Quantification of Uncultured <i>Ruminococcus obeum</i> -Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

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<jats:title>ABSTRACT</jats:title> <jats:p> A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured <jats:italic>Ruminococcus obeum</jats:italic> -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 <jats:italic>Ruminococcus</jats:italic> 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 <jats:italic>Clostridium coccoides-Eubacterium rectale</jats:italic> group, a group that includes the uncultured <jats:italic>R. obeum</jats:italic> -like bacteria. Quantification of the uncultured <jats:italic>R. obeum</jats:italic> -like bacteria and the <jats:italic>C. coccoides-E. rectale</jats:italic> 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 <jats:italic>R. obeum</jats:italic> -like bacteria comprise about 16% of the <jats:italic>C. coccoides-E. rectale</jats:italic> group. These results indicate that the uncultured <jats:italic>R. obeum</jats:italic> -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. </jats:p>

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