The Human Microbiome: The

Undiscovered Country

Walter J. Coyle, MD, FACP,FACG

Movement of the Talk

Describe the Microbiome Microbiology 101 Review the diversity of human gut

flora Understand how our bacteria

influence our metabolism and obesity

Review the plethora of probiotics Describe prebiotics and their

potential uses Conclusions and the Future

The Human Microbiome

Definitions: Microbiome: Aggregate of all gut

species Microbiota: Individual bacterial species

in the biome Over 100 trillion organisms (1014)

Passengers in the mobile colonic petri dish

Over 500 species identified so far (70 divisions)

90% of the cells in our body our microbial!

100 fold more genes in our gut then in us

Our flora are an integral part of our genetic landscape and evolution

Anatomic Regions of the Gut

Upper GI tract: 102 – 104 cells/ml Lactobacilli, streptococci, H pylori

Ileum: 106-1012 cells /ml, upper bacteria plus Faculative anaerobes: Enterobacteriaceae Obligate anaerobes: Bacteroides,

Veillonella, Fusobacterium and Clostridium species

Colon: distal human colon is the most biodense natural ecosystem known (1010-1012 cells/ml) Complex and diverse Comprise most of our bacterial biomass

Microbes and Humans

Dethlefsen Nature 2007; 449:812-818

Micro 101

Pace in Science 1997 276:735-740

The Human Gut Flora

DiBiase, et al. Mayo Clin Proc 2008;83:460-469

Mouse intestine, mucus gel layerHome to billions of organisms

Sonnenburg J, et al. in NatureImmunology 2004:5

The Microbiome: Who’s there?

Early gut colonization has four phases Phase 1: Sterile gut Phase 2: Initial acquisition: vagina,

feces, hospital Phase 3: Breast feeding or bottle-

feeding (different) Breast fed more bifidobacteria (up to 90%

of flora) Bottle fed more diverse; more Bacteroides ,

and Clostridial species Phase 4: Start of solids; move to adult

flora Bifidobacteria remain key flora into

adulthood

Ley, Peterson, Gordon. Cell 2006 ;124:837Ley, et al. PNAS. 2005, 102: 11070 Edwards, et al. Br J Nutr. 2002

The Microbiome: Who’s there?

Adult Microbiome: Increasing diversity of flora as we age In some newer PCR (16S rRNA) studies,

up to 92% of the flora in adults were “novel” species

Serial stool collections show remarkable stability by an individual

Greatest concordance with twins Less concordance with households

Host genetic influence unexplored.McCartney and Gibson in Gastrointestinal Microbiology, 51-73, 2006

The Microbiome: Who’s there?

Multiple species: eukaryotic, bacterial, archael

Firmicutes (Gram +/ motile) and Bacteroidetes (Gram -) : Majority These two groups comprise > 90% Anaerobes / aerobes: 1000:1

Far fewer species than the environment

High levels of interpersonal diversity My stool is not your stool

Ley, Peterson, Gordon. Cell 2006 ;124:837Ley, et al. Science 2005; 307: 1915

Ley R, et al in Cell 2006:124:837-848

Gut Flora and Metabolism

Metabolomics

Study of the metabolites and small molecules that the body and gut bacteria produce.

New area of science Broader than proteonomics

Includes bacteria products with our own genetic products

Pioneered by Jeremy Nicholson and Jeff Gordon

Gut Flora and Metabolism

Microbial genomes enhance our metabolic activity May indirectly or directly effect our

metabolism The colon is very active metabolically

20-70 gms of carbos and 5-20 gms of protein/day

Over 100 kcal per day!

Mass of colonic microbiome = single kidney Metabolically as active as the liverHooper, et al. Annu Rev

Nutr, 2002

Gut Flora and Metabolism

Energy salvage: esp via the short-chain fatty acids

Acetate, butyrate, propionate (SCFAs) Absorbed into body and used by liver and

others organs Acetate and propionate modulate glucose

metabolism in the liver and adipocytes (glycemic index)

50-70% of colonic cell energy derived from butyrate

Number and diversity of organisms key

Do we acquire flora that program us for obesity or normal weight?

