To our knowledge, this is the first study directed specifically at establishing the identities of the butyrate-producing bacteria in the human gut. In one respect the diversity of the butyrate-producing strains as revealed by phylogenetic analysis of 16S rDNA sequences was less than might have been anticipated. All isolates were related to gram-positive bacteria, 80% of which belonged to cluster XIVa, and none were related to gram-negative bacteria, since the few F. prauznitzii-like isolates reported here are in fact also related to gram-positive bacteria at the genotypic level. On the other hand, the RFLP analyses revealed extraordinary diversity and variability of the major strain types recovered between the different individuals and sampling times. Much more extensive work would be required to establish whether this diversity reflects differences in diet, age, or genotype between the individuals tested or simply large fluctuations in the predominant types within individuals between different times of sampling. While it is likely that the bacteria isolated include many of the predominant butyrate producers from the human colon, further work will be required to establish the occurrence of related strains in a larger number of individuals. This could be approached by using specific molecular probes to avoid possible cultural bias.
Phylogenetic analysis shows that the butyrate-producing strains isolated that belong to cluster XIVa are widely distributed within this cluster (Fig. ). The majority of isolates appear to be broadly related to known species, but only 8 out of the 24 sequenced strains had greater than 95% 16S rDNA identity with type strains. This and the lack of recognized species type strains in some branches of the tree indicate that new uncharacterized phylogenetic groups are represented among our isolates. Of the three clusters, the Eubacterium rectale-R. cecicola group comprises the highest proportion of sequenced isolates (10 out of 24, 42%) while overall ribotypes 1 and 4, whose sequenced representatives fall within this group, account for 27 out of the 74 (36%) isolates examined. Within the main grouping the Eubacterium rectale subgroup appears to contain isolates from all of the individuals sampled whereas the R. cecicola subgroup was found mainly in infant samples. It would be of particular interest to establish in future work whether R. cecicola-related strains belonging to ribotype 1 are characteristic of preweaned infants.
A number of molecular approaches are now available for rapid bacterial strain typing. RFLP analyses based on hybridization with 16S rDNA probes have been widely used and depend on detection of variations in chromosomal sequences flanking multiple copies of rRNA target genes (2
). The approach used here is quite different in that it depends on PCR amplification of internal 16S rDNA fragments and cleavage with restriction enzymes (50
). This approach proved very valuable here in revealing the diversity of butyrate-producing strains, and two ribotypes, 1 and 7, conformed well to branches within the phylogenetic tree obtained subsequently from sequence analysis. In general, however, it must be recognized that PCR-RFLP analysis based on a single enzyme will often group together genetically distant strains since extensive sequence changes can occur between strains without affecting sites for a particular restriction enzyme. The RFLP classes must therefore be seen as a useful comparative method for examining diversity rather than as a definitive phylogenetic typing approach, unless results from many enzymes are combined (33
Fifty percent of butyrate-producing isolates examined here showed some net utilization of acetate in M2GSC medium. Such utilization is consistent with the operation of the butyryl coenzyme A-acetyl coenzyme A transferase route for butyrate synthesis (20
). On average across the strains studied in vitro here, approximately 1 mol of acetate initially present in the medium was utilized for every 2 mol of butyrate produced. We were not able to estimate how much additional acetate may have been produced and utilized by each strain for butyrate synthesis. Some acetate may, of course, be used for biosynthetic reactions rather than butyrate synthesis. The high proportion of acetate-utilizing strains seen here suggests that a significant fraction of colonic acetate may be rerouted into butyrate in the human colon. Preliminary studies involving 13
C-labeled acetate suggest that appreciable butyrate may be derived from exogenous acetate in mixed fecal fermentations (S. H. Duncan et al., unpublished data). Since for some of the strains isolated here growth was absolutely dependent on inclusion of acetate in the medium (S. H. Duncan et al., unpublished data), these strains would not have been recovered through isolations with media lacking acetate. The alternative butyrate kinase pathway is known to operate in many Clostridium
spp. and in certain ruminal B. fibrisolvens
strains, including the type strain 2221, and strains depending on this pathway would not be expected to utilize acetate (13
). It is also worth noting that only 1% of the non-butyrate-producing isolates examined showed net acetate utilization and that nearly all (95%) of the acetate utilizers recovered were butyrate producers.
Recent work by Diez-Gonzalez et al. (13
) suggested that rumen butyrate-producing Butyrivibrio
species could be divided into two distinct groups, based upon pathways that they utilized to produce butyrate and that these could be distinguished by production or consumption of acetate. Rumen Butyrivibrio
strains have also been classified as lactate producing or non-lactate producing (42
). None of the human isolates obtained in this study fell within either group of Butyrivibrio
strains. However, the present work does confirm that one isolate, B. fibrisolvens
16.4, obtained previously from fermentor studies with the human fecal flora (38
), is related to the group of ruminal B. fibrisolvens
strains that includes B. fibrisolvens
1.230 and 2223.
We found no simple correlation between the phylogenetic position of the butyrate-producing isolates examined here and their metabolic behavior. Thus, for example, ribotype 1 strains included one acetate producer (L1-810) in addition to the predominant acetate-utilizing strains. In addition, we were able to detect significant production of lactate both in acetate-producing strains (e.g., L1-810) and in acetate-utilizing strains (e.g., L1-952) from human feces. On the other hand it is worth noting that ribotypes 1 and 7 contained particularly high proportions of strains that produced >10 mM butyrate in vitro (11 out of 14 [78%] and 6 out of 9 [67%], respectively, compared to 24 out of 74 [32%] butyrate producers over all ribotypes).
Recent investigations by fluorescent in situ hybridization with group-specific probes (18
) showed that the highest proportion of human fecal organisms detected fell within the Clostridium coccoides-Eubacterium rectale
group (7.2 × 1010
cells/g [dry weight] of feces), which forms part of clostridial cluster XIVa. We have shown here that most of the butyrate-producing isolates in this study fall into cluster XIVa and that they are related to the Clostridium coccoides-Eubacterium rectale
group. Further understanding of the phylogeny and physiology of this group of organisms will be crucial in understanding the role of the anaerobic microflora in colonic metabolism and gut health.