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

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.

Ulcerative Colitis

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.

Digestive Tract Crohn’s Disease Ulcerative Colitis

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

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

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

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.

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

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.

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.

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

Clone Libraries

T-RFLP Size List

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

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

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

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

IBD free IBD

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.

IBD free IBD

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

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.

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.

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

References: "Ulcerative Colitis." Mayo Clinic. Mayo Clinic, n.d. Web. 21 Jun 2011. <http://www.mayoclinic.org/ulcerative-colitis/>. "Healthy Colon Vs Nonhealthy Colon." Virtual Medical Center. Web. 21 Jun 2011.<http://www.virtualmedicalcentre.com/uploads/VMC/DiseaseImages/650_Ulcerative_Colon.JPG>. Oberhuber G, . "Focally enhanced gastritis: a frequent type of gastritis in patients with Crohn's disease.." Medscape (1997): n. pag. Web. 21 Jun 2011. <http://www.medscape.com/medline/abstract/9041230> "Chron's Disease." Mayo Clinic. Mayo Clinic, n.d. Web. 21 Jun 2011. <http://www.mayoclinic.org/crohns/>. "Digestive Tract." Generic Look . Web. 21 Jun 2011. <http://www.genericlook.com/img/uploads/anatomy/digestive-system.jpg>