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Infect Immun. May 1994; 62(5): 1889–1895.
PMCID: PMC186432

Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis.


To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes, including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.

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  • Amann R, Springer N, Ludwig W, Görtz HD, Schleifer KH. Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature. 1991 May 9;351(6322):161–164. [PubMed]
  • Amann RI, Krumholz L, Stahl DA. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. [PMC free article] [PubMed]
  • Armitage GC, Dickinson WR, Jenderseck RS, Levine SM, Chambers DW. Relationship between the percentage of subgingival spirochetes and the severity of periodontal disease. J Periodontol. 1982 Sep;53(9):550–556. [PubMed]
  • Chen K, Neimark H, Rumore P, Steinman CR. Broad range DNA probes for detecting and amplifying eubacterial nucleic acids. FEMS Microbiol Lett. 1989 Jan 1;48(1):19–24. [PubMed]
  • Chuba PJ, Pelz K, Krekeler G, de Isele TS, Göbel U. Synthetic oligodeoxynucleotide probes for the rapid detection of bacteria associated with human periodontitis. J Gen Microbiol. 1988 Jul;134(7):1931–1938. [PubMed]
  • DeLong EF, Wickham GS, Pace NR. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. [PubMed]
  • Dix K, Watanabe SM, McArdle S, Lee DI, Randolph C, Moncla B, Schwartz DE. Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria. J Clin Microbiol. 1990 Feb;28(2):319–323. [PMC free article] [PubMed]
  • Fuhrman JA, McCallum K, Davis AA. Novel major archaebacterial group from marine plankton. Nature. 1992 Mar 12;356(6365):148–149. [PubMed]
  • Gersdorf H, Meissner A, Pelz K, Krekeler G, Göbel UB. Identification of Bacteroides forsythus in subgingival plaque from patients with advanced periodontitis. J Clin Microbiol. 1993 Apr;31(4):941–946. [PMC free article] [PubMed]
  • Gersdorf H, Pelz K, Göbel UB. Fluorescence in situ hybridization for direct visualization of gram-negative anaerobes in subgingival plaque samples. FEMS Immunol Med Microbiol. 1993 Mar;6(2-3):109–114. [PubMed]
  • Giovannoni SJ, Britschgi TB, Moyer CL, Field KG. Genetic diversity in Sargasso Sea bacterioplankton. Nature. 1990 May 3;345(6270):60–63. [PubMed]
  • Giovannoni SJ, DeLong EF, Olsen GJ, Pace NR. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988 Feb;170(2):720–726. [PMC free article] [PubMed]
  • Göbel UB, Geiser A, Stanbridge EJ. Oligonucleotide probes complementary to variable regions of ribosomal RNA discriminate between Mycoplasma species. J Gen Microbiol. 1987 Jul;133(7):1969–1974. [PubMed]
  • Liesack W, Stackebrandt E. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol. 1992 Aug;174(15):5072–5078. [PMC free article] [PubMed]
  • Listgarten MA. Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study. J Periodontol. 1976 Jan;47(1):1–18. [PubMed]
  • Listgarten MA, Helldén L. Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J Clin Periodontol. 1978 May;5(2):115–132. [PubMed]
  • Loesche WJ. The role of spirochetes in periodontal disease. Adv Dent Res. 1988 Nov;2(2):275–283. [PubMed]
  • Lukehart SA, Tam MR, Hom J, Baker-Zander SA, Holmes KK, Nowinski RC. Characterization of monoclonal antibodies to Treponema pallidum. J Immunol. 1985 Jan;134(1):585–592. [PubMed]
  • Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene. 1988 Nov 30;71(2):491–499. [PubMed]
  • Meyerhans A, Vartanian JP, Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res. 1990 Apr 11;18(7):1687–1691. [PMC free article] [PubMed]
  • Moore WE. Microbiology of periodontal disease. J Periodontal Res. 1987 Sep;22(5):335–341. [PubMed]
  • Moore WE, Holdeman LV, Cato EP, Smibert RM, Burmeister JA, Ranney RR. Bacteriology of moderate (chronic) periodontitis in mature adult humans. Infect Immun. 1983 Nov;42(2):510–515. [PMC free article] [PubMed]
  • Moore WE, Holdeman LV, Smibert RM, Hash DE, Burmeister JA, Ranney RR. Bacteriology of severe periodontitis in young adult humans. Infect Immun. 1982 Dec;38(3):1137–1148. [PMC free article] [PubMed]
  • Olsen GJ. Microbial ecology. Variation among the masses. Nature. 1990 May 3;345(6270):20–20. [PubMed]
  • Olsen GJ, Overbeek R, Larsen N, Marsh TL, McCaughey MJ, Maciukenas MA, Kuan WM, Macke TJ, Xing Y, Woese CR. The Ribosomal Database Project. Nucleic Acids Res. 1992 May 11;20 (Suppl):2199–2200. [PMC free article] [PubMed]
  • Paster BJ, Dewhirst FE, Weisburg WG, Tordoff LA, Fraser GJ, Hespell RB, Stanton TB, Zablen L, Mandelco L, Woese CR. Phylogenetic analysis of the spirochetes. J Bacteriol. 1991 Oct;173(19):6101–6109. [PMC free article] [PubMed]
  • Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med. 1990 Dec 6;323(23):1573–1580. [PubMed]
  • Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the uncultured bacillus of Whipple's disease. N Engl J Med. 1992 Jul 30;327(5):293–301. [PubMed]
  • Riviere GR, Wagoner MA, Baker-Zander SA, Weisz KS, Adams DF, Simonson L, Lukehart SA. Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis. N Engl J Med. 1991 Aug 22;325(8):539–543. [PubMed]
  • Riviere GR, Weisz KS, Adams DF, Thomas DD. Pathogen-related oral spirochetes from dental plaque are invasive. Infect Immun. 1991 Oct;59(10):3377–3380. [PMC free article] [PubMed]
  • Riviere GR, Weisz KS, Simonson LG, Lukehart SA. Pathogen-related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis. Infect Immun. 1991 Aug;59(8):2653–2657. [PMC free article] [PubMed]
  • Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. [PubMed]
  • Schmidt TM, DeLong EF, Pace NR. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol. 1991 Jul;173(14):4371–4378. [PMC free article] [PubMed]
  • Slots J. Subgingival microflora and periodontal disease. J Clin Periodontol. 1979 Oct;6(5):351–382. [PubMed]
  • Söder PO, Frithiof L, Söder B. Spirochaetes and granulocytes at sites involved in periodontal disease. Arch Oral Biol. 1990;35 (Suppl):197S–200S. [PubMed]
  • Stackebrandt E, Liesack W, Goebel BM. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J. 1993 Jan;7(1):232–236. [PubMed]
  • Ward DM, Weller R, Bateson MM. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990 May 3;345(6270):63–65. [PubMed]
  • Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 Jan;173(2):697–703. [PMC free article] [PubMed]
  • Wilson KH, Blitchington R, Frothingham R, Wilson JA. Phylogeny of the Whipple's-disease-associated bacterium. Lancet. 1991 Aug 24;338(8765):474–475. [PubMed]
  • Woese CR. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. [PMC free article] [PubMed]

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