First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063)

Abstract

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. , 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.

Signature of Microbial Dysbiosis in Periodontitis

“…The genera include well-identified species (Tannerella forsythia, Treponema denticola, C. rectus, and E. nodatum) that are strongly associated with disease (3,(20)(21)(22). It should be noted that some species, such as the newly cultivated Tannerella clone BU063 (23,24), which is thought to be health associated, are also found in active periodontal sites (25); therefore, this species is still controversial. Despite a significant difference in the abundance of the Porphyromonas genus (including P. gingivalis, which is strongly associated with periodontitis) between healthy samples (3.35%) and CP samples (13%), the genus was excluded in the first dysbiosis ratio because of its similar prevalence rates.…”

Cultivation strategies for growth of uncultivated bacteria

“…In brief, targeted approaches were used for the cultivation of specific uncultivated targets [19, 64] and open-ended approaches were used for cultivation of multiple uncultivated bacteria, in anticipation of a higher-throughput yield [33, 65]. …”

“…In an effort to increase productivity, a novel in-vitro cultivation method using an open-ended approach and combination of strategies was devised in the Wade lab [33, 65]. A brief description of the protocol is provided in Fig 1 B.…”

Genomics of the Uncultivated, Periodontitis-Associated Bacterium Tannerella sp. BU045 (Oral Taxon 808)

“…forsythia strain 92A2 but had little synteny beyond operon level and were quite different in GC content (9). Recently, BU063 has been cultivated; however, it required proximity to other bacteria for efficient growth (10). The genome for cultivated BU063 (strain W11667) has recently been deposited in sequence databases (e.g., GenBank accession number CP017038).…”