OUTLINE OF TALKOUTLINE OF TALK What is meant by Human MicrobiomeWhat is meant by Human Microbiome

Describe the factors and processes that influence Describe the factors and processes that influence

community assembly and compositioncommunity assembly and composition

Importance of Human microbiomeImportance of Human microbiome

Gut Microbiota and its importanceGut Microbiota and its importance

Clostridium difficileClostridium difficile

Fecal TransplantationFecal Transplantation

ConclusionConclusion

• To make you all generally conversant in the language of microbiomes and metagenomics

• To provide examples of how microbial communities affect health and cure diseases

• To give practical examples of how medical interventions interact with the microbiome and change outcomes

Goal of my Talk

THE THE HUMAN HUMAN

MICROBIOMMICROBIOMEE

The Human Microbiome: The Undiscovered Country

What is the Human MicrobiomeMicrobe : Tiny living organism, such as bacterium, fungus, protozoan, or virus

Microbiome : Collectively all the microbes in the human body; a community of microbes

Biofilm : A community of microbes that live together on a surfaceA metagenome: the collection of genes/genomes in an environmental

The Human Genome Project started in 1990

Provides a basis to understand how the human genome relates to health and diseaseHowever, the number of microbial cells is 10 times more than human cells in a human being

The Problem : Who am I Vs. Who are we

MICROBES ARE ALL OVER USMICROBES ARE ALL OVER US

There are millions of microbes per square inch on your body

Thousands of different species on the skin alone some thrive on dry patches of the elbow, others thrive in moist environment of armpit

It is estimated that there are more microbes in your intestine there are human cells in your body

Sites that harbor a normal floraSites that harbor a normal flora::

Skin and mucous Skin and mucous membranesmembranes

Upper respiratory tractUpper respiratory tract

Gastrointestinal tractGastrointestinal tract

Outer opening of urethraOuter opening of urethra

External genitaliaExternal genitalia

VaginaVagina

External ear canalExternal ear canal

External eye (lids, External eye (lids, conjunctiva)conjunctiva)

We are Covered with Microbes ?

External Ear Nose

Sinuses Eyes

Mouth Pharynx

Skin Enteric tract

Vagina

How dense they are?Who are they?Where did they come from?Do they all matter?Democracy? Dictatorship? Socialism Can they be moved? Transplanted? Adjusted?Do they change when I am sick?When they change do they make me sick?

What factors determine the species composition of normal flora in the human body?

Skin regions are like geographic regions of Earth

HOW MANY DIFFERENT HOW MANY DIFFERENT ORGANISMS ARE NORMALLY IN ORGANISMS ARE NORMALLY IN

OUR BODY?OUR BODY?

• Mouth ; > 600 SpeciesMouth ; > 600 Species

• Skin : > 600 SpeciesSkin : > 600 Species

• Intestine : (Cecum/ colon) : 8,000 Intestine : (Cecum/ colon) : 8,000

generagenera

• Vagina : > 200 SpeciesVagina : > 200 Species

A microbe’s view of us

Skin Bacterial cells outnumber your body

cells 10:1 and comprise up to 4-6 lbs of your

body mass

1. Synthesize and excrete vitamins Vitamin K and Vitamin B12

2. Prevent colonization by pathogens competing for attachment sites or for essential nutrients 

3. May antagonize other bacteria the production of substances which inhibit or kill non-indigenous species(nonspecific fatty acids, peroxides, bacteriocins).

4. Stimulate the development of certain tissuesi.e., intestines, certain lymphatic tissues, capillary density

5. Stimulate the production of cross-reactive antibodies. Low levels of antibodies produced against components of the normal flora are known to cross react with certain related pathogens, and thereby prevent infection or invasion.

Benefits of the normal flora

YesHow? No

Why ?

• Microbes can communicate each other by chemical language

• They interact each other by signals and respond to the signals by using chemical language

• This Phenomenon is called “ Quorum Sensing”• Quorum Sensing signals are called auto inducers• All auto inducers are chemical language signals to one

another.

