Diversity of the butyrate-producing microflora of the human gut.

Butyrate, a C4 short-chain fatty acid, is one of the principal products of bacterial fermentations in the human colon and is the preferred energy source of the colonic epithelial cells in a healthy colon (Roediger, 1980). Potential protective roles have been suggested for butyrate as an anti-cancer and anti-inflammatory therapeutic treatment for the colon (Hague et al. 1997). Despite this, rather little is known about the main groups of butyrate-producing bacteria present in the human colon, their metabolic activities and their phylogenetic relationships. Butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult following an omnivorous diet and an adult vegetarian. Isolations were performed anaerobically in a rumen fluid based medium. A second isolation was performed on the same individuals one year later to detect any changes in butyrate-producing flora composition between individuals as well as within individuals. A total of 613 bacterial isolates were analyzed by conventional microbiology tests; of these isolates 74 produced significant amounts of butyrate. Phenotypic tests highlighted similarities of the isolates with the genera Eubacterium. Roseburia and Butyrivibrio, but these genera are not readily distinguishable at phenotypic level and few culture collection isolates are available. Most of the isolates degraded starch and their growth was stimulated by maltose. Considerable variation was observed in the levels of butyrate produced, acetate utilised and response obtained with human butyrate-producing isolates to various growth media. The majority of isolates showed significant utilization of acetate present in their growth medium, and many required acetate for growth. A non-acetate utilizing isolate, L2-50, required other SCFAs for growth. Butyrate-producing isolates were grouped by 16S rDNA PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) with the restriction enzyme Alu-I, which proved to give the best ribotype discrimination from a total selection of four restriction enzymes. A total of 18 Alu-I PCR-RFLP ribotypes were obtained for the 74 butyrate-producing isolates. The results indicate that the butyrate-producing flora of each individual changed significantly between the two isolations and although some of the subjects had a few ribotypes in common, the majority of PCR-RFLP profiles for each subject were unique. Presumptive identification of the isolates to species level by ribotyping was possible only for two species, Eubacterium ventriosum and Fusobacterium prausnitzii. Selected isolates were grouped by phylogenetic analysis after determination of their 16S rDNA sequences. Most of the butyrate-producing isolates fell into three main groups. Group 1 related to Eubacterium rectale -Roseburia cecicola (the most abundant), group 2 related to Eubacterium halli-Eubacterium ventriosum and group 3 related to Fusobacterium prausnitzii. The results indicate that most of the strains (groups 1 and 2) fall within Clostridium cluster XlVa of Gram-positive bacteria, as defined by Collins et al. (1994) and this is supported by bootstrap values. However, group 3 is phylogenetically placed between Clostridium clusters III and IV (Collins et al. 1994, Wang et al. 1996a). This indicates that despite the phylogenetic diversity of human butyrate-producing species, Clostridium cluster XIVa seems to be predominant in the isolates of this thesis. Most of the ribotypes fall within distinct branches in the tree, but some strains that were grouped together by PCR-RFLP analysis are separated in the tree, presumably because extensive sequence changes can occur between strains without affecting sites for a particular restriction enzyme. The RFLP classes must be therefore seen as a useful comparative method for examining diversity rather than a definitive typing approach, unless results from several enzymes are combined. Species-specifie PCR primers were designed from the sequences of the most abundant ribotypes and in most cases gave specific amplification. The results obtained in this thesis by 16S rDNA PCR-RFLP cataloguing, and by species-specific primers, are of potential importance and could be valuable in population studies for determining the predominance of different butyrate-producing bacteria between individuals with different diets and health conditions. Further investigations appear worthwhile on dietary factors and probiotic species as a natural means of increasing in vivo the delivery of butyrate in the human distal colon.

Grantor dc:publisher.institution

Robert Gordon University

Year dc:date.issued

1999

Author dc:creator

• Barcenilla, Adela

Advisor dc:contributor.advisor

• H. Flint, C. Stewart and C. Henderson

• Butyrate-producing bacteria
• Human colon
• Epithelial cells
• Phenotypic tests
• Eubacterium ventriosum
• Fusobacterium prausnitzii
• Ribotyping

Language dc:language

en

Identifier

oai:rgu-repository.worktribe.com:2807291
https://doi.org/10.48526/rgu-wt-2807291

OAI identifier oai:identifier

oai:rgu-repository.worktribe.com:2807291