Diversity and morphology of members of the phylum "synergistetes" in periodontal health and disease - PubMed (original) (raw)

Diversity and morphology of members of the phylum "synergistetes" in periodontal health and disease

S R Vartoukian et al. Appl Environ Microbiol. 2009 Jun.

Abstract

Members of the phylum "Synergistetes" have frequently been detected in the human oral cavity at sites of dental disease, but they have rarely been detected in studies of oral health. Only two oral "Synergistetes" taxa are cultivable. The aims of this study were to investigate the diversity of "Synergistetes" in the oral cavity, to establish whether "Synergistetes" taxa are more strongly associated with periodontitis than with oral health, and to visualize unculturable "Synergistetes" in situ. Sixty samples (saliva, dental plaque, and mucosal swabs) were collected from five subjects with periodontitis and five periodontally healthy controls. Using phylum-specific 16S rRNA gene primers, "Synergistetes" were identified by PCR, cloning, and sequencing of 48 clones per PCR-positive sample. Subgingival plaque samples were labeled with probes targeting rRNA of unculturable oral "Synergistetes" using fluorescent in situ hybridization (FISH). Analysis of 1,664 clones revealed 12 "Synergistetes" operational taxonomic units (OTUs) at the 99% sequence identity level, 5 of which were novel. "Synergistetes" OTU 4.2 was found in significantly more subjects with periodontitis than controls (P = 0.048) and was more abundant in subgingival plaque at diseased sites than at healthy sites in subjects with periodontitis (P = 0.019) or controls (P = 0.019). FISH analysis revealed that unculturable oral "Synergistetes" cells were large curved bacilli. The human oral cavity harbors a diverse population of "Synergistetes." "Synergistetes" OTU 4.2 is associated with periodontitis and may have a pathogenic role.

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Figures

FIG. 1.

FIG. 1.

Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the relationships between novel “_Synergistetes_” taxa identified in this study and related species. The tree was constructed using the neighbor-joining method following distance analysis of aligned sequences using the Jukes-Cantor correction and was rooted with Treponema socranskii (accession no. AF033307). The novel sequences were around 750 bp long and were compared in a pairwise manner with virtually full-length sequences of reference strains with unaligned bases for each pair deleted. The numbers at the nodes are bootstrap values for the branches based on data for 500 trees. The accession number for the 16S rRNA sequence is given for each strain. The scale bar indicates 0.05 nucleotide substitutions per site.

FIG. 2.

FIG. 2.

Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the variation among representatives of “_Synergistetes_” OTU 3.3 in a single sample (saliva from subject A7A). The tree was constructed using the neighbor-joining method following distance analysis of aligned sequences using the Jukes-Cantor correction, was rooted with “_Synergistetes_” phylotype D084, and was based on 435 unambiguously aligned bases.

FIG. 3.

FIG. 3.

Detection of 12 “_Synergistetes_” OTUs in cases and controls.

FIG. 4.

FIG. 4.

Percentage of clones representing each “_Synergistetes_” OTU as a proportion of the total “_Synergistetes_” clones from all sites for cases and controls.

FIG. 5.

FIG. 5.

Percentage of clones representing each “_Synergistetes_” OTU in subgingival plaque samples from cases and controls.

FIG. 6.

FIG. 6.

Confocal FISH micrographs of subgingival plaque showing (a) “_Synergistetes_” OTU 3.3 (green) (probe 3.3_65 and Alexa Fluor 488), (b) cluster A “_Synergistetes_” (red) (probe A_487 and Cy3), and (c) an overlay for “_Synergistetes_” OTU 3.3 (yellow), cluster A “_Synergistetes_” (yellow and pink), and total bacteria (blue) (probe EUB338 and Cy5).

FIG. 7.

FIG. 7.

FISH images of subgingival plaque showing (a) “_Synergistetes_” OTU 3.3 (green) (probe 3.3_65 and Cy3) and (b) an overlay of “_Synergistetes_” OTU 3.3 (yellow or green) with total bacteria (red) (probe EUB338 and Cy5).

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