Project description:Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum, have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola, and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities. GO (gene ontology) term enrichment analysis showed up-regulation of transporters, pathogenicity related traits and hemin/heme uptake mechanisms for all three species in vivo. Differential gene expression analysis revealed that cysteine proteases, transporters and hemin/heme-binding proteins were highly up-regulated in the periodontal niche, while genes involved in DNA modification were down-regulated. The data suggest strong interactions between those three species regarding protein degradation, iron up-take, and mobility in vivo, explaining their enhanced synergistic pathogenicity. We discovered a strikingly high frequency of Single Nucleotide Polymorphisms (SNPs) in vivo. For F. nucleatum we discovered a total of 127,729 SNPs in periodontal niche transcripts, which were found in similar frequency in health and disease and covered the entire genome, suggesting continuous evolution in the host. We conclude that metabolic interactions shape gene expression in vivo. Great caution is required when inferring pathogenicity of microbes from laboratory data, and microdiversity is an important adaptive trait of natural communities.
Project description:Periodontal disease, the most prevalent infectious disease in the world, is caused by biofilms formed in periodontal pockets. No specific bacterial species that can cause periodontitis alone has been found in any study to date. Several periodontopathic bacteria are associated with the progress of periodontal disease. Consequently, it is hypothesized that dysbiosis of subgingival microbiota may be a cause of periodontal disease. This study aimed to investigate the relationship between the subgingival microbiota and the clinical status of periodontal pockets in a quantitative and clinically applicable way with the newly developed Oral Care Chip. The Oral Care Chip is a DNA microarray tool with improved quantitative performance, that can be used in combination with competitive PCR to quantitatively detect 17 species of subgingival bacteria. Cluster analysis based on the similarity of each bacterial quantity was performed on 204 subgingival plaque samples collected from periodontitis patients and healthy volunteers. A significant difference in the number of total bacteria, Treponema denticola, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus intermedia bacteria in any combination of the three clusters indicated that these bacteria gradually increased in number from the stage before the pocket depth deepened. Conversely, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Streptococcus constellatus, which had significant differences only in limited clusters, were thought to increase in number as the pocket depth deepened, after periodontal pocket formation. Furthermore, in clusters where healthy or mild periodontal disease sites were classified, there was no statistically significant difference in pocket depth, but the number of bacteria gradually increased from the stage before the pocket depth increased. This means that quantitative changes in these bacteria can be a predictor of the progress of periodontal tissue destruction, and this novel microbiological test using the Oral Care Chip could be effective at detecting dysbiosis.
Project description:Periodontal infection involves a complex interplay between oral biofilms, gingival tissues and cells of the immune system in a dynamic microenvironment. A humanized in vitro model that reduces the need for experimental animal models, while recapitulating key biological events in a periodontal pocket, would constitute a technical advancement in the study of periodontal disease. The aim of this study was to use a dynamic perfusion bioreactor in order to develop a gingival epithelial-fibroblast-monocyte organotypic co-culture on collagen sponges. An 11 species subgingival biofilm was used to challenge the generated tissue in the bioreactor for a period of 24 h. The histological and scanning electron microscopy analysis displayed an epithelial-like layer on the surface of the collagen sponge, supported by the underlying ingrowth of gingival fibroblasts, while monocytic cells were also found within the sponge mass. Bacterial quantification of the biofilm showed that in the presence of the organotypic tissue, the growth of selected biofilm species, especially Campylobacter rectus, Actinomyces oris, Streptococcus anginosus, Veillonella dispar, and Porphyromonas gingivalis, was suppressed, indicating a potential antimicrobial effect by the tissue. Multiplex immunoassay analysis of cytokine secretion showed that interleukin (IL)-1 ?, IL-2, IL-4, and tumor necrosis factor (TNF)-? levels in cell culture supernatants were significantly up-regulated in presence of the biofilm, indicating a positive inflammatory response of the organotypic tissue to the biofilm challenge. In conclusion, this novel host-biofilm interaction organotypic model might resemble the periodontal pocket and have an important impact on the study of periodontal infections, by minimizing the need for the use of experimental animal models.
Project description:Periodontitis is a progressive disease of the periodontium with a complex, polymicrobial etiology. Recent Next-Generation Sequencing (NGS) studies of the microbial diversity associated with periodontitis have revealed strong, community-level differences in bacterial assemblages associated with healthy or diseased periodontal sites. In this study, we used NGS approaches to characterize changes in periodontal pocket bacterial diversity after standard periodontal treatment. Despite consistent changes in the abundance of certain taxa in individuals whose condition improved with treatment, post-treatment samples retained the highest similarity to pre-treatment samples from the same individual. Deeper phylogenetic analysis of periodontal pathogen-containing genera Prevotella and Fusobacterium found both unexpected diversity and differential treatment response among species. Our results highlight how understanding interpersonal variability among microbiomes is necessary for determining how polymicrobial diseases respond to treatment and disturbance.
Project description:The periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues.A significant unbalance in protein expression between healthy and pathological sites was recorded. Thirty-two protein spots were overall identified, and four proteins (S100A9, HSPB1, LEG7 and 14-3-3) were selected for Western blot analysis of both periodontally-affected and healthy patients. The four selected proteins resulted over-expressed in periodontal pocket tissue when compared with the corresponding tissue of periodontally-healthy patients. The results of Western blot analysis are congruent with the defensive and the regenerative reaction of injured periodontal tissues.The proteomic analysis was performed for the first time directly on periodontal pocket tissue. The proteomic network highlighted in this study enhances the understanding of periodontal disease pathogenesis necessary for specific therapeutic strategies setting.
