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Nitric oxide (NO) produced in the oral cavity is a powerful resource for the human body, especially when NO-syntethase production is not adequate. The role of oral microbiome in determining blood pressure levels has been linked to the active role of some bacterial species involved in the nitro-reducing process. In the present study we investigated the correlation between selected oral microbiome characteristics, nitric oxide (NO) concentration in saliva and their association with hypertension.A case-control study including 48 (25 normotensive and 23 hypertensive subjects), subjects between 50 and 70 years old, was carried out at the dental clinic of an Italian teaching hospital. Characteristics of participants have been evaluated by means of a physical examination, and by an assisted interview. A real-time polymerase chain reaction in samples of saliva and plaque was used to detect Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola, Streptococcus mutans, Streptococcus sanguinis, Veillonella dispar and Neisseria subflava as well as total bacterial count. Nitric oxide in saliva was evaluated by the ELISA method.Normotensive subjects, compared with hypertensive subjects, had significantly higher concentration of NO (165.77 ± 61.7 vs 57.49 ± 19.61 μmol/l; p = 0.023), and higher bacterial concentration of the supragingival plaque (4.73E+07 ± 4.33+07 vs 4.02E+07 ± 4.00+07; p = 0.024). Bacterial species, usually associated to good oral health status, such as Neisseria subflava, were significantly more present in normotensive subjects than in hypertensive ones (9090.88 ± 5481.49 vs 4791.35 ± 4349.37; p 0.001). considering the concentration of bacteria as a biomarker of the development of hypertension.The results support the association between hypertension, oral microbiome and salivary nitric oxide, in fact do the results allow us to establish any biomarkers (microbial or biochemical, NO) that allow early therapeutic intervention.

Periodontitis, which is a chronic inflammation caused by biofilm from numerous gram-negative and -positive oral bacterial species between gingiva and tooth, is known to have a poor prognosis. Susceptibility of standard strains (19 strains) and clinical isolates (90 strains) of aerobic and anaerobic oral bacteria, including a recently recovered as novel pathogens for periodontitis called Filifactor alocis, was tested to minocycline (MINO) by using the agar dilution method according to the Clinical and Laboratory Standards Institute. MINO is a well-used therapeutic antibiotic for periodontits. In this study, minimum inhibitory concentrations (MICs) of MINO against Aggregatibacter actinomycetemcomitans (n = 1), Porphyromonas gingivalis (n = 5), Prevotella intermedia (n = 1), Tannerella forsythia (n = 1) and F. alocis (n = 1), were 0.12, ≤0.016–0.03, ≤0.016, 0.03, and 2 μg/mL, respectively. MICs range of MINO against clinical isolates (10 isolates each) Streptococcus intermedius, P. gingivalis, P. intermedia, Fusobacterum nucleatum, Parvimonas micra were 0.06–16, ≤0.016–0.03, ≤0.016–1, ≤0.016–0.12, and ≤0.016–0.25 μg/mL, respectively. These results showed that MINO has superior in vitro activities against to known and recent recovered oral bacteria. Moreover, low prevalence in non-susceptible bacteria was observed to MINO.

Objectives Administration of systemic antimicrobials as an adjunct to mechanical treatment of periodontitis and sites with adverse clinical results leads to improved outcomes. This study aimed to assess the antimicrobial susceptibility of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia isolated from periodontitis patients to amoxicillin, metronidazole, azithromycin and moxifloxacin. Methods A total of 76 patients diagnosed with generalised periodontitis were included in the study. Subgingival samples were processed by culture. Etest was used to determine susceptibility to amoxicillin, metronidazole, azithromycin and moxifloxacin. Results A total of 141 isolates from 76 patients were evaluated, including 61 P. gingivalis, 43 T. forsythia and 37 A. actinomycetemcomitans. Etest results showed complete susceptibility of A. actinomycetemcomitans, P. gingivalis and T. forsythia to moxifloxacin. However, the isolates presented reduced susceptibility to the other antimicrobial agents investigated. Of the A. actinomycetemcomitans isolates, 70.3%, 40.5% and 89.2% were resistant to amoxicillin, azithromycin and metronidazole, respectively. The P. gingivalis samples showed relatively similar rates of resistance to amoxicillin (24.6%), azithromycin (21.3%) and metronidazole (24.6%). Similarly, 25.6%, 21.0% and 25.6% of the T. forsythia isolates were resistant to amoxicillin, azithromycin, and metronidazole, respectively. Conclusion These findings show that moxifloxacin may be a promising antimicrobial agent against P. gingivalis, T. forsythia and A. actinomycetemcomitans for the treatment of periodontitis. However, amoxicillin, azithromycin and metronidazole were less effective, especially against A. actinomycetemcomitans in vitro.

Periodontal disease is a chronic infectious disease, which is characterized by the damaged dental hard tissue by lactic acid generated by microorganisms after the fermentation of carbohydrates rich diet. The risk of periodontal disease is known to be higher in diabetic patients. We compared the diversity of five commonly occurring dental bacteria including Porphyromonas gingivalis, Tannerella forsythia, Capnocytophaga ochracea, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans in 14 type-2 diabetic patients and equal numbers of healthy controls. The subgingival samples were collected using sterile paper points. We used 16S rRNA sequence specific primers for PCR-based identification of dental bacteria. Our results showed that A. actinomycetemcomitans was completely absent in control subjects but present in 43% of diabetic patients. C. ochracea was highly prevalent in diabetic patients (100%) as compared to controls (28.5%). The frequency of other three bacterial species was also higher in diabetic patients than control subjects. These findings indicate that dental bacteria are highly prevalent in subgingival pockets of diabetic patients. Therefore, proper monitoring of diabetic patients for dental care is important to prevent bacterial growth and its sequela in risky individuals. Further case-control studies using larger sample size would help in validating the association between oral diseases and diabetes. Keywords: Dental bacteria, Identification, PCR, Diabetes

Introduction The design of the orthodontic bracket or appliance is 1 of the most important factors for creating retentive areas for biofilm formation. In orthodontics, this would be the first study to compare the microbial level changes in 3 different types of orthodontic appliances using checkerboard DNA-DNA hybridization technique. The purpose of this study was to evaluate and compare the extent of appearance of orange and red microbial complexes in patients undergoing orthodontic treatment using aligners, conventional metallic fixed labial appliances, and lingual fixed appliances. Methods A total of 60 patients, of which 20 patients were undergoing treatment with aligners, 20 patients with labial fixed appliances, and 20 patients with lingual fixed appliances, were included in our study. After 30 days, debonded brackets and rinsed aligners were stored and processed for analysis with checkerboard DNA-DNA hybridization. Results Most bacterial species showed moderate counts, with the exception of Treponema denticola, which showed a higher count in all 3 types of appliances. Fusobacterium nucleatum, Porphyromonas gingivalis, and T denticola were present in a higher percentage in the lingual appliance. Fusobacterium periodontium and Prevotella intermedia were present in a higher percentage in the labial fixed appliance. Campylobacter rectus, Tannerella forsythia, and Prevotella melaninogenica counts were moderate in all 3 appliances, with the first 2 microbes showing slightly higher counts in aligners. The association between all the microorganisms were statistically insignificant, with the exception of F nucleatum, which showed a strong statistically significant association in all 3 types of appliances. Conclusions The microbial contamination in metallic brackets was higher than that of aligners, when used for a month. Lingual fixed appliances showed more microbial contamination than labial fixed appliances followed by aligners.

