Multimodal inhibitory effect of matcha on Porphyromonas gingivalis.

Porphyromonas gingivalis has been associated with progression of periodontitis, characterized by inflammation and destruction of periodontal tissues. Here, we report that matcha, a product of Camellia sinensis, hampers the adherence and survival of P. gingivalis through multiple tactics. Matcha extract (ME) inhibited the growth not only of P. gingivalis but also of Prevotella nigrescens and Fusobacterium nucleatum, while it did not inhibit growth of nine species of oral streptococci and Aggregatibacter actinomycetemcomitans. ME-mediated P. gingivalis growth inhibition was characterized by both morphological and physiological changes at the bacterial envelope, which were accompanied by nano-particle formation and decreased membrane fluidity/permeability without loss of membrane integrity. ME also triggered autoaggregation of P. gingivalis in a major fimbriae (FimA)-dependent manner. In addition, adherence of P. gingivalis was dramatically inhibited by ME, irrespective of fimbriae. Furthermore, a structure-activity relationship study tested a series of catechins isolated from ME and identified the pyrogallol-type B-ring of catechins as essential for P. gingivalis growth inhibition. In a clinical study to assess the microbiological and therapeutic effects of matcha mouthwash in patients with periodontitis, the P. gingivalis number in saliva was significantly reduced by matcha mouthwash compared to the pre-intervention level. A tendency toward improvement in probing pocket depth was observed in the matcha group, although the difference was not statistically significant. Taken together, we present a proof of concept, based on the multimodal inhibitory effect of matcha against P. gingivalis, and that matcha may have clinical applicability for prevention and treatment of periodontitis. IMPORTANCE: Periodontitis, a multifactorial inflammatory disease of the oral cavity, results in alveolar bone destruction, and is a major cause of tooth loss of humans. In addition, emerging evidence has demonstrated associations between periodontitis and a wide range of other chronic inflammation-driven disorders, including diabetes mellitus, preterm birth, cardiovascular disease, aspiration pneumonia, rheumatoid arthritis, cognitive disorder, and cancer. In the present study, we report that matcha, a product of Camellia sinensis, hampers Porphyromonas gingivalis, a major periodontal pathobiont, in not only a series of in vitro experiments but also a pilot intervention clinical trial of patients with periodontitis, in which matcha mouthwash statistically significantly reduced the P. gingivalis number in saliva, as compared to the pre-intervention level. Taken together, we suggest that matcha may have clinical applicability for prevention and treatment of periodontitis.

Essential Oil from Psidium cattleianum Sabine (Myrtaceae) Fresh Leaves: Chemical Characterization and in vitro Antibacterial Activity Against Endodontic Pathogens

Abstract Endodontic infections result from oral pathogenic bacteria which reach and infect dental pulp, as well as surrounding tissues, through cracks, unrepaired caries and failed caries restorations. This study aims to determine the chemical composition of essential oil from Psidium cattleianum leaves (PC-EO) and to assess its antibacterial activity against endodontic bacteria. Antibacterial activity of PC-EO was evaluated in terms of its minimum inhibitory concentration (MIC) values by the broth microdilution method on 96-well microplates. Bacteria Porphyromonas gingivalis (MIC = 20 µg/mL), Prevotella nigrescens (MIC = 62.5 µg/mL), Fusobacterium nucleatum (MIC = 12.5 µg/mL), Actinomyces naeslundii (MIC = 50 µg/mL), Bacteroides fragilis (MIC = 12.5 µg/mL), Aggregatibacter actinomycetemcomitans (MIC = 6.25 µg/mL) and Peptostreptococcus anaerobius (MIC = 62.5 µg/mL) were evaluated and compared to chlorhexidine dihydrochloride (CDH), the positive control. PC-EO was obtained by hydrodistillation with the use of a Clevenger-type apparatus whereas its chemical composition was analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Viridiflorol (17.9%), β-caryophyllene (11.8%), 1,8-cineole (10.8%) and β-selinene (8.6%) were the major constituents found in PC-EO, which exhibited high antibacterial activity against all endodontic pathogens under investigation. Therefore, PC-EO, a promising source of bioactive compounds, may provide therapeutic solutions for the field of endodontics.

