Bacterial diversity within the equine large intestine as revealed by molecular analysis of cloned 16S rRNA genes

Abstract

The molecular diversity of the microflora present within the equine large intestine was investigated through the analysis of PCR-amplified 16S ribosomal RNA gene sequences. Total genomic DNA, recovered from samples of large intestinal wall tissue and lumen contents, was used to generate 272 random clones that were subjected to comparative phylogenetic analysis. The 272 sequences were classified into 168 operational taxonomic units/molecular species (at least 97% similarity), with 92% of recovered sequences being placed within two major phyla: low %G+C Gram-positive bacteria (72%) and Cytophaga– Flexibacter– Bacteroides (20%). Over one-third (37%) of all sequences were affiliated with one clostridial group, cluster XIVa. The remaining sequences were associated with Spirochaetaceae (3%), Verrucomicrobiales (3%), high %G+C Gram-positive bacteria (<1%) and Proteobacteria (<1%). Within the recovered equine clone population only 5% of the sequences corresponded to known organisms whose sequences are available in public databases. A further 6% corresponded to unidentified sequences retrieved in similar 16S rDNA PCR-based studies. The vast majority of sequences recovered (89%) did not correspond to any recorded sequences suggesting that the anaerobic microflora of the equine large intestine is severely underrepresented in the public domain and the lack of recognised sequences in many branches of the phylogenetic tree suggests the equine flora may contain many novel bacterial species.