Transcript Correlation Between Bacteria and Inflammatory Bowel

Correlation Between
Bacteria and Inflammatory
Bowel Diseases
Scott Belozer
BB major at OSU
L a r r y H o d g e s & D r. Wa l t R e a m
M i c ro b i o l og y @ O S U
Amnon Sonnenberg, MD
P o r t l a n d VA M e d i c a l C e n t e r & O H S U
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
Generate clone
libraries
Sequence cloned 16S
rRNA genes
Use BLAST to identify
bacterial species;
correlate sequences
with T-RFLP profiles
DNA extraction
PCR amplify
bacterial 16S
ribosomal RNA
genes
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
Fluorescent intensity
Veillonella
Prevotella
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
 Walt
Ream
 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/DiseaseImag
es/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/digestivesystem.jpg>