Identification of Candidate Periodontal Pathogens and Beneficial Species by Quantitative 16S Clonal Analysis

Abstract

Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted DNA approaches, and so it is likely that pathogens remain undiscovered. The purpose of this study was to use culture-independent, quantitative analysis of biofilms associated with chronic periodontitis and periodontal health to identify pathogens and beneficial species. Samples from subjects with periodontitis and controls were analyzed using ribosomal 16S cloning and sequencing. Several genera, many of them uncultivated, were associated with periodontitis, the most numerous of which were gram positive, including Peptostreptococcus and Filifactor. The genera Megasphaera and Desulfobulbus were elevated in periodontitis, and the levels of several species or phylotypes of Campylobacter, Selenomonas, Deferribacteres, Dialister, Catonella, Tannerella, Streptococcus, Atopobium, Eubacterium, and Treponema were elevated in disease. Streptococcus and Veillonella spp. were found in high numbers in all samples and accounted for a significantly greater fraction of the microbial community in healthy subjects than in those with periodontitis. The microbial profile of periodontal health also included the less-abundant genera Campylobacter, Abiotrophia, Gemella, Capnocytophaga, and Neisseria. These newly identified candidates outnumbered Porphyromonas gingivalis and other species previously implicated as periodontopathogens, and it is not clear if newly identified and more numerous species may play a more important role in pathogenesis. Finally, more differences were found in the bacterial profile between subjects with periodontitis and healthy subjects than between deep and shallow sites within the same subject. This suggests that chronic periodontitis is the result of a global perturbation of the oral bacterial ecology rather than a disease-site specific microbial shift.There is considerable evidence to show that bacterial plaque is the etiologic agent in chronic periodontitis. No single species has been implicated as a primary pathogen, and the available evidence is consistent with a polymicrobial disease etiology. Nearly all studies on the bacterial etiology of periodontitis have used either culture-based or directed DNA approaches, targeting known species. The prevailing paradigm that implicates minor constituents of the subgingival community, the gram-negative bacteria Porphyromonas gingivalis, Tanerella forsythensis, and Treponema denticola (24), as periodontopathogens is based on such approaches. However, culturing is not representative of the composition of a microbial community, since it is often too selective, especially for fastidious and as-yet-uncultivable species. Even culture-independent targeted approaches are limited to detecting the presence and levels of known species. Obviously cultivation will not detect uncultivated species, but the limitations of closed-ended molecular approaches such as PCR or hybridization assays such as checkerboard and microarrays are not as widely appreciated. Using these approaches it...

Response of Subgingival Bacteria to Smoking Cessation

“…There is growing evidence from molecular investigations, including the present study, that members of the genus Veillonella form a major component of the subgingival microbiome in periodontal health (1,13,14). This ability of Veillonella to saturate the subgingival niche could play an important role in preventing colonization by pathogens and deserves further investigation as an indicator of clinical health.…”

“…The subgingival microbiome is complex, with several uncultivated and as-yet-unrecognized members (13,17), and hence, cultivation-based approaches have not been able to comprehensively examine the effects of smoking cessation on this ecosystem. We have previously reported that smoking cessation alters subgingival microbial profiles, determined using a comprehensive, open-ended molecular approach (7).…”

“…The reverse primer was modified to contain a 5Ј biotin, 12-carbon linker and five phosphorothioate bonds (Integrated DNA Technologies, Skokie, IL). The cycling conditions and protocols have been described previously (13). Our previous experiments using 16S rRNA cloning and sequencing have provided us with a clone library of 16S rRNA genes (1,500 bp) from cultivated and as-yet-uncultivated organisms.…”

A study of the control of oral plaque biofilms via antibacterial photodynamic therapy

“…Dental plaque is a biofilm, consisting of a diversity of co-aggregated bacteria (Al Ahmad et al, 2007;Dige et al, 2007). The commensal bacteria of dental plaque microflora (early colonisation) are important for the preservation of oral health and avoidance of oral disease (Socranscy et al, 2002;Kumar et al, 2005). Ecological shifts of commensal dental microflora (colonisation of early colonisers by late colonisers) due to environmental perturbations, such as excessive sugar intake, plaque over accumulation, can lead from dental health to caries and periodontal diseases (Allaker and Douglas, 2009).…”

Dysbiosis and Alterations in Predicted Functions of the Subgingival Microbiome in Chronic Periodontitis

“…Recently, culture-independent molecular methods targeting small-subunit rRNA sequences (16S rRNA) in combination with high-throughput DNA sequencing have emerged as a popular approach to profile microbial flora from diverse sites of the human body. By amplification and deep sequencing of 16S rRNA gene segments, this approach has provided an unprecedented view of human-associated microbial communities and has uncovered novel or previously unrecognized microbes associated with health and disease (16,(28)(29)(30)(31). The Human Microbiome Project (HMP) data set is currently the largest reference data set (143 individuals) for the subgingival microbiome (32).…”