Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes

Abstract

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

FIG. 1

Phase-contrast and epifluorescence micrographs of pure culture and fecal samples. (A and B) Pure culture of E. barkeri hybridized with Cy3-labeled E.bar1237 (S-S-E.bar-1237-a-A-18) probe. Panels A and B show the same microscopic field viewed by phase-contrast and epifluorescence microscopy, respectively. (C and D) Detection of E. biforme in a fecal sample with E.bif462 (S-S-E.bif-0462-a-A-18). Panels C and D show the same microscopic field viewed by phase-contrast and epifluorescence microscopy, respectively. (E and F) Detection of E. hadrum in a fecal sample with E.had579 (S-S-E.had-0579-a-A-20) and cross-hybridization to an unknown coccoid-shaped bacterium. Panels E and F show the same microscopic field viewed by phase-contrast and epifluorescence microscopy, respectively. In panel E the unknown coccoid-shaped bacterium is marked with an arrow. Both visible bacteria (coccoid shaped and rod shaped) were detected with E.had579.

FIG. 2

PCR products obtained with genomic DNA of E. limosum from different cell numbers in autoclaved feces and pure culture on 1% agarose gel. Lanes M are the molecular marker (1-kb ladder). Lanes 1 to 9 show the PCR products obtained with cell numbers in the range from 10⁸ to 10⁰ from pure culture. Lanes 10 to 17 show PCR products obtained with cell numbers in the range from 10⁸ to 10¹ which were added to autoclaved feces.