Exploring the antimicrobial efficacy of tea tree essential oil and chitosan against oral pathogens to overcome antimicrobial resistance.

BACKGROUND: Considering that antimicrobial resistance among oral pathogens is a significant concern in dental practice, with broader implications for overall health due to the oral microbiota serving as a reservoir for antibiotic resistance genes (ARGs), research into natural products is crucial for addressing this issue. OBJECTIVE: This study aimed to evaluate tea tree oil (TTO) and chitosan (CH) performance against oral pathogens, including mixed-species biofilm, and its effects on bacteria growth, in addition to chemical characterization and cytotoxicity of TTO. METHODS: Tea Tree Oil and low molecular weight chitosan were used in this study. The chemical composition of TTO was analyzed using gas chromatography coupled with mass spectrometry (GC-MS). To evaluate TTO's antimicrobial properties, time-kill and cell viability assays were conducted. Additionally, minimum inhibitory concentration (MIC), minimum microbiocidal concentration (MMC), checkerboard, and biofilm assays were performed using TTO and CH alone and in combination. RESULTS: TTO chromatography peaks found consistent with the standard ISO4730:2017 and literature. TTO and CH exhibited inhibitory activity against all tested microorganisms. The predominantly microbiostatic activity of TTO is probably related to terpinen-4-ol associated with terpinene. The oil at MIC value was able to delay the log phase of Aggregatibacter actinomycetemcomitans growth. Fibroblasts (L929) viability remained above 70 % during 24 h for TTO concentrations ranging from 0.5 to 0.0625 mg/ml. TTO-CH combination showed a synergistic activity (FIC = 0.5) against A. actinomycetemcomitans and Streptococcus sanguinis, at a concentration of 0,25MIC for both species. The compounds at MIC concentration inhibited both monospecies and mixed-species biofilms studied bacteria to the same extent as the azithromycin control. CONCLUSION: TTO and CH demonstrated efficacy in combating oral pathogens and TTO-CH combination offers a promising approach to confront microbial resistance in the oral environment.

The efficacy of diode laser and subgingival air polishing with erythritol in treatment of periodontitis (clinical and microbiological study).

BACKGROUND: There is insufficient clinical and microbiological evidence to support the use of diode laser and air-polishing with erythritol as supplements to scaling and root planning(SRP). The aim of the current study is to evaluate the clinical and microbiologic efficacy of erythritol subgingival air polishing and diode laser in treatment of periodontitis. METHODS: The study encompassed twenty-four individuals seeking periodontal therapy and diagnosed with stage I and stage II periodontitis. Eight patients simply underwent SRP. Eight more patients had SRP followed by erythritol subgingival air polishing, and eight patients had SRP followed by diode laser application. At baseline and six weeks, clinical periodontal parameters were measured, including Plaque Index (PI), Gingival Index (GI), periodontal Probing Depth (PPD), and Clinical Attachment Level (CAL). The bacterial count of Aggregatibacter actinomycetemcomitans(A.A), Porphyromonas gingivalis (P.G) was evaluated at different points of time. RESULTS: The microbiological assessment revealed significant differences in the count of A.A. between the laser and erythritol groups immediately after treatment, indicating a potential impact on microbial levels. However, the microbial levels showed fluctuations over the subsequent weeks, without statistically significant differences. Plaque indices significantly decreased post-treatment in all groups, with no significant inter-group differences. Gingival indices decreased, and the laser group showed lower values than erythritol and control groups. PPD and CAL decreased significantly across all groups, with the laser group exhibiting the lowest values. CONCLUSION: The supplementary use of diode laser and erythritol air polishing, alongside SRP, represents an expedited periodontal treatment modality. This approach leads to a reduction in bacteria and improvement in periodontal health. TRIAL REGISTRATION: This clinical trial was registered on Clinical Trials.gov (Registration ID: NCT06209554) and released on 08/01/2024.

Production and characterization of the lipopeptide with anti-adhesion for oral biofilm on the surface of titanium for dental implants.