Mackowiak, NEJM, 1982; 307:83-93 Hooper, et al. Annu Rev Nutr, 2002

Gut Flora Metabolic Reactions

Metabolic reactions

Goldin BR in Gastrointestinal Microbiology 138-154, 2006

Gut Flora and the Brain

Collins and Bercik in GASTRO 2009;136:2003–2014

Obesity and the Gut Flora

Ley, et al. PNAS. 2005, 102: 11070-75

The Microbiome: Changes in flora

Mice and humans have different gut flora but the two largest divisions are shared in common Bacteroidetes (Gram -) Firmicutes (Gram +)

These flora change in response to diet and obesity of host

Ley, et al. PNAS. 2005, 102: 11070-75

Ley, et al. PNAS. 2005, 102: 11070-75

The Microbiome: Changes in flora

Obesity changes the relative proportions of divisions.

Obese mice AND obese people have more Firmicutes than Bacteroidetes: dieting and weight gain shifts these proportions.

What are the implications of the change in our colonic metabolic machine Chicken or the egg?Ley, et al. PNAS. 2005,

102: 11070-75

Gut Flora and Obesity

Germ free mice studied by Gordon, et al

Control mice (remaining germ free) were changed from low fat diet to “Western” diet

Mice given normal gut flora (conventialized mice) also had same diet change.

Low fat diet: 5% lipids “Western” diet: 41% lipids, 41%

carbos (with simple sugars), 18% protein

Gordon J, et al, PNAS 2007;104:983

Gut Flora and Obesity

Mice then studied for change in weight and fat pad size

Assessed response to fatty meal loading

Assessed locomotion of mice via gastrocnemius muscle activity.

Results: See Figures

Gordon J, et al, PNAS 2007;104:983

Gordon J, et al, PNAS 2007;104:983

Gut Flora and Obesity

Gut Flora and Obesity GF mice are “resistant” to the

Western diet Gut flora are responsible for

increasing energy production from food “Doctor, I don’t eat” May be

partially true! Gut flora also influence lipid

production, adipose cell storage, and appear to influence mouse locomotor activity “Couch potato” gut flora My gut flora made me do it!

Gordon J, et al, PNAS 2007;104:983

Hippocrates

“Let food be thy medicine and

medicine be thy food.”

The Microbiome: transplant experiments

Turnbaugh, et al. Nature 2006; 444:1027

Ob Ob mouseWild type mouse

The Microbiome: transplant experiments

Germ-free mice given ob/ob or wild-type flora

Chow consumption and exercise the same for both groups

Both sets had similar starting weight and % BF.

The ob/ob microbiota promote host adiposity

Turnbaugh, et al. Nature 2006; 444:1027

Ley R, et al in Nature 2006:444

Human Flora and Obesity

Mice data links gut flora with obesity and diet

Studied 12 obese human subjects; low calorie diet for one year Fat-restricted (FAT-R) Carbohydrate restricted (CARB-R)

Microbiota studied for one year (16s rRNA)

Firmicutes and bacteroidetes dominated (92.6%)

Ley R, et al in Nature 2006:444

Human Flora and Obesity

Ley R, et al in Nature 2006:444

Human Flora and Obesity

Firmicutes dominate in obese subjects

All subjects (no diff with diet) had bloom of all Bacteroidetes with decline in Firmicutes

CARB-R: Change began at 2% wgt loss

FAT-R: Change began at 6% wgt loss Dynamic link between obesity and

gut flora exists in humans (proven 1st in mice)

Proposed Mechanisms in Obesity

DiBiase, et al. Mayo Clin Proc 2008;83:460-469

Microbiome: regulators

Archae: 1-2 % of mouse and human flora Represent a major microbial group in

gut flora Increased in obese mice Many are methanogenic :

Methanobacter smithii Converts CO2 and H2 gas to methane By decreasing the partial pressure of

H2 gas these bacteria can drive bacterial metabolism

The flora of obese mice are more efficient at extracting energy: “The Energy Harvest”

The Gut and Obesity: Options

Diet may manipulate flora Low carbs, high fat High carbs, low fat

Probiotics Prebiotics Stool transplants

“Guarantee weight loss with thin people’s feces!”

