Microbiome analysis: from concept to completion Matthew Stoll MD,PhD,MSCS January 26, 2015
We re surrounded by bugs Human body contains 100 trillion microbes Out-number human cells 10:1 Human gut alone: ~ 99% of all of the microbiota 3 pounds of bacteria 50% of fecal volume ~ 1000 different species 3 million different genes (100x human host) Scher, Nat Rev Rheumatol 2011;7:569 De Cruz, Inflamm Bowel Dis 2012;18:372
Are they important? Germ-free state results in altered / absent disease expression in several models of autoimmune diseases. IBD Spondyloarthritis with colitis Rheumatoid arthritis Type I Diabetes Multiple sclerosis Wu, Gut Microbes 2012;3:1
Microbiota and human inflammatory diseases Inflammatory bowel disease Type I DM Arthritis Allergies / asthma Cancer Celiac disease Clemente, Cell 2012;148:1258
Microbiota may impact noninflammatory diseases Obesity 1 Metabolic syndrome 2 Non-alcoholic steatohepatosis 3 Fatty liver Autism 4 1 Turnbaugh, Nature 2006;444:11070 2 Vrieze, Gastroenterol 2012; 143:913 3 Paolella, World J Gastroenterol 2014;20:15518 4 Hsiao, Cell 2013;155:1451
Getting started Talk with someone who s done it Contact Casey, Elliot Important decisions at outset Habitat (e.g. skin, gut, oral, genitourinary) Controls: may already exist Sample size / power analyses
Bacteria composition differs by site HMP Consortium, Nature 2012;486:207
Skin has topographically distinct microbiota Heel Elbow Back Findley, Nature 2013;498:367
So does the mouth Buccal mucosa Keratinized gingiva Hard palate Throat Tonsils Tongue Dorsum Saliva Segata, Genome Biol 2012;13:R42 Supragingival plaque Subgingival plaque
Genitourinary Send subject home with cotton swab
Intestinal microbiota: Feces or colonoscopy What do you have access to Bias in selection of patients / controls Affect of washout? Jalanka, Gut 2014;ePub
Fecal microbiota Need to overcome the eeew! factor Patient prepares own sample Usually at home Shipment Money helps $25 payment $50 payment Received / Promised 27 / 62 (44%) 128 / 178 (72%)
Stool collection Place over toilet
Stool collection Scoop
Stool collection Add media Tape it up
Stool collection Open Therapak box Insert biohazard bag Add cold pack
Stool collection Mail in Fed Ex ClinPak Dispose of trash
Getting started Stool collected from Three children with ERA One child with psoriasis + joint pain Two adult controls 16S sequencing performed
Kingdom 1 2 3 4 5 6 ERA ERA Psor. ERA Ctrl Ctrl
SpA SpA Psor. SpA Ctrl Ctrl Phylum Verrucomicrobia
Family Verrucomicrobiaceae (Akkermansia muciniphila) SpA SpA Psor. SpA Ctrl Ctrl
Start of a story 6 yo female with psoriasis and joint pain Subsequently developed abdominal pain Diagnosed with IBD A. muciniphila chews up protective mucin layer in intestines Mucin-deficient mice develop colitis (van der Sluis, Gastroenterology 2006;131:117)
Next set of data 16S sequencing performed on 3 pediatric subjects with SpA 4 healthy control subjects
Increased bacteroidacaea 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% SpA SpA SpA Ctrl Ctrl Ctrl Ctrl
Preliminary data => grant Power calculations were performed using the Dirichlet-Multinomial test a sample size of n = 25 / group would yield statistical power of 0.85 Alternatively, could use prelim data to power to a limited number of organisms using standard approaches (t-test)
Next study Feature SpA Controls n 27 13 Age (years;median,range) 14, 7 19 13, 5.8 18 Male:female 13:14 6:7 BMI (median,range) 20.4, 16 41 23.7, 14 43 Disease duration 2.4, 0 8.2 N/A (years;median,range) Immunosuppresive therapy 20/27 (74%) 0/13
Unsupervised cluster analysis
Table Species SpA Cluster 1 SpA Cluster 2 p-value n 8 19 N/A F. prausnitzii (% of total bacteria, median) Bacteroides (% of total bacteria, median) Akkermansia (fraction 2%) 3.2% 4.4% 0.897 41% 13% < 0.001 0/8 8/19 0.061
Summary of findings Decreased Faecalibacterium prausnitzii Increased in two bacteria identified as being potentially relevant in the preliminary data Bacteroides genus Akkermansia muciniphila
Next directions Is dysbiosis an epiphenomenon? Transfer microbiota to arthritis model in gnotobiotic facility (NIH / NIEHS) Does treatment alter the microbiota Collaborative study of newly diagnosed subjects (Friends of CARRA) Immunologic affects of microbiota Are there functional alterations? Metagenomics Metabolomics (Steve Barnes)
Summary / conclusions Meet with stats in advance, also with researchers studying same microbiota mstoll@peds.uab.edu Incentives help! With funding agencies, be prepared to answer the so what question, what comes next after you show differences