Quantification of the flavonoid-degrading bacterium Eubacterium ramulus in human fecal samples with a species-specific oligonucleotide hybridization probe

Abstract

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E. ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 x 10(7) to 2.0 x 10(9) cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.

FIG. 1

Epifluorescence and phase-contrast micrographs of a mixed culture and fecal sample. (A and B) Mixed culture of Butyrivibrio fibrisolvens, Eubacterium fissicatena, E. ramulus, E. rectale, Eubacterium uniforme, and Streptococcus pleomorphus hybridized with Cy3-labeled S-S-E.ram-0997-a-A-18. Panels A and B show the same microscopic field as viewed by epifluorescence and phase-contrast microscopy, respectively; (C and D) human fecal sample hybridized with Cy3-labeled S-S-E.ram-0997-a-A-18. Panels C and D show the same microscopic field, viewed by epifluorescence and phase-contrast microscopy, respectively.

FIG. 2

RAPD profiles obtained with genomic DNA of E. rectale, B. fragilis, and different strains of E. ramulus by using the M13-core as the random primer. Lanes: M, molecular mass marker (1-kb ladder; Gibco-BRL); 1, E. ramulus ATCC 29099; 2, E. ramulus WK1; 3, E. ramulus WK5; 4, E. ramulus WKBK; 5, E. ramulus WKDT; 6, E. ramulus WKJN; 7, E. ramulus WKRS; 8, E. rectale; 9, B. fragilis; 10, DNA-free control (H₂O). No amplification products were observed in the absence of template DNA (H₂O control).