Titanium implants are subject to bacterial adhesion and peri-implantitis induction, and biosurfactants bring a new alternative to the fight against infections. This work aimed to produce and characterize the biosurfactant from Bacillus subtilis ATCC 19,659, its anti-adhesion and antimicrobial activity, and cell viability. Anti-adhesion studies were carried out against Streptococcus sanguinis, Staphylococcus aureus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Proteus mirabilis as the minimum inhibitory concentration and the minimum bactericidal concentration. Cell viability was measured against osteoblast and fibroblast cells. The biosurfactant was classified as lipopeptide, with critical micelle concentration at 40 µg mL- 1, and made the titanium surface less hydrophobic. The anti-adhesion effect was observed for Staphylococcus aureus and Streptococcus sanguinis with 54% growth inhibition and presented a minimum inhibitory concentration of 15.7 µg mL- 1 for Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans. The lipopeptide had no cytotoxic effect and demonstrated high potential application against bacterial biofilms.

Decision-making on systemic antibiotics in the management of periodontitis: A retrospective comparison of two concepts.

AIM: To retrospectively compare two approaches for the adjunctive use of systemic antibiotics in non-surgical periodontal therapy: one based on the detection of Aggregatibacter actinomycetemcomitans (Aa) and the other on age and severity of periodontitis (Age & PPD). We also assessed the additional benefit of antibiotics in reducing the need for further surgical therapy in each group. MATERIALS AND METHODS: Patients of the Department of Periodontology, Goethe University Frankfurt, Germany, were screened for microbiological testing between 2008 and 2018. Patients were categorized by their microbiological result (Aa+/-) and demographic/clinical data (Age & PPD+/-). Agreement on antibiotic indication was tested. The clinical evaluation focussed on teeth with probing pocket depths (PPDs) ≥ 6 mm. RESULTS: Analysis of 425 patients revealed 30% categorized as Age & PPD+ and 34% as Aa+. Sixty-three percent had consistent antibiotic recommendations (phi coefficient 0.14, p = .004). Patients in the Age & PPD+ group receiving antibiotics showed the most substantial reduction in the number of teeth with PPD ≥ 6 mm after non-surgical periodontal therapy. CONCLUSIONS: Both strategies resulted in a significant clinical improvement compared with those without antibiotic treatment and restricted antibiotic use similarly, but targeted different patient groups. Younger individuals with severe periodontitis benefited most from antibiotics, reducing the need for additional surgeries. The study was registered in an international trial register (German Clinical Trial Register number DRKS00028768, registration date 27 April 2022, https://drks.de/search/en/trial/DRKS00028768).

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.

Layer-by-layer self-assembly of PLL/CPP-ACP multilayer on SLA titanium surface: Enhancing osseointegration and antibacterial activity in vitro and in vivo.

Dental Implants are expected to possess both excellent osteointegration and antibacterial activity because poor osseointegration and infection are two major causes of titanium implant failure. In this study, we constructed layer-by-layer self-assembly films consisting of anionic casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) and cationic poly (L-lysine) (PLL) on sandblasted and acid etched (SLA) titanium surfaces and evaluated their osseointegration and antibacterial performance in vitro and in vivo. The surface properties were examined, including microstructure, elemental composition, wettability, and Ca2+ ion release. The impact the surfaces had on the adhesion, proliferation and differentiation abilities of MC3T3-E1 cells were investigated, as well as the material's antibacterial performance after exposure to the oral microorganisms such as Porphyromonas gingivalis (P. g) and Actinobacillus actinomycetemcomitans (A. a). For the in vivo studies, SLA and Ti (PLL/CA-3.0)10 implants were inserted into the extraction socket immediately after extracting the rabbit mandibular anterior teeth with or without exposure to mixed bacteria solution (P. g & A. a). Three rabbits in each group were sacrificed to collect samples at 2, 4, and 6 weeks of post-implantation, respectively. Radiographic and histomorphometry examinations were performed to evaluate the implant osseointegration. The modified titanium surfaces were successfully prepared and appeared as a compact nano-structure with high hydrophilicity. In particular, the Ti (PLL/CA-3.0)10 surface was able to continuously release Ca2+ ions. From the in vitro and in vivo studies, the modified titanium surfaces expressed enhanced osteogenic and antibacterial properties. Hence, the PLL/CPP-ACP multilayer coating on titanium surfaces was constructed via a layer-by-layer self-assembly technology, possibly improving the biofunctionalization of Ti-based dental implants.

