was 0.03 mg/mL while that of CHX ranged from 0.12 to 0.06 mg/mL. MTZ and CHX exerted a significant inhibitory effect on P. gingivalis growth in a dose-dependent manner. MTZ at a low and ineffective concentration of 0.015 mg/mL, associated with a suboptimal concentration of CHX (0.03 mg/mL), exhibited a significant synergistic inhibitory effect on bacterial growth (50% inhibition vs. control) (p < 0.001), and the effect was more remarkable with 0.06 mg/mL CHX (75% inhibition vs. control). Conclusions: CHX and MTZ showed a significant synergistic effect against P. gingivalis growth. A non-effective concentration of MTZ (0.015 mg/mL) combined with suboptimal concentrations of CHX (0.03 mg/mL and 0.06 mg/mL) were related to a 50% growth in the inhibition and 99.99% death of P. gingivalis, respectively. The applicability of the clinical use of these concentrations should be tested in randomized controlled trials.

Glutamate is a key nutrient for Porphyromonas gingivalis growth and survival during intracellular autophagic life under nutritionally limited conditions.

This study reveals that P. gingivalis heavily depends on preferential utilization of Glutamate (Glu) for autophagic vacuolar growth and survival in human GECs. Several novel observations are made to support this: (i) GdhA of P. gingivalis is highly enriched in these vacuoles, (ii) P. gingivalis induces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu in P. gingivalis wild-type strain infection which is opposite in a Δ gdhA strain, (iv) P. gingivalis uptakes 14 C-Glu and preferentially utilizes Glu-Glu dipeptide, (v) similarly, wild-type strain shows growth increase in a nutritionally reduced bacterial culture media, and (vi) finally, Glu-Glu supplementation increases bacterial cell-volume of P. gingivalis wild-type but not Δ gdhA strain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu for P. gingivalis growth-rate, biomass induction and survival in nutritionally limited host subcellular environments.

Silica nanoparticles containing nano-silver and chlorhexidine to suppress Porphyromonas gingivalis biofilm and modulate multispecies biofilms toward healthy tendency

ABSTRACT Objectives This research first investigated the effect of mesoporous silica nanoparticles (nMS) carrying chlorhexidine and silver (nMS-nAg-Chx) on periodontitis-related biofilms. This study aimed to investigate (1) the antibacterial activity on Porphyromonas gingivalis (P. gingivalis) biofilm; (2) the suppressing effect on virulence of P. gingivalis biofilm; (3) the regulating effect on periodontitis-related multispecies biofilm. Methods Silver nanoparticles (nAg) and chlorhexidine (Chx) were co-loaded into nMS to form nMS-nAg-Chx. Inhibitory zone test and minimum inhibitory concentration (MIC) against P. gingivalis were tested. Growth curves, crystal violet (CV) staining, live/dead staining and scanning electron microscopy (SEM) observation were performed. Biofilm virulence was assessed. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Quantitative Real Time-PCR (qPCR) were performed to validate the activity and composition changes of multispecies biofilm (P. gingivalis, Streptococcus gordonii and Streptococcus sanguinis). Results nMS-nAg-Chx inhibited P. gingivalis biofilm dose-dependently (p<0.05), with MIC of 18.75 µg/mL. There were fewer live bacteria, less biomass and less virulence in nMS-nAg-Chx groups (p<0.05). nMS-nAg-Chx inhibited and modified periodontitis-related biofilms. The proportion of pathogenic bacteria decreased from 16.08 to 1.07% and that of helpful bacteria increased from 82.65 to 94.31% in 25 μg/mL nMS-nAg-Chx group for 72 h. Conclusions nMS-nAg-Chx inhibited P. gingivalis growth, decreased biofilm virulence and modulated periodontitis-related multispecies biofilms toward healthy tendency. pH-sensitive nMS-nAg-Chx inhibit the pathogens and regulate oral microecology, showing great potential in periodontitis adjunctive therapy.

Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells.

