First cultivation of health-associated Tannerella sp. HOT-286 (BU063)

Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet over a third of the approximately 700 different bacterial taxa found in the human oral cavity are as-yet-uncultivated in vitro. One such taxon, Tannerella sp. HOT-286 (phylotype BU063), is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbour, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily-diluted sample of subgingival plaque was inoculated onto culture plates, supplemented with siderophores (pyoverdines-Fe-complex or desferricoprogen) or a neat plaque suspension. After eight days of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Sub-cultured colonies were identified by 16S rRNA-gene-sequencing, and purity confirmed by sequencing 20 clones/library prepared from a single colony. Three of the colonies of interest (derived from pyoverdines- and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes or Prevotella intermedia for stimulation, with best growth on membranes over ‘helper’ lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel ‘periodontal health-associated’ taxon with T. forsythia will be valuable in investigating virulence factors of the latter, and possible health benefits of the former.