Objective This study aimed to investigate the antimicrobial activity of β-caryophyllene against periodontopathogens as well as its inhibitory effects on the expression of inflammatory cytokines and production of volatile sulfur compounds by lipopolysaccharide and periodontopathogenic enzymes, respectively. Design The antimicrobial activity of β-caryophyllene againstPorphyromonas gingivalis, Tannerella forsythia, and Treponema denticola was investigated via a susceptibility assay using a microplate reader. THP-1 cells were treated with lipopolysaccharide in the presence or the absence of β-caryophyllene, and the expression and production of inflammatory cytokines were then analyzed by a real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. After fluorescence-labelling lipopolysaccharide, the effect of β-caryophyllene on the binding of lipopolysaccharide to the cell wall was investigated via flow cytometry. The spent culture media of P. gingivalis was shaken with or without β-caryophyllene and gaseous volatile sulfur compounds (VSCs) were measured by a gas chromatograph. Results β-caryophyllene showed strong the antimicrobial activity against periodontopathogens. It also reduced lipopolysaccharide-induced expression and production of cytokines, thereby inhibiting the binding of lipopolysaccharide-binding to toll-like receptors by interfering with the complex of lipopolysaccharide and CD14 or lipopolysaccharide-binding protein. β-caryophyllene also inhibited the emission of gaseous VSCs produced byP. gingivalis. Conclusions β-caryophyllene may improve periodontal health via antimicrobial activity against periodontopathogens, reducing inflammation caused by lipopolysaccharide, and by neutralizing VSCs.

The development of treatment strategies for periodontitis that maximise the effectiveness of antibiotics is highly desirable. Azithromycin is proving to be an effective antibiotic for treatment of refractory periodontitis which works by binding to the outer membrane of Gram-negative bacteria and subsequently inhibits protein synthesis. Lactoferrin is a membrane-active host antimicrobial protein and so the objective of this study was to determine whether the effect of azithromycin (AZM) against example periodontopathogens (Porphyromonas gingivalis and Tannerella forsythia) could be potentiated by lactoferrin. Two strains of P. gingivalis and T. forsythia were exposed to lactoferrin (LF; up to 10 mg/ml) and AZM (up to 5 µg/ml) for 0 – 72 h. The MICs for AZM were established using E-Test strips and by agar diffusion. Susceptibility to LF and LF + AZM was evaluated using diffusion assays, with and without iron depletion. The range of MIC values of AZM for P. gingivalis strains and T. forsythia was 0.16 - 0.63 µg/ml and 0.50 - 0.63 µg/ml, respectively. However, no inhibition was observed with iron saturated lactoferrin at any concentration or under iron depletion conditions nor was any effect observed on the AZM MIC by its presence. P. gingivalis and T. forsythia were inhibited by AZM but were not affected by Lf and there was no synergism between AZM and Lf.

Background T. forsythia a gram negative, anaerobe inhabits the mature biofilm present at sites expressing progressive periodontitis. It is a part of “red complex” group which contributes to the pathogenesis of periodontitis. The BspA protein and prtH gene encoded cysteine protease play a vital role in the virulence of T. forsythia. The present study aims to detect the two genotypes (bspA and prtH) in periodontitis and healthy subjects. Materials & Method Subgingival plaque samples were collected from periodontitis patients and healthy subjects (Chronic Periodontitis n = 128, Aggressive Periodontitis n = 72, healthy subjects n = 200). The samples were screened for the presence of T. forsythia 16S rRNA, bspA and prtH genotypes by Polymerase Chain Reaction. The prevalence of the genotypes between periodontitis patients and healthy subjects was compared with Pearson’s Chi-square test. A P value of Results The prevalence for T. forsythia in Chronic Periodontitis (n = 128), Aggressive Periodontitis (n = 72) and health (n = 200) was 73.4%, 59.7% and 10.5% respectively. The prevalence of T.forsythia bspA/prtH genotypes was 81.90%/43.60%, 88.40%/53.50% and 33.30%/14.3% in Chronic Periodontitis, aggressive Periodontitis and health respectively. Compared to healthy subjects, the odds of detecting T.forsythia 16S rRNA was 18.53 times high in individuals with periodontitis (P = 0.0001). Conclusion The high odds ratio of T.forsythia 16S rRNA among periodontitis strongly suggests its role in periodontitis. In addition, the high prevalence of T. forsythia bspA genotype among Chronic Periodontitis signifies it as a useful marker for chronic periodontitis.

Background and objectives Pathogenic infections caused by Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia can result in the production of volatile sulfur compounds (VSC’s) and other toxic compounds from methionine catabolism that can lead to halitosis and periodontitis. Our aim is to block the activity of methionine gammalyase-deaminase (Mgld) of methionine catabolism to prevent halitosis/periodontitis. Designs Cloned, expressed, Mgld protein was tested for purity by SDS-PAGE and western blotting. Mgld activity was tested by UV–vis spectroscopy and DTNB assay. Effects of Mgld inhibitor propargylglycine (PGLY) was tested on P. gingivalis growth by turbidity measurements. The effects of PGLY on oral epithelial and periodontal ligament cells in culture at different concentrations and time were tested for cell viability by MTT and Live-Dead assays. Amino acid comparisons of Mgld from different oral pathogens were done using standard bioinformatics program. Results Propargylglycine (PGLY) inhibited purified Mgld activity completely. In vivo, PGLY is a potent inhibitor on the growth of the P. gingivalis over 24 h, grown at 25 °C and 37 °C. Correspondingly in vivo Mgld activity was also affected by PGLY. Amino acid comparisons of oral pathogens showed 100% identity on the key residues of Mgld catalysis. Mammalian oral cell lines with PGLY, showed no difference in cell death over untreated controls assessed by MTT and Live-Dead assays. Conclusions PGLY arrest’s VSC’s production by P. gingivalis. Since initial Mgld activity is inhibited subsequent enzymatic and nonenzymatic products formed will be prevented. PGLY showed no toxicity towards cultured mammalian oral cells. Thus, PGLY can serve as a mouthwash ingredient to prevent halitosis/periodontitis.

High throughput, reliable and low cost methodologies for parallel pathogen counting are demanded in the diagnosis of infections, but still technically challenging. This work proposed a precise, simultaneous process of enumerating multiplex periodontal pathogens by using multiplex polymer chain reaction (mPCR) and quantitative capillary electrophoresis (qCE). A self-developed qCE protocol, which was time modified internal standard (TMIS) method, was employed to quantitatively study mPCR reactions. The optimal mPCR conditions were obtained for the amplification of three important periodontal pathogens, Porphyromonas gingivalis (P.g.), Treponema denticola (T.d.) and Tannerella forsythia (T.f.). The simultaneous quantitative determination of genome copies of P.g., T.d. and T.f. was demonstrated with a dynamic range from 0.2 to 1.5 × 105 unit/μL. Results showed that this mPCR-qCE assay had run-to-run and day-to-day variations of 6.1–10.9% and 2.3–17.4%, respectively. As little as 4 pathogen cells would be sufficient to have a positive result. Finally, a clinical sample was determined and its P.g., T.d. and T.f. concentrations were 39720 unit/μL, 5214 unit/μL, and 1140 unit/μL, respectively. This mPCR-qCE assay was proved to have comparable precision and sensitivity with quantitative PCR technology. Besides, it was more cost-effective, time saving and more capable of simultaneous detection.