Frankincense is produced by Boswellia trees, which can be found throughout the Middle East and parts of Africa and Asia. Boswellia serrata extract has been shown to have anti-cancer, anti-inflammatory, and antimicrobial effects. Periodontitis is an oral chronic inflammatory disease that affects nearly half of the US population. We investigated the antimicrobial effects of B. serrata extract on two oral pathogens associated with periodontitis. Using the minimum inhibitory concentration and crystal violet staining methods, we demonstrated that Porphyromonas gingivalis growth and biofilm formation were impaired by treatment with B. serrata extracts. However, the effects on Fusobacterium nucleatum growth and biofilm formation were not significant. Using quantification of colony-forming units and microscopy techniques, we also showed that concentrations of B. serrata that were not toxic for host cells decreased intracellular P. gingivalis infection in human gingival epithelial cells. Our results show antimicrobial activity of a natural product extracted from Boswellia trees (B. serrata) against periodontopathogens. Thus, B. serrata has the potential for preventing and/or treating periodontal diseases. Future studies will identify the molecular components of B. serrata extracts responsible for the beneficial effects.

Potential of 10% propolis-based toothpaste on the inhibition of biofilm-forming bacteria growth in vitro

Aim of the study: To investigate the potential of 10% propolis-based toothpaste on inhibiting biofilm-forming bacteria growth in vitro. Material and method: Organoleptic properties are evaluated, considering color, odor, and taste. Antibacterial tests use a disc diffusion method against Streptococcus mutans, Staphylococcus aureus and Porphyromonas gingivalis bacteria, while cytotoxicity is assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on fibroblast cells. Statistical analysis involves mean ± standard deviation. The data were then tested using a one-way analysis of variance and Kruskal-Wallis, followed by post-hoc test (p &amp;lt; 0.05). Results: The organoleptic evaluation of 10% propolis toothpaste reveals a visually clear appearance, consistent orange flavor, and aroma lasting 30 days. Based on the antibacterial results, a 10% level of propolis toothpaste sample inhibited the growth of Streptococcus mutans, Staphylococcus aureus and Porphyromonas gingivalis bacteria. The post-hoc test showed that toothpaste demonstrated significant inhibition on S. mutans and S. aureus compared to the negative control (p &amp;lt; 0.05). The toothpaste showed a larger inhibitory zone towards P. gingivalis compared to the adverse control; however, no significant differences were observed (p &amp;gt; 0.05). Cytotoxicity assessment on fibroblast cells shows a high percentage (85.31%) of viable cells. The findings highlight the 10% of propolis toothpaste’s potential and non-toxic as oral care product. Conclusions: 10% propolis toothpaste inhibits S. mutans, S. aureus, P. gingivalis growth, and not toxic on fibroblast.

Retraction Note: A Highly Porous Co-MOF for Cyanosilylation Reaction and Inhibition on P. Gingivalis Growth and rgp and kgp Expression for Periodontal Treatment

Retraction Note: A Highly Porous Co-MOF for Cyanosilylation Reaction and Inhibition on P. Gingivalis Growth and rgp and kgp Expression for Periodontal Treatment

Antibacterial activities of phytochemicals against Porphyromonas gingivalis with and without experimental fluoride varnish for periodontal disease prevention.

We aimed to evaluate the antibacterial activity of phytochemicals with or without an experimental fluoride varnish against Porphyromonas gingivalis. Five phytochemicals, chrysophanol (CHR), emodin (EMO), anthrarufin (ANT), bavachalcone (BCC), and isobavachromene (IBC), were tested using agar diffusion, minimal inhibition concentration (MIC), and minimum bacterial concentration (MBC) assays. We also assessed the cell viability and cytotoxicity of phytochemicals. All phytochemicals showed clear inhibition zones in the agar diffusion test. The inhibition zones of all phytochemical-containing fluoride varnishes were similar to or larger than that of the positive control, excluding that of 1 mM EMO. With or without the fluoride varnish, BCC exhibited the lowest MIC and MBC levels. Cell viability was high in the presence of all phytochemicals except 200 μM EMO. In conclusion, BCC was most effective as a phytochemical alone, while all phytochemical-containing fluoride varnishes inhibited P. gingivalis growth without cytotoxicity.

