Spirochetes are thought to remain motile in environments (such as intercellular spaces) that immobilize extracellularly flagellated eubacteria. This attribute suggests that the viscosity of the milieu is of importance to locomotion. We sought to determine the interdependence of oral spirochete locomotion with media viscosity. Video time-lapse microscopy using darkfield optics was used. The motility of the spirochetes in media of different viscosities (various concentrations of Noble agar) was measured. Treponema denticola exhibited the fastest speed (18.7 +/- 4.4 microns/min) at a viscosity of 30 mPa.s. The highest speeds for Treponema vincentii and Treponema socranskii were 41.9 +/- 14.9 and 33.4 +/- 13.2 microns/min, respectively, at 88 mPa.s. These data show that optimal migration of spirochetes is viscosity-dependent. The results support the hypothesis that such viscosity-dependent locomotion could be a virulence factor that enables oral spirochetes to initiate and sustain periodontal disease.

Colony-forming units or cells in suspension of oral anaerobic spirochetes (Treponema denticola, Treponema vincentii and Treponema socranskii) bind hemin and Congo red. Hemin or Congo red binds to a hydrophobic polypeptide receptor that is located in the outer membrane of the bacterial cells and it has a relative molecular mass of 47 kDa. These oral spirochetes also lyse sheep erythrocytes to produce beta-hemolytic zones around colony-forming units. The oral spirochetes may acquire iron for growth when they lyse erythrocytes and bind heme from which they may sequester and transport iron into the cells.

Spirochetes are markedly prevalent in periodontal disease but are not included as predominant cultivable organisms because of the inability to quantify them by viable count. A successful method was developed for enumerating viable oral spirochetes as colony-forming units (CFU) in an agarose-based medium. Treponema denticola, Treponema vincentii and Treponema socranskii in log-phase growth in new oral spirochete (NOS) broth were used for evaluation of the method. Critical components of the method include enzyme-free low temperature-gelling (37 degrees C) agarose in NOS medium in small tissue-culture flasks into which the spirochetes were seeded and diluted. The flasks were anaerobically incubated in a glove-box. Reliable, consistent and reproducible viable counts of pure spirochete cultures were obtained. The injurious effects of spirochete temperature-sensitivity were averted by using molten agarose at 37 degrees C. Distinctive colony morphologies of spirochete species could be compared from pure cultures. Addition of rifampin into the medium showed no decrease in spirochete CFU count. The method as described allows for selection of mutants and detection of biochemical activity and is potentially useful for enumeration of spirochetes from periodontal pockets as members of the predominant cultivable flora.

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6-kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6-kb plasmid from T. denticola e' was shown to be similar to pTD1, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e' share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2-kb plasmid in 4 of these strains.