Multiple aPDT sessions on periodontitis in rats treated with chemotherapy: histomorphometrical, immunohistochemical, immunological and microbiological analyses.

BACKGROUND: The aim of this study was to evaluate the effect of multiple sessions of antimicrobial photodynamic therapy (aPDT) on the treatment of experimental periodontitis (EP) in rats treated with chemotherapy. METHODS: Chemotherapy using 5-fluorouracil (5-FU) consisted of intraperitoneal administration of 60 and 40 mg/kg of 5-FU. 120 rats were subjected to chemotherapy with 5-FU and divided into groups: PT (periodontal treatment); PT+1aPDT (PT and single aPDT session); PT+4aPDT(PT and 4 sessions of aPDT); 1aPDT (single aPDT session); 4aPDT(4 sessions of aPDT). EP was induced in the mandibular molars via ligature placement. The alveolar bone loss (ABL) area in the furcation region was analysed histometrically. Proliferating cell nuclear antigen (PCNA), tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and cleaved caspase-3 (CC3) were analysed by immunohistochemistry. Prostaglandin E2 was quantified using an ELISA, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assessed using a multiplex method. The prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Fusobacterium nucleatum was assessed using PCR. The data were statistically analysed (P < 0.05). RESULTS: The PT+4aPDT group showed lower ABL than the PT or 1aPDT groups on day 7. Rats treated with aPDT showed a higher number of PCNA-positive cells with reduced immunolabeling of RANKL. Significant reductions in Prevotella nigrescens were observed in the PT+4aPDT group and in Aggregatibacter actinomycetemcomitans for the 1aPDT and 4aPDT groups. CONCLUSION: Repeated sessions of aPDT as an adjunct or alternative therapy were effective at reducing ABL, regulating bone metabolism, and reducing Prevotella nigrescens and Aggregatibacter actinomycetemcomitans.

Copaifera reticulata oleoresin: Chemical characterization and antibacterial properties against oral pathogens.

Oral infections such as periodontitis and tooth decay are the most common diseases of humankind. Oleoresins from different copaifera species display antimicrobial and anti-inflammatory activities. Copaifera reticulata is the commonest tree of this genus and grows abundantly in several Brazilian states, such as Pará, Amazonas, and Ceará. The present study has evaluated the chemical composition and antimicrobial potential of the Copaifera reticulata oleoresin (CRO) against the causative agents of tooth decay and periodontitis and has assessed the CRO cytotoxic potential. Cutting edge analytical techniques (GC-MS and LC-MS) aided the chemical characterization of CRO. Antimicrobial assays included determination of the Minimum Inhibitory Concentration (MIC), determination of the Minimum Bactericidal Concentration (MBC), determination of the Minimum Inhibitory Concentration of Biofilm (MICB50), Time Kill Assay, and Checkerboard Dilution. Conduction of XTT assays on human lung fibroblasts (GM07492-A cells) helped to examine the CRO cytotoxic potential. Chromatographic analyses revealed that the major constituents of CRO were ß-bisabolene, trans-α-bergamotene, ß-selinene, α-selinene, and the terpene acids ent-agathic-15-methyl ester, ent-copalic acid, and ent-polyalthic acid. MIC and MBC results ranged from 6.25 to 200 µg/mL against the tested bacteria. The time-kill assay conducted with CRO at concentrations between 50 and 100 µg/mL showed bactericidal activity against Fusobacterium nucleatum (ATCC 25586) and Streptococcus mitis (ATCC 49456) after 4 h, Prevotella nigrescens (ATCC 33563) after 6 h, Porphyromonas gingivalis (ATCC 33277) and Lactobacillus casei (clinical isolate) after 12 h, and Streptococcus salivarius (ATCC 25975) and Streptococcus mutans (ATCC 25175) after 18 h. The fractional inhibitory concentration indexes (FICIs) revealed antagonistic interaction for Lactobacillus casei (clinical isolate), indifferent effect for Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586), Prevotella nigrescens (ATCC 33563), and Streptococcus salivarius (ATCC 25975), and additive effect for Streptococcus mutans (ATCC 25175) and Streptococcus mitis (ATCC 49456). Treatment of GM07492-A cells with CRO demonstrated that concentrations up to 39 µg/mL significantly reduced cell viability as compared to the negative control, being IC50 equal to 51.85 ± 5.4 µg/mL. These results indicated that CRO plays an important part in the search for novel sources of agents that can act against oral pathogens.