Project description:Periodontal abscess is an oral infective disease caused by various kinds of bacteria. We aimed to characterize the microbiota composition of periodontal abscesses by metagenomic methods and compare it to that of the corresponding pocket and healthy gingival crevice to investigate the specific bacteria associated with this disease. Samples from abscess pus (AB), periodontal pocket coronally above the abscess (PO), and the gingival crevice of the periodontal healthy tooth were obtained from 20 periodontal abscess patients. Furthermore, healthy gingival crevice samples were obtained from 25 healthy individuals. Bacterial DNA was extracted and 16S rRNA gene fragments were sequenced to characterize the microbiota and determine taxonomic classification. The beta-diversity analysis results showed that the AB and PO groups had similar compositions. Porphyromonas gingivalis, Prevotella intermedia, and other Prevotella spp. were the predominant bacteria of human periodontal abscesses. The abundances of Filifactor alocis and Atopobium rimae were significantly higher in periodontal abscesses than in the periodontal pocket, suggesting their association with periodontal abscess formation. In conclusion, we characterized the microbiota in periodontal abscess and identified some species that are positively associated with this disease. This provides a better understanding of the components of periodontal abscesses, which will help facilitate the development of antibiotic therapy strategies.
Project description:The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ~90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
Project description:INTRODUCTION:Periodontitis is a chronic inflammatory condition initiated by microorganisms and is positively linked to systemic conditions such as cancer, cardiovascular disease, and diabetes mellitus. OBJECTIVES:To prospectively investigate associations between empirically derived clusters of IgG antibodies against 19 selected periodontal microorganisms and cancer mortality in a representative sample of the US population. METHODS:We evaluated 6,491 participants aged ?40 y from the Third National Health and Nutrition Examination Survey (1988 to 1994), who had complete data on IgG antibody titers against 19 selected periodontal microorganisms and were free of cardiovascular disease and cancer. In a prior study, antibodies were categorized into 4 mutually exclusive groups via cluster analysis: red-green, orange-red, yellow-orange, and orange-blue. Cluster scores were estimated by summing z scores of the antibody titers making up each cluster. Participants were followed up to death until December 31, 2011. Cox proportional hazard models were applied to estimate hazard ratios (HRs) and 95% CIs for all-cancer mortality by tertiles of cluster scores. RESULTS:During follow-up for a median of 15.9 y, there were 2,702 deaths (31.3%), including 631 cancer-related deaths (8.1%). After adjusting for multiple confounders, the orange-blue cluster was inversely associated with cancer mortality (tertile 2 vs. tertile 1: HR = 0.67, 95% CI = 0.54 to 0.84; tertile 3 vs tertile 1: HR = 0.62, 95% CI = 0.46 to 0.84). The association between the yellow-orange cluster and all-cancer mortality was also inverse but not significant, and the orange-red cluster and the red-green cluster were not associated with all-cancer mortality. CONCLUSIONS:Antibodies against Eubacterium nodatum and Actinomyces naeslundii may be novel predictors of cancer mortality. If further studies establish a causal relationship between these antibodies and cancer mortality, they could be targets to prevent possible systemic effects of periodontal disease with potential interventions to raise their levels. KNOWLEDGE TRANSFER STATEMENT:Periodontal antibodies against Eubacterium nodatum and Actinomyces naeslundii were inversely associated with cancer mortality among adults followed up for an average of 16 y. Periodontal antibodies may predict cancer mortality.
Project description:Objectives:To improve understanding of periodontitis pathology, we need more profound knowledge of relative abundances of single prokaryotic species and colonization dynamics between habitats. Thus, we quantified oral microbes from two oral habitats to gain insights into colonization variability and correlation to the clinical periodontal status. Methods:We analyzed tongue scrapings and subgingival pocket samples from 237 subjects (35-54 years) with at least 10 teeth and no recent periodontal treatment from the 11-year follow-up of the Study of Health in Pomerania. Relative abundances of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus sanguinis, total bacteria, and Archaea were correlated to clinically assessed pocket depths (PD) and clinical attachment levels (CAL). Results:Increased relative abundances of P. gingivalis, A. actinomycetemcomitans, and F. nucleatum were linked to increased levels of PD and CAL (i) on the subject level (mean PD, mean CAL) and (ii) in subgingival pockets. Relative abundances of Archaea from tongue samples correlated negatively with mean PD or mean CAL. Detection and quantity of bacterial species correlated weakly to moderately between the tongue and subgingival pocket, except for Archaea. Conclusions:Relative abundances of specific oral species correlated weakly to moderately between habitats. Single species, total bacteria, and Archaea were linked to clinically assessed severity of periodontitis in a habitat-dependent manner.
Project description:To quantify the digit preference effect for three manual periodontal probes and to calculate correction values to enable comparison of studies with equal recording protocols, but different periodontal probes.A prospective in vivo crossover study was conducted with a six-sequence three-period design. Six examiners assessed attachment loss (AL), probing pocket depth (PD) and gingiva height (GH) at four surfaces, full-mouth, in six generally healthy subjects using three manual probes: PCP11 (3-3-3-2 mm increments), PCP2 (2 mm increments), and PCPUNC15 (1 mm increments).Distributions of AL, PD and GH differed between probes (p < 0.001). Compared with PCPUNC15, periodontal measurements coinciding with probe markings of PCP11 and PCP2, respectively, were preferentially named by examiners. Digit preference was most pronounced for PD, but less for AL and GH. In multilevel models, PD differed significantly between all three probes (p < 0.05); probe- and examiner-related effects were also observed for AL and GH. Correction values for pairwise combinations of probes were determined.We provided empirical evidence and quantified the effect of probe type on periodontal measurements. Differences in probe type should be considered when comparing periodontal data within and between epidemiological studies and appropriate corrections, provided here, should be applied.