Efficacy of a mouthwash containing ε-poly-L-lysine, funme peptides and domiphen in reducing halitosis and supragingival plaque: a randomized clinical trial.

OBJECTIVE: To evaluate the antibacterial effectiveness of a combination of ε-poly-L-lysine (ε-PL), funme peptide (FP) as well as domiphen against oral pathogens, and assess the efficacy of a BOP® mouthwash supplemented with this combination in reducing halitosis and supragingival plaque in a clinical trial. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans were determined by the gradient dilution method. Subsequently, the CCK-8 assay was used to detect the toxicity of mouthwash on human gingival fibroblastst, and the effectiveness in reducing halitosis and supragingival plaque of the mouthwash supplemented with the combination was analyzed by a randomized, double-blind, parallel-controlled clinical trial. RESULTS: The combination exhibited significant inhibitory effects on tested oral pathogens with the MIC < 1.56% (v/v) and the MBC < 3.13% (v/v), and the mouthwash containing this combination did not inhibit the viability of human gingival fibroblasts at the test concentrations. The clinical trial showed that the test group displayed notably lower volatile sulfur compounds (VSCs) at 0, 10, 24 h, and 7 d post-mouthwash (P < 0.05), compared with the baseline. After 7 days, the VSC levels of the and control groups were reduced by 50.27% and 32.12%, respectively, and notably cutting severe halitosis by 57.03% in the test group. Additionally, the Plaque Index (PLI) of the test and control group decreased by 54.55% and 8.38%, respectively, and there was a significant difference in PLI between the two groups after 7 days (P < 0.01). CONCLUSIONS: The combination of ε-PL, FP and domiphen demonstrated potent inhibitory and bactericidal effects against the tested oral pathogens, and the newly formulated mouthwash added with the combination exhibited anti-dental plaque and anti-halitosis properties in a clinical trial and was safe. TRIAL REGISTRATION: The randomized controlled clinical trial was registered on Chinese Clinical Trial Registry (No. ChiCTR2300073816, Date: 21/07/2023).

Quorum-quenching enzyme Est816 assisted antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in rats.

Introduction: Quorum-quenching enzyme Est816 hydrolyzes the lactone rings of N-acyl homoserine lactones, effectively blocking the biofilm formation and development of Gram-negative bacteria. However, its applications in the oral field is limited. This study aimed to evaluate the efficacy of enzyme Est816 in combination with antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in vitro and in vivo. Methods: The antimicrobial efficacy of enzyme Est816 in combination with minocycline, metronidazole, and amoxicillin was determined using the minimum inhibitory concentration test. The anti-biofilm effect of enzyme Est816 was assessed using scanning electron microscopy, live/dead bacterial staining, crystal violet staining, and real-time quantitative PCR. Biocompatibility of enzyme Est816 was assessed in human gingival fibroblasts (HGF) by staining. A rat model of periodontitis was established to evaluate the effect of enzyme Est816 combined with minocycline using micro-computed tomography and histological staining. Results: Compared to minocycline, metronidazole, and amoxicillin treatment alone, simultaneous treatment with enzyme Est816 increased the sensitivity of biofilm bacteria to antibiotics. Enzyme Est816 with minocycline exhibited the highest rate of biofilm clearance and high biocompatibility. Moreover, the combination of enzyme Est816 with antibiotics improved the antibiofilm effects of the antibiotics synergistically, reducing the expression of the virulence factor leukotoxin gene (ltxA) and fimbria-associated gene (rcpA). Likewise, the combination of enzyme Est816 with minocycline exhibited a remarkable inhibitory effect on bone resorption and inflammation damage in a rat model of periodontitis. Discussion: The combination of enzyme Est816 with antibiotics represents a prospective anti-biofilm strategy with the potential to treat periodontitis.

