Analysis of the large bowel microbiota of colitic mice using PCR/DGGE

Authors


R. Bibiloni, Department of Agricultural, Food and Nutritional Science, University of Alberta, Room 4-10 Ag/For Centre, Edmonton, AB, Canada T6G 2P5 (e-mail: bibiloni@ualberta.ca).

Abstract

Aim:  To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis.

Methods and Results:  The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations (‘Bacteroides sp.’, Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed.

Conclusions:  PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated.

Significance and Impact of the Study:  PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.

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