Relationships Among Gingival Crevicular Fluid Biomarkers, Clinical Parameters of Periodontal Disease, and the Subgingival Microbiota

Background The objectives were to measure the levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy and periodontitis subjects in order to explore relations among these biomarkers, the subgingival microbiota, and clinical parameters of periodontal disease. Material and methods Clinical periodontal parameters were measured at 6 sites per tooth in 20 periodontitis and 20 periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesiobuccal aspect of every tooth. GCF levels of interleukin-1β (IL-1β), matrix metalloproteinase-8 (MMP-8) and IL-8 were measured using checkerboard immunoblotting and the levels of 40 bacterial taxa quantified using checkerboard DNA-DNA hybridization. A subset of “clinically healthy” (CH) sites from each group was analyzed separately. Significance of differences between groups was determined using the unpaired t-test or the Mann-Whitney test. Correlations among immunological, microbiological and clinical data were determined using the Spearman rank correlation coefficient. Results There were positive correlations among mean clinical parameters and mean levels of the 3 biomarkers and proportions of Orange and Red complex species (p\u3c0.05). CH sites from periodontitis subjects had higher levels of IL-1β and IL-8 and higher proportions of Orange and Red complex species (p\u3c0.05) than CH sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (p\u3c0.05), while Purple and Yellow complex species had negative correlations with IL-1β and IL-8 (p\u3c0.05). Conclusions CH sites from periodontitis subjects present higher levels of GCF biomarkers and periodontal pathogens than CH sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers

RNA-Oligonucleotide Quantification Technique (ROQT) for the Enumeration of Uncultivated Bacterial Species in Subgingival Biofilms

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 104 cells. Fusobacterium nucleatum ss polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples

Microbial Shifts During Dental Biofilm Re-Development in the Absence of Oral Hygiene in Periodontal Health and Disease

Aim to monitor microbial shifts during dental biofilm re-development Methods Supra and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects at baseline and immediately after tooth cleaning. Samples were taken again from 7 teeth in randomly selected quadrants during 1, 2, 4 and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Species counts were averaged within subjects at each time point. Significant differences in counts between healthy and periodontitis subjects were sought using the Mann-Whitney test. Results Total supra and subgingival counts were significantly higher in periodontitis on entry and reached or exceeded baseline values after day 2. Supragingival counts of Veillonella parvula, Fusobacterium nucleatum ss vincentii and Neisseria mucosa increased from 2 to 7 days. Subgingival counts were greater for Actinomyces, green and orange complex species. Significant differences between groups in supragingival counts occurred for 17 of 41 species at entry, 0 at day 7; for subgingival plaque these values were 39/41 taxa at entry, 17/41 at day 7. Conclusions Supragingival plaque re-development was similar in periodontitis and health, but subgingival species recolonization was more marked in periodontitis

Early Microbial Succession in Re-Developing Dental Biofilms in Periodontal Health and Disease

Objective To determine the order of bacterial species succession in re-developing supra and subgingival biofilms. Methods Supra and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from 7 teeth in randomly selected quadrants after 1, 2, 4 and 7 days of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. % DNA probe counts were averaged within subjects at each time point. Ecological succession was determined using a modified moving window analysis. Results Succession in supragingival biofilms from periodontitis and health was similar. At 1 day, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 days. At 4–7 days, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 days by Capnocytophaga sputigena and P. nigrescens. No significant increase in proportions of periodontal pathogens was observed in any of the clinical groups or locations. Conclusions There is a defined order in bacterial species succession in early supra and subgingival biofilm re-development after professional cleaning

Microbiota of Deciduous Endodontic Infections Analyzed by MDA and Checkerboard DNA-DNA Hybridization

Aims To evaluate the microbiota of endodontic infections in deciduous teeth by checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). Methodology Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interadicular bone resorption were collected and 32 were analyzed, with 3 individuals contributing 2 samples; these were MDA- amplified and analyzed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species; and the mean proportion of each bacterial taxon present across all samples were computed. Results The mean amount of DNA in the samples prior to amplification was 5.2 (± 4.7) ng and 6.1 (± 2.3) μg after MDA. The mean number of species detected per sample was 19 (± 4) (range: 3–66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. Conclusion Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens among the most prominent species detected

Comparison of Microbial Changes in Early Re-Developing Biofilms on Natural Teeth and Dentures

