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,more than a third of the approximately 700 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 neighbor,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 8 d 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. subcultured colonies were identified by 16s rrna gene sequencing,and purity was confirmed by sequencing 20 clones per library prepared from a single colony. three 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 and 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. © international & american associations for dental research 2016.