Effects of a tart cherry (Prunus cerasus L.) phenolic extract on Porphyromonas gingivalis and its ability to impair the oral epithelial barrier.

Periodontal diseases, including gingivitis and periodontitis, are a global oral health problem. Porphyromonas gingivalis, a key pathogen involved in the onset of periodontitis, is able to colonize the subgingival epithelium and invade the underlying connective tissue due to the contribution of cysteine proteases known as gingipains. In this study, we investigated the effects of a phenolic extract prepared from tart cherry (Prunus cerasus L.) juice on the growth, adherence, and protease activity of P. gingivalis. We also assessed the protective effect of the tart cherry extract on the disruption of the oral epithelial barrier induced by P. gingivalis. The tart cherry extract that contains procyanidins and quercetin and its derivatives (rutinoside, glucoside) as the most important phenolic compounds attenuated P. gingivalis growth, reduced adherence to an experimental basement membrane matrix model, and decreased the protease activities of P. gingivalis. The tart cherry extract also exerted a protective effect on the integrity of the oral epithelial barrier in an in vitro model infected with P. gingivalis. More specifically, the extract prevented a decrease in transepithelial electrical resistance as well as the destruction of tight junction proteins (zonula occludens-1 and occludin). These results suggest that the tart cherry phenolic extract may be a promising natural product for the treatment of periodontitis through its ability to attenuate the virulence properties of P. gingivalis and curtail the ability of this pathogen to impair the oral epithelial barrier.

A screening system using minimal media identifies a flavin-competing inhibitor of Porphyromonas gingivalis growth.

Chronic periodontitis is caused by dysbiosis of human oral commensals and especially by increase in Porphyromonas gingivalis. Inhibitors of P. gingivalis growth are expected to serve as effective drugs for the periodontal therapy. In the present study, we isolated new growth inhibitors of P. gingivalis using minimal media for P. gingivalis. The minimal media included the previously reported Globulin-Albumin (GA) and the newly developed Lactalbumin-Ferric chloride (LF) and Globulin-Calcium chloride (GC); all supported growth of the wild-type strain of P. gingivalis but did not support the growth of a mutant defective for a type IX secretion system. GC contains CaCl2, indicating that P. gingivalis requires a calcium ion for growth. Using LF and GA, we screened about 100 000 compounds and identified 73 that strongly inhibited the growth of P. gingivalis. More than half of these candidates would not have been obtained if these minimal media had not been used in our screen. One of our candidate inhibitors was diphenyleneiodonium chloride (DPIC), which showed strong bactericidal activity against P. gingivalis. Excess amounts of flavin adenine dinucleotide or flavin mononucleotide suppressed the inhibitory activity of DPIC, suggesting that DPIC would be a novel potent growth inhibitor.

Effect of Photodynamic Therapy on Microorganisms Responsible for Dental Caries: A Systematic Review and Meta-Analysis.

The aim of this study was to perform a systematic review of the literature followed by a meta-analysis about the efficacy of photodynamic therapy (PDT) on the microorganisms responsible for dental caries. The research question and the keywords were constructed according to the PICO strategy. The article search was done in Embase, Lilacs, Scielo, Medline, Scopus, Cochrane Library, Web of Science, Science Direct, and Pubmed databases. Randomized clinical trials and in vitro studies were selected in the review. The study was conducted according the PRISMA guideline for systematic review. A total of 34 articles were included in the qualitative analysis and four articles were divided into two subgroups to perform the meta-analysis. Few studies have achieved an effective microbial reduction in microorganisms associated with the pathogenesis of dental caries. The results highlight that there is no consensus about the study protocols for PDT against cariogenic microorganisms, although the results showed the PDT could be a good alternative for the treatment of dental caries.

Effects of Lactobacillus salivarius WB21 combined with green tea catechins on dental caries, periodontitis, and oral malodor.

