Molecular Analysis of the Intestinal Microflora in IBD Gerald W. Tannock Department of Microbiology & Immunology, University of Otago, Dunedin, New Zealand
Commensalism
The uncultured bacterial world is greater than the cultured world. Bacterial small ribosomal subunit RNA (16S rrna) or its gene. Phylogenetic signatures contained in nucleotide base sequences. ATTCGCTTCATCGCGIAATAMTTAGCBACTEROIDESTATACTGTCCG ATTCGCTTGCAACTGIGCTAMCCATACLOSTRIDIUMCATGGTGTCCG
Phylogenetic catalogues based on 16S rrna (gene) sequences Reference to ribosomal database project II permits phylogenetic classification of sequences. Determine biodiversity. Unclassified Bacteroidetes clone frequencies detected in association with bowel biopsies from NEWLY DIAGNOSED, UNTREATED patients (CD = 20, UC = 15) and controls (14). 12.5 10.0 7.5 5.0 P <0.01 2.5 0.0 CONTROLS UC CD Clones = (63) (234) (276) GROUP Bibiloni et al., 2006, Journal of Medical Microbiology 55:1141-1149.
Fluorescence in situ hybridization (FISH)/Flow cytometry (FC) Six oligonucleotide probes that target specific sequences in 16S rrna. The probes detect phylogenetic groups of bacteria commonly present in faeces. Labeled with fluorescent dyes. FC counts the number of fluorescing cells. Bacterial groups expressed as % of total community (Eub338 probe). The six probes cover ~75% of the bowel community of healthy humans. The remaining ~25% is referred to as the phylogenetic gap.
Lay, C., et al. 2005. Applied & Environmental Microbiology 71:4153-4155. %; n = 91 European adults. Kah Weng Mah et al., 2007, Pediatric Research, in press. %; n= 17 formula-fed infants. Bifidobacteria Lab158 100 90 Atopobium group Bacteroides- Prevotella Phylogenetic gap 80 70 60 50 40 30 20 10 Clostridium cluster IV Clostridium cluster XIVa 0 0 50 100 150 200 250 300 350
Inter-subject variability = a normal range of microbiota compositions. Fecal microbiota composition determined by FISH/FACS. Ninety one healthy, European subjects. Proportions of 12 bacterial groups colorcoded.
Ileal pouch-anal anastomosis. 60 50 40 30 20 10 0 CP ANTIBIOTICS CP NO ANTIBIOTICS FAP NO ANTIBIOTICS n = 17 n = 15 www.hopkinskimmelcancercenter.org Pouchitis - can be treated with antibiotics. Total bacterial numbers the same whether CP no antibiotic, CP antibiotic, or FAP (log 10 7-8 per gram of stool). Patient group The phylogenetic gap is greater in pouchitis. This means that CP pouches contain bacteria not usually predominant in normal feces. Antibiotic treatment reduces the number of these bacteria (P <0.0001).
Community snapshots. DGGE TTGE 13 14 107 116 TI RC TC LC REC TI RC TC LC REC RC TC LC REC RC TC LC REC A 112 139 162 N In N In N In 79 80 121 124 N In N In N In N In Bibiloni et al., 2006, Journal of Medical Microbiology 55:1141-1149. B C
50 60 70 80 90 100 E37-CR Bowel biopsies reveal nationality? Albertans (Canadian) Mexican n = 36 n = 62 BACTEROIDES SPECIES 74% E44-UC E16-CR E42-CR M2-H M32-UC M39-H M16-UC M17-UC M18-UC M20-UC M19-CR M21-H E64-UC M3-H M30-H M31-H M25-H M1-UC M8-UC M34-H M36-H M37-H M40-H M22-H M27-H M38-CR M23-H M26-UC M14-UC M6-UC M35-UC M9-UC M11-H M13-H E20-CR E33-CR E29-H E56-UC E62-UC E17-H E8-CR E49-UC E9-UC E11-H E7-H E50-UC E5-H E13-CR E54-CR E25-H E26-H E23-UC E28-H E30-CR E1-CR E3-H E4-H E63-UC E2-CR E21-H E12-UC E18-CR E19-UC E22-H E31-H E34-CR E36-UC E38-H E40-H E35-H E32-H E41-CR E43-CR E6-H E61-UC E45-UC E46-CR E47-UC E55-CR E57-UC E59-UC E10-H E39-H E27-CR E51-CR E53-CR E14-CR E58-UC E15-CR E52-CR E48-UC M24-UC M28-UC M33-H M15-UC M4-UC Bibiloni et al., 2007, J. Medical Microbiology in press. M29-H
