Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes.
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.
Zoetendal
Erwin G
EG
Wageningen Centre for Food Sciences, Laboratory of Microbiology, Wageningen University, The Netherlands. egzoetendal@hotmail.com
Ben-Amor
Kaouther
K
Harmsen
Hermie J M
HJ
Schut
Frits
F
Akkermans
Antoon D L
AD
de Vos
Willem M
WM
D003013:Clostridium / Q000235:genetics / Q000502:physiology*; D015342:DNA Probes; D005243:Feces / Q000382:microbiology*; D005434:Flow Cytometry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D015203:Reproducibility of Results
D015342:DNA Probes; D012336:RNA, Ribosomal, 16S