Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes

Abstract

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.

FIG. 1.

Rooted neighbor-joining tree showing the phylogenetic relationships among the 16S rDNA sequences of the uncultured R. obeum-like bacteria (indicated by boldface type) and some closely and distantly related bacteria. The phylogenetic tree was generated from the tree of life of the ARB software package. The Bacteroides group was used as an outgroup. Accession numbers of the 16S rDNA sequences are indicated. Bar = 10% sequence divergence. The target sequences of the Urobe63 probe and the Erec482 probe are indicated.

FIG. 2.

Photographs of fecal cells in one microscopic field hybridized with the Cy3-labeled Urobe63 probe (A), the FITC-labeled Erec482 probe (B), and DAPI (C). (D) Phase-contrast photograph. Bar = 5 μm.

FIG. 3.

Flow cytometric analysis of fecal samples, showing the dot plots representing the Urobe63-Cy5 fluorescence intensity and Erec482-FITC fluorescence intensity of unhybridized bacteria (A) and bacteria hybridized with Urobe63 plus Erec482 in feces (B). Cells labeled with both probes (indicated by the arrow in panel B) were marked with a dark color and appeared as small clusters in dot plots of the forward-angle light scatter (FSC) versus side-angle light scatter (SSC) of the total community (C), indicating limited morphological variability among these bacteria.