Molecular epidemiology of oral treponemes associated with periodontal disease

Abstract

Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone. Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role. Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable. To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. Subgingival plaque specimens taken from diseased sites (n = 200) and healthy control sites (n = 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. Spirochetes were found in all patients, but their distributions varied considerably. Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency. These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated. All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g., Treponema maltophilum, or uncultivable phylotypes. Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.

FIG. 1

Dot blot hybridizations of identical membranes with group-specific probe TRE I (a) and species-specific probe TVIN (b). The strains were kindly provided by R. Mutters, Marburg, Germany (identified as RM); C. Wyss, Zürich, Switzerland (identified as CW); and B. Wilske, Munich, Germany (identified as BW). In columns 1 to 8 PCR products of the following strains were applied as controls: the putative oral pathogens Actinobacillus actinomycetemcomitans MCCM 02638 (A1) (RM), Capnocytophaga gingivalis MCCM 00858 (A2), (RM), Capnocytophaga ochracea MCCM 00238 (A3) (RM), Eubacterium lentum ATCC 25559^T (A4) (RM), Fusobacterium nucleatum ATCC 25586^T (A5) (RM), Porphyromonas gingivalis ATCC 33277 (A6) (RM), and Prevotella intermedia MCCM 00407 (A7) (RM); the cultivable treponema species T. vincentii ATCC 35580 (B1), T. denticola ATCC 35405^T (B2), T. socranskii subsp. socranskii ATCC 35536 (B3), T. socranskii subsp. buccale ATCC 35534 (B4), T. maltophilum ATCC 51939^T (B5) (CW), and T. phagedenis subsp. reiterii (B6) (BW); a clinical isolate (CW) (B8; highest degree of homology to clone NZM 3142), and T. pectinovorum ATCC 33768^T (E1); group I recombinant clones NZM3D292 (C1), NZM3D464 (C5), NZM3112 (C6; sequence 100% homologue to probe TVIN), NZM3142 (D2), NZM3147 (D4), and NZM3166 (D7); group II recombinant clones NZM3106 (C7) and NZM3158 (D6); group III recombinant clones NZM3143 (D3), NZM3D298 (C3), and NZM3D527 (C4); group IV recombinant clones NZM3122 (C8), NZM3D505 (C2), and NZM3125 (D8); group V recombinant clones NZM3124 (D1) and NZM3155 (D5); the group VI recombinant clone NZM3104 (E2); and the group VII recombinant clone NZM3D384 (E3). In columns 9 to 15 PCR products from subgingival plaque samples were applied: lanes A to D, PCR products from deep periodontal pockets; lane E, PCR products from the respective controls.

FIG. 2

Presence of cultivable versus uncultivable oral treponemes in RPP patients revealed by dot blot hybridization with group-specific probes (probes TRE I, TRE II, and TRE IV) and species-specific probes (probes TVIN, TDEN, and TMAL).

FIG. 3

Fluorescence in situ hybridization of subgingival plaque material from an RPP patient. (a) Microphotograph showing simultaneous hybridization with EUB338_FITC (green) and TRE I_Cy3 (yellow). The eubacterial probe reveals the different morphotypes of subgingival plaque bacteria and the spherical bodies of the treponemes (arrows), as described by Wecke et al. (33). (b) Microphotograph showing hybridization with TRE I_FITC (green) and TRE II_Cy3 (yellow). Note the different morphologies of the treponemes detected with the group-specific probes.