Objective The antimicrobial efficacy of zinc- (ZnCl 2 ) and cetylpyridinium-chloride (CPC) and their inhibition capacity on volatile sulfur compound (VSC) production by oral bacterial strains were investigated. Design Minimum inhibitory concentrations (MIC) and growth curves were determined for ZnCl 2 , CPC, and CPC with ZnCl 2 solutions against eight oral microorganisms ( Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum, Porphyromonas gingivalis , Prevotella intermedia , Treponema denticola , Tannerella forsythia , Staphylococcus aureus and Streptococcus mutans ) known to be involved in the pathophysiology of both halitosis and periodontal disease. Gas chromatography was applied to measure VSCs (H 2 S, CH 3 SH, (CH 3 ) 2 S) production levels of each strains following exposure to the solutions. Results ZnCl 2 and CPC effectively inhibited growth of all eight strains. ZnCl 2 was generally more effective than CPC in suppressing bacterial growth excluding A. actinomycetemcomitans , P. intermedia , and T. forsythia . Synergism between CPC and ZnCl 2 was shown in A. actinomycetemcomitans . The MIC for CPC was significantly lower than ZnCl 2 . VSC production was detected in five bacterial strains ( A. actinomycetemcomitans , F. nucleatum , P. gingivalis , T. denticola , and T. forsythia ). Each bacterial strain showed unique VSCs production profiles. H 2 S was produced by F. nucleatum , P. gingivalis , and T. denticola , CH 3 SH by all five strains and (CH 3 ) 2 S by A. actinomycetemcomitans , F. nucleatum , P. gingivalis , and T. denticola . Production of CH 3 SH, the most malodorous component among the three major VSCs from mouth air was evident in F. nucleatum and T. forsythia . Conclusion Both ZnCl 2 and CPC effectively inhibit bacterial growth causative of halitosis and periodontal disease, resulting in a direct decrease of bacterial VSCs production.

Objective This review intended to evaluate the in vitro antibacterial effect of natural plant extracts against periodontal pathogens responsible for periodontal disease. Methods The research was conducted in five English databases (PubMed, Cochrane Library, Scopus, EBSCO, web of science) from January 2000 to June 2019. Only articles in English and French were retrieved. The inclusion criteria concerned all in vitro studies of the antibacterial activity of plant extracts on periodontal bacteria. The Reports on in vivo trials, essential oils, pure compounds have been excluded. The risk of bias was assessed using a standard approach. Results 51 studies were selected in this review. The outcomes of selected studies expressed by the diameter of the zone of inhibition, the values of the minimum inhibitory concentration and the minimum bactericidal concentration were included. Out of the 51 studies, 25 have shown the antibacterial effect of plant extracts against Aggregatibacter actinomycetemcomitans; 33 out of the 51 studies reported on the effects against Porphyromanas gingivalis; 17 studies against Prevotella intermedia names; 13 studies on Fusobacterium nucleatum; three studies on Tannerella forsythia; and only one study against Treponema denticola. Conclusion Although there is a high heterogeneity in the results of the minimum inhibitory concentration from one study to another, the results showed that plant extracts had an antimicrobial activity on the major periodontal pathogens. Thus, they can serve as alternatives to chemical antiseptic agents for the prevention and treatment of periodontal diseases.

Objective To detect the distribution and expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes in periapical lesions and to analyse the possible microbial stimuli outside of teeth. Design The distribution of NLRP3 and AIM2 inflammasomes in sixteen periapical lesions was investigated by immunohistochemistry. Meanwhile, the relative gene expression levels of NLRP3 and AIM2 in sixteen periapical lesions and three health periodontal tissue were quantified by real-time polymerase chain reaction (PCR). Moreover, forty-seven teeth without sinus tracts were obtained in the clinic and included in bacterial analysis using PCR. Then, the mRNA levels of apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-1β (IL-1β), NLRP3 and AIM2 in THP-1-derived macrophages treated with lipopolysaccharides (LPS) of Porphyromonas were also quantified by real-time PCR, and the IL-1β secretion level was investigated using enzyme-linked immunosorbent assay (ELISA). Results NLRP3 and AIM2 were positively expressed in periapical lesions and were mainly distributed in inflammatory cells. Most of the samples that demonstrated up-regulation of NLRP3 mRNA also demonstrated up-regulation of caspase-1 mRNA. Microbial analysis revealed that Porphyromonas endodontalis was the most commonly detected species and was detected in 27 of 47 cases (57.4%), followed by Fusobacterium nucleatum (20/47, 42.6%), Porphyromonas gingivalis (19/47, 40.4%), Tannerella forsythia (19/47, 40.4%), Actinomyces sp. (17/47, 36.17%), Treponema denticola (10/47,21.28%), Actinomyces israelii (9/47,19.15%), Prevotella intermedia (6/47, 12.77%), Enterococcus faecalis (1/47,2.13%) and Enterococcus faecium (0/47,0). Furthermore, we found that LPS of P. gingivalis induced THP-1 cells to produce IL-1β and to activate NLRP3 and AIM2 inflammasomes. Conclusions Our results suggest that the NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis. Anaerobes, such as P. endodontalis, P. gingivalis, F. nucleatum and T. forsythia, were the main detected microbial stimuli that might activate inflammasomes in periapical tissues.

The objective of this study was to assess antibiotic susceptibility among predominant Gram-negative anaerobic bacteria isolated from periodontitis patients who 5 years prior had been subject to mechanical therapy with or without adjunctive metronidazole. One pooled sample was taken from the 5 deepest sites of each of 161 patients that completed the 5 year follow-up after therapy. The samples were analyzed by culture. A total number of 85 anaerobic strains were isolated from the predominant subgingival flora of 65/161 patient samples, identified, and tested for antibiotic susceptibility by MIC determination. E-tests against metronidazole, penicillin, amoxicillin, amoxicillin + clavulanic acid and clindamycin were employed. The 73/85 strains were Gram-negative rods (21 Porphyromonas spp., 22 Prevotella/Bacteroides spp., 23 Fusobacterium/Filifactor spp., 3 Campylobacter spp. and 4 Tannerella forsythia). These were all isolated from the treated patients irrespective of therapy procedures (+/−metronidazole) 5 years prior. Three strains (Bifidobacterium spp., Propionibacterium propionicum, Parvimonas micra) showed MIC values for metronidazole over the European Committee on Antimicrobial Susceptibility Testing break point of >4 μg/mL. All Porphyromonas and Tannerella strains were highly susceptible. Metronidazole resistant Gram-negative strains were not found, while a few showed resistance against beta-lactam antibiotics. In this population of 161 patients who had been subject to mechanical periodontal therapy with or without adjunct metronidazole 5 years prior, no cultivable antibiotic resistant anaerobes were found in the predominant subgingival microbiota.

The aim of the present study was to evaluate the effectiveness of photodynamic therapy (PDT) on clinical gingival inflammatory parameters, bacterial load, proinflammatory cytokine status, and pain scores in adolescent patients undergoing fixed orthodontic treatment with gingivitis.Thirty adolescent undergoing fixed orthodontic treatment with gingivitis were randomly divided into two groups: Group A - patients undergoing dental scaling (DS) with adjunctive photodynamic therapy (PDT) and Group BDS alone. Clinical gingival parameters including plaque scores, (PS), bleeding on probing (BOP) and probing depth (PD) were assessed. Visual analogue scale (VAS) and McGill Pain Questionnaire (MPQ) were used to report the values of present pain intensity. Crevicular fluid was sampled to quantify the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α using enzyme-linked immunosorbent assay. The total bacterial counts for Porphyromonas gingivalis (P. gingivalis) and Tannerella forsythia (T. forsythia) were assessed. All examinations were performed at baseline, 1 week and 4 weeks.Both the treatment regimens reported statistically significant reduction in PS and BOP (p0.05). However, no significant difference was observed in PS and PD values when Group B was compared with Group A (p0.05). There was a statistically significant reduction noted for BOP in Group B when compared to Group A (p0.05). There was a statistically significant reduction in the microbial counts of T. forsythia in Group A when compared to Group B at week-1 (p0.05). During the 4-week follow-up, the counts for T. forsythia significantly increased. No significant changes could be seen in either of the pain scores in both the groups (p0.05). Both groups showed statistically significant reduction in the IL-6 and TNF-α levels. However, IL-6 was significantly reduced at 1 week, while TNF-a significantly reduced at 4 weeks of follow up among the participants in Group B.PDT has a positive effect in significantly reducing the periodontal microbial load in established gingivitis in adolescent patients undergoing fixed orthodontic treatment.