Phylloquinone is preferable over menadione as a growth factor for Porphyromonas gingivalis

ObjectivesPorphyromonas gingivalis is the etiological agent of chronic periodontitis. Menadione (vitamin K3) and phylloquinone (vitamin K1) are well-known growth factors for P. gingivalis, while menadione is widely used in growth experiments. Here we attempted to determine the differences in phylloquinone and menadione in P. gingivalis growth experiments, which have not been well studied to date. MethodsWe investigated the effects of menadione and phylloquinone on the growth of two W83 strains and seven ATCC 33277 strains of P. gingivalis. ResultsThe ATCC 33277 strains grew well with phylloquinone at 2.9 μM in a complex medium (nutrient medium) and at 29 μM in two minimal media. In contrast, the W83 strains grew well without menadione or phylloquinone in three different culture media. Menadione at 2.9 μM, the conventionally used concentration for culturing P. gingivalis, supported the growth of most ATCC 33277 strains but inhibited the growth of some W83 and ATCC 33277 strains. Furthermore, menadione at 14.5 μM frequently inhibited cell growth, while phylloquinone at 145 μM promoted cell growth. ConclusionsThese results indicate that menadione and phylloquinone act as growth factors for ATCC 33277 but that menadione also can inhibit P. gingivalis growth. Thus, we propose that phylloquinone be used instead of menadione in P. gingivalis growth experiments requiring vitamin K.

Efek potensiasi kombinasi sefadroksil dan ekstrak camellia sinensis terhadap pertumbuhan aggregatibacter actinomycetemcomitans Dan porphyromonas gingivalis

Background: Cefadroxil, betalactam antibiotic belonging to cephalosporin class, is a drug of choice for treating periodontal infection. Previous studies reported that addition of green tea (Camellia sinensis) extract to betalactam antibiotics has shown synergistic antibacterial effect. Objective: To examine the antibacterial effect of GTE combined with cefadroxil on A. actinomycetemcomitans and P. gingivalis growth. Methods: In vitro experimental with post-test-only control design, conducted at MiCORE Laboratory, Trisakti University Faculty of Dentistry. There were 8 treatments: combination of cefadroxil 8 µg/mL with GTE 3,125 mg/mL, 6,25 mg/mL, and 12,5 mg/mL, GTE 3,125 mg/mL, 6,25 mg/mL, and 12,5 mg/mL, cefadroxil 16 µg/mL, and NaOH 0,1 N. Results: In treatment of solely GTE and NaOH 0,1, no inhibition zones were found. Meanwhile, combination of cefadroxil 8 µg/mL with GTE 3,125 mg/mL, 6,25 mg/mL, and 12,5 mg/mL, and cefadroxil 16 µg/mL inhibited both bacteria’s growth. Treatment of cefadroxil 8 µg/mL combined with GTE 3,125 mg/mL resulted in the largest inhibition zone. One-way ANOVA test showed significant difference, then post hoc test showed that combination of cefadroxil 8 µg/mL with GTE 3,125 mg/mL and 6,25 mg/mL were not significantly different from the positive control. Conclusion: Potentiation effect occurred on combination of cefadroxil 8 µg/mL with GTE 3,125 mg/mL and 6,25 mg/mL against A. actinomycetemcomitans and P. gingivalis growth. Green tea extract could increase cefadroxil antibacterial effect up to twice larger.&#x0D;

In Vitro Antimicrobial and Antibiofilm Properties and Bioaccessibility after Oral Digestion of Chemically Characterized Extracts Obtained from Cistus × incanus L., Scutellaria lateriflora L., and Their Combination.

Periodontal diseases are oral inflammatory diseases ranging from gingivitis to chronic periodontitis. Porphyromonas gingivalis is one of the major pathogens responsible for severe and chronic periodontitis. Plant extracts with antimicrobial activity could be considered possible alternatives to chlorhexidine, an antiseptic substance used in oral hygiene thatcan cause bacteria resistance. Here, two commercial extracts obtained from Cistus × incanus L. and Scutellaria lateriflora L. were chemically characterized usingUltra-High-Performance Liquid Chromatography (UHPLC) coupled with a Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. The extracts were studied for their bioaccessibility after simulated in vitro oral digestion, their antimicrobial activity against P. gingivalis, their protective effects against cellular invasion by P. gingivalis, and their antibiofilm activity. The extracts were found to contain very complex mixtures of polyphenols, which were quite stable after in vitro simulated oral digestion and demonstrated mild, dose-dependent inhibitory activity against P. gingivalis growth. This activity increased with the combination of the two extracts. Moreover, the combination of the extracts induced a reduction in P. gingivalis HaCaT invasiveness, and the reduction in biofilm came to around 80%. In conclusion, a combination of C. incanus and S. lateriflora showed promising effects useful in the treatment of gingivitis.

