Design and evaluation of group-specific oligonucleotide probes for quantitative analysis of intestinal ecosystems: their application to assessment of equine colonic microflora.
Nine oligonucleotide probes complementary to conserved regions of small subunit rRNA from phylogenetically defined clusters of intestinal anaerobic bacteria were designed and evaluated for use in quantitative analysis of intestinal microflora. Optimum wash temperatures (T(w)) were determined according to the temperature of dissociation (T(d)) of each probe and target group specificity was demonstrated by comparing hybridisation to target and non-target rRNA immobilised on nylon membranes. Three probes are targeted to phylogenetic clusters of Clostridiaceae, clusters III, IV and IX, with three probes designed to target previously undefined clusters within the low %G+C Gram-positive phyla. The remaining three probes encompass the Cytophaga-Flexibacter-Bacteroides assemblage, the Bacillus-Lactobacillus-Streptococcus group and the Spirochaetaceae. Application of these probes, alongside available probes targeted to other intestinal bacterial groups, against rRNA extracted from equine colonic samples has provided the first quantitative data on the predominant bacterial populations inhabiting the equine large intestine. Results show the Spirochaetaceae, the Cytophaga-Flexibacter-Bacteroides assemblage, the Eubacterium rectale-Clostridium coccoides group, and 'Unknown cluster C' of the Clostridiaceae to be the largest populations in the equine gut, each comprising 10-30% of the total microflora in each horse sampled. Other detected notable populations were the Bacillus-Lactobacillus-Streptococcus group, Fibrobacter and 'Unknown cluster B', each comprising 1-10% of the total flora. Average coverage using the suite of probes described here exceeded 75% of the total microbial community.