Introduction to Microbiome & Discussion of He, et al. Paer

1. Introduction to Microbiome & Discussion of He, et al. Paer Amir Zarrinpar, MD, PhD Assistant Professor Division of Gastroenterology Institute for Diabetes and Metabolic Health Center for Microbiome Innovation VA San Diego GI Obesity and NAFLD/NASH Clinic May 2019

2. The Human Super Organism • The human body is comprised of ~40 trillion cells. • The number of organisms that live on and within us: 43 trillion. • 50% of cells in our body are microbial. • We have 100x more microbial genes than human genes. • Microbial genes turn on and off in response to what we do. • Our genes turn on and off in response to what our microbiota do. • Can adapt to new stimuli in the order of hours.

3. The Human Microbiome - Definitions • Microbiota: microorganisms • Microbiome: microorganisms and their genomes • Metagenome: collective genes of microorganisms • Metabolome: metabolites • Metaproteome: Proteins • Metatranscriptome: the genes (mRNA) that are being expressed by the bacteria

4. The Human Microbiome - Definitions • Microbiota: Who’s there? • Metagenome: What can they do? • Metabolome: What have they changed? • Metaproteome: What have they made? • Metatranscriptome: What are they doing?

5. Why the microbiome research boom? • In the past, to study microbes, you had to be able to grow it in a lab. • Bacteria characterized by: • Colony characteristic • Growth media requirements. • Oxygen use • Staining method. • 1980s, sequencing technology started • Too impractical to fully sequence every microbe. • Would lead to unknown sequences from undiscovered microbes.

6. Development of a Bacterial Marker • Need DNA sequences that can be used as markers to categorize organisms into phylogeny. • The more related the taxonomic unit for two organisms, the more similar the DNA marker will be. • Kingdom/phylum/class/order/family/genus/species.

7. 16S rDNA is That Marker • 16S ribosomal DNA encodes 16S ribosomal RNA which is a component of 30S small subunit of prokaryotic ribosomes. • Used to determine bacterial phylogeny.

8. 16S rDNA Regions • Conserved regions – Same DNA sequence for all known bacteria. • Variable regions – Different sequences depending on the kind of bacteria. • Amplify 16S rDNA genes using primers directed at conserved regions but flanking variable regions. • By convention, members of a species share 97% of sequence. • Operational taxonomic unit (OTU).

9. The Microbiome Boom • Combination of factors: • 16S rDNA phylogeny • Cheaper sequencing • Better bioinformatic tools • Faster computers • But only study Bacteria. • New techniques being developed to study fungi. • Internal transcribed spacer (ITS) region primers. • Viruses? Archea?

10. 16S rDNA Approach Morgan & Huttenhower, PLoS Comp Bio 2012

11. Shotgun metagenomic approach Morgan & Huttenhower, PLoS Comp Bio 2012

12. Germ Free/Gnotobiotic Mice • Germ Free mice are born and raised in sterile conditions. • They are removed from the mother by Caesarean section • Live in the isolators with germ-free foster mothers. • Investigators must perform all experiments using gloves attached to the isolators so that the animals never come into accidental contact with germs. • Gnotobiotic mice – where bacteria in intestine is known (includes germ-free mice).

13. Physiological changes in Germ-Free Mice • Animals reared in a gnotobiotic colonies have: • Poorly developed immune systems (especially T-reg) • Lower cardiac output • Thin intestinal walls • Malnourished • High susceptibility to infectious pathogens • Elevated corticosterone levels

14. What does the gut microbiome do? Grenham S et al. Front Physiol, 2011

15. Gut Microbiome - Niches • Small intestine has various niches which attract different kinds of bacteria. • Lumen • Mucous • Villa • Crypts • Abundance • Diversity • Oral cavity • Esophagus • Stomach • Small intestine • Colon • Niche affected by pH, bile, digestive enzymes, pO2, transit time, nutrients available. • Fecal and mucosal microbiome are vastly different Small intestine:

16. What changes the gut microbiome? • Factors that affect gut microbiome include: • Age • Genetics • Geography • Disease state • BMI • Diet • Environmental • Household chemicals • Medications Quigley, Nat Rev Gastro & Hep 2017

17. Intestinal Dysbiosis • Dysbiosis – when there is imbalance in microbiome. • Some use “bacterial overgrowth”… but that is an inaccurate term. • Pathobiont vs. Symbiont • Increasingly being recognized as a risk factor for disease development: • Obesity • Diabetes • Atopic dermatitis • Inflammatory bowel disease • Irritable bowel syndrome • Autism? • Parkinson’s disease?

18. Probiotics/Prebiotics • Probiotics: Non-pathogenic organisms which provide beneficial effects to the host. • Beyond their inherent nutrition if provided in adequate quantities. • Prebiotics: chemicals that induce the growth and/or activity of commensal microorganisms (e.g., bacteria and fungi). • Contribute to the well-being of their host. • Polysaccharides like inulin.

19. Problems with clinical studies • It’s not clear what is normal • Do not account for diet and many other confounders • Single point in time • State vs. trait • Influence of therapy • Sampling • There is a difference between mucosal and fecal microbiome (Ringel Gut microbes 2015) • Lack of reproducibility, even within the same lab

20. Establishing a More Causative Role of the Microbiome in Disease • Homogenous phenotype • Control for diet and other external factors (e.g. fiber intake, alcohol use, smoking, PPIs, other exposures). • Standardize sampling, storage, analytical techniques • Longitudinal rather than single-point-in-time studies. • Especially if changes in phenotype occur • Sampling microbiota at the site of action. • Define bacterial function using multi-omic approach. • Show transfer of phenotype in gnotobiotic mice. • Observe symptomatic improvement with a therapy directed at specifically particular pathway identified.

21. The future • Define Microbiota changes that are truly linked to disease • Mechanistic studies linking bacterial function to phenotype • Microbiota in diagnostics • New disease categories • Predict therapeutic responses • Smarter approach to therapeutics – niche modulation and functional change • Engineered bacteria • Synbiotics • Prebiotics

22. Summary • Microbiota is important in health and disease • Host-microbiome interactions in man are complex and far from completely understood. • Diet is a major modulator of the microbiome • Associations with disease are tantalizing but it remains to be shown that they are causal • Many possibilities for new therapeutics.

23. Today’s paper discussion…

24. Background • Commercial microbiota tests compare composition of microbiome to a “normal” cohort. • But what does “normal” mean, and what is dysbiosis in this setting. • Regional differences • Dietary differences • Need incredibly large sample sizes (e.g. American Gut) • Additional challenge: variability of technical replicates > variability within a population

25. Guangdong province

27. Overview of sampling regions with GDP

28. Regional variation is important

29. Heatmap showing maximal fold change differences in OTUs among different locations and metabolic disorders. Fold changes weregrouped as one to two, two to five and more than five.

30. Evaluating cross-applicability of gut microbiota–based disease models among locations.

32. Illustration of the difficulty gradient used to interpolate andextrapolate the MetS model

33. Thank You!