OBJECTIVE: To evaluate the combined use of Lactobacillus salivarius WB21 and (-)-epigallocatechin gallate (EGCg) for oral health maintenance. DESIGN: The effects of L. salivarius WB21 on growth of Streptococcus mutans, the insoluble glucan produced by S. mutans, and on growth of Porphyromonas gingivalis were evaluated in vitro. In addition, the susceptibility of five oral pathogenic bacteria and L. salivarius WB21 to EGCg, the inhibiting effect of EGCg on methyl mercaptan, and the effects of L. salivarius WB21 and EGCg in combination on growth of P. gingivalis were examined. RESULTS: Lactobacillus salivarius WB21 showed concentration-dependent inhibition of the growth of S. mutans. Addition of L. salivarius WB21 inhibited production of the insoluble glucan by S. mutans (p < 0.001). A filtrate of L. salivarius WB21 culture solution inhibited growth of P. gingivalis (p < 0.001 vs. control), and this effect was enhanced when it was used in combination with EGCg (p < 0.001 vs. the addition of L. salivarius WB21). In addition, EGCg directly inhibited methyl mercaptan in a concentration-dependent manner (p < 0.001). Concerning bacterial susceptibility to EGCg, growth of P. gingivalis, Prevotella intermedia, and Fusobacterium nucleatum was inhibited at 2.5 mg/mL of EGCg, while that of L. salivarius WB21 was inhibited at 25 mg/mL EGCg. CONCLUSIONS: Our results imply that L. salivarius WB21 may be useful for controlling dental caries, periodontitis, and oral malodor. In addition, the effects of L. salivarius WB21 on periodontitis and oral malodor may be synergistically enhanced by use in combination with EGCg.

Kaurenoic acid and its sodium salt derivative: antibacterial activity against Porphyromonas gingivalis and their mechanism of action.

AIM: To evaluate the antibacterial activity of 12 kaurane-type diterpenes against a panel of bacteria that cause endodontic infection. METHODS & MATERIALS: We conducted tests against bacteria in the planktonic or in the sessile mode, cytotoxic assays for the most promising compounds against human normal lung fibroblast cells, and Porphyromonas gingivalis (ATCC 33277) proteomic analysis. RESULTS & CONCLUSION: Kaurenoic acid and its salt exhibited satisfactory antibacterial action against the evaluated bacteria. Proteomic analysis suggested that these compounds might interfere in bacterial metabolism and virulence factor expression. Kaurane-type diterpenes are an important class of natural products and should be considered in the search for new irrigating solutions to treat endodontic infections.

Litsea japonica Leaf Extract Suppresses Proinflammatory Cytokine Production in Periodontal Ligament Fibroblasts Stimulated with Oral Pathogenic Bacteria or Interleukin-1ß.

Periodontal disease, a chronic disease caused by bacterial infection, eventually progresses to severe inflammation and bone loss. Regulating excessive inflammation of inflamed periodontal tissues is critical in treating periodontal diseases. The periodontal ligament (PDL) is primarily a connective tissue attachment between the root and alveolar bone. PDL fibroblasts (PDLFs) produce pro-inflammatory cytokines in response to bacterial infection, which could further adversely affect the tissue and cause bone loss. In this study, we determined the ability of Litsea japonica leaf extract (LJLE) to inhibit pro-inflammatory cytokine production in PDLFs in response to various stimulants. First, we found that LJLE treatment reduced lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (interleukin-6 and interleukin-8) mRNA and protein expression in PDLFs without cytotoxicity. Next, we observed the anti-inflammatory effect of LJLE in PDLFs after infection with various oral bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. These anti-inflammatory effects of LJLE were dose-dependent, and the extract was effective following both pretreatment and posttreatment. Moreover, we found that LJLE suppressed the effect of interleukin-1 beta-induced pro-inflammatory cytokine production in PDLFs. Taken together, these results indicate that LJLE has anti-inflammatory activity that could be exploited to prevent and treat human periodontitis by controlling inflammation.

New Application of Psoralen and Angelicin on Periodontitis With Anti-bacterial, Anti-inflammatory, and Osteogenesis Effects.