• Quorum Sensing is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population .(wiki)

GOALS OF THE HUMAN MICROBIOME GOALS OF THE HUMAN MICROBIOME PROJECTPROJECT

• Determine if there are sets of microbes Determine if there are sets of microbes

common to each humancommon to each human

• Understand if changes in our microbiota Understand if changes in our microbiota

result in different states of health or diseaseresult in different states of health or disease

• Develop new technologies for studying Develop new technologies for studying

complex microbial and studying complex complex microbial and studying complex

microbial systems within their natural microbial systems within their natural

environments environments

• ••Begin to deal with the legal ethical and Begin to deal with the legal ethical and

social complications that may arise with social complications that may arise with

human microbiome human microbiome

THE CONCEPT OF A CORE THE CONCEPT OF A CORE MICROBIOMEMICROBIOME

WHAT DO THE MICROBES DO WHAT DO THE MICROBES DO FOR US? FOR US?

• Provide the ability to harvest nutrients and Provide the ability to harvest nutrients and

• Produce additional energy otherwise Produce additional energy otherwise

inaccessible to the host.inaccessible to the host.

• Produce vitaminsProduce vitamins

• Metabolize xenobiotics Metabolize xenobiotics

• Provide resistance to tumor and cancer Provide resistance to tumor and cancer

leading neoplasms leading neoplasms

• Assist in developing a mature immune systemAssist in developing a mature immune system

Let’s focus on the microbial community in the gut:How is the community assembled?How does community composition affect function?

INTESTINAL MICROBIOMEINTESTINAL MICROBIOME

• >>1,000 species but most in adults are 1,000 species but most in adults are

from 2 phyla: Firmicutes and from 2 phyla: Firmicutes and

BacteroidetesBacteroidetes

• Outnumber human somatic cells by Outnumber human somatic cells by

factor of 10factor of 1022

• Total Weight: 1-2 kg Total Weight: 1-2 kg

• 60% of total fecal content60% of total fecal content

• Concentration: ~1012/gram in colon Concentration: ~1012/gram in colon

• ••Total #: ~1014Total #: ~1014

O’Hara and Shanahan (2006) EMBO Reports

Where are all the organisms?

THE HUMAN GUT FLORATHE HUMAN GUT FLORA

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

When Microbes entered in the humans body?

Is it starts in the Mother’s Womb? Or? after the birth

?

THE MICROBIOME: WHO’S THE MICROBIOME: WHO’S THERE?THERE?

• Early gut colonization has four phasesEarly gut colonization has four phases

• Phase 1: Sterile gut Phase 1: Sterile gut

• Phase 2: Initial acquisition: vagina, feces, hospitalPhase 2: Initial acquisition: vagina, feces, hospital

• Phase 3: Breast feeding or bottle-feeding (different)Phase 3: Breast feeding or bottle-feeding (different)

• Breast fed more bifidobacteria (up to 90% of flora)Breast fed more bifidobacteria (up to 90% of flora)

• Bottle fed more diverse; more Bottle fed more diverse; more BacteroidesBacteroides , and , and

Clostridial speciesClostridial species

• Phase 4: Start of solids; move to adult flora Phase 4: Start of solids; move to adult flora

• Bifidobacteria remain key flora into adulthoodBifidobacteria 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 gut environment selects its microbiota

Primary Succesion in the

Lina.et,al 2012

MATURATION OF THE MATURATION OF THE MICROBIOME DURING THE 1MICROBIOME DURING THE 1STST

YEAR OF LIFEYEAR OF LIFE

Development of the Human Infant Intestinal Microbiota Palmer C,Bik EM,DiGiulio DB,Relman

THE MICROBIOME: WHO’S THE MICROBIOME: WHO’S THERE?THERE?

• Adult Microbiome:Adult Microbiome:

−Increasing diversity of flora as we ageIncreasing diversity of flora as we age

−In some newer PCR (16S rRNA) studies, up to 92% In some newer PCR (16S rRNA) studies, up to 92%

of the flora in adults were “novel” speciesof the flora in adults were “novel” species

• Serial stool collections show remarkable stability by Serial stool collections show remarkable stability by

an individualan individual

−Greatest concordance with twinsGreatest concordance with twins

−Less concordance with householdsLess concordance with households

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

METABOLOMICSMETABOLOMICS

• Study of the metabolites and small molecules Study of the metabolites and small molecules

that the body and that the body and gut bacteria producegut bacteria produce..

