Cultivation of a Synergistetes strain representing a previously uncultivated lineage

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.

Fig. 1

Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1221 aligned bases showing relationship between Synergistetes strain SGP1, Cluster A Synergistetes and representatives of other genera and groups within the phylum Synergistetes. Tree was constructed using the neighbour-joining method following distance analysis of aligned sequences. Numbers represent bootstrap values for each branch based on data for 500 trees. Accession numbers for 16S rRNA sequences are given for each strain. Scale bar shows number of nucleotide substitutions per site.

Fig. 2

Cluster A Synergistetes 16S rRNA gene copies in CMM broths inoculated with a mixed culture of sub-gingival plaque.

Fig. 4

Cluster A Synergistetes in CMM/serum broth visualized by FISH with probe A_487, Cy3 (yellow) and total bacteria with probe EUB338, Cy5 (red) at days: (A) 1, (B) 14 and (C) 33 of growth. Bars, 10 µm.

Fig. 3

Proportion of Cluster A Synergistetes within the total bacteria in CMM broths inoculated with a mixed culture of sub-gingival plaque.

Fig. 6

Cells harvested from a colony hybridization-positive site on streak plate G at day 15 of culture: (A) Gram stain and (B) FISH analysis of same slide after Gram-staining; overlay of Cluster A Synergistetes in yellow (probe A_487, Cy3) with total bacteria in red (probe EUB338, Cy5). Bars, 10 µm.

Fig. 5

Blot from streak plate G hybridized at day 14 with probe 3.3_65; position of S. aureus streak indicated by line.

Fig. 8

Ten-day cultures of Synergistetes strain SGP1: (A) No donor streak, (B) P. micra donor.

Fig. 7

Gram-stained cells from an isolated 16-day colony of Synergistetes SGP1. Bar, 10 µm.

Fig. 9

Transmission electron micrograph of an ultrathin section showing cells of Synergistetes SGP1 harvested from 10-day colonies cultured in the presence of F. nucleatum. Bar, 0.5 µm.