Susan E. Pryde

Phylogenetic Relationships of Butyrate-Producing Bacteria from the Human Gut

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.

Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis

Abstract The bacterial species diversity of three colonic tissue samples from elderly people was investigated by sequence analysis of randomly cloned eubacterial 16S rDNA. The majority of sequences (87%) clustered within three bacterial groups: (1) Bacteroides; (2) low G+C content Gram-positives related to Clostridium coccoides (cluster XIVa); (3) Gram-positives related to Clostridium leptum (cluster IV). These groups have been shown to dominate the human faecal flora. Only 25% of sequences were closely related (>97%) to current species type strains, and 28% were less than 97% related to any database entry. 19% of sequences were most closely related to recently isolated butyrate-producing bacteria belonging to clusters XIVa and IV, with a further 18% of the sequences most closely related to Ruminococcus obeum and Ruminococcus torques (members of cluster XIVa). These results provide the first molecular information on the microbial diversity present in human colonic samples.

Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine

ABSTRACT Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.

Challenges in the identification of species of canned fish

Abstract The identification of fish species becomes a problem when the usual identifying characteristics are removed on processing and only a portion of flesh is available. When the flesh is raw or cooked under normal conditions, the species is readily established by electrophoresis of the muscle proteins. The procedure cannot be used for heat-sterilised canned fish as the proteins are severely denatured. DNA is also degraded but techniques are now available for targeting and amplifying species-specific fragments. The amplified products can then be analysed by a range of techniques some of which are suitable for food control laboratories.

Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns

Analysis of single strand conformation polymorphism (SSCP) of an amplicon (123 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrome b gene was used to identify the fish species in canned tuna. Single-stranded DNA (ssDNA) was separated by polyacrylamide gel electrophoresis, and visualised by silver staining. The reliability of the method was tested by a collaborative study in which eight European laboratories participated. Seven unknown samples (five from individual species and two mixtures of two tuna species) of canned tuna had to be identified by comparison with reference material. From a total of 72 cases, 65 (90.3%) were assigned correctly. Intra-species variability of SSCIP patterns was found in the case of Katsuwonus pelamis and Sarda sarda. As specimens from various fishing grounds gave two or three different patterns of ssDNA, the possibility of some variability of the DNA patterns has to be considered in SSCP analysis of these species.

MiniReviewThe microbiology of butyrate formation in the human colon

Butyrate arising from microbial fermentation is important for the energy metabolism and normal development of colonic epithelial cells and has a mainly protective role in relation to colonic disease. While certain dietary substrates such as resistant starch appear to be butyrogenic in the colon, it is not known to what extent these stimulate butyrate production directly, e.g. by promoting amylolytic species, or indirectly, e.g. through cross-feeding of fermentation products. Cultural and molecular studies indicate that the most numerous butyrate-producing bacteria found in human faeces are highly oxygen-sensitive anaerobes belonging to the Clostridial clusters IV and XIVa. These include many previously undescribed species related to Eubacterium, Roseburia, Faecalibacterium and Coprococcus whose distribution and metabolic characteristics are under investigation. A better understanding of the microbial ecology of colonic butyrate-producing bacteria will help to explain the influence of diet upon butyrate supply, and to suggest new approaches for optimising microbial activity in the large intestine.