• New area of science New area of science

• Broader than Broader than proteomicsproteomics

• Includes bacteria products with our own genetic Includes bacteria products with our own genetic

productsproducts

• Pioneered by Jeremy Nicholson and Jeff GordonPioneered by Jeremy Nicholson and Jeff Gordon

GUT FLORA AND GUT FLORA AND METABOLISMMETABOLISM

• Microbial genomes enhance our metabolic Microbial genomes enhance our metabolic activityactivity

• May indirectly or directly effect our May indirectly or directly effect our metabolismmetabolism

• The colon is very active metabolicallyThe colon is very active metabolically• 20-70 gms of carbons and 5-20 gms of 20-70 gms of carbons and 5-20 gms of

protein/dayprotein/day• Over 100 kcal per day!Over 100 kcal per day!

• Mass of colonic microbiome = single kidneyMass of colonic microbiome = single kidney• Metabolically as active as the liverMetabolically as active as the liver

Hooper, et al. Annu Rev Nutr, 2002

GUT FLORA AND THE BRAINGUT FLORA AND THE BRAIN

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

Fecal Microbiota TransplantsFecal Microbiota Transplants(FMT)(FMT)

Clostridium difficile also known as "CDF/cdf", or "C. diff", is a species of Gram-positive spore-forming bacterium that is best known for causing antibiotic-associated diarrhea(AAD).

While it can be a minor normal component of colonic flora, the bacterium is thought to cause disease when competing bacteria in the gut have been wiped out by antibiotic treatment.

In severe cases, C. difficile can cause "pseudomembranous colitis," a severe inflammation of the colon.

Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants

The classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process.

Inflammation

LITTLE-KNOWN FECAL LITTLE-KNOWN FECAL TRANSPLANT CURES WOMAN'S TRANSPLANT CURES WOMAN'S

BACTERIAL INFECTIONBACTERIAL INFECTION““After surviving a near-fatal car accident, Kaitlin Hunter found herself After surviving a near-fatal car accident, Kaitlin Hunter found herself

battling a devastating bacterial infection in her colon that also threatened her battling a devastating bacterial infection in her colon that also threatened her life.life.

The persistent infection was beaten through a little-known technique involving The persistent infection was beaten through a little-known technique involving the transplant of fecal matter from Hunter's mother...the transplant of fecal matter from Hunter's mother...

Following the July procedure, "I've been so happy," said Hunter, 20, of Following the July procedure, "I've been so happy," said Hunter, 20, of Marietta, Georgia. "I'm cured."Marietta, Georgia. "I'm cured."

Why did this work?

What happened in Katie’s

colon?

FMTFMTProcedure:

Donar History (Similar to blood donation )

Obtain stool sample, homogenize with saline and filter

How to Administer

Nasogastric Tube

Enema

Colonoscope

Perform 6-24 hrs of obtaining the sample

Future: Frozen Samples, Lyophilized Powders, Capsules..??

TJ.BOOD ET.AL

• FecalFecal microbiota transplantationmicrobiota transplantation ( (FMTFMT) also ) also

known as a known as a stool transplantstool transplant is the process of is the process of

transplantation of fecal bacteria from a healthy transplantation of fecal bacteria from a healthy

individual into a recipient. It has been proven to be a individual into a recipient. It has been proven to be a

highly effective treatment for patients suffering from highly effective treatment for patients suffering from

Clostridium difficile infection (CDI),, which produces which produces

effects ranging from diarrhea to effects ranging from diarrhea to

pseudomembranous colitis..

BACTERIOTHERAPYBACTERIOTHERAPY

Clostridium difficile-associated diarrhea (CDAD)- usually results from prior antibiotic treatment and persistant disruption of gut microbiota- can be severe, even causing death

J Clin Gastroenterology (2010) 44:354-360

CONCLUSIONSCONCLUSIONS

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

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

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

CONCLUSIONSCONCLUSIONS

• Future studies must focus on the mechanisms Future studies must focus on the mechanisms

that influence of our gut flora.that influence of our gut flora.

• Studies must be place to controlled and high Studies must be place to controlled and high

quality research should be done.quality research should be done.

• Truly need translational science to work at Truly need translational science to work at

the levels of the petri dish, genomics, and the levels of the petri dish, genomics, and

clinical outcomes.clinical outcomes.

• Hope much much more to come!Hope much much more to come!