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R(2) and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively.

Chronic periodontitis has a polymicrobial biofilm aetiology. Polymicrobial biofilms are complex, dynamic microbial communities formed by two or more bacterial species that are important for the persistence and proliferation of participating microbes in the environment. Interspecies adherence, which often involves bacterial surface-associated molecules, and communications are essential in the spatial and temporal development of a polymicrobial biofilm, which in turn is necessary for the overall fitness of a well-organized multispecies biofilm community. In the oral cavity, interactions between key oral bacterial species, including Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, are essential for the progression of chronic periodontitis. In vivo, P. gingivalis and T. denticola are frequently found to co-exist in deep periodontal pockets and have been co-localized to the superficial layers of subgingival plaque as microcolony blooms adjacent to the pocket epithelium, suggesting possible interbacterial interactions that contribute towards disease. The motility and chemotactic ability of T. denticola, although not considered as classic virulence factors, are likely to be important in the synergistic biofilm formation with P. gingivalis. In vitro, P. gingivalis and T. denticola display a symbiotic relationship in nutrient utilization and growth promotion. Together these data suggest there is an intimate relationship between these two species that has evolved to enhance their survival and virulence.

Objectives The frequency of periodontitis among elderly is increasing in Japan. This study aimed to quantify the periodontitis-associated bacteria in subgingival plaques from elderly patients with periodontitis and from periodontally healthy subjects. Methods Subgingival plaque samples were collected from independent subjects (mean age=71.1 years, n =518). All samples were subjected to real-time PCR analysis to assess the presence of Porphyromonas gingivalis . In addition, we tested for Tannerella forsythia , Eubacterium saphenum and Streptococcus oralis in the earliest 95 samples; and performed fimA gene classification of P. gingivalis in the latest 49 samples. Results P. gingivalis and T. forsythia comprised a significantly higher proportion of total bacteria in subjects with periodontitis (1.1% and 5.1%) than periodontally healthy subjects (0.3% and 1.4%, respectively). The proportion of E. saphenum was low in both groups, whereas that of S. oralis was higher in periodontally healthy subjects. In 24 of 49 samples, fimA genotypes were detected and classified. Genotypes Ib ( n =5) and II ( n =7) were identified in those of subjects with periodontitis ( n =15); while those of healthy subjects ( n =9) were found to belong to genotypes I ( n =2), II ( n =2), III ( n =2) and IV ( n =3). In 4 out of the 5 subjects in whom P. gingivalis was detected at healthy sites, the fimA genotypes were identical between periodontitis and healthy sites, but the mean proportion of P. gingivalis was significantly higher at periodontitis sites (3.0%) than at healthy sites (0.5%) ( P Conclusions This study suggests that an increase in P. gingivalis and T. forsythia may be associated with periodontitis in the elderly, and we have identified characteristic pathogenic fimA genotypes that target this vulnerable group.

Periodontal disease initiated by subgingival pathogens is linked with diminished secretion of saliva, and implies pathogenic bacteria dissemination to or affects secondary sites such as the salivary glands. MicroRNAs activated in response to bacteria may modulate immune responses against pathogens. Therefore, Sprague-Dawley rats were infected by oral lavage consisting of polymicrobial inocula, namely Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, or sham-infected for 12 weeks (n = 6). We quantified inflammatory miRNA expression levels of miRNA-132, miR-146a, and miR-155 at secondary sites to the primary infection of the gingiva, including submandibular salivary glands, lacrimal glands, and pancreas. The presence of bacteria was detected in situ at secondary sites. Infected rat gingiva showed increased relative expression of miR-155. In contrast, miRNA-155 expression was decreased in submandibular salivary glands, along with positive identification of P. gingivalis in 2/6 and T. denticola in 1/6 rat salivary glands. Furthermore, miRNA-132 and miRNA-146a were significantly decreased in the pancreas of infected rats. This study is the first to show primary periodontal infections can alter miRNA profiles in secondary sites such as the salivary gland and pancreas. Whether these alterations contribute to pathologies of salivary glands in Sjögren's syndrome or of pancreas in diabetes warrants further investigation.

The aim of the present study was to quantify pathogenic bacteria isolated from the subgingival oral-biofilm samples collected from cigarette-smokers and ENDS-users with periodontitis, when compared to non-smokers with and without periodontitis.Demographic data was collected using a questionnaire. Periodontal parameters (plaque [PI] and gingival [GI] indices, clinical attachment loss [CAL], probing depth [PD] and marginal bone loss [MBL]) were measured. Subgingival oral bio-film samples were collected and assessed for periodontopathogenic bacteria (Aggregatibacter actinomycetemcomitans [A. actinomycetemcomitans], Prevotella intermedia [P. intermedia], Porphyromonas gingivalis [P. gingivalis], Tannerella forsythia [T. forsythia] and Treponema denticola [T. denticola]). Group-comparisons were performed; and P 0.01 were considered statistically significant.All cigarette-smokers, ENDS-users and non-smokers with periodontitis had Grade-B periodontitis. The CFU/mL of A. actinomycetemcomitans (P 0.001) and P. gingivalis (P 0.001) were significantly higher among cigarette-smokers (P 0.01) and ENDS-users (P 0.01) than non-smokers with periodontitis. The CFU/mL of T. denticola were significantly higher among cigarette-smokers (P 0.001), ENDS-users (P 0.001) and non-smokers with periodontitis (P 0.001) compared with non-smokers without periodontitis. There was no statistically significant difference in the CFU/mL of P. intermedia and T. denticola among cigarette-smokers, ENDS-users and non-smokers with periodontitis.Counts of periodontopathogenic bacteria in the subgingival oral-biofilm are comparable among cigarette-smokers and individuals using ENDS.

Aim We evaluated the efficacy of antimicrobial photodynamic therapy (A-PDT) in the reduction of pain intensity and performed cytokine profiling and measurement of microbiological parameters in patients with acute pericoronitis. Methods Sixty patients with age range 14–19 years were randomized into two groups: ‘A-PDT group’ which received additional A-PDT with conventional protocol, and ‘conventional protocol group’ (CPG) without A-PDT. Present pain intensity (PPI) were investigated using McGill Pain Questionnaire (MPQ) and Visual Analogue Scale (VAS). Gingival crevicular fluid were sampled to evaluate the levels of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) and quantified using enzyme-linked immunosorbent assay. Microbial plaque was collected to evaluate total counts of Porphyromonas gingivalis and Tannerella forsythia expressed as log colony forming unit (CFU)/mL. Level of pain intensity, cytokine profiling and microbial load were assessed at baseline (B0), and post treatment F7 (Day 7) and F14 (Day 14). Results A total of 59 patients (30 CPG and 29 A-PDT) completed the clinical trial. Present pain intensity scores statistically significantly reduced from B0 to F7 in both treatment groups (p 0.05). Only TNF-α showed statistically significant reduction with A-PDT compared to CPG at both F7 and F14. CPG group showed significant reduction for only T. forsythia at both follow-up points. The microbial counts for P. gingivalis and T. forsythia significantly reduced in A-PDT groups compared to CPG group at both F7 and F14. Conclusion The results of this study indicate that A-PDT may be a reliable treatment option in pericoronitis for the reduction of TNF-α and microbial load. However, A-PDT does not have any effect in the reduction of pain.

