Methods: Samples of subgingival plaque from pockets > 8 mm in depth were collected from subjects with periodontitis patients and used to inoculate hydroxyapatite-coated pegs immersed in Periodontitis Peg Medium broth (PPB) in the Calgary Biofilm Device (CBD). The CBD was incubated anaerobically and the medium changed every 3.5 days. PCR primers and oligonucleotide probes specific for LachnospiraceaeHOT 500 were designed and validated. Colony hybridisation with DIG-labelled probes was performed on Periodontitis Peg Medium agar plates (PPA) inoculated with biofilms harvested from CBD biofilms incubated anaerobically for 10 days. Hybridisation-positive areas of the plates were subcultured onto fresh media to enrich for the target.
Results: Lachnospiraceae HOT 500-positive regions were seen on colony hybridisation blots from CBD culture plates and, after enrichment, a simple community was seen consisting of Veillonella parvula, Parvimonas micra and tiny colonies growing in close proximity to the Veillonella parvula colonies. 16S rRNA gene sequence analysis identified the tiny colonies, designated strain SP1_1, as be Lachnospiraceae HOT 500. SP1_1 was found to be entirely dependent on V. parvula for growth. Growth stimulation was also seen with Fusobacterium nucleatum and Propionibacterium acnes. The addition of V. parvulaculture sonicates allowed SP1_1 to be cultured in PPB.
Conclusion: A strain representative of the previously uncultivated human oral taxon Lachnospiraceae 500 has been successfully cultured using a colony hybridisation directed enrichment approach.