Could this be the future?

Coyle’s Corollary

It is better to be a stool donor than a recipient.

Probiotics

Probiotics Definition: Live microorganisms which

when ingested in adequate amounts confer a health benefit on the host.

Majority of probiotics are Gram +, lactic acid producers Bifidobacterial species and Lactobacillus

species Survive transit through stomach and

duodenum Others include: non-pathogenic

streptococci, enterococci, E coli Nissle 1917, Saccharomyces boulardii (yeast)Fooks, et al. Int Dairy J, 1999

Sheil, et al. In Gastrointestinal Microbiology, 2006

Common Probiotics

Khedkar and Ouwehand in Gastrointest Microbiology 315-334, 2006

Probiotics

VSL #3 4 lactobacilli

L. plantarum, casei, acidopholus, delbrueckii spp

3 bidifobacteria B. infantis, breve,

longum 1 streptococcus

Streptococcus salivarius ssp. thermophilus

Rand, PC studies have shown efficacy in pouchitis and IBS

Probiotics

Digestive Advantage Ganeden BC30

Bacillus coagulans Erythritol Cellulose Other minor

ingredients

Probiotics

Bifidobacterium infantis 35624 aka Bifantis

“Patented” strain of probiotic in Align

Decreased symptoms in two large trials in subjects with IBS*

*Whorwell P, et al. Am J Gastro 2006; 101O’Mahoney L, et al. Gastro 2005;128

Probiotics

Saccharomyces boulardii

Other minor ingredients

Shown in Rand / PC trials to help prevent recurrent C. difficile infection

Probiotics in Food (Actimel)

L. casei Immunitas™

Claim it is scientifically proven to be effective

“Each bottle contains 10 billion live” bacteria “that survive and remain active in thedigestive tract.”

Prebiotics

Prebiotics Ingested substances that selectively

stimulate the proliferation and/or activity of desirable bacterial populations present in the host intestinal tract.

Usually target bifidobacteria and lactobacilli Bifidogenic or bifidus factors explored

in the 50s Usually are non-digestible

oligosaccharides (NDOs) Lactulose, galacto-oligosaccharides,

lactosucrose…

Crittenden and Playne. In Gastrointestinal Microbiology, 2006, pg 285-314.

Prebiotics

Inulin: plant polymers mainly comprising fructose units, use have a terminal glucose

Indigestable fiber Gut flora produce

H2, CO2, methane gas from inulin

Prebiotics

Is is possible to design a food, sugar, protein, or fat that would alter your gut flora to promote weight loss?

More likely possibility is to give a prebiotic that decreases your “Energy Harvest” of colonic bacteria ie. lose weight by making your gut flora

less efficient at digesting your left over food

Designing Probiotics: The Future?

GASTROENTEROLOGY 2009;136:2015–2031

Conclusions

The human microbiome and the Microbiome Project: research just beginning…

Gut flora by their genes, by-products, and metabolic activity influence our metabolism, weight, activity, immunity, health and disease.

Manipulation of gut flora may be an integral part of weight loss programs and different disease treatments in the future.

Conclusions

Future studies must focus on the mechanisms of influence of our gut flora.

Studies must be placebo controlled and high quality.

Truly need translational science to work at the levels of the petri dish, genomics, and clinical outcomes.

Much more to come!

The Future

Define who is there: in healthy subjects and those with disease Study at Scripps in subjects with diet

change, NASH, and IBD Elucidate the gut flora host

interaction Design trials that truly assess the

potential for probiotics and prebiotics to make a difference in health and disease.

Questions