Psoralen and angelicin are two effective compounds isolated from psoraleae, a traditional Chinese medicine. They have a wide range of applications for bone disease treatment and immune modulation. In this study, we explored their new applications for the treatment of periodontal diseases. This study aimed to investigate the effects of psoralen and angelicin on Porphyromonas gingivalis growth and P. gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation, and further to evaluate their effects on osteogenesis. Finally, the effects of angelicin on a mouse model of periodontitis were also investigated. The results showed that psoralen and angelicin had beneficial dose-dependent effects regarding the inhibition of planktonic P. gingivalis and biofilms of P. gingivalis. There were no significant differences in the viability of monocyte-like THP-1 cells and human periodontal ligament cells (hPDLCs) treated with either psoralen or angelicin compared to the untreated control cells. Psoralen and angelicin also markedly decreased the mRNA expression and release of inflammatory cytokines (interleukin [IL]-1ß and IL-8) by THP-1 cells in a dose-dependent manner. They significantly enhanced the alkaline phosphatase (ALP) activity of hPDLCs and up-regulated the expression of osteogenic proteins (runt-related transcription factor 2 [RUNX2], distal-less homeobox 5 [DLX5], and osteopontin [OPN]). Angelicin significantly attenuated alveolar bone loss and inflammation response in the mice with periodontitis. In conclusion, our data demonstrated that psoralen and angelicin could inhibit the growth of planktonic P. gingivalis and P. gingivalis biofilm. It is also the first report on the anti-inflammatory effect of psoralen and angelicin against Pg-LPS. They also had an osteogenesis-potentiating effect on hPDLCs. The in vivo study also indicated the effect of angelicin regarding protection against periodontitis. Our study highlighted the potential ability of psoralen and angelicin to act as novel natural agents to prevent and treat periodontitis.

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis.

The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.

The Efficacy of a Chewing Gum Containing Phyllanthus emblica Fruit Extract in Improving Oral Health.

Phyllanthus emblica: (PE) fruit extract has pharmacological activity and exert anti-bacterial, anti-oxidative, anti-inflammatory and anti-cancer effects, but few study exist for evaluating its improved effects on the imbalance of oral ecology, which may contribute to series of oral diseases. In this study, an examiner-blinded, randomized, and gum-base-controlled crossover manner was conducted to evaluate the efficacy of a sugar-free chewing gum containing PE fruit extract in changing the oral microbiome. Twenty healthy young adults were randomly instructed to chew either PE gum or placebo gum. Saliva samples were collected at baseline and from 0 to 2, 2 to 5, 5 to 10, 10 to 15, and 75 to 80 min after each intervention. The following outcomes were measured: (i) salivary flow rate and pH value; (ii) total bacteria, Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) counts; and (iii) volatile sulfur compound (VSC) concentrations. The results showed similar data between groups at baseline and significantly higher salivary flow rates and pH levels in the PE fruit gum group after 0-2, 2-5, and 5-10 min of chewing. Assessment of total bacteria, S. mutans, P. gingivalis, and VSC levels revealed significant differences between the PE and control gum groups at 75-80 min. No adverse effects were registered. The present finding indicated chewing gum containing PE fruit extract stimulated salivary flow and significantly reduced clinical test indexes in the short term. Chewing PE gum might be a safe means of improving oral hygiene.

Role of PCSK9 in the Development of Mouse Periodontitis Before and After Treatment: A Double-Edged Sword.

Periodontitis is a highly prevalent infectious disease associated genetically with coronary heart disease (CHD). The effects of proprotein convertase subtilisin/kexin type 9 (PCSK9), a critical regulator of CHD, on periodontitis have not been studied to date. Here, we found that PCSK9 expression was increased in periodontitis patients and Porphyromonas gingivalis (Pg)-infected mice. Loss of PCSK9 attenuated Pg-induced periodontal bone loss in mice. First, PCSK9 deficiency reduced the release of inflammation-associated cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin 1ß, in vitro and in vivo. Second, its deficiency enhanced Pg and endotoxin clearance during Pg invasion in part by upregulating CD36 and low-density lipoprotein receptor (LDLR), respectively. However, after berberine treatment, periodontal bone regeneration in the PCSK9 knockout group was significantly lower than that in wild-type. This was because PCSK9 overexpression promoted osteogenic differentiation of periodontal ligament stem cells (PDLCs) prechallenged by TNF-α. Furthermore, PCSK9 could rescue PDLC osteogenesis by repressing the NF-κB signaling pathway by interacting with TRAF2. These results suggest that PCSK9 may be a potent drug target for treating periodontitis.

