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

This research introduces the development and assessment of oligonucleotide probes, a tool used to identify and measure bacteria in a horse’s large intestine. The probe design focuses on anaerobic bacteria groups, and findings show possible major and minor bacteria populations in sampled horses.

Development and Evaluation of Oligonucleotide Probes

• The researchers developed nine oligonucleotide probes that correspond to shared parts of small subunit rRNA from unique clusters of intestinal anaerobic bacteria, which do not require oxygen to live.
• The probes’ efficacy and specificity were tested by comparing their binding, or hybridization, to their intended target rRNA and other non-target rRNA coated on nylon membranes. This process helps ensure the probes bind to the correct microbial species.
• The best wash temperatures for each test probe were identified based on the temperature at which each probe dissociates, or separates, from its target.

Probes Targeted to Various Bacterial Clusters

• Three probes target Clostridiaceae family clusters, a bacteria group associated with diseases in humans and animals when found in excess.
• Another three probes target ‘unknown’ clusters within the low G+C Gram-positive phyla. These are bacteria with a specific type of cell wall structure and a particular DNA composition.
• The remaining three probes target the Cytophaga-Flexibacter-Bacteroides group, the Bacillus-Lactobacillus-Streptococcus group, and the Spirochaetaceae family, bacteria associated with various maladies in humans and animals if their populations become dysregulated.

Application of Probes and Initial Findings

• The suite of nine developed probes was applied to rRNA extracted from horse colon samples, along with other previously developed probes targeting different intestinal bacteria groups.
• This application gave the first quantitative data about the predominant bacterial populations in the horse’s large intestine.
• Significant bacterial populations were from the Spirochaetaceae family, the Cytophaga-Flexibacter-Bacteroides group, the Eubacterium rectale-Clostridium coccoides group, and ‘Unknown cluster C’ of the Clostridiaceae, each making up 10-30% of each horse’s total gut microflora.
• Other notable detected populations included the Bacillus-Lactobacillus-Streptococcus group, Fibrobacter, and ‘Unknown cluster B’, each making up 1-10% of the total gut flora.
• The coverage from the suite of probes described in this study exceeded 75% of the total microbial community in the horses’ large intestines, suggesting the majority of the microbial variety can be accounted for using these probes.