1. Correlation Between Bacteria and Inflammatory Bowel Diseases
Scott Belozer
BB major at OSU
Larry Hodges & Dr. Walt Ream OSU Amnon Sonnenberg, MD Portland VA Medical Center & OHSU

2. Ulcerative Colitis
Definition: Inflammatory Bowel Disease (IBD) results in chronic inflammation of the digestive tract.
Area affected: Usually only affects the innermost lining of the large intestine (colon) and rectum.

3. Ulcerative Colitis

4. Crohn’s Disease
Definition: An IBD that results in chronic inflammation of the lining of the digestive tract.
Area affected: Can occur in patches anywhere in the digestive tract.

5. Digestive Tract
Crohn’s Disease
Ulcerative Colitis

6. Occurrence of IBD Occurs mainly in developed countries
Onset usually between ages 15-30
About 1.4 million Americans have IBD
About 30,000 Americans each year learn
they suffer from IBD

7. Significance No effective treatment for Crohn’s Disease.
-Ulcerative Colitis requires surgery
Identifying the cause of IBD may lead to more effective treatment.

8. Focally Enhanced Gastritis & IBD
Background:
~76% of Crohn’s patients have focally enhanced gastritis (FEG)
~20% of ulcerative colitis patients have FEG
< 1% of the general population (IBD free) have FEG

9. Hypothesis/Rationale
Hypothesis: Pathogen causing inflammation in the stomach may cause inflammation in the colon.
Rationale: Stomach microbiota are much simpler than microbial populations in the colon.

10. Methods to Study Gastric Bacteria
Receive gastric biopsies
DNA extraction
Generate clone libraries
PCR amplify bacterial 16S ribosomal RNA genes
Sequence cloned 16S rRNA genes
Use BLAST to identify bacterial species; correlate sequences with T-RFLP profiles
T-RFLP analysis

11. Bacterial 16S rRNA All bacteria have 16S ribosomal RNA (rRNA).
Bacterial 16S rRNA genes have highly conserved regions and variable regions.
Large database of 16S rRNA gene sequences.

12. Terminal Restriction Fragment Length Polymorphism Analysis
T-RFLP analysis measures length and relative quantity of each labeled (terminal) restriction fragment.
A mixture of end-labeled 16S rDNAs amplified by PCR are digested with a restriction endonuclease.
The size of each labeled fragment is measured using a DNA sequencer.

13. T-RFLP Example Fluorescent intensity IBD 3 1uL pcr1 Rsa
Size of Fragments

14. Clone Libraries

15. T-RFLP Size List

16. Final Steps Get sequences of cloned 16S rRNA genes
Do a BLAST search on NCBI database
Identify major T-RFLP peaks
Determine abundance of bacterial species in patients

17. Results for Patient 3 Predominant genera present in sample: Prevotella
Streptococcus
Veillonella
Identified an unknown peak also found in other patients:
Streptococcus mitis strain GCSS 1303

18. Patient 3 Msp T-RFLP Diagram
Veillonella
Prevotella
Fluorescent intensity
Streptococcus ?
Strep. mitis GCSS 1303
Size of Fragments

19. Overall Results
Most IBD patients have larger gastric bacterial populations than IBD-free patients.

20. IBD free
IBD

21. Overall Results
Most IBD patients have larger gastric bacterial populations than IBD-free patients.
Bacterial populations differ widely between patients.
Streptococcus, Prevotella, and Veillonella species are present in almost all patients.
-Streptococcus and Prevotella species are usually the most abundant
Large populations of Prevotella (30-85%) occur in four of eleven patients with active or severe IBD.

22. IBD free
IBD

23. Summary
Most IBD patients have larger gastric bacterial populations than IBD-free patients.
Bacterial populations differ widely between patients.
Streptococcus, Prevotella, and Veillonella species are present in almost all patients.
-Streptococcus and Prevotella species are usually the most abundant

24. Summary
Large populations of Prevotella (30-85%) occur in four of eleven patients with active or severe IBD.
Species of Prevotella differ between patients, and strain differences may account for inflammatory disease in some patients.

25. Future Work
Analyze gastric microbiota in 2nd gastric biopsy from patient 10, who has severe Crohn’s disease.
Culture Prevotella melaninogenica from IBD patient 10 and controls.
Use genome sequencing to examine differences between strains.

26. Acknowledgements HHMI Program Kevin Ahern Dr. Walt Ream’s Lab Group
Larry Hodges
Sarah Layoun
Michael Wiens
Joe Sexton
Takeda Pharmaceutical

27. References:
"Ulcerative Colitis." Mayo Clinic. Mayo Clinic, n.d. Web. 21 Jun <
"Healthy Colon Vs Nonhealthy Colon." Virtual Medical Center. Web. 21 Jun 2011.<
Oberhuber G, . "Focally enhanced gastritis: a frequent type of gastritis in patients with Crohn's disease.." Medscape (1997): n. pag. Web. 21 Jun <
"Chron's Disease." Mayo Clinic. Mayo Clinic, n.d. Web. 21 Jun <
"Digestive Tract." Generic Look . Web. 21 Jun <