Efficacy of amoxicillin and metronidazole combination for the management of generalized aggressive periodontitis.

BACKGROUND: The aim of this study is to evaluate the effects of metronidazole-amoxicillin combination on clinical and microbiologic parameters in patients with generalized aggressive periodontitis. METHODS: Twenty-eight patients were randomly included. The test group (n = 12) received amoxicillin-metronidazole combination and scaling and root planing; the control group (n = 16) received scaling and root planing alone. In addition to the clinical examinations, subgingival plaque samples were analyzed for total cultivable bacteria and the presence of Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), Treponema denticola, Prevotella intermedia, Prevotella nigrescens, Prevotella pallens, and Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) using polymerase chain reaction. RESULTS: All clinical parameters improved significantly compared to baseline (P <0.05) in both groups. There was a statistically significant reduction of pockets and clinical attachment gain in the combined group compared to the control group (P <0.05). Total counts of bacteria also decreased significantly at 3 and 6 months in both groups (P <0.05). T. denticola and T. forsythia were the most prevalent bacteria throughout the study. T. denticola showed a continuous decrease over 6 months in the test group, whereas no change was seen in the control group beyond 3 months. P. gingivalis decreased significantly at 3 months (P <0.05), whereas T. forsythia was the only pathogen decreased below detection limits by the combination therapy with a significant difference compared to the control group (P <0.05). CONCLUSIONS: The results from this study suggest that combined amoxicillin and metronidazole use as an adjunct to scaling and root planing leads to better clinical healing compared to mechanical treatment alone. The polypharmaceutical approach used results in a significant and substantial decrease in T. forsythia and prevents its recolonization for 6 months, suggesting that T. forsythia may determine the long-term stability of periodontal treatment outcomes.

Antimicrobial effect of propolis and other substances against selected endodontic pathogens.

OBJECTIVE: The aim of this study was to determine the antimicrobial effect of ethanol extract of propolis (EEP) and intracanal medicaments calcium hydroxide, camphorated paramonochlorophenol, and formocresol by means of the macrodilution method using the reinforced clostridial medium (RCM) and brucella and brain heart infusion media. STUDY DESIGN: The antimicrobial agents were sequentially diluted and tested against anaerobic bacteria Prevotella nigrescens, Fusobacterium nucleatum, Actinomyces israelii, and Clostridium perfringens and against Enterococcus faecalis, with the 5 x 10(5) CFU/mL standardized inocula. The tubes were anaerobically incubated and the minimum inhibitory concentration was detected. Blood agar RCM subcultures were performed to provide minimum bactericidal concentration. The results were analyzed by analysis of variance test. RESULTS: All drugs were effective against all tested strains, without statistical differences. E. faecalis was the less susceptible strain, and RCM broth promoted faster bacterial growth, but there were no significant differences in these results. Ethanol did not influence the antimicrobial effect of EEP.

Effect of calcium hydroxide-treated Prevotella nigrescens on the gene expression of matrix metalloproteinase and its inhibitor in MG63 cells.

