Novel Method for Enrichment and Isolation of New TM7-Host Pairs

Objectives: Out of 688 bacterial species inhabiting the human oral cavity, 224 taxa remain as-yet-uncultivated. Saccharibacteria (Candidate phylum TM7), often associated with host inflammatory mucosal diseases, have been considered intractable to culture axenically. Successful cultivation and maintenance of TM7x as an epibiont of Actinomyces odontolyticus strain XH001 was recently reported by He et al., 2014. To obtain additional TM7-taxa-/-host-species pairs representing other uncultured oral TM7 taxa, we explored novel bait-taxa cocultivation techniques.
Methods: Human plaque samples were added to RPMI 1640 medium containing 10% fetal calf serum and gentamicin (100 μg/ml). After ~48 hr of anaerobic growth the culture was vortexed, and larger bacteria were spun down gently. The supernatant was passed through a 0.45 μm filter and centrifuged at 20,000xg, 1h to yield a TM7 enriched pellet. A portion of the pellet was analyzed by 16S rRNA Illumina sequencing and revealed the presence of 10 different TM7 species. The TM7-enriched pellet was resuspended and used to infect potential new host strains grown in a combination broth medium, including Actinomyces sp. HOT-171, Propionibacterium propionicum, Propionibacterium acnes, Lachnoanerobaculum sabbureum, and Corynebacterium matruchotti. Cultures were incubated anaerobically and the presence of TM7 was monitored by PCR using TM7-specific primers. Positive cultures were passaged and levels of TM7 were quantified by qPCR. Dark field microscopy and electron microscopy were used to visualize potential TM7-infected pairs.
Results: Using this protocol we were able to successfully isolate at least three novel TM7-host pairs. Co-culture and passaging showed that several TM7-strains can be co-cultivated for a few passages; however long-term passage was achieved only for a TM7-HOT-488/P. propionicum pair.
Conclusions: This work showed previously uncultivated members of the 13 known TM7 human oral taxa can co-cultured with targeted host-bacterial species, though, stable long-term passage of co-cultured pairs remains a challenge.