Antibacterial and antibiofilm activities of eugenol from essential oil of Syzygium aromaticum (L.) Merr. & L. M. Perry (clove) leaf against periodontal pathogen Porphyromonas gingivalis.

The antibacterial effect and mechanism of eugenol from Syzygium aromaticum (L.) Merr. & L. M. Perry (clove) leaf essential oil (CLEO) against oral anaerobe Porphyromonas gingivalis were investigated. The results showed that eugenol, with content of 90.84% in CLEO, exhibited antibacterial activity against P. gingivalis at a concentration of 31.25 µM. Cell shrink and lysis caused by eugenol were observed with Scanning Electron Microscopy (SEM). The release of macromolecules and uptake of fluorescent dye indicated that the antibacterial activity was due to the ability of eugenol to permeabilize the cell membrane and destroy the integrity of plasmatic membrane irreversibly. In addition, eugenol inhibited biofilm formation and reduced preformed biofilm of P. gingivalis at different concentrations. The down-regulation of virulence factor genes related to biofilm (fimA, hagA, hagB, rgpA, rgpB, kgp) explained that eugenol suppressed biofilm formation at the initial stage. These findings suggest that eugenol and CLEO may be potential additives in food and personal healthcare products as a prophylactic approach to periodontitis.

Combined antioxidant effects of Neem extract, bacteria, red blood cells and Lysozyme: possible relation to periodontal disease.

BACKGROUND: The common usage of chewing sticks prepared from Neem tree (Azadirachta indica) in India suggests its potential efficacy in periodontal diseases. The objective of this study is to explore the antibacterial effects of Neem leaf extract on the periodontophatic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and its antioxidant capacities alone and in combination with bacteria and polycationic peptides that may be at the site of inflammation. METHODS: Neem leaf extract was prepared by ethanol extraction. The growth kinetics of P. gingivalis and F. nucleatum under anaerobic conditions in the presence of Neem leaf extract were measured. Broth microdilution test was used to determine the Minimal Inhibitory Concentration (MIC) of Neem leaf extract against each bacterial strain. The effect of Neem leaf extract on the coaggregation of the bacteria was assessed by a visual semi-quantitative assay. The antioxidant capacities of Neem leaf extract alone and in combination with bacteria, with the addition of red blood cells or the polycationic peptides chlorhexidine and lisozyme, were determined using a chemiluminescence assay. RESULTS: Neem leaf extract showed prominent dose-dependent antibacterial activity against P. gingivalis, however, had no effect on the growth of F. nucleatum nor on the coaggregation of the two bacteria. Yet, it showed intense antioxidant activity, which was amplified following adherence to bacteria and with the addition of red blood cells or the polycationic peptides. CONCLUSIONS: Neem leaf extract, containing polyphenols that adhere to oral surfaces, have the potential to provide long-lasting antibacterial as well as synergic antioxidant activities when in complex with bacteria, red blood cells and lisozyme. Thus, it might be especially effective in periodontal diseases.

Preventive effects of the novel antimicrobial peptide Nal-P-113 in a rat Periodontitis model by limiting the growth of Porphyromonas gingivalis and modulating IL-1ß and TNF-α production.

