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. 2000 Oct;66(10):4523-7.
doi: 10.1128/AEM.66.10.4523-4527.2000.

Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups

H J Harmsen  1 , A C Wildeboer-Veloo, J Grijpstra, J Knol, J E Degener, G W Welling
Affiliations

Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups

H J Harmsen et al. Appl Environ Microbiol. 2000 Oct.

Abstract

Two 16S rRNA-targeted probes were developed: one for the Coriobacterium group and the other for the Atopobium cluster (which comprises most of the Coriobacteriaceae species, including the Coriobacterium group). The new probes were based on sequences of three new Coriobacteriaceae strains isolated from human feces and clinical material and sequences from databases. Application of the probes to fecal samples showed that formula-fed infants had higher numbers of Coriobacterium group cells in their feces than breast-fed infants. In addition, based on the presented results, it is hypothesized that with the increasing age of a person, the diversity of Atopobium cluster species present in the feces increases.

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Figures

FIG. 1
FIG. 1
Tree based on 16S rRNA sequences showing the phylogenetic relationship of the newly isolated strains G118, H818, and EKSO3 with the genus Collinsella and Coriobacterium glomerans, the family of Coriobacteriaceae, and three distant bacteria as an outgroup. Accession numbers of the used sequences are after the species names. ∗, sequence is available from the Ribosomal Database Project. (A), specificity of the ATO291 probe; (CA), the strain hybridizes with both ATO291 and COR653 probes. Numbers next to the branch nodes indicate bootstrap values (%); only values more than 90% are shown. Bar, 10% sequence divergence.
FIG. 2
FIG. 2
FISH of a fecal sample from a formula-fed newborn infant at day 20. Two images were taken from the same microscopic field. Left, a phase-contrast image of all bacteria; right, the epifluorescence image of the bacteria that hybridized with 16S rRNA-based oligonucleotide probes COR653 (specific for Coriobacterium group) and ATO291 (specific for the Atopobium cluster). The yellowish fluorescence is a combination of the green light of the fluorescein-labeled COR653 probe and the red light of the rhodamine-labeled ATO291 probe, indicating that these are Coriobacterium group cells. Bar, 5 μm.
FIG. 3
FIG. 3
FISH of a fecal sample from a young volunteer (age, 10 years). The micrograph shows merged images of the epifluorescence image after DAPI staining and hybridization with two 16S rRNA-based oligonucleotide probes. The yellowish-white cells indicated with a white arrow show fluorescence with the fluorescein-labeled probe COR653, the rhodamine-labeled probe ATO291, and DAPI fluorescence, indicating that they are Collinsella group cells. The red bacteria (green arrow) show only fluorescence of the probe ATO291 and DAPI fluorescence, indicating that they belong to the Atopobium cluster and that they are not Coriobacterium group cells. The blue background cells are only DAPI stained. Bar, 5 μm.
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