Dr Robert Smibert from the Virginia Polytechnic Institute, USA, isolated and collected over 200 strains of oral treponemes over a 20-year period. Dr Smibert, Dr W. E. C. Moore and Dr L. V. Moore separated these isolates and reference strains into different groups on the basis of cellular fatty acid analysis. In this study, the 16S rRNA genes were sequenced for 47 strains that were representative of these groups. Five distinct species were identified on the basis of 16S rRNA sequence comparisons; two of these species are newly named and three have not yet been characterized. The first species, designated Smibert-1, was represented by the single strain D4B-1 and was later identified as the newly described However, strain D4B-1 possessed a different flagellar arrangement to that of The second species, Smibert-2, was represented by nine isolates that possessed identical 16S rRNA gene sequences. The closest relatives of this species were Smibert-3 and Smibert-4 at approximately 90% sequence similarity. Within Smibert-2, there was no correlation between phylogenetic analysis and cellular fatty acid analysis since six different cellular fatty acid groups represented the nine strains. Smibert-3 (strain D36ER-1) and Smibert-4 (D62CR-12) were each represented by only a single strain and were closely related to each other at 98% sequence similarity. Strain D36ER-1 of Smibert-3 was identified as belonging to the not-yet-cultivated phylotype 20 [Choi, B. K., Paster, B. J., Dewhirst, F. E. & Göbel, U. B. (1994). 62,1889–1895]. Strain D62CR-12 of -4 was nearly identical in sequence to the newly described amylovorum. The fifth species, Smibert-5, was represented by a single strain, D120CR-1, and was closely related at about 98% sequence similarity to the three subspecies of The phylogenetic analyses of strains of and of subspecies of are also reported. The closest oral relatives of were at 98·7% sequence similarity and at 91·5% sequence similarity. subspp. and formed three separate phylogenetic branches with sequence similarities of about 98% to each other. The closest relative of the subspecies of and of Smibert-5 was Smibert-2 at about 86% sequence similarity. Historic reference strains Fuji, , Fm, lchelson-2, N-39, TD2, TRRD, MRB, IPP, Jethro and T32A, as well as an unknown strain designated only as , were identified as strains of Reference strains Fuji, Jethro, T32A and IPP plus three isolates of the Smibert collection were also contaminated with a mycoplasma as determined by 16S rRNA comparative analysis. Consequently, spirochaetal cultures should be screened for mycoplasmas. There are presently at least ten species of cultivable oral species of with the cut-off for separate species


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