Phylogenetic analysis of pathogen-related oral spirochetes

Recently, Riviere et al. reported as yet uncultivable invasive oral spirochetes that cross-reacted with monoclonal antibodies (MAbs) specific for Treponema pallidum (G. R. Riviere, K. S. Elliot, D. F. Adams, L. G. Simonson, L. B. Forgas, A. M. Nilius, and S. A. Lukehart, J. Periodontol. 63:131-136, 1992; G. R. Riviere, M. A. Wagoner, S. A. Baker-Zander, K. S. Weisz, D. F. Adams, L. Simonson, and S. A. Lukehart, N. Engl. J. Med. 325:539-543, 1991; G. R. Riviere, K. S. Weisz, D. F. Adams, and D. D. Thomas, Infect. Immun. 59:3377-3380, 1991; G. R. Riviere, K. S. Weisz, L. G. Simonson, and S. A. Lukehart, Infect. Immun. 59:2653-2657, 1991). In an attempt to phylogenetically analyze these pathogen-related oral spirochetes, we used immunomagnetic separation, combined with comparative sequence analysis of 16S rRNA genes amplified in vitro by the PCR. The bacteria were immunomagnetically enriched from subgingival plaque samples of patients with rapidly progressive periodontitis by using MAb H9-2 specific for the 37-kDa endoflagellum sheath protein of T. pallidum. After PCR amplification with universal eubacterial primers 16S rRNA gene fragments were cloned into Escherichia coli. A total of 20 randomly selected recombinants were analyzed by sequencing about 200 to 300 bases of the 500-bp inserts. All the spirochetal 16S rRNA sequences clustered to previously described, as yet uncultivable cluster 7 treponemes of group I (B. K. Choi, B. J. Paster, F. E. Dewhirst, and U. B. Göbel, Infect. Immun. 62:1889-1895, 1994). With a sequence similarity of 96.4% the most closely related cultivable treponeme was Treponema vincentii, which also belongs to the group I treponemes. Subsequent immunological analysis of cultured treponemes with MAb H9-2 revealed that only T. vincentii strains showed specific immunofluorescence or a characteristic 37-kDa band in immunoblots. We therefore conclude that pathogen-related oral spirochetes constitute a heterogeneous population of treponemes comprising T. vincentii and T. vincentii-related organisms that have common epitopes cross-reacting with MAb H9-2.