High glucose levels contribute to vascular fibrosis via the activation of the endothelial-to-mesenchymal transition in periodontitis.

To determine the changes of Porphyromonas gingivalis (P.gingivalis) growth and metabolism and identify whether the vascular epithelium change could be induced in diabetic periodontitis. Maintaining favourable vascular function is a precondition for periodontal regeneration. In diabetic periodontitis, high glucose levels could enhance the metabolism of pathogens, and a complex condition involving inflammation and high glucose levels would disrupt homeostasis of the epithelium and promote fibrosis by endothelial-to-mesenchymal transition (EndMT). Porphyromonas gingivalis was cultured with glucose to judge its metabolic activity. Human umbilical vein endothelial cells (HUVECs) were treated with P. gingivalis-lipopolysaccharide (LPS) (10μg/ml) and/or high glucose concentrations (25 mM), and transforming growth factor (TGF)-β inhibitor was used to block EndMT. Inflammation level was assessed by flow cytometry. Multiple biological functions including EndMT, angiopoiesis, and cell migration were analysed. Additionally, gene expressions and protein levels were determined with qPCR and western blot, respectively. Finally, blood vessels were cultured ex vivo, and EndMT and fibrosis markers were detected by immunohistochemistry. Glucose could promote P. gingivalis growth and biofilm formation as well as the expression of virulence factor genes including FimA, RgpA, RgpB, and Kgp. P. gingivalis-LPS and glucose could increase intracellular reactive oxygen species (ROS) and promote fibrosis via EndMT in HUVECs, along with attenuating angiopoiesis and cell migration, which could be resumed by blocking EndMT with TGF-β inhibitor. Vascular fibrosis was observed after the addition of glucose via EndMT regulation. Glucose augmented the growth and metabolism of P. gingivalis and promoted fibrosis by the activation of EndMT, as well as the inhibition of angiopoiesis and cell migration.

The inhibitory effect of gourami fish scale (Osphronemus gouramy) extract-curcumin combination on Porphyromonas gingivalis

Background: Gourami fish scales, often considered as a waste, turned out to be an alternative source for antibacterial components. Its extract contains alkaloid, an antibacterial agent, while also contains hydrogen bonds which have the effect of increasing the interaction with other substances. Curcumin is one of the active ingredients of turmeric, a spice commonly used as an herbal medicine in Indonesia. Curcumin could suppress bacterial proliferation by inhibiting virulence agents. The combination of these substances was expected to help inhibit Porphyromonas gingivalis growth, one of the key pathogens in periodontitis patients. Objective: This study aims to determine the inhibitory effect of the gourami fish scale extract-curcumin combination on the growth of P. gingivalis bacteria. Method: This research is an in vitro experimental analytical laboratory research. The concentrations used are 100%; 50%; 25%; 12.5%; 6.25%; 3.125%; 1.56%; and 0.78%. The inhibition testing on P. gingivalis bacteria cultures was carried out by spectrophotometric methods and colony counts. Results: Gourami fish scale extract-curcumin combination with a concentration of 12.5%; 25%; 50%; and 100% killed colonies of P. gingivalis bacteria resulting in 0 colonies. A concentration of 6.25% inhibits bacterial growth by 92.9%. The statistical test result showed a significant difference (p &lt; 0.05). Conclusion: Gourami fish scale extract-curcumin combination has an inhibitory effect on the growth of P. gingivalis bacteria with MIC value at the concentration of 6.25% and MBC of 12.5%.

SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of Porphyromonas gingivalis

