3rd Annual BMRP Investigator Meeting - Abstract
Real-Time PCR Quantification of Suspect Bacteria in Inflammatory Bowel Disease Mucosa
Chin Wen Png1, Sharise P. Heazlewood1,2, Diana Story2, Anthony Fowler1,2, Linda L. Blackall1, Michael A. McGuckin3 and Timothy H. J. Florin2,3,a
1School of Molecular and Microbial Sciences and 2Department of Medicine, University of Queensland (South Brisbane, Queensland, Australia); 3Mater Medical Research Institute (South Brisbane, Queensland, Australia)
In animal models of inflammatory bowel disease (IBD), some bacteria are protective while others are pathogenic.
Our group hypothesized a possible role in IBD for mucosal Ruminococcus gnavus and mucosal Bacteroides vulgatus on the basis of an extensive 16S rDNA cloning technique that, although only semi-quantitative, permitted an accurate description of the diversity of the intestinal mucosal bacteria, most of which had never been identified by traditional culture methods.
Aim & Methods: To measure quantitatively mucosal R. gnavus and B. vulgatus by real-time PCR with SYBR® Green I and optimized primers for (universal domain) bacteria, GAPDH (internal human gene standard), R. gnavus and B. vulgatus rDNA in inflamed and non-inflamed colonic and ileal mucosa of 26 Crohn’s (CD) and 20 ulcerative colitis (UC), no antibiotics for > 16 weeks, compared with matched sites in 10 healthy controls (HC).
Results: GAPDH/ biopsy was not significantly different between groups. There were 5-fold more mucosal bacteria 16S rRNA in UC and CD (P < 0.001) and 2-fold more in non-inflamed UC vs. inflamed colonic mucosa (P = 0.04). Mucosal R. gnavus was increased in IBD. This was significant for CD when normalized for GAPDH (P = 0.023), but not when normalized for bacteria. There was reduced mucosal B. vulgatus in IBD, which was statistically significant for UC normalized for GAPDH or bacteria (P < 0.016) and for CD normalized for bacteria (P = 0.03). B. vulgatus were 2.7 x 104-fold more abundant than R. gnavus in HC, but the ratio of these bacteria was an order less in IBD mucosa: 2.4 x 103-fold in CD (P = 0.019) and 2.1 x 103-fold in UC (P= 0.005).
Conclusions: Both inflamed and non-inflamed IBD mucosa holds more bacteria. (FISH/Alcian Blue showed them to be mainly in mucin). This is the first study to confirm an association with real-time PCR of specific mucosal bacteria with IBD. B. vulgatus is the numerically predominant species in the human colon, but is not a versatile utilizer of polysaccharides. R. gnavus has glycosidases that degrade mucin oligosaccharides and glycosphingolipids. An imbalance of these bacteria could critically affect the integrity of the mucus layer. A defective mucin barrier is believed to be an early event in IBD pathogenesis.
aPrincipal Investigator