Mucosal Flora in IBD. Alexander Swidsinski. Supported by Broad Medical Research Program

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1 Mucosal Flora in IBD Alexander Swidsinski Supported by Broad Medical Research Program

2 Mean± SD (x10 3 cfu/μl) of Mucosal Bacteria Asymptomatic Controls (n=40) Self-limiting Colitis (n=28) Total anaerobes 0.18± ±5.3 NS Bacteroides 0.02± ±0.6 NS Total aerobes 0.003± ±0.4 NS Enterobacteriaceae 0.002± ±0.5 P < 0.06 Indeterminate Colitis (n=104) 3.41±16 P < ±2.1 P < ±0.5 P < ±0.3 P < UC (n=156) 3.8±11 P < ±9 P< ±0.6 P < ±0.5 P <0.047 CD (n=82) 9.1±18 P < ±5.5 P < ±0.8 P < ±0.8 P < P as compared to controls

3 Mucosal bacteria (x10 3 cfu/μl) and clinical data UC (n=156) CD (n=82) All patients age < > age at the time of man ifestation 30 < > UC CD (n=156) (n=82) All duration of disease in years < > f m ,5 2,6 2,5 24 3, ,9 3,5 1,5 < >40 UC Crohn

4 Mucosal bacteria, disease activity and therapy UC CD (n=156) (n=82) All UC CD (n=156) (n=82) All remission activity exacerbated fistula 20.1 no 4.8 colonic surgery without surgery no antibiotics in last 12 months on antibiotics after antibiotics (1-4 weeks) no azathioprine azathioprine corticosteroids without ,8 5,5 2,9 2,9 8,2 Remission aktiv Schub 5ASA in gramm , > UC Crohn

5 Multicellular bacteria forming stromatolith in Australian salt lakes

7 FISH analysis of the mucosal biofilm using r-rna targeted probes

8 Mucus gap between the colonic wall and feces in healthy mouse

9 Mucus gap between the colonic wall and feces in healthy mouse

10 Human colonic wall covered with mucus omitting bacteria

11 Human ileal wall covered with mucus omitting bacteria

12 intact mucus is partially denuded, the biopsy is covered by bacteria

13 Fecal contamination on submucosal parts of the biopsy

14 bacteria on the peritoneal side of the mouse intestine/ biases

15 Island of bacteria nestled within mucin and intimately connected with epithelial surface

16 Island of bacteria nestled within mucin and intimately connected with epithelial surface (colon)

17 Island of bacteria nestled within mucin and intimately connected with epithelial surface (ileum)

18 Lawns of bacteria covering the epithelial surface

19 Lawns of bacteria covering the epithelial surface (colon)

20 Lawns of bacteria covering the epithelial surface (ileum)

21 Differential focusing of bacterial lawns covering the epithelial surface

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39 Goblet cell empting

40 Goblet cell empting

41 Goblet cell empting

42 Goblet cell empting

43 Goblet cell empting

44 Goblet cell empting

45 Goblet cell empting

46 Goblet cell empting

47 Goblet cell empting

48 Goblet cell empting

49 Goblet cell empting

50 Goblet cell empting.

51 Onionlayer like biofilm structure

52 Airbubbles like biofilm structure.

53 Table 1 FISH probes Name Target Eub338 virtually all Bacteria, Kingdom (Eu)Bacteria Arch915 Archaea Alf1b Alpha group of Proteobacteria: Rhodobacter, Acetobacter, Paracoccus, some Pseudomonas etc. Beta42a Beta subclass of Proteobacteria: Rhodocyclus, Bordetella, Neisseria,Thiobacillus, Alcaligenes and other Gam42a gamma subclass of Proteobacteria: Enterobacteriaceae, Proteus, Legionella,Azotobacter Ec1531 Escherichia coli Srb385 sulfate reducing bacteria, the main component of the delta subclass of Proteobacteria Hpy-1 Helicobacter pylori epsilon subclass of Proteobacteria Arc1430 Arcobacter ssp. epsilon subclass of Proteobacteria HGC Gram positive bacteria with high G+C content LGC Gram positive bacteria with low G+C content Sfb Segmented filamentous bacteria Erec482 Clostridium coccoides - Eubacterium rectale group Chis150 Clostridium histolyticum group Clit135 C. lituseburense group Lab158 Lactobacillus and Enterococcus group Strc493 Streptococcus Ecyl Eubacterium biforme, Clostridium innocuum and other Phasco Acidaminococcus fermentans and other Veil Veillonella group Rbvo,Rfla Ruminococcus flavefaciens, Clostridium leptum Bif164 Bifidobacterium Ato291 Atopobium, Coriobacterium, Eggerthella and Collinsella spp CF319a Cytophaga-Flavobacterium group Bac303 Bacteroides/Prevotella group Bfra602 Bacteroides fragilis group Bdis656 B. distasonis group

54 FISH signals simulating intracellular bacteria

55 FISH signals simulating intracellular bacteria

56 FISH signals simulating intracellular bacteria (SAMP mouse)

57 Intraepithelial bacteria located perinuclear at different levels

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70 The mucosa of patients with IBD is covered with a complex spatially structured multispecies biofilm. This biofilm is organized in islands, patches and lawns of multispecies bacteria. Biofilm patches and layers penetrate mucus leading to direct contact between fecal flora and epithelia. They are ideal for luminal antigens and toxins to reach the unshielded epithelial surface and to trigger cascades of host responses. The peculiarities of individual immunity and genetic disposition may explain the rest.

71 We thank Broad Medical Research Program BMRP for supporting this project

Spatial organization of bacterial flora in normal and inflamed intestine: A fluorescence in situ hybridization study in mice

Spatial organization of bacterial flora in normal and inflamed intestine: A fluorescence in situ hybridization study in mice PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(8):1131-1140 www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327 wjg@wjgnet.com LSVIR 2005 The WJG Press and lsevier Inc. All