Three reference strains of Fusobacterium nucleatum and 32 human oral isolates were compared by a variety of physiological tests, enzyme electrophoretic profiles, SDS-PAGE patterns, DNA base composition and hybridization to test their possible site specificity and frequency of the recently described subspecies of F. nucleatum. Nine of the 11 isolates assigned to F. nucleatum subspecies nucleatum were from diseased sites, whereas isolates from healthy sites were all identified as F. nucleatum subspecies polymorphum or F. nucleatum subspecies fusiforme. Strains of the latter subspecies were the least frequently isolated (2 of 32). These results, although still inconclusive because of the relatively small sample size, nevertheless confirmed the heterogeneity of F. nucleatum and indicate that most human oral isolates from subgingival sites probably belong to F. nucleatum subspecies nucleatum.

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.

The periodontal pocket provides a unique structural site for studies on host/bacterial interactions. The pocket is colonized by a complex but characteristic anaerobic bacterial flora. Many new taxa have been described that have now been supported by comparative rRNA sequence analysis. Within recent years, several molecular approaches have been used to describe both cultivable and noncultivable species, and new genotypes have been reported. Microbial activity rather than the mere presence of a microorganism at a site must be a major factor in the process of disease development. Information on metabolic activities of species and identification of substrates are therefore essential to elucidate the complex interactions that are likely to occur in vivo. We have been using a variety of analytical procedures such as 13C substrate-enrichment nuclear magnetic resonance, 14C isotopic labeling experiments, enzyme assays, impedance measurements, and various chromatographic and electrophoretic procedures to study key species of this predominantly asaccharolytic flora. Results have so far indicated a major role for cationic and anionic acids as sources of energy, but the mechanisms of substrate processing may differ significantly between species. In this ecosystem, crevicular fluid is the likely source of nutrients for species. Components of this fluid appear to have a role in the selection of species in subgingival sites.