The aim of this study was designed to assess the peri-implant oral hygiene parameters, clinical, radiographic, host-derived immune biomarkers and microbiological levels after photochemotherapy (PCT) and local antibiotic therapy (LAT) in peri-implantitis lesions among cigarette smokers.Fifty current cigarette smokers with peri-implantitis were divided into two groups: PCT and LAT. Test implants received PCT that consisted of toluidine blue photosensitizer and application of 660 nm diode laser with a total of 100 mW power and 124.3 W/cmPlaque scores reduced in both groups (p 0.05). Mean BOP percentage significantly increased in both the groups at 1-month follow-up compared to baseline. Following this period, BOP showed reduction from 1-month to consecutive follow-up periods. The PD significantly reduced in both the groups with no statistically significant difference when compared between PCT and LAT groups at follow-up (p 0.05). CAL did not change over the period and between both groups (p 0.05). The differences from baseline to 12 months and between the groups for mesial and distal crestal bone levels did not show any statistical significance (p 0.05). The levels of IL-1β significantly dropped from baseline to 12 months in the LAT group (p 0.05). However, for PCT groups, the levels of IL-1β significantly reduced only at 12-month visit follow-up (p 0.05). MMP-1 showed statistically significant reduction at 9 and 12-months compared with baseline for LAT group. Both P. gingivalis and T. forsythia showed statistically significant reduction in both the groups when values were compared from baseline to 3-, 6- and 12-months follow-up (p 0.05). However, these differences were not significant when compared between the groups (p 0.05).Both PCT and LAT showed equal efficacies in improving clinical, host-derived immune biomarkers and microbiological parameters in peri-implant infection in cigarette smokers.

Objective To analyze the change of six periodontal pathogens around short locking-taper implants and adjacent teeth in patients with different periodontal conditions for three years. Methods Sixty implants and 62 adjacent teeth from 24 patients with different periodontal conditions were included: 5 patients with history of aggressive periodontitis (AgP group), 14 patients with history of chronic periodontitis (CP group), and 5 patients with healthy condition or slight gingivitis (H group). Subgingival samples were collected at five timepoints: before implant placement (T1); before second stage operation (T2); one month after restoration (T3); one year after functional loading (T4) and two years after functional loading (T5). Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans were detected by polymerase chain reaction (PCR). Results Pathogens were hardly found around implants or adjacent teeth until T4. The detection rates of five pathogens other than A. actinomycetemcomitans raised up from T3 to T5. F. nucleatum and P. gingivalis were mostly detected followed by P. intermedia, T. forsythia, and T. denticola. The detection rate of P. gingivalis in implants were higher than natural teeth. There was significant correlation between pathogenic bacteria from implants and adjacent teeth. A. actinomycetemcomitans were only detected positively in peri-implant sites of AgP group. Peri-implantitis sites showed significantly higher detection rates of T. denticola, F. nucleatum at T4, and P. gingivalis, F. nucleatum at T5 than peri-implant mucositis and healthy groups. Conclusion This three-year longitudinal study demonstrated that periodontal pathogens accumulate over time around short locking-taper implants and adjacent natural teeth after restoration. Adjacent teeth may become the microbial reservoir for peri-implant bacteria. Therefore, periodontally compromised patients may face higher risk for peri-implant disease. Clinical significance Plaque control of implant should be intensified with time instead of diminished. Patients with history of periodontitis need more frequent and individualized implant maintenance. Treatment and maintenance for adjacent teeth is as important as for implants..

Evidence indicates that patients with type 2 diabetes mellitus (DM) exhibit impaired wound healing and are at higher risk for periodontal disease. In DM patients, adjunctive periodontal treatment provides small but statistically significant benefits in terms of reductions in clinical periodontal parameters. This study incorporates antimicrobial and immune modulatory treatment approach specifically targeted at these patients.To evaluate and compare the clinical periodontal, microbiological and HbA1c levels with the use of photodynamic therapy (PDT) and probiotic therapy (PT) as adjunct to root surface debridement (RSD) in the treatment of periodontitis in DM.Demographic data was collected using a questionnaire. Treatment-wise, chronic periodontitis with 2DM patients were subdivided into: (i) Group-A: Patients that underwent RSD with adjunct PDT; (ii) Group-B: Patients that underwent RSD with adjunct PT and; (iii) Group-C: RSD alone. In all groups, probing depth (PD), plaque scores (PS), bleeding on probing (BOP) and clinical attachment level (CAL) gain were measured at baseline and 3 months. Microbiological data consisted the assessment of detection percentage of Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola.A total of 58 patients completed the trial. All clinical and microbiological parameters showed statistically significant reduction from baseline to 3 months in all groups (p 0.05). On inter-group comparison, the proportion of PD with ≥4 mm and ≥5 mm showed statistically significant reduction for Group-A compared to Group-B and Group-C (p 0.01). Only Group-A showed statistically significant reduction in percentage of HbA1c levels from baseline to 3 months (p 0.05). Groups A and B showed significantly higher reductions for all the three bacteria compared to Group-C (p 0.05). However, this reduction was comparable between Groups-A and B, respectively (p 0.05).PDT showed additional benefit in deep periodontal pockets and slightly modest reduction in HbA1c levels in DM patients. Further clinical trials are required with large sample size and longer follow up duration to ascertain the findings of the present clinical study.

To evaluate clinical periodontal and microbiological parameters after the treatment with adjunctive antimicrobial photodynamic therapy (APDT) among HIV seropositive and seronegative patients with necrotizing ulcerative periodontitis (NUP).Seropositive patients (Group-I) and health controls (Group-II) with NUP were randomized into two groups and further underwent APDT and scaling and root planing (SRP), respectively. Clinical periodontal parameters including full mouth plaque index (FMPI), bleeding on probing (FMBOP), probing depth (PD) and clinical attachment level (CAL) gain were assessed. Levels of bacteria including Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf) were assessed using PCR technique. All assessments were done at baseline, 3 months and 6 months.All periodontal parameters including FMPI, FMBOP, PD and CAL significantly improved in both HIV seropositive and seronegative patients. The reduction in mean PD was higher only with the APDT treatment among group II patients compared to group I patients at follow-up (p 0.05). The gain in mean CAL was higher only with the APDT treatment among group I and group II patients at follow-up (p 0.05). All bacterial levels reduced from baseline to follow-up with both APDT and SRP treatments in both groups (p 0.05). APDT showed significantly reduced counts of Aa and Tf at 3 months, and only Aa at 6 months among HIV seropositive patients, while the levels of Pg and Tf significantly reduced at 3 months and only Aa at 6 months in the seronegative patients, respectively (p 0.05).Application of antimicrobial photodynamic therapy as an adjunct to scaling and root planing was effective in improving clinical periodontal parameters and bacterial levels among HIV positive patients with NUP. However, the improvement was not greater when compared with HIV seronegative patients.

BackgroundUnique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed.MethodsLipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI–MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium.ResultsT. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2.ConclusionThe study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol.General significanceThe effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets.

Objective To date, no information on the distribution of periodontal microorganisms among family members of Chinese patients with aggressive peridontitis (AgP) is available. The aim of the present study was to investigate the probability of transmission of eight periodontal microorganisms between patients with aggressive periodontitis and their family members. Design Saliva and pooled subgingival plaque samples were collected from 103 participants from 41 nuclear families (including 41 AgP probands, 19 mothers, 22 fathers, 21 siblings). Eight periodontal microorganisms, including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum were detected in these samples by the polymerase chain reaction (PCR). In addition, the distribution of fimA genotypes was assessed in P. gingivalis-positive individuals by PCR. Results P. gingivalis, T. forsythia, T. denticola, C. rectus and F. nucleatum were the most frequently detected species both in AgP probands and in their relatives. Kappa statistical analysis revealed that the detection of A. actinomycetemcomitans (Kappa = 0.503) and F. nucleatum (Kappa = 0.565) in probands was highly consistent with that in their relatives. Most probands shared the identical fimA genotype of P. gingivalis with their relatives. Conclusions Our results suggested that the intrafamilial transmission of periodontal microorganisms may occur between Chinese patients with aggressive periodontitis and their relatives.

All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10(4) (cathepsin G) to 7.1 × 10(5) m(-1)s(-1) (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1' site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.

