Abstract
Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.
Highlights
Bacteria belonging to the recently described phylum Synergistetes (Jumas-Bilak et al, 2009) are widespread in the environment and in animals (Godon et al, 2005)
Of the primers targeting Synergistetes, 763F/827R demonstrated the broadest range of linearity (10-1–10-8), the highest linear regression coefficient (R2 value of 0.999), a large cycle threshold (Ct) value of 33 for the no-template control and a distinct melting curve profile confirming reaction specificity
Sequence analysis of clone libraries prepared from the pooled products of two replicate end-point PCR reactions with primers 763F/ 827R, mixed DNA template A6G and an annealing temperature of 57°C showed that all 42 clones sequenced were members of the Cluster A Synergistetes confirming the validity of the primers
Summary
Bacteria belonging to the recently described phylum Synergistetes (Jumas-Bilak et al, 2009) are widespread in the environment and in animals (Godon et al, 2005). The Synergistetes Cluster A comprises 22 taxa, related to the TG5 group (Hugenholtz et al, 2009) (Fig. 1) These taxa are exclusively oral but none have ever been cultivated in the laboratory, despite their widespread prevalence when molecular methods are used for their detection (Vartoukian et al, 2009). It is estimated that half of the approximately 700 taxa found in the human oral cavity have yet to be cultured (Paster et al, 2006). The study of such bacteria is clearly important in order to improve our understanding of this complex environment, its bacterial inhabitants, their interactions and their potential role in disease. Bacteria must be cultivated in purity before they can be fully characterized with regard to their phenotypic and physiological properties, and their virulence potential and susceptibility to antimicrobials
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