Microbiological Effects of Sodium Hypochlorite/-Amino Acids and Cross-linked Hyaluronic Acid Adjunctive to Non-surgical Periodontal Treatment.

PURPOSE: To investigate the microbiological outcomes obtained with either subgingival debridement (SD) in conjunction with a gel containing sodium hypochlorite and amino acids followed by subsequent application of a cross-linked hyaluronic acid gel (xHyA) gel, or with SD alone. MATERIALS AND METHODS: Forty-eight patients diagnosed with stages II-III (grades A/B) generalised periodontitis were randomly treated with either SD (control) or SD plus adjunctive sodium hypochlorite/amino acids and xHyA gel (test). Subgingival plaque samples were collected from the deepest site per quadrant in each patient at baseline and after 3 and 6 months. Pooled sample analysis was performed using a multiplex polymerase chain reaction (PCR)-based method for the identification of detection frequencies and changes in numbers of the following bacteria: Aggregatibacter actinomycetemcomitans (A.a), Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f), Treponema denticola (T.d), and Prevotella intermedia (P.i). RESULTS: In terms of detection frequency, in the test group, statistically significant reductions were found for P.g, T.f, T.d and P.i (p < 0.05) after 6 months. In the control group, the detection frequencies of all investigated bacterial species at 6 months were comparable to the baseline values (p > 0.05). The comparison of the test and control groups revealed statistically significant differences in detection frequency for P.g (p = 0.034), T.d (p < 0.01) and P.i (p = 0.02) after 6 months, favouring the test group. Regarding reduction in detection frequency scores, at 6 months, statistically significant differences in favour of the test group were observed for all investigated bacterial species: A.a (p = 0.028), P.g (p = 0.028), T.f (p = 0.004), T.d (p <0.001), and P.i (p = 0.003). CONCLUSIONS: The present microbiological results, which are related to short-term outcomes up to 6 months post-treatment, support the adjunctive subgingival application of sodium hypochlorite/amino acids and xHyA to subgingival debridement in the treatment of periodontitis.

Toxin-triggered liposomes for the controlled release of antibiotics to treat infections associated with the gram-negative bacterium, Aggregatibacter actinomycetemcomitans.

Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.

Comparison of the effectiveness of Morus alba and chlorhexidine gels as an adjunct to scaling and root planing on stage II periodontitis - A randomized controlled clinical trial.

OBJECTIVES: The present study aimed to assess and compare the effect of Morus alba and chlorhexidine gel as an adjunct to scaling and root planing (SRP) in treating stage II periodontitis. METHODS: A single-blind, randomized controlled trial was conducted on 180 patients with stage II periodontitis who received full-mouth SRP. They were randomly assigned to receive chlorhexidine digluconate (CHX) gel, Morus alba (MA) and placebo gel for Groups A, B and C, respectively, at the baseline, 15 days and 30 days. Plaque index (PI), Gingival index (GI), periodontal pocket depth (PPD) and quantitative analysis (culture) of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia were assessed at baseline and 45 days. Analysis of variance was used to compare the significant difference in PI, GI, PPD and microbiological parameters between the three groups after the intervention, followed by post hoc Mann-Whitney U and Tukey's HSD test for clinical and microbiological parameters, respectively. RESULTS: Intergroup comparison of the PI, GI and microbiological parameters between the MA and CHX groups at the end of 45 days did not show a statistically significant difference (p > 0.05), whereas a statistically significant difference was observed for PPD between MA and CHX groups with the mean difference of 0.18 mm (p = 0.002). CONCLUSION: Morus alba gel was found to be effective in decreasing PPD. However, there was no difference between Morus alba and chlorhexidine gel as an adjunct to SRP in treating stage II periodontitis.