Objective: Previous studies have suggested positive associations between periodontal infection and cardiovascular disease. We aimed to investigate the associations of circulating antibodies against periodontal pathogens with 1-year cardiovascular outcome, as well as the extent of coronary atherosclerosis, plaque vulnerability and lesion remodeling on intravascular ultrasound (IVUS) imaging. Methods: Between 2008 and 2011, radiofrequency IVUS imaging of a non-culprit coronary artery was performed in 581 patients who underwent coronary angiography. Immunoglobulin G (IgG) and A (IgA) against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Prevotella intermedia were measured in plasma. Results: None of the antibody levels were associated with coronary plaque burden, radiofreqeuncy-IVUS-derived thin-cap fibroatheroma lesion morphology or 1-year incidence of major adverse cardiac events (MACE), which included all-cause mortality, acute coronary syndrome and unplanned coronary revascularization. IgA against A. actinomycetemcomitans, T. forsythia and P. intermedia were inversely associated with extent of positive lesion remodeling (OR for highest versus lowest tertile 0.55, 95% CI 0.35-0.88, p = 0.012; 0.53, 95% CI 0.32-0.87, p = 0.012; and 0.64, 95% CI 0.40-1.02, p = 0.061, respectively). In diabetic patients specifically, IgG against P. gingivalis tended to be associated with coronary plaque burden (p = 0.080), while IgA against P. gingivalis tended to be associated with incident MACE (p = 0.060). Conclusion: Plasma IgG and IgA against major periodontal pathogens were not associated with the extent of coronary atherosclerosis (with the exception of a trend in diabetics) nor with coronary plaque vulnerability. IgA against periodontal pathogens were inversely associated with extent of coronary remodeling. Altogether, these results do not add evidence for a substantial role of systemic exposure to periodontal pathogens in coronary artery disease. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Objectives The aim of this study was to investigate the mandibular third molar pericoronitis flora by using real-time polymerase chain reaction (PCR). Materials and methods The quantitative values of Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia (Tf) were evaluated in comparison with the healthy third molar flora by using real time PCR. Results Aa, Cr, Pg, and Pi were not statistically significant but numerically higher than the pericoronitis group. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study revealed the strong relation between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times relative risk of pericoronitis as the individuals with an absence of Tf in their samples. Conclusion Tf plays an important role in the development of clinical symptoms related to pericoronitis.

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress in affected cats. Treatment methods are currently very limited. The aims of this study were to assess the feline innate immune response by investigating the levels of cytokine and Toll-like receptor (TLR) mRNAs in tissue biopsies of cats with and without FCGS, and to relate this to the presence or absence of putative oral pathogens identified previously within these cats. Mucosal biopsies were collected from 28 cats with FCGS and eight healthy cats. The levels of TLR (TLR2, TLR3, TLR4, TLR7, TLR9) and cytokine (IL-1β, IL-4, IL-6, IL-10, IL-12, TNF-α, IFN-γ) mRNA was determined using quantitative PCR. In the FCGS group a statistically significant increase was seen in TLR2, TLR7, TNF-α, IFN-γ, IL-1β and IL-6 mRNA levels compared to the healthy group. In cats where Tannerella forsythia was present, statistically significant increases were seen in TLR2, TLR4, TLR7, TLR9, TNF-α and IL-1β mRNA levels compared to cats where this putative pathogen was absent. Statistically significant increases in mRNA expression were also seen in cats harbouring feline calicivirus (FCV) (TLR2, IL-1β, IL-6, IFN-γ) and Porphyromonas circumdentaria (TLR2, TLR3) compared to cats where these putative pathogens were absent. Pasteurella multocida subsp. multocida and Pseudomonas sp. did not significantly alter the expression of any TLR or cytokine mRNAs when compared to animals who tested negative for these species, while cats colonised with P. multocida subsp. septica demonstrated a statistically significant reduction in the expression of TLR7, TNF-α and IFN-γ mRNAs compared to cats free of this species. The expression of mRNA for several TLRs and cytokines is elevated in FCGS. A positive correlation was observed between clinical disease severity and the presence of FCV (p=0.001; Rho=0.58). Although the number of cats harbouring T. forsythia was low by comparison, 80% of samples in which it was present were from cases with the highest clinical disease severity. Positive correlations with clinical disease severity were seen for TLR2 (p=0.00086), TLR7 (p=0.049), TNF-α (p=0.027), IFN-γ (p=0.0015), IL-1β (p=0.004) and IL-6 (p=0.00001) mRNAs. The putative pathogens FCV and T. forsythia may be important in stimulating a host immune response to FCGS and may play a role in the pathogenesis of this disease.

Periodontitis is initiated by bacteria in subgingival biofilms, which are composed mostly of Gram-negative anaerobes. Autoinducer 2 (AI-2) is a universal quorum sensing (QS) molecule that mediates intergeneric signalling in multispecies bacterial communities and may induce biofilm formation. As Fusobacterium nucleatum is the major coaggregation bridge organism that links early colonising commensals and late pathogenic colonisers in dental biofilms via the accretion of periodontopathogens, we hypothesised that AI-2 of F. nucleatum contributes to this interspecies interaction, and interruption of this signalling could result in the inhibition of biofilm formation of periodontopathogens. To test this hypothesis, we evaluated the effect of partially purified F. nucleatum AI-2 on monospecies biofilm as well as mutualistic interactions between F. nucleatum and the so-called 'red complex' (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia). Then we tested the effect of two QS inhibitors (QSIs), (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (furanone compound) and d-ribose, on AI-2-induced biofilm formation and coaggregation. F. nucleatum AI-2 remarkably induced biofilm growth of single and dual species and coaggregation between F. nucleatum and each species of the 'red complex', all of which were inhibited by the QSIs. F. nucleatum AI-2 induced the expression of the representative adhesion molecules of the periodontopathogens, which were inhibited by the QSIs. Our results demonstrate that F. nucleatum AI-2 plays an important role in inter- and intraspecies interactions between periodontopathogens via enhanced expression of adhesion molecules and may be a target for the inhibition of pathogenic dental biofilm formation.

Objective Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. Design Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis , Porphyromonas gulae , Treponema denticola , Tannerella forsythia , Capnocytophaga ochracea , Capnocytophaga sputigena , Prevotella intermedia , Prevotella nigrescens , Aggregatibacter actinomycetemcomitans , Campylobacter rectus , and Eikenella corrodens were performed. Results P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. Conclusions These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

Objective The aim of this study was to investigate the association between angiographically verified coronary artery disease (CAD) and salivary levels of four major periodontal pathogens. Methods The study population ( n = 492) was composed of 179 (36.4%) patients with stable CAD, 166 (33.7%) with acute coronary syndrome (ACS), and 119 (24.2%) showing no pathological findings by coronary angiography. All patients were subjected to a detailed oral health examination. The saliva samples were analyzed for lipopolysaccharide activity as well as for Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , Prevotella intermedia , and Tannerella forsythia by quantitative PCR. Serum antibodies levels against A. actinomycetemcomitans were analyzed. Results The level of bacterial burden was linearly associated with alveolar bone loss ( p p = 0.015). The median salivary levels of A. actinomycetemcomitans in pathogen-positive patients were significantly higher in the “Stable CAD” ( p = 0.014) and the “ACS” ( p = 0.044) groups when compared to “No significant CAD” patients. In logistic regression models, a 10-fold increase in the salivary A. actinomycetemcomitans levels was associated with a risk for stable CAD and ACS with odds ratios (ORs) of 7.47 (95% confidence interval [CI]: 1.57–35.5, p = 0.012) and 4.31 (95% CI: 1.06–17.5, p = 0.041), respectively. The OR for the association of IgA-class antibody levels against A. actinomycetemcomitans with ACS risk was 3.13 (95% CI: 1.38–7.12, p = 0.006)/log 10 unit increase. Conclusions High salivary levels of A. actinomycetemcomitans and systemic exposure to the bacterium were associated with increased risk for CAD. These findings emphasize the importance of oral microbiota in cardiovascular risk assessment and therapeutics.