Background and objective Surfaces and fluids can affect oral bacterial colonization. The aim of this study was to compare re-developing biofilms on natural teeth and dentures. Methods Supragingival plaque samples were taken from 55 dentate subjects and the denture teeth of 62 edentulous subjects before and after professional cleaning. Also, samples from 7 “teeth” in randomly selected quadrants were collected after 1, 2, 4 and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Counts and proportions of 41 bacterial taxa were determined at each time point and significant differences were sought using the Mann-Whitney test. Ecological succession was determined using a modified moving window analysis. Results Mean total DNA probe counts were similar pre-cleaning but were higher in dentate subjects at all post-cleaning visits (pStreptococcus mitis, Streptococcus oralisand Streptococcus mutans, whereas dentate subjects had higher proportions of Tannerella forsythia, Selenomonas noxia and Neisseria mucosa. By 2 days, mean counts of all taxa were higher in natural teeth and most remained higher at 7 days (pS. mitis and S. oralis by 1 day. N. mucosa, Veillonella parvula and Eikenella corrodens increased in both groups but later in edentate samples. Conclusions “Mature” natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm re-development is more rapid and more complex on natural than denture teeth

Effects of Hypericum Perforatum, in a rodent model of periodontitis

<p>Abstract</p> <p>Background</p> <p><it>Hypericum perforatum </it>is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. In this study we evaluate the effect of <it>Hypericum perforatum </it>in animal model of periodontitis.</p> <p>Methods</p> <p>Periodontitis was induced in adult male Sprague-Dawley rats by placing a nylon thread ligature around the lower 1st molars. Hypericum perforatum was administered at the dose of 2 mg/kg os, daily for eight days. At day 8, the gingivomucosal tissue encircling the mandibular first molar was removed.</p> <p>Results</p> <p>Periodontitis in rats resulted in an inflammatory process characterized by edema, neutrophil infiltration and cytokine production that was followed by the recruitment of other inflammatory cells, production of a range of inflammatory mediators such as NF-κB and iNOS expression, the nitration of tyrosine residues and activation of the nuclear enzyme poly (ADP-ribose) polymerase; apoptosis and the degree of gingivomucosal tissues injury. We report here that Hypericum perforatum exerts potent anti-inflammatory effects significantly reducing all of the parameters of inflammation as described above.</p> <p>Conclusions</p> <p>Taken together, our results clearly demonstrate that treatment with Hypericum reduces the development of inflammation and tissue injury, events associated with periodontitis.</p

Effect of smoking on subgingival microflora of patients with periodontitis in Japan

<p>Abstract</p> <p>Background</p> <p>Smoking is a risk factor for periodontitis. To clarify the contribution of smoking to periodontitis, it is essential to assess the relationship between smoking and the subgingival microflora. The aim of this study was to gain an insight into the influence of smoking on the microflora of Japanese patients with periodontitis.</p> <p>Methods</p> <p>Sixty-seven Japanese patients with chronic periodontitis (19 to 83 years old, 23 women and 44 men) were enrolled in the present study. They consisted of 30 smokers and 37 non-smokers. Periodontal parameters including probing pocket depth (PPD) and bleeding on probing (BOP) and oral hygiene status were recorded. Detection of <it>Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum/periodonticum, Treponema denticola </it>and <it>Campylobacter rectus </it>in subgingival plaque samples was performed by polymerase chain reaction. Association between the detection of periodontopathic bacteria and smoking status was analyzed by multiple logistic regression analysis and chi-square test.</p> <p>Results</p> <p>A statistically significant association was found between having a PPD ≥ 4 mm and detection of <it>T. denticola, P. intermedia, T. forsythia</it>, or <it>C. rectus</it>, with odds ratios ranging from 2.17 to 3.54. A significant association was noted between BOP and the detection of <it>C. rectus </it>or <it>P. intermedia</it>, and smoking, with odds ratios ranging from 1.99 to 5.62. Prevalence of <it>C. rectus </it>was higher in smokers than non-smokers, whereas that of <it>A. actinomycetemcomitans </it>was lower in smokers.</p> <p>Conclusions</p> <p>Within limits, the analysis of the subgingival microbial flora in smokers and non-smokers with chronic periodontitis suggests a relevant association between smoking and colonization by the specific periodontal pathogens including <it>C. rectus</it>.</p

Correlation Network Analysis Applied to Complex Biofilm Communities

The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc)