Culture-independent identification of periodontitis-associated Porphyromonas and Tannerella populations by targeted molecular analysis

Abstract

Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections.

FIG. 1.

Phylogenetic tree based on 16S rRNA gene sequence comparisons over 898 aligned bases showing the relationship between members of the genera Porphyromonas and Tannerella and novel phylotype Porphyromonas P3. The tree was constructed by the neighbor-joining method following distance analysis of the aligned sequences. Numbers represent bootstrap values for each branch based on data for 100 trees; only estimates >50 are shown. The scale bar shows the number of nucleotide substitutions per site.

FIG. 2.

Proportions of T. forsythensis (a), P. endodontalis (b), P. catoniae (c), Tannerella BU63 (d), P. gingivalis (e), and Porphyromonas P3 (f) in plaque from shallow and deep pockets in patients with periodontitis and healthy controls. The horizontal lines indicate the medians.