This present study evaluated the subgingival microbiota of the Cebus apella with different periodontal conditions kept by the Tufted Capuchin Monkey Procreation Center (São Paulo State University - UNESP) or free-ranging monkeys. For this purpose, clinical specimens of subgingival biofilm were collected from 52 monkeys, of both genders, 40 kept in captivity and 12 free-ranging monkeys. The primates were submitted to periodontal evaluation and biofilm samples were transferred to VMGA III transport medium and ultrapure water. The microbiota was cultivated in selective and non-selective culture media and microbial DNA was extracted and the presence of periodontal pathogens was evaluated using PCR and real-time PCR. The actinomycetes, fusobacteria, Campylobacter rectus, Eikenella corrodens, black-pigmented Gram-negative anaerobic rods, Tannerella forsythia, staphylococci and streptococci represent the predominantly detected microorganisms. Aggregatibacter actinomycetemcomitans, Dialister pneumosintes and Prevotella nigrescens were rarely observed, whereas Treponema denticola was not found. Populations of C. rectus, E. corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, T. forsythia and the total microbial load were significantly higher in animals with bone loss and, in smaller extension, in animals with gingival bleeding.

The bovine-casein-derived peptide κ-casein (109–137) has been shown to inhibit the major proteases of the periodontal pathogen Porphyromonas gingivalis. The casein peptides αS1-casein (11–23), β-casein (193–209), κ-casein (106–169) and κ-casein (109–137) were analysed for their inhibitory activity against the whole cell proteolytic activity of the three bacterial pathogens highly associated with chronic periodontitis, P. gingivalis, Treponema denticola and Tannerella forsythia. The peptides β-casein (193–209) and κ-casein (106–169) inhibited the major proteolytic activity of all three pathogens, κ-casein (109–137) inhibited P. gingivalis and T. denticola proteolytic activities while αS1-casein (11–23) only inhibited proteolytic activity associated with P. gingivalis. The peptides β-casein (193–209) and κ-casein (106–169) exhibited synergism with Zn2+ in the inhibition of the P. gingivalis Arg- and Lys-specific proteases.

Objective Along with conventional non-surgical periodontal therapy (NSPT) systemic antimicrobials may provide more effective treatment for chronic periodontitis by targeting tissue-invasive bacteria. The aim of this randomized, placebo-controlled, double-masked clinical trial was to evaluate the adjunctive effects of oral clarithromycin (CLM) to non-surgical periodontal therapy for chronic periodontitis. Methods 40 patients were categorized into two groups: test group – scaling and root planing (SRP) plus CLM (500 mg b.i.d. for 3 days) and control group – SRP plus placebo. Clinical parameters were recorded at baseline and at 1, 3, 6, and 9 months. They included gingival index (GI), probing depth (PD), and clinical attachment level (CAL). Also microbial analysis of dental plaque was done at baseline, 3 and 9 months to estimate the levels of periodontopathic organisms using polymerase chain reaction. ANOVA, Chi-square and Bonferroni tests were used for statistical analysis. Results Thirty-seven subjects completed the study and the results demonstrated that both groups displayed clinical improvements. Using a subject-based analysis, patients treated with SRP + CLM showed enhanced reductions in PD and gains in CAL (p

Objectives The aims of this study were to evaluate periodontal conditions and identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia, and four different species of Candida (C. albicans, C. dubliniensis, C. glabrata and C. tropicalis) in periodontal pockets and furcation sites of insulin-dependent type 2 diabetic and non-diabetic patients with generalised chronic periodontitis. Design Clinical parameters, including oral status assessed using plaque index, gingival index, probing depth, gingival recession and clinical attachment level and systemic conditions with fasting glucose level or glycosylated haemoglobin were measured in diabetic and non-diabetic patients with chronic periodontitis. Samples of subgingival biofilm were obtained from the periodontal pockets and furcation sites and submitted to phenol-chloroform DNA extraction and PCR analysis using specific primers. Results Clinical conditions of diabetic and non-diabetic patients were similar, without statistical differences in both periodontal indexes and glucose levels (p > 0.05). Diabetics had a higher prevalence of Candida spp., mainly C. albicans and C. dubliniensis, and a lower frequency of T. forsythia, when compared to non-diabetic patients, for both periodontal sites. C. glabrata and C. tropicalis were not found in periodontal pockets and furcation sites of non-diabetic patients. Conclusion The results demonstrated a strong colonisation of Candida spp. in the periodontal sites of diabetic patients that have generalised chronic periodontitis with a higher prevalence of C. dubliniensis followed by C. albicans.

Objective In this study of patients with chronic periodontitis (CP), the severity of the disease and the main periodontal pathogens identified in patients with chronic kidney disease (CKD) were compared with those detected in individuals without systemic disease. Design Nineteen patients with CP without evidence of systemic disease (control group), 25 patients with CP and CKD who were in the pre-dialysis stages (pre-dialysis group), and 22 patients with CP and CKD who were on renal replacement therapy (RRT group) were examined. The severity of CP was based on the investigation of probing depth (PD) and clinical attachment level (CAL). The definition and stage of CKD were based on the criteria proposed by the Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation. Glomerular filtration rate (GFR) was estimated using the equation of Modification of Diet in Renal Disease and the identification of microorganisms in subgingival plaque was performed using polymerase chain reaction (PCR). Results Candida albicans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were more common in patients who were on RRT and pre-dialysis than in control subjects. CP was more severe in patients with CKD. A strong association was observed between the frequency of C. albicans (P = 0.056), P.gingivalis (P = 0.008), T. denticola (P = 0.013) and CAL, when CKD patients were compared with the control group. Conclusion CP is more severe and is associated with increased frequency of C. albicans, P. gingivalis, T. forsythia, and T. denticola in patients with CKD.

Introduction Epidemiological studies indicate an association between periodontitis and cardiovascular disease, but the underlying mechanisms are poorly understood. Vasodilator-stimulated phosphoprotein (VASP) in its phosphorylated form represents a regulator of platelet function and an indicator for the sensitivity of platelets towards physiologically relevant antagonists of platelet function. As platelets and their activation state play a central role in the development of cardiovascular disease, this study aimed to investigate the influence of periodontal disease and periodontal pathogens on intraplatelet VASP-phosphorylation and platelet function. Material and Methods Besides several markers of platelet activation, basal and PGE 1 induced intracellular VASP-phosphorylation were determined in platelets of periodontitis patients (n=26) and healthy donors (n=19). Furthermore, platelets from healthy donors were incubated with distinct periodontal pathogens and basal and PGE 1 induced VASP-phosphorylation was determined. Results Compared to controls, platelets of periodontitis patients showed a significant decrease in basal and PGE 1 induced VASP-phosphorylation. VASP-phosphorylation in platelets from periodontitis patients positive for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or Tannerella forsythia was significantly decreased compared to patients that were negative for these bacteria. Furthermore, VASP-phosphorylation in platelets isolated from healthy donors was affected by incubation with these periodontal pathogens. Conclusions Our results provide evidence that periodontitis interferes with VASP-phosphorylation in human platelets, presumably as a consequence of a direct effect of periodontitis-associated bacteria. Decreased basal and PGE 1 induced VASP-phosphorylation might represent a mechanism responsible for enhanced platelet activation in periodontitis.