Rodents (such as IL-10 KO) with dysfunctional immune systems as models of inflammatory diseases. SPF microbiota = disease Germfree = absence of disease 3 2 1 0 Bibiloni et al., 2005. Analysis of the large bowel microbiota of colitic mice using PCR/DGGE. Letters in Applied Microbiology 41 :45-51. Mouse group
WT mice IL-10-deficient mice 1 3 6 weeks after exposure to WT microbiota Bacteroides sp. 18 6 Lactobacillus Acidophilus group Eubacterium ventriosum 5 22 10 19 Clostridium cocleatum 9 Enterococcus sp. 16 Lactobacillus murinus 4 3 2 8, 12 Bifidobacterium animalis
Are the bifidobacteria involved in pathogenesis? Germfree wild type 129S6/SvEv mice and IL-10-deficient mice Monoassociated with Bifidobacterium animalis subspecies animalis ATCC 25527 Histology, colon explants (IL-12) 3.5 DUO CECUM PROX COLON TRANS COLON DIST COLON 2 Colon explants 3.0 P <0.005 2.5 1 2.0 1.5 1.0 0.5 0 WT IL-10 KO 0.0 WT KO MOUSE GROUP (n = 10-19) Mouse group Moran et al., 2006. Bifidobacterium animalis causes extensive duodenitis and mild colonic inflammation in interleukin-10 knockout mice. Abstract at Digestive Diseases Week meeting.
Ankylosing spondylitis (AS) and bowel inflammation. Inflammation mainly of axial, particularly the sacroiliac, joints. Most patients are HLA-B27 positive. Between 5 and 10% of cases of AS are associated with IBD, either CD or UC. A much larger percentage of AS patients have subclinical gut inflammation manifested either by endoscopic findings or by histology. Rudwaleit & Baeten, 2006, Best Practice & Research Clinical Rheumatology 20:451-471. 1988 1998
HLA-B27 transgenic rats Rath et al.,1999, Infection & Immunity 67:2969-2974. Commensals Colitis & spondyloarthritis Germfree Absence of colitis & spondyloarthritis P <0.001
Clostridium cluster XIVa Atopobium group Bifidbacterium Clostridium cluster IV Bacteroides-Prevotella Patients, n = 15 P >0.05 Sulphate-reducing bacteria more persistent over three months in patients than controls. Klebsiella not detected. Stebbings et al., 2002,Rheumatology 41:1395-1401. Controls, n = 15
Comparison of B. vulgatus populations (DNA probe). 6 5 B. vulgatus as % total microbiota P >0.05 4 3 2 1 0 Patients Group Controls AS immune systems react differently to resident Bacteroides than controls?
AS and matched controls (21 of each) Stool sample Randomly pick 10 Bacteroides fragilis group cultures Lymphocyte proliferation 3 H-thymidine Blood sample Cytokines (IL-1β, IFN-γ, TNF-α, IL-6, IL-8, IL-17, IL-10)
AS IL10 2500 2000 IL-10 1500 1000 500 0 CAS IL10 2500 2000 IL-10 1500 1000 500 0
IL-10 production during T-cell proliferation. Bacteroides SEA 2500 3500 2000 3000 2500 1500 2000 1000 500 1500 1000 500 0 0 PATIENTS CONTROLS PATIENTS CONTROLS P <0.0001 P >0.05 AS lack anti-inflammatory activity with regard to Bacteroides?
In summary - Nucleic acid-based analytical methods have potential in discriminating between microbiota of different compositions. Experimental animal and human studies point to Bacteroides as worthy of further study in IBD and related conditions, especially where the objective is to obtain functional rather than phylogenetic knowledge.
Supertechs: Karen Munro Mary Alice Simon Corinda Taylor Postdocs: Rodrigo Bibiloni Christophe Lay Academic associates: Margaret Baird Clinical associates: Lori Baladay Chris Daynes Richard Fedorak John Highton Robin McLeod Balfour Sartor Simon Stebbings Puneeta Tandon Mark Thompson-Fawcett Bee Wah Florencia Vargas-Voracka Sponsors: Crohns and Colitis Foundation of America. Crohns and Colitis Foundation of Canada. Lottery Grants Board HealthCare Otago Charitable Trust Otago Medical Research Foundation Otago University Research Grant Otago School of Medical Sciences/Dunedin Medical School