Synergistic antibacterial activity of herbal extracts with antibiotics on bacteria responsible for periodontitis.

INTRODUCTION: Development of bacterial resistance and antimicrobial side-effect has shifted the focus of research toward Ethnopharmacology. A biologically active compound derived from the plants may increase the effectiveness of antibiotic when used in combination. The present study aims to determine the synergistic antibacterial effect of ethanolic extracts of Punica granatum (pericarp), Commiphora molmol, Azadirachta indica (bark) in combination with amoxicillin, metronidazole, tetracycline, and azithromycin on periodontopathic bacteria: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans. METHODOLOGY: Periodontopathic bacterial strains were isolated from the plaque sample that was collected from periodontitis patients and grown under favorable conditions. Susceptibility of bacteria to the antibiotics and extracts was determined by disc diffusion method by measuring the diameter of the inhibition zones. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts were evaluated against each bacterium. Synergistic effect of plant extract in combination with antibiotics was tested against each bacterium by measuring the diameter of zone of inhibition (ZOI). RESULTS: Findings revealed that all plant extracts exhibited an inhibitory effects on the proliferation and growth of periodontopathic bacteria. The maximum antibacterial effect was exhibited by C. molmol on P. gingivalis (ZOI = 20 ± 0.55 mm, MIC = 0.53 ± 0.24 mg/mL and MBC = 5.21 ± 1.81 mg/mL) (p < 0.05), meanwhile, no antibacterial activity was exhibited by P. granatum on T. forsythia. Synergistic antibacterial effect was recorded when plant extracts were used in combination with antibiotics. The best synergism was exhibited by P. granatum with amoxicillin against A. actinomycetemcomitans (24 ± 1.00 mm) (p < 0.05). CONCLUSIONS: The synergistic test showed significant antibacterial activity when plant extracts were combined with antibiotics against all the experimented bacteria.

Effect of cranberry juice deacidification on its antibacterial activity against periodontal pathogens and its anti-inflammatory properties in an oral epithelial cell model.

Cranberries are widely recognized as a functional food that can promote oral health. However, the high concentration of organic acids in cranberry juice can cause tooth enamel erosion. Electrodialysis with bipolar membrane (EDBM) is a process used for the deacidification of cranberry juice. The present study investigated whether the removal of organic acids (0%, 19%, 42%, 60%, and 79%) from cranberry juice by EDBM affects its antibacterial activity against major periodontopathogens as well as its anti-inflammatory properties in an oral epithelial cell model. A deacidification rate ≥60% attenuated the bactericidal effect against planktonic and biofilm-embedded Aggregatibacter actinomycetemcomitans but had no impact on Porphyromonas gingivalis and Fusobacterium nucleatum. Cranberry juice increased the adherence of A. actinomycetemcomitans and P. gingivalis to oral epithelial cells, but reduced the adherence of F. nucleatum by half regardless of the deacidification rate. F. nucleatum produced more hydrogen sulfide when it was exposed to deacidified cranberry juice with a deacidification rate ≥42% compared to the raw beverage. Interestingly, the removal of organic acids from cranberry juice lowered the cytotoxicity of the beverage for oral epithelial cells. Deacidification attenuated the anti-inflammatory effect of cranberry juice in an in vitro oral epithelial cell model. The secretion of IL-6 by lipopolysaccharide (LPS)-stimulated oral epithelial cells exposed to cranberry juice increased proportionally with the deacidification rate. No such effect was observed with respect to the production of IL-8. This study provided evidence that organic acids, just like phenolic compounds, might contribute to the health benefits of cranberry juice against periodontitis.

Epigallocatechin gallate alters leukotoxin secretion and Aggregatibacter actinomycetemcomitans virulence.

OBJECTIVES: We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. METHODS: We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. KEY FINDINGS: EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. CONCLUSIONS: Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.

Development of an enzyme-enhancer system to improve laccase biological activities.

