Design and evaluation of group-specific oligonucleotide probes for quantitative analysis of intestinal ecosystems: their application to assessment of equine colonic microflora

Abstract

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-FlexibacterBacteroides 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.

Composition of the Landfill Microbial Community as Determined by Application of Domain- and Group-Specific 16S and 18S rRNA-Targeted Oligonucleotide Probes

“…The relative abundance of Clostridium cluster XIV, as determined using probe Erec 482, varied from 8 to 27% for the rumen samples (Table 2). These data are in agreement with the study of Daly and Shirazi-Beechey (8), in which the abundance of cluster XIV as determined with the same probe (Erec 482) was found to represent the highest proportion of rRNA found in any one of the equine intestinal samples studied (28%). The abundance of cluster XIV clostridia was found to vary greatly in their study, and this was also the case here.…”

“…2), a very low hybridization signal was obtained for rumen fluid samples. Clostridium cluster III has been quantified in equine colonic samples, and the relative abundance varied from 1.3% to 3.0% of the total rRNA (8). It may be, therefore, that the relative proportion of cluster III clostridia is also low in the rumen fluid samples analyzed here and abundances of 1 to 3% are below the threshold for accurate quantification.…”

“…32 P]ATP (Perkin Elmer) (8). Membranes were placed in glass hybridization tubes (Hybaid) and 100 l hybridization buffer (0.9 M NaCl, 50 mM NaH 2 PO 4 [pH 7.0], 5 mM EDTA, 10ϫ Denhardt's solution, 0.5% SDS, and 0.1 mg poly[A] ml Ϫ1 ) added per cm 2 of membrane.…”

Molecular Approaches for the Study of Genetic Diversity in Microflora of Poultry Gastrointestinal Tract

“…This method has been used to quantify rRNA from samples of human, horse and rumen (Sghir et al, 2000;Marteau et al, 2001;Daly and Shirazi -Beechey, 2003). Because PCR has no relation to other amplification methods, quantification determination is more accurate, and therefore, this method has a good reputation and is widely used.…”

Understanding the equine cecum-colon ecosystem: current knowledge and future perspectives

“…Using molecular analysis, Daly et al (2001) reported that 89% of the recovered sequences did not correspond to any recorded ones, suggesting that the anaerobic microflora of the equine large intestine are underrepresented and that the equine flora may contain many novel bacterial species. In a latter study, Daly and Shirazi-Beechey (2003), using specific oligonucleotide probes, quantitatively analyzed the intestinal microflora and obtained the first data of predominant bacterial populations in the large intestine of horses. The results showed that the strains Spirochaetaceae, Cytophaga-Flexibacter-Bacteroides, the group EubacteriumClostridium rectal Coccoidea and an 'unknown cluster C' of clostridiaceae were the largest populations in the cecumcolon, each group comprising 10% to 30% of the total microflora in examined horses.…”