Stability and Resilience in the Human : Response of Gut to Colonic Lavage Laurie Rumker, Dr. Les Dethlefsen, Dr. David Relman Department of and Immunology, School of Medicine

Microbiota and the Human Body Ecosystem Experimental Design Methodology Study Aims

• = Fecal and Urine ! Aim 1. Compare roune unperturbed dynamics to varied • Bacteria contribute 90% of cells, Samples Taken ! responses to lavage; characterize effect of disturbance 99% of genes in human body Colonic • Roles of microbiota in health: Lavage! • Aim 2. Evaluate applicaon of ecological principles to human gut - Pathogen resistance microbiota in context of colonic lavage disturbance - Metabolizaon - Xenobioc modificaons Baseline Stability! Disturbance! Recovery Dynamics! (Weekly Sampling)! (Daily Sampling)! (Weekly Sampling)! • Aim 3. Idenfy features characterisc of resilient communies - Immune system regulaon • Contributes to malnutrion, obesity, heart disease, • Study Subjects: 8 healthy volunteer adult parcipants Body Sites and Predominant inflammatory bowel disease, • Fecal and urine samples at each me point analyzed three ways: Proof of Concept: Anbioc Disturbance Taxa of the Microbiota Crohn’s disease, many more (Dethlefsen and Relman, 2007) Fluctuaons of α-Diversity Metrics in Response to an Anbioc Series • Marker gene amplified, sequenced 16S rDNA • Conserved idenficaon tag Anbiocs Anbiocs Research Quesons Sequencing • Reveals changes in taxonomic 80 composion, relave abundances 60 • How consistently does the microbiome retain its composion and 40 acvity over me? 20 • Fragmented, sequenced whole Relave Diversity • 0 How easily can the health benefits conferred upon a host by the Shotgun genome microbiota be lost due to a disturbance of the community? 0 50 100 150 200 250 300 350 Metagenomic • Reveals relave gene frequencies Day of Sampling Sequencing • Computaonal reassembly associates Phylogenec Diversity Applying Macroecological Theory genes with specific taxa Taxonomic Diversity (Shannon Index, Values *10) OTU Richness (Values/10) • Stability = ability of system to return to equilibrium quickly, with • Anbioc series triggered dramac temporary decrease in α- minimal deviaon aer temporary disturbance • Idenfies small molecules produced diversity metrics based on 16S rDNA data (Dethlefsen, 2011) • Equilibrium = set of defined values for relevant key variables Metabolomic Mass • Present study expands upon this one with longer mescale, Spectrometry • (Analyses performed by collaborators • Resilience = when challenged with disturbance, ecosystem ability at University College London) greater sampling depth, use of metagenomics and metabolomics to adapt and retain funcon, structure, identy, and feedbacks • Colonic lavage is less selecve disturbance causing greater • Disturbance could induce regime shi to new stable state decrease in total biomass

Ecosystem Project Status

Variaon Around Stable Equilibrium Alternave Subjects DNA Send Stable State Design Study Recruit Metagenomic Data Processing Sample Extracon 16S Metabolomics and Obtain and Enroll Sequencing and with QIIME and and from Fecal Sequencing Urine and Fecal Ecosystem Stable States or “Basins of Aracon” IRB Approval Subjects Reassembly Phyloseq Cleanout Samples Samples • Possible predictors of microbiome resilience: - Taxonomic diversity - Intermediate stability week-to-week or day-to-day 3 parcipants currently sampling, 5 parcipants’ samples DNA extracted, 16S sequenced - Funconal redundancy (the Insurance Hypothesis) about to undergo cleanout, will complete on HISeq2500 with 150nt paired-reads, produced - Specific constellaon of taxa sampling December 2014 >98 million reads, metagenomics preparaons ongoing