The present investigation reports an in-vitro study using combination of laccase and an enhancer capable of inhibiting the growth of pathogenic microorganisms, preventing biofilm formation, and whitening teeth. Laccase-cinnamic acid system remarkably inhibited the growth of Aggregatibacter actinomycetemcomitans, Candida albicans, S. aureus, and Streptococcus mutans whilst showed no significant effects on Gram-negative bacteria. Data presented that cinnamic acid (10 mM) with laccase (0.125 U ml-1) led to a maximum decrease of about 90%, in S. mutans biofilm formation. The confocal laser scanning microscopy showed considerable detachment of S. mutans cells from glass substratum. The combined laccase-cinnamic acid system could remove teeth discoloration caused by coffee. SEM of the teeth surface exhibited no damages such as surface cracking or fracture. Liquid chromatography-tandem mass spectrometry (LC-MS) and cyclic voltammetry (CV) studies showed that laccase can catalyze the one-electron oxidation of cinnamic acid to the respective radical. This radical can then undergo several fates, including recombination with another radical to form a dimeric species, dismutation of the radical back to cinnamic acid or decarboxylation to give various reduced oxygen species. Therefore, the redox potential values of phenolic monomers/oligomers are related with their biological activities.

Long-term antibacterial activity of a composite coating on titanium for dental implant application.

Dental implants are the most innovative and superior treatment modality for tooth replacement. However, titanium implants still suffer from insufficient antibacterial capability and peri-implant diseases remain one of the most common and intractable complications. To prevent peri-implant diseases, a composite coating containing a new antibacterial agent, (Z-)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) was fabricated on titanium. This study was designed to investigate the antibacterial activity of the composite coating against two common peri-implant pathogens (Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans). The morphology of the composite coating showed that BBF-loaded poly(L-lactic acid) nanospheres were well-distributed in the pores of the microarc oxidation coating, and cross-linked with each other and the wall pores by gelatin. A release study indicated that the antibacterial coating could sustain the release of BBF for 60 d, with a slight initial burst release occurring during the first 4 h. The antibacterial rate of the composite coating for adhering bacteria was the highest (over 97%) after 1 d and over 90% throughout a 30-day incubation period. The total fluorescence intensity of the composite coating was the lowest, and the vast majority of the fluorescence was red (dead bacteria). Moreover, real-time polymerase chain reaction analysis confirmed that the relative gene expression of the adherent bacteria on the composite coating was down-regulated. It was therefore concluded that the composite coating fabricated on titanium, which showed excellent and relatively long-term antibacterial activity against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, is a potential and promising strategy to be applied on dental implants for the prevention of peri-implant diseases.

Antibacterial activity of nitric oxide-releasing carboxymethylcellulose against periodontal pathogens.

The prevalence of periodontal disease poses a significant global health burden. Treatments for these diseases, primarily focused on removal and eradication of dental plaque biofilms, are challenging due to limited access to periodontal pockets where these oral pathogens reside. Herein, we report on the development and characterization of nitric oxide (NO)-releasing carboxymethylcellulose (CMC) derivatives and evaluate their in vitro bactericidal efficacy against planktonic Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, two prominent periodontopathogens. Bactericidal exposure assays revealed that three of the synthesized NO-releasing polymers were capable of reducing bacterial viability of both species by 99.9% in 2 hr at concentrations of 4 mg ml-1 or lower, reflecting NO's potent and rapid bactericidal action. The NO-releasing CMCs elicited minimal toxicity to human gingival fibroblasts at their bactericidal concentrations following 24-hr exposure.

Maillard reaction products inhibit the periodontal pathogen Aggregatibacter actinomycetemcomitans by chelating iron.