The purpose of this study was to monitor the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by an osteoblastic cell line MG63 stimulated with Prevotella nigrescens lipopolysaccharides (LPS), and to compare the level of secretion before and after the P. nigrescens LPS was treated with calcium hydroxide [Ca(OH)2]. The underlying hypothesis is that the balance between MMP and TIMP secretion is the key to an understanding of the host degradative pathways involved in the pathogenesis of bacterial derived pulpal and periapical diseases. Confluent monolayers of MG63 human osteosarcoma cells were exposed to varying concentrations of P. nigrescens or Escherichia coli LPS. Alternately, confluent cultures were exposed to 10 microg/ml of bacterial LPS pretreated with Ca(OH)2 (12.5 mg/ml) for 72 hours. At the end of the experimental period, total RNA was extracted and real-time quantitative polymerase chain reaction (PCR) was performed for MMP-1, TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that the expression of MMP-1 mRNA was low and invariant for the experimental period in the negative controls. However, exposure to P. nigrescens LPS increased expression after 48 hours. Expression of TIMP-1 mRNA was highly increased at 24 and 48 hours with lower concentrations of LPS in contrast to a suppression with a concentration of 10 microg/ml. Treatment of P. nigrescens LPS with Ca(OH)2 resulted in a down-regulation of MMP-1, whereas pretreated E. coli LPS demonstrated no stimulatory activity for MMP-1 gene expression. Both types of LPS when pretreated with Ca(OH)2 induced slightly up-regulated expression of TIMP-1.

[Bacterial spectra and antibiotics in odontogenic infections. Renaissance of the penicillins?]. / Keimspektren und Antibiotika bei odontogenen Infektionen. Renaissance der Penicilline?

BACKGROUND: The role played by odontogenic infection in dental, oral, and maxillofacial surgery is not to be underestimated even at the present time. An extensive, standardized, prospective study was performed with the intention of verifying the bacterial spectrum of odontogenic infections to evaluate antibiotic sensitivity. MATERIAL AND METHODS: Bacterial spectra and resistograms of 65 patients with an odontogenic infection were analyzed in a prospective study under standardized conditions for specimen collection and transport. RESULTS: A total of 226 bacterial strains were analyzed. The ratio between anaerobes and aerobes was approximately 2:1. The most frequent aerobes were members of the genera Streptococcus (46 isolates), Staphylococcus (10 isolates), and Neisseria (9 isolates), respectively. The anaerobic gram-positive spectrum was dominated by members of the genera Eubacterium (19 isolates), Peptostreptococcus (16 isolates), and Actinomyces (12 isolates). The most frequently isolated gram-negative anaerobes were Prevotella (46 isolates), and Fusobacterium (21 isolates). The overall resistance to antibiotics was very low: only 7.3% of all bacteria were resistant to penicillin G/V, and 8.8% showed resistance to ampicillin. The resistance rates to other beta-lactam antibiotics were 4.4% to piperacillin and 0.6% to imipenem, respectively. Penicillin G presented the highest antimicrobial activity among aerobes: only 4.5% of anaerobic strains were resistant of penicillin G. The other resistance rates of anaerobic bacteria to antibiotics were as follows: ampicillin 24%, doxycycline 34%, erythromycin 18%, and clindamycin 9.3%. Penicillin G was also highly antimicrobially active to anaerobes. The resistance rates were: penicillin G 8.1%, ampicillin 2.6%, doxycycline 9.2%, erythromycin 10.2%, and clindamycin 1.4%, respectively.

Effects of zinc and copper on adhesion and hemagglutination of Prevotella intermedia and Prevotella nigrescens.

This study investigated the mechanism of protein attachment to the surface of the putative periodontal pathogens Prevotella intermedia and Prevotella nigrescens in artificial gingival crevicular fluid, and ways to increase protein attachment to the bacterial cells. The effects of cations on protein attachment, bacterial adhesion, and hemagglutination were examined, and cation-binding components on both bacterial species were identified. The presence of cations, especially zinc, copper and cerium, increased attachment of human serum proteins to both bacterial species. In contrast, the presence of hydrophobic inhibitors or sugars had little effect. Protein attachment was reduced by heat treatment of the bacterial cells. Pretreatment of bacteria with human serum proteins inhibited adhesion of both species to buccal epithelial cells and hemagglutination. These effects were enhanced by the presence of zinc and copper during pretreatment. Using a chelating column, specific zinc- and copper-binding proteins were identified on the surfaces of both bacterial species.