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. 2000 Apr;66(4):1654-61.
doi: 10.1128/AEM.66.4.1654-1661.2000.

Phylogenetic relationships of butyrate-producing bacteria from the human gut

A Barcenilla  1 , S E Pryde, J C Martin, S H Duncan, C S Stewart, C Henderson, H J Flint
Affiliations

Phylogenetic relationships of butyrate-producing bacteria from the human gut

A Barcenilla et al. Appl Environ Microbiol. 2000 Apr.

Abstract

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812-826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195-199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.

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Figures

FIG. 1
FIG. 1
Summary of butyrate synthesis and acetate utilization in a selection of human colonic butyrate-producing isolates. Key to strains: 1, A1-86; 2, A1-189; 3, A1-816; 4, L2-6; 5, L2-21; 6, A2-204; 7, A2-207; 8, L2-15; 9, A1-87; 10, A1-815; 11, L2-10; 12, A2-225; 13, L1-81; 14, L1-83; 15, A2-194; 16, L2-7; 17, L1-92; 18, A2-215; 19, L1-93; 20, L2-39; 21, L2-61; 22, L1-911; 23, A2-223; 24, L1-872; 25, A2-173; 26, A2-165; 27, L1-910; 28, A2-227; 29, L1-94; 30, L1-9171; 31, L1-8151; 32, L1-97; 33, A2-181; 34, L1-91; 35, L1-82; 36, A2-183; and 37, L1-952.
FIG. 2
FIG. 2
PCR-RFLP profiles of a selection of human butyrate-producing isolates and standard strains digested with the restriction endonuclease AluI. (A) Lanes 1 to 14 contain human colonic isolates A2-165 (ribotype 7), A2-168 (ribotype 11), A2-166 (ribotype 11), A2-228 (ribotype 14), A2-175 (ribotype 14), A2-178 (ribotype 14), T2-132 (ribotype 14), A1-89 (ribotype 4), A2-171 (ribotype 4), A2-181 (ribotype 4), A2-183 (ribotype 4), T2-87 (ribotype 17), T2-145 (ribotype 18), and T1-815 (ribotype 5), respectively. (B) Lanes 1 to 12 contain human colonic isolates L2-6 (ribotype 7), L2-12 (ribotype 10), L2-10 (ribotype 9), L2-21 (ribotype 3b), L1-92 (ribotype 3a), L2-9 (ribotype 3b), L2-16 (ribotype 3b), L1-81 (ribotype 1), L2-7 (ribotype 8), L2-65 (ribotype 8), L1-83 (ribotype 2), and B. fibrisolvens 16.4, respectively. Lanes 13 and 14 contain rumen isolates B. fibrisolvens 1.230 and B. fibrisolvens 2221 (ATCC 19971), respectively. Size markers (1-kb ladder; Promega) are shown in lanes marked “M.” The 610-bp band in panel A, lanes 4 to 7, and in panel B, lanes 4, 7, and 8 and the 400-bp fragment in panel B, lane 11, represent partial products. Ribotype groups 6, 12, 13, 15, and 16 are not represented in this figure. Their AluI banding profiles (in base pairs) are 610, 475, 210, 190, and 50 (ribotype 6); 610, 240, and 220 (ribotype 12); 475, 265, 220, and 190 (ribotype 13); 400, 265, 220, 190, and 127 (ribotype 15); and 797, 610, 265, and 190 (ribotype 16). The band pattern for ribotype 6 is similar to that in panel A, lanes 8 to 11, while that for ribotype 12 is similar to that in panel A, lanes 4 to 7. The band pattern for ribotype 13 is similar to that in panel A, lane 12, while that for ribotype 15 is similar to that in panel B, lane 13. The band pattern for ribotype 16 is similar to that in panel A, lanes 4 to 7.
FIG. 3
FIG. 3
Phylogenetic tree showing the relationships of 16S rDNA sequences from human butyrate-producing isolates falling within cluster XIVa of the Clostridium subphylum of low-G+C-content gram-positive bacteria (7, 48). The scale bar represents genetic distance (10 substitutions per 100 nucleotides). The tree was constructed using the neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment. Bootstrap values (expressed as percentages of the value for 100 replications) are shown at branch points; values of 97% or more were considered significant. Sequences derived from the database are shown in italics (e.g., B. fibrisolvens). Atopobium minutum is used as the outgroup sequence.
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