IntroductionSerum amyloid P component (SAP) regulates the innate immune system and microbial diseases. Periodontitis is an inflammatory oral disease developed by the host immune system's interaction with the dysbiotic oral microbiome, thereby SAP could play a role in periodontitis pathogenicity. ObjectivesTo investigate the role of SAP in oral microbiome modulation and peridontitis pathogenicity. MethodsIn this study, wildtype and SAP-knockout (KO) mice were used. Ligature-based periodontitis was developed in mice. Oral microbiome diversity was analyzed by 16 s rRNA sequencing. Macrophages and Porphyromonas gingivalis (P. gingivalis) co-culture system analyzed the effect of SAP in macrophage phagocytosis of P. gingivalis. ResultsThe level of SAP was upregulated in the periodontitis-affected periodontium of humans and mice but not in the liver and blood circulation. Periodontal macrophages were the key source of upregulated SAP in periodontitis. SAP-KO aggravated periodontal inflammation, periodontitis, and a higher number of M1-type inflammatory macrophage infiltration in the periodontium. The oral microbiome of SAP-KO periodontitis mice was altered with a higher abundance of Porphyromonas at the genus level. SAP-KO macrophages showed compromised phagocytosis of P. gingivalis in the co-culture system. Co-culture of SAP-KO macrophages and P. gingivalis induced the C5a expression and exogenous SAP treatment nullified this effect. Exogenous recombinant SAP treatment did not affect P. gingivalis growth and opsonization. PMX205, an antagonist of C5a, treatment robustly enhanced P. gingivalis phagocytosis by SAP-KO macrophages, indicating the involvement of the C5a-C5aR signaling in the compromised P. gingivalis phagocytosis by SAP-KO macrophages. ConclusionSAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of P. gingivalis. A higher abundance of P. gingivalis during SAP deficiency could promote M1 macrophage polarization and periodontitis. This finding suggests the possible protecting role of elevated levels of periodontal SAP against periodontitis progression.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3′,5′)-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe–microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Antiplanktonic and Antibiofilm Activity of Rheum palmatum against Streptococcus oralis and Porphyromonas gingivalis.

Periodontitis and peri-implantitis are inflammatory conditions with a high global prevalence. Oral pathogens such as Porphyromonas gingivalis play a crucial role in the development of dysbiotic biofilms associated with both diseases. The aim of our study was to identify plant-derived substances which mainly inhibit the growth of “disease promoting bacteria”, by comparing the effect of Rheum palmatum root extract against P. gingivalis and the commensal species Streptococcus oralis. Antiplanktonic activity was determined by measuring optical density and metabolic activity. Antibiofilm activity was quantified using metabolic activity assays and live/dead fluorescence staining combined with confocal laser scanning microscopy. At concentrations of 3.9 mg/L, R. palmatum root extract selectively inhibited planktonic growth of the oral pathogen P. gingivalis, while not inhibiting growth of S. oralis. Selective effects also occurred in mature biofilms, as P. gingivalis was significantly more stressed and inhibited than S. oralis. Our studies show that low concentrations of R. palmatum root extract specifically inhibit P. gingivalis growth, and offer a promising approach for the development of a potential topical agent to prevent alterations in the microbiome due to overgrowth of pathogenic P. gingivalis.

Glucose-sensitive delivery of tannic acid by a photo-crosslinked chitosan hydrogel film for antibacterial and anti-inflammatory therapy

A glucose-sensitive antibacterial and anti-inflammatory hydrogel film with controlled release of tannic acid (TA) was synthesized using chitosan (CS). Specifically, the photo-crosslinked CS hydrogel was first obtained and then immersed in TA solution to generate composite hydrogel film with enhanced mechanical properties. Subsequently, N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide based coupling chemistry was used to covalently crosslink glucose oxidase (GOx) to CS to obtain glucose sensitivity. The physicochemical properties, including chemical composition, enzyme-related characteristics, glucose responsiveness, and mechanical strength, were thoroughly investigated, followed by the cytotoxicity, antibacterial and anti-inflammatory tests. The results showed that the GOx immobilized on the film surface by covalent bonding gave better stability than those that were physically adsorbed. In addition, it could quickly and correspondingly modify its inner pore structure in response to the glucose stimulus and then control the loaded TA release. Meanwhile, the TA addition could enhance the film's mechanical properties. The composite hydrogel film demonstrated adequate biocompatibility and can inhibit NO, IL-6, and TNF-α production in stimulated macrophages, as well as Porphyromonas gingivalis growth, demonstrating effective antibacterial and anti-inflammatory activity.

