The aetiology of periodontal disease has been a field of intensive research in the past decades. Along with a variety of other putative pathogens, different members of the genus Selenomonas have repeatedly been associated with both generalized aggressive periodontitis and chronic periodontitis. For the present study, a specific oligonucleotide probe targeting the majority of all oral Selenomonas spp. was designed. Their prevalence was determined, using dot-blot hybridization, in a total of 742 subgingival samples collected from patients with generalized aggressive (n=62) and chronic periodontitis (n=82), and from periodontitis-resistant subjects (n=19). In addition, fluorescence in situ hybridization (FISH) and electron microscopy were performed to analyze the spatial arrangement of Selenomonas in subgingival biofilms collected from patients with generalized aggressive periodontitis. In the samples from patients, Selenomonas spp. showed a lower prevalence in both diseased groups compared with other putative pathogens, and a relatively high prevalence in the periodontitis-resistant group. Consequently, Selenomonas spp. do not seem to be suitable diagnostic marker organisms for periodontal disease. By contrast, FISH and electron microscopic analysis of periodontal carriers revealed that Selenomonas spp. appeared in large numbers in all parts of the collected biofilms and seemed, if present in a site from patients, to make a relevant contribution to their structural organization.
Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.
Periodontitis is one of the most common chronic inflammatory diseases. A number of putative bacterial pathogens have been associated with the disease and are used as diagnostic markers. In the present study, we compared the prevalence of oral bacterial species in the subgingival biofilm of generalized aggressive periodontitis (GAP) (n = 44) and chronic periodontitis (CP) (n = 46) patients with that of a periodontitis-resistant control group (PR) (n = 21). The control group consisted of subjects at least 65 years of age with only minimal or no periodontitis and no history of periodontal treatment. A total of 555 samples from 111 subjects were included in this study. The samples were analyzed by PCR of 16S rRNA gene fragments and subsequent dot blot hybridization using oligonucleotide probes specific for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, a Treponema denticola-like phylogroup (Treponema phylogroup II), Treponema lecithinolyticum, Campylobacter rectus, Fusobacterium spp., and Fusobacterium nucleatum, as well as Capnocytophaga ochracea. Our data confirm a high prevalence of the putative periodontal pathogens P. gingivalis, P. intermedia, and T. forsythia in the periodontitis groups. However, these species were also frequently detected in the PR group. For most of the species tested, the prevalence was more associated with increased probing depth than with the subject group. T. lecithinolyticum was the only periodontopathogenic species showing significant differences both between GAP and CP patients and between GAP patients and PR subjects. C. ochracea was associated with the PR subjects, regardless of the probing depth. These results indicate that T. lecithinolyticum may be a diagnostic marker for GAP and C. ochracea for periodontal health. They also suggest that current presumptions of the association of specific bacteria with periodontal health and disease require further evaluation.
The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.
The role of oral bacterial infections including periodontal disease in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Among the major periodontal pathogens, Porphyromonas gingivalis has been mostly associated with RA pathogenesis. The aim of this study was to analyze the effect of P. gingivalis total lipid (TL) fraction and dihydroceramides, as potent virulence factors, on human primary chondrocytes. Primary chondrocyte cultures were incubated with P. gingivalis phosphoglycerol dihydroceramide (PG DHC) lipids, the TL fraction or phosphoethanolamine dihydroceramide. Cell morphology changes were determined by phase contrast light microscopy. Early and late apoptosis cell analysis was performed by Annexin-V, active caspases, and 7-Aminoactinomycin D staining, and examined by flow cytometry, and cell necrosis was evaluated by lactate dehydrogenase release. Procaspase-3 activation was determined by Western blot analysis. Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with P. gingivalis TLs and PG DHC lipids. Flow cytometry demonstrated an increase of Annexin-V positive and active caspases positive chondrocytes after incubation with TL and PG DHC fractions but not after phosphoethanolamine dihydroceramide (control lipid) treatment or in untreated control cells. Furthermore, Western blot analysis showed an early cleavage of procaspase-3 after 1 hr. Significant lactate dehydrogenase release following incubation with P. gingivalis lipids was demonstrated. The present data demonstrate that P. gingivalis lipids promote apoptosis in primary human chondrocytes, and thereby may contribute to the joint damage seen in the pathogenesis of RA.
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