Introduction to Microbiome & Discussion of He, et al. - PowerPoint Presentation

Slide1

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

Slide2

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.

Slide3

The Human Microbiome - Definitions

Microbiota

: microorganisms

Microbiome: microorganisms and their genomesMetagenome: collective genes of microorganismsMetabolome: metabolitesMetaproteome: Proteins

Metatranscriptome:

the genes (mRNA) that are being expressed by the bacteria

Slide4

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?

Slide5

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 characteristicGrowth media requirements.Oxygen use Staining method.1980s, sequencing technology startedToo impractical to fully sequence every microbe.Would lead to unknown sequences from undiscovered microbes.

Slide6

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.

Slide7

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.

Slide8

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).

Slide9

The Microbiome Boom

Combination of factors:

16S rDNA phylogeny

Cheaper sequencingBetter bioinformatic toolsFaster computersBut only study Bacteria.New techniques being developed to study fungi.Internal transcribed spacer (ITS) region primers.Viruses? Archea

?

Slide10

16S rDNA Approach

Morgan &

Huttenhower

,

PLoS

Comp Bio 2012

Slide11

Shotgun metagenomic approach

Morgan &

Huttenhower

,

PLoS

Comp Bio 2012

Slide12

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).

Slide13

Physiological changes in Germ-Free Mice

Animals reared in a gnotobiotic colonies have:

Poorly developed immune systems (especially T-

reg)Lower cardiac outputThin intestinal wallsMalnourishedHigh susceptibility to infectious pathogensElevated corticosterone

levels

Slide14

What does the gut microbiome do?

Grenham

S et al. Front

Physiol

, 2011

Slide15

Gut Microbiome - Niches

Small intestine has various niches which attract different kinds of bacteria.

Lumen

MucousVillaCryptsAbundance

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:

Slide16

What changes the gut microbiome?

Factors that affect gut microbiome include:

Age

GeneticsGeographyDisease stateBMIDietEnvironmentalHousehold chemicalsMedications

Quigley, Nat Rev Gastro & Hep 2017

Slide17

Intestinal Dysbiosis

Dysbiosis – when there is imbalance in microbiome.

Some use “bacterial overgrowth”… but that is an inaccurate term.

Pathobiont vs. SymbiontIncreasingly being recognized as a risk factor for disease development:ObesityDiabetesAtopic dermatitis

Inflammatory bowel disease

Irritable bowel syndrome

Autism?

Parkinson’s disease?

Slide18

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.

Slide19

Problems with clinical studies

It’s not clear what is normal

Do not account for diet and many other confounders

Single point in timeState vs. traitInfluence of therapySamplingThere is a difference between mucosal and fecal microbiome (

Ringel

Gut microbes 2015)

Lack of reproducibility, even within the same lab

Slide20

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.

Slide21

The future

Define Microbiota changes that are truly linked to disease

Mechanistic studies linking bacterial function to phenotype

Microbiota in diagnosticsNew disease categoriesPredict therapeutic responsesSmarter approach to therapeutics – niche modulation and functional change

Engineered bacteria

Synbiotics

Prebiotics

Slide22

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 microbiomeAssociations with disease are tantalizing but it remains to be shown that they are causalMany possibilities for new therapeutics.

Slide23

Today’s paper discussion…

Slide24

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 differencesDietary differencesNeed incredibly large sample sizes (e.g. American Gut)Additional challenge: variability of technical replicates > variability within a population

Slide25

Guangdong province

Slide26

Slide27

Overview of sampling regions with GDP

Slide28

Regional variation is important

Slide29

Heatmap showing maximal fold change differences in OTUs among different locations and metabolic disorders. Fold changes were

grouped as one to two, two to five and more than five.

Slide30

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

Slide31

Slide32

Illustration of the difficulty gradient used to interpolate and

extrapolate the

MetS

model

Slide33

Thank You!