BACKGROUND: P-113 (AKRHHGYKRKFH-NH2) is a 12-amino-acid histidine-rich peptide derived from histatin 5 that is highly degradable in high salt concentrations and biological fluids such as serum, plasma and saliva. Nal-P-113, a novel antimicrobial peptide whose histidine residues are replaced by the bulky amino acids ß-naphthylalanine, causes the antimicrobial peptide to retain its bactericidal activity even in physiological environments. This study evaluated the effect of the novel antimicrobial peptide Nal-P-113 in a rat periodontitis model and the mechanisms of action of Nal-P-113 for suppressing periodontitis. METHODS: Periodontitis was induced in mandibular first molars in rats receiving a ligature and infected with Porphyromonas gingivalis. Animals were randomly divided into six groups: a, P. gingivalis W83 alone; b, P. gingivalis W83 with 6.25 µg/mL of Nal-P-113; c, P. gingivalis W83 with 25 µg/mL of Nal-P-113; d, P. gingivalis W83 with 100 µg/mL of Nal-P-113; e, P. gingivalis W83 with 400 µg/mL of Nal-P-113; and f, control without P. gingivalis W83 or Nal-P-113. Morphometric analysis was used to evaluate alveolar bone loss. Microbiological assessment of the presence of Porphyromonas gingivalis and total bacteria was performed using absolute quantitative real-time PCR and scanning electron microscopy. Gingival tissue was collected for western blot and immunohistochemical assays of IL-1ß and TNF-α levels. RESULTS: Alveolar bone loss was inhibited by 100 µg/mL or 400 µg/mL of Nal-P-113 compared to the control group (P < 0.05). Lower amounts of P. gingivalis and total bacteria were found in groups d and e compared with group a (P < 0.05). A decrease in the levels of IL-1ß and TNF-α was detected in group d and group e compared to the control group (P < 0.05). The amount of P. gingivalis was positively correlated with IL-1ß and TNF-α expression in periodontal tissue (P < 0.05). CONCLUSIONS: Nal-P-113 exhibited protective effects on Porphyromonas gingivalis-induced periodontitis in rats by limiting the amount of bacteria and modulating IL-1ß and TNF-α production. The use of Nal-P-113 in vivo might serve as a beneficial preventive or therapeutic approach for periodontitis.

Efficiency of Nanotube Surface-Treated Dental Implants Loaded with Doxycycline on Growth Reduction of Porphyromonas gingivalis.

PURPOSE: The prevalence of peri-implant infection in patients with dental implants has been shown to range from 28% to 56%. A nanotube-modified implant surface can deliver antibiotics locally and suppress periodontal pathogenic bacterial growth. The aim of this study was to evaluate the deliverability of antibiotics via a nanotube-modified implant. MATERIALS AND METHODS: Dental implants with a nanotube surface were fabricated and loaded with doxycycline. Afterward, each dental implant with a nanotube surface was placed into 2-mL tubes, removed from solution, and placed in a fresh solution daily for 28 days. Experimental samples from 1, 2, 4, 16, 24, and 28 days were used for this evaluation. The concentration of doxycycline was measured using spectrophotometric analysis at 273-nm absorbance. The antibacterial effect of doxycycline was evaluated by supplementing Porphyromonas gingivalis (P gingivalis) growth media with the solution collected from the dental implants at the aforementioned time intervals for a period of 48 hours under anaerobic conditions. A bacterial viability assay was used to evaluate P gingivalis growth at 550-nm absorbance. RESULTS: Doxycycline concentration varied from 0.33 to 1.22 µg/mL from day 1 to day 28, respectively. A bacterial viability assay showed the highest P gingivalis growth at day 1 (2 nm) and the lowest at day 4 (0.17 nm), with a gradual reduction from day 1 to day 4 of approximately 87.5%. The subsequent growth pattern was maintained and slightly increased from baseline in approximately 48.3% from day 1 to day 24. The final P gingivalis growth measured at day 28 was 29.4% less than the baseline growth. CONCLUSION: P gingivalis growth was suppressed in media supplemented with solution collected from dental implants with a nanotube surface loaded with doxycycline during a 28-day time interval.

In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

ABSTRACT Objective Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. Material and Methods The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. Results The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). Conclusion M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

Two new limonoids isolated from the fuits of Melia toosendan.

