Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale

Abstract

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation.

FIG. 1.

Phylogenetic tree, constructed by the neighbor-joining method, based on 16S rRNA sequences of Roseburia- and E. rectale-related clones and species. Isolation of strains T1-815, L2-21, A1-86, L1-8151, L1-81, L1-82^T, L1-952, L1-93, A2-181, A2-183^T, L1-82, and A2-194^T is described in reference , and isolation of strains M104/1, M72/1, M88/1, M6/1^T, and M50/1 is described in reference ; for S2Ss2/7 and S2Ss2/2, see the text. 16S rRNA sequence accession numbers are given in parentheses. Numbers above each node are confidence levels (percent) generated from 1,000 bootstrap trials. The Escherichia coli sequence is used as the outgroup to root the tree. The scale bar refers to fixed nucleotide substitutions per sequence position. For the sake of space, the tree is presented in a schematic form that retains the general topology. The branch length of E. coli is not to scale. The probe coverage is shown on the extreme right.

FIG. 2.

Dual probing of a fecal sample from donor 9 (Table 3) with Rint623 plus helper (green label) and Rrec584 (red label) probes for Roseburia species and E. rectale. Orange cells have hybridized with both probes. A bacterium that has hybridized with Rint623 only, indicating that it is either a Roseburia inulinivorans or a Eubacterium ramulus cell, can be seen in the center.