Single-cell enumeration of an uncultivated TM7 subgroup in the human subgingival crevice

Abstract

Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells from healthy site samples.

FIG. 1.

(A) FISH stringency tests with clones pSBG1, pSBG1a, pMF1, and A. aquaticus and probe TM7-905 at 0 to 20% formamide concentrations. All green panels refer to the use of the nonspecific YO-PRO-1 DNA stain, and red panels refer to the use of the Cy3-labeled TM7-905-specific probe. (B) Staining with TM7-905-Cy3 (10% formamide; clone pSBG1a) or IO25-136-Cy3 (20% formamide; pSBG1), demonstrating, in addition to YO-PRO-1 (green) and probe-Cy3 (red) labels, the positive-control Bac338-Cy5 (blue) probe. The last TM7-905 panel shows colocalization of the three stains with heterologous 16S rRNA at the cell poles. Scale bar = 5 μm for all panels.

FIG. 2.

FISH on dental plaque samples from human subgingival sites of moderate chronic periodontitis (A to E) and from healthy sites (F). (A.1 to A.3) Staining with YO-PRO-1 (green) general stain (A.1) and TM7-905-Cy5 (blue) (A.2) and colocalization of these two stains (A.3). Asterisks, two segments of the TM7 filament that are not labeled by the probe. In all other panels, YO-PRO-1 and probe signals are merged as in panel A.3. (B and C) TM7-905 probe and YO-PRO-1-labeled cells (blue-green); (D to F) TM7-905 probe-labeled cells (solid arrowhead, dark blue) plus IO25-136 probe-labeled cells (open arrowhead, light blue) and cells labeled with YO-PRO-1 alone (green). Scale bar = 5 μm in all panels.