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

Objective The red complex bacteria (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) have been implicated, either singly or in combination, in the development of various forms of periodontal diseases. The aim of this study was to investigate the presence of “red complex” in acute periradicular abscesses by real-time polymerase chain reaction (PCR) method. Study design Microbial samples were collected by aspiration from 32 cases diagnosed as acute periradicular abscess. DNA was extracted from the samples by using a QIAamp DNA mini-kit and analyzed with real-time PCR. Results At least 1 member of the red complex was found in 84% of the cases. In general T. denticola, P. gingivalis, and T. forsythia were detected in 65.6%, 43.7%, and 40.6% of the cases, respectively. Red complex was detected in 15.6% of samples taken from acute periradicular abscesses. Conclusions Our findings suggest that “red complex” can participate in the pathogenesis of acute periradicular abscesses.

Objective The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. Methods The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl 2 , and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. Results The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5 mM CaCl 2 was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N -ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. Conclusion The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.

In this study, we investigated the antibacterial activity of eight antimicrobial peptides (AMPs), comprising four human beta-defensins (HBDs), three human neutrophil defensins (HNPs) and the cathelicidin LL-37, against two representative periodontopathogens, Prevotella intermedia and Tannerella forsythia. The neutralising effect of these AMPs on expression of interleukin (IL)-1beta, IL-8 and intercellular adhesion molecule 1 (ICAM-1) induced by lipopolysaccharide (LPS) from P. intermedia and T. forsythia was also tested in THP-1 cells and human gingival fibroblasts. Prevotella intermedia was susceptible to HBD-3 and LL-37 but was resistant to HBD-1, HBD-2, HBD-4, HNP-1, HNP-2 and HNP-3 at concentrations up to 10microM. However, all of the AMPs except HNP-2 at 5microM significantly inhibited the expression of IL-1beta, IL-8 and ICAM-1 induced by P. intermedia LPS. Tannerella forsythia showed marked susceptibility to the AMPs tested in the following order: LL-37, HBD-3, HBD-2, HBD-1, HNP-1 and HBD-4. All of the AMPs except HNP-3 had significant neutralising effects on T. forsythia LPS activity. The AMPs showing LPS-neutralising activity inhibited LPS binding to the cells. These results suggest that AMPs may be considered as preventive and therapeutic agents against mixed bacterial infections such as periodontitis by eliminating the pathogens themselves as well as reducing the activity of LPS.

Introduction Orthodontic appliances hinder mechanical plaque control. In this study, we evaluated the effect of self-performed supragingival plaque removal with ultrasonic, electric, and manual toothbrushes on subgingival plaque composition in orthodontically banded molars. Methods Twenty-one patients wearing fixed orthodontic appliances were assigned to this single-blind crossover study. Samples of subgingival plaque were collected from banded molars, before and after each toothbrush usage period, for quantification of 22 bacterial species by the checkerboard DNA-DNA hybridization method. For each crossover, patients used a toothbrush for 30 days, followed by a washout period of 14 days. Results The prevalence of Tannerella forsythia decreased significantly after a month of electric brush usage. In the manual brush group, the prevalences of Selenomonas noxia, Streptococcus sanguinis, and Prevotella melaninogenica also decreased significantly. However, there were no significant differences in the prevalences and levels of bacteria after usage of the ultrasonic brush. Intergroup comparisons showed no statistical differences among the 3 brushes for the microbiologic parameters. Conclusions All 3 brushes generally reduced bacterial prevalences, and, although electric and manual toothbrushes showed some isolated significant variations, we found no superiority with any toothbrush type when used three times daily for 2 minutes on microbiologic parameters in orthodontically banded molars.

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P. gingivalis, T. forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P. gingivalis), bspA (T. forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, P. gingivalis, T. forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P. gingivalis and T. forsythia levels ranged from 5.65 10 6 to 1.20 10 2 and from 5.76 10 6 to 1.35 10 1 . T. forsythia was highly prevalent and numerous in the study groups, whereas P. gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P. gingivalis and T. forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009;35:1518‐1524)

Treponema denticola is a Gram-negative, motile, asaccharolytic, anaerobic spirochaete which along with Porphyromonas gingivalis and Tannerella forsythia has been shown to form a bacterial consortium called the Red Complex that is strongly associated with the clinical progression of chronic periodontitis. T. denticola was grown in continuous culture in a complex medium with a mean generation time of 15.75 h. Samples from two different membrane-enriched preparations and a cytoplasm-enriched preparation were separated by two-dimensional gel electrophoresis and the proteins identified by MALDI-TOF/TOF mass spectrometry. In total, 219 non-redundant proteins were identified including numerous virulence factors, lipoproteins, ABC transporter proteins and enzymes involved in the metabolism of nine different amino acids of which glycine seems to be of particular importance. Novel findings include the identification of several abundant peptide uptake systems, and the identification of three flagellar filament outer layer proteins. Two-dimensional Western blot analysis using sera from mice immunized with formalin-killed T. denticola cells suggested that Msp, PrcA, OppA, OppA10, MglB, TmpC and several flagellar filament proteins are antigenic.

Objective Saliva may become a potential source of contamination through vertical and horizontal transmissions as well as cross-infections. This study aims to use saliva as a screening tool to detect putative periodontal pathogens in a young population with fairly good oral hygiene. Materials and methods Stimulated saliva samples were obtained from 134 dental students (20.5 ± 1 years, range 18–22 years). Among those, 77 subjects also completed a periodontal examination including attachment loss, modified dental, gingival and plaque indices (AL, mDI, GI and PI). The test bacteria were identified using a 16S rRNA-based PCR detection method. Results One or more of the test bacteria was found in 67% of the subjects. Prevotella nigrescens was detected as single bacterium in 16% of the subjects followed by Treponema denticola (4%), Porphyromonas gingivalis (2%), Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans (1%) and Tannerella forsythia (1%). Two or more pathogens were detected in 42% of the subjects. Clinical examination revealed health with no attachment loss (AL) in 84% of the students. In no AL group, 38% of the students were pathogen free while this was 25% for students in localized AL group (p > 0.05). There was a statistically significant association between the detection of salivary periodontal pathogen in general and higher PI (p = 0.018) and GI (p = 0.043). Conclusion Within the limits of this study, it is possible to detect all six periodontal pathogens in the saliva of dental students. Although a correlation can be observed between the presence of salivary periodontal pathogen and clinical signs of inflammation such as plaque accumulation and gingival bleeding, detection of specific bacteria in saliva is not related to the presence of localized AL based on the presented study population.

Background Chronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1. Materials and methods J774.1 cells were incubated with and without bacteria for 24 h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1β and tumor necrosis factor-α. The cytokine concentrations were determined using a standard curve prepared for each assay. Results Mixed infection with P. gingivalis and either T. forsythia or T. denticola at 105 CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1β or tumor necrosis factor-α production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 105 CFU/ml of mixed bacteria. Conclusion These results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.

Introduction: Prophylactic programs to prevent dental biofilm accumulation must be implemented to minimize the risk for periodontal diseases in orthodontic patients. Therefore, we assessed the possible periodontal and microbiologic changes resulting from the use of 2 methods of orthodontic archwire ligation: elastomeric rings and steel ligatures. Methods: The following parameters were measured: plaque index, gingival bleeding index, probing depth, and biofilm samples from the maxillary second premolars and the mandibular lateral incisors were evaluated in 14 subjects without clinical signs of gingival inflammation before orthodontic appliance placement and after 6 months of treatment. Each orthodontic arch was fixed with elastomeric rings on 1 side of the midline, and steel ligatures were used on the opposite side. Polymerase chain reaction analysis was used to detect Porphyromonas gingivalis , Tannerella forsythia , Actinobacillus actinomycetemcomitans , Prevotella intermedia , and P nigrescens . Results: The elastomeric rings were associated with a higher score for plaque index and bleeding than steel ligatures, as well as many positive sites of T forsythia and P nigrescens ( P Conclusions: Elastomeric rings favored these 2 periodontopathogens and harmed gingival conditions.