In the present study, two new limonoids, 1α, 7α-dihydroxyl-3α-acetoxyl-12α-ethoxylnimbolinin (1) and 1α-tigloyloxy-3α-acetoxyl-7α-hydroxyl-12ß-ethoxylnimbolinin (2), together with other four known limonoids (3-6), were isolated from the fruits of Melia toosendan. Their structures were elucidated by means of extensive spectroscopic analyses (NMR and ESI-MS) and comparisons with the data reported in the literature. The isolated compounds were evaluated for their antibacterial activities. Compound 4 exhibited significant antibacterial activity against an oral pathogen, Porphyromonas gingivalis ATCC 33277, with an MIC value of 15.2 µg·mL(-1). Compound 2 was also active against P. gingivalis ATCC 33277, with an MIC value of 31.25 µg·mL(-1). In conlcusion, our resutls indicate that these compounds may provide a basis for future development of novel antibiotics.

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.

Green tea catechins potentiate the effect of antibiotics and modulate adherence and gene expression in Porphyromonas gingivalis.

OBJECTIVES: A number of studies have brought evidence that green tea catechins may contribute to periodontal health. The objective of this study was to investigate the ability of a green tea extract and its principal constituent epigallocatechin-3-gallate (EGCG) to potentiate the antibacterial effects of antibiotics (metronidazole, tetracycline) against Porphyromonas gingivalis, and to modulate the adherence to oral epithelial cells and expression of genes coding for virulence factors and the high temperature requirement A (HtrA) stress protein in P. gingivalis. METHODS: A broth microdilution assay was used to determine the antibacterial activity of the green tea extract and EGCG. The synergistic effects of either compounds in association with metronidazole or tetracycline were evaluated using the checkerboard technique. A fluorescent assay was used to determine bacterial adherence to oral epithelial cells. The modulation of gene expression in P. gingivalis was evaluated by quantitative RT-PCR. The Vibrio harveyi bioassay was used for monitoring quorum sensing inhibitory activity. RESULTS: The MIC values of the green tea extract on P. gingivalis ranged from 250 to 1000 µg/ml, while those of EGCG ranged from 125 to 500 µg/ml. A marked synergistic effect on P. gingivalis growth was observed for the green tea extract or EGCG in combination with metronidazole. Both the green tea extract and EGCG caused a dose-dependent inhibition of P. gingivalis adherence to oral epithelial cells. On the one hand, green tea extract and EGCG dose-dependently inhibited the expression of several P. gingivalis genes involved in host colonization (fimA, hagA, hagB), tissue destruction (rgpA, kgp), and heme acquisition (hem). On the other hand, both compounds increased the expression of the stress protein htrA gene. The ability of the green tea extract and EGCG to inhibit quorum sensing may contribute to the modulation of gene expression. CONCLUSIONS: This study explored the preventive and therapeutic potential of green tea catechins against periodontal disease. In addition to inhibit growth and adherence of P. gingivalis, a green tea extract and its main constituent EGCG was found to decrease the expression of genes coding for the major virulence factors.

In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria.

OBJECTIVE: Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. MATERIAL AND METHODS: The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. RESULTS: The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). CONCLUSION: M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

Evaluation of metronidazole-loaded poly(3-hydroxybutyrate) membranes to potential application in periodontitis treatment.

Guided tissue regeneration is a technique used for periodontium reconstruction. This technique uses barrier membranes, which prevent epithelial growth in the wound site and may also be used to release antibiotics, to protect the wound against opportunistic infections. Periodontal poly(3-hydroxybutyrate) membranes containing metronidazole (a drug used to help in infection control) were produced and characterized. The kinetic mechanism of the metronidazole delivery of leached and nonleached membrane as well as its cytotoxicity and structural integrity were evaluated. Poly(3-hydroxybutyrate) membranes containing 0.5-2 wt % of the drug and 20 wt % of the plasticizer were manufactured via compression molding. Based on morphological analysis, membranes loaded with 2% metronidazole were considered for detailed studies. The results revealed that metronidazole delivery by the leached membranes seemed to follow the Fick's law. Membranes were noncytotoxic. The amount of metronidazole delivered was in the range of the minimal inhibitory concentration for Porphyromonas gingivalis, and the membranes inhibited the proliferation of these bacteria. Besides, they maintained their mechanical resistance after 30 days of immersion in phosphate buffer at pH 7.4.