Anti-biofilm activities of coumarin as quorum sensing inhibitor for Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is a keystone pathogen in periodontitis, a biofilm-mediated infection disease. This research aimed to investigate the effect of coumarin on P. gingivalis biofilm formation. We detected the antimicrobial effect on P. gingivalis planktonic growth, observed membrane structure and morphological change by TEM, and quantified membrane permeability by calcein-AM staining. The cell surface hydrophobicity, aggregation, and attachment were assessed. We also investigated different sub-MIC concentrations of coumarin on biofilm formation, and observed biofilm structureby confocal laser scanning microscopy. The biofilm-related gene expression was evaluated using real-time PCR. The results showed that coumarin inhibited P. gingivalis growth and damaged the cell morphology above 400 μM concentration. Coumarin did not affect cell surface hydrophobicity, aggregation, attachment, and the early stage of biofilm formation at sub-MIC concentrations. Still, it exhibited anti-biofilm effects for the late-stage and pre-formed biofilms dispersion. The biofilms after coumarin treatment became interspersed, and biofilm-related gene expression was downregulated. Coumarin also inhibited AI-2 activity and interacted with the HmuY protein by molecular docking analysis. Our research demonstrated that coumarin inhibited P. gingivalis biofilm formation through a quorum sensing system.

Expanded substrate specificity supported by P1′ and P2′ residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1′ and P2′ residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

Anti-Periodontitis Effect of Ethanol Extracts of Alpinia Katsumadai Seeds.

Oral microbes are intimately associated with many oral and systemic diseases. Ongoing research is seeking to elucidate drugs that prevent and treat microbial diseases. Various functions of Alpinia Katsumadai seed extracts have been reported such as their anti-viral, anti-oxidant, anti-inflammatory, anti-puritic, anti-emetic, and cytoprotective effects. Here, we investigated the anti-periodontitis effect of an ethanol extract of Alpinia Katsumadai seeds (EEAKSs) on dental plaque bacteria (DPB)-induced inflammation and bone resorption. DPB and Porphyromonas gingivalis (P. gingivalis) were cultured and lipopolysaccharide (LPS) was extracted. Prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX-2) levels were estimated using ELISA. Cytotoxicity was also verified. Proteases were screened using a protease antibody array method. Osteoclastic bone resorption was also investigated. EEAKSs suppressed P. gingivalis growth on agar plates. LPS prepared from dental plaque bacteria (DPB-LPS) and P. gingivalis (PG-LPS) significantly increased PGE2 and COX2 levels in immortalized gingival fibroblasts (IGFs), immortalized human oral keratinocytes (IHOKs), and RAW264.7 macrophage cells. However, DPB-LPS and PG-LPS-induced PGE2 and COX-2 increases were effectively abolished by EEAKS treatment at non-cytotoxic concentrations. In the protease antibody array, matrix metalloproteinase (MMP)-2, MMP-3, MMP-7, kallikrein 10, cathepsin D, and cathepsin V levels were increased by PG-LPS stimulation. However, increases in protease levels except for cathepsin D were suppressed by EEAKS treatment. In addition, RANKL-induced osteoclast differentiation was significantly inhibited by EEAKS treatment, leading to reductions in resorption pit formation. These results suggest that EEAKSs exerted a beneficial oral health effect to help prevent DPB-mediated periodontal disease.

Antimicrobial Mechanisms of Leucocyte- and Platelet Rich Fibrin Exudate Against Planktonic Porphyromonas gingivalis and Within Multi-Species Biofilm: A Pilot Study.

Leucocyte- and platelet rich fibrin (L-PRF) is an autologous biomaterial used in regenerative procedures. It has an antimicrobial activity against P. gingivalis although the mechanism is not fully understood. It was hypothesized that L-PRF exudate releases hydrogen peroxide and antimicrobial peptides that inhibit P. gingivalis growth. Agar plate and planktonic culture experiments showed that the antimicrobial effect of L-PRF exudate against P. gingivalis was supressed by peroxidase or pepsin exposure. In developing multi-species biofilms, the antimicrobial effect of L-PRF exudate was blocked only by peroxidase, increasing P. gingivalis growth with 1.3 log genome equivalents. However, no effect was shown on other bacteria. Pre-formed multi-species biofilm trials showed no antimicrobial effect of L-PRF exudate against P. gingivalis or other species. Our findings showed that L-PRF exudate may release peroxide and peptides, which may be responsible for its antimicrobial effect against P. gingivalis. In addition, L-PRF exudate had an antimicrobial effect against P. gingivalis in an in vitro developing multi-species biofilm.