OBJECTIVE: To determine the mechanism of growth inhibition of Aggregatibacter actinomycetemcomitans by Maillard reaction products (MRP). DESIGN: Growth and cell viabilities in the presence or absence of MRP were measured for both the rough and smooth variants of the bacteria. Effects of addition of ferrous and ferric ions on the inhibition of the bacteria by MRP were determined. RESULTS: MRPs decreased the extent of complex formation of Chrome Azurol S with iron suggesting that MRPs can chelate iron effectively. The chelation causes growth inhibition of both the rough and smooth strains. At low concentrations of the inhibitor, lag time was extended by approximately 12 h while at high concentrations, cells were killed, decreasing cell viability by up to 8 orders of magnitude. Growth of both the rough and smooth strains could be restored to original level by addition of iron. For the rough strain, both ferrous and ferric ions could relieve the inhibition by MRP while for the smooth strain only ferrous ion was effective. CONCLUSION: MRPs inhibit the growth of A. actinomycetemcomitans by chelating iron and the inhibition can be relieved by addition of iron.

Weissella cibaria CMU exerts an anti­inflammatory effect by inhibiting Aggregatibacter actinomycetemcomitans­induced NF­κB activation in macrophages.

Periodontitis is a chronic inflammatory disease caused by various periodontal pathogens. Weissella cibaria CMU (oraCMU) is a probiotic that promotes oral health. However, its anti­inflammatory effects against periodontal pathogens have not yet been investigated. The present study evaluated the anti­inflammatory effects of live oraCMU against stimulation with the formalin­inactivated periodontal pathogen Aggregatibacter actinomycetemcomitans in RAW 264.7 macrophages. Cell viability was analyzed by the MTS assay in a dose­dependent manner (at multiplicities of infection of 0.1, 1, 10, 100 and 1,000). Nitric oxide (NO) was monitored using the Griess test. The mRNA expression of proinflammatory cytokines such as interleukin (IL)1ß and IL6 was assessed by reverse transcription­quantitative PCR. Western blotting was used to examine the effects of oraCMU on the phosphorylation of NF­κB inhibitor α (IκBα) and IκBα kinase (IKK), the nuclear translocation of the NF­κB subunit p65 and the expression of inducible NO synthase (iNOS). Live oraCMU had no cytotoxic effects on RAW 264.7 macrophages. In A. actinomycetemcomitans­stimulated RAW 264.7 macrophages, oraCMU reduced NO production by suppressing iNOS expression and downregulating the mRNA expression of proinflammatory cytokines in a dose­dependent manner. IKK phosphorylation and IκBα degradation were dose­dependently inhibited by oraCMU and the nuclear translocation of p65 via the canonical NF­κB pathway was simultaneously reduced. The results indicated that oraCMU possessed anti­inflammatory activity associated with the inhibition of NF­κB signal activation in response to periodontal pathogens. This suggests that oraCMU is a beneficial anti­inflammatory probiotic that can aid in the maintenance of oral health.

An Antibacterial Strategy of Mg-Cu Bone Grafting in Infection-Mediated Periodontics.

Periodontal diseases are mainly the results of infections and inflammation of the gum and bone that surround and support the teeth. In this study, the alveolar bone destruction in periodontitis is hypothesized to be treated with novel Mg-Cu alloy grafts due to their antimicrobial and osteopromotive properties. In order to study this new strategy using Mg-Cu alloy grafts as a periodontal bone substitute, the in vitro degradation and antibacterial performance were examined. The pH variation and Mg2+ and Cu2+ release of Mg-Cu alloy extracts were measured. Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), two common bacteria associated with periodontal disease, were cultured in Mg-Cu alloy extracts, and bacterial survival rate was evaluated. The changes of bacterial biofilm and its structure were revealed by scanning electron microscopy (SEM) and transmission electronic microscopy (TEM), respectively. The results showed that the Mg-Cu alloy could significantly decrease the survival rates of both P. gingivalis and A. actinomycetemcomitans. Furthermore, the bacterial biofilms were completely destroyed in Mg-Cu alloy extracts, and the bacterial cell membranes were damaged, finally leading to bacterial apoptosis. These results indicate that the Mg-Cu alloy can effectively eliminate periodontal pathogens, and the use of Mg-Cu in periodontal bone grafts has a great potential to prevent infections after periodontal surgery.