Intestinal Microflora

(1)

The Developing Intestinal

Microbial Ecosystem:

Relationship to Subsequent

Health and Disease

Josef Neu, M.D.

University of Florida

Extra! Scientists Discover a

New Organ in Humans

!

Citation: Gross L (2007) Human Gut Hosts a Dynamically Evolving Microbial Ecosystem. PLoS Biol 5(7): e199

Intestinal Microflora

The

Microbiome

•

• Microbiome

Microbiome

–

The Microbiome is the

full collection of microbes (bacteria,

fungi, viruses, etc.) that naturally

exist within the human body.

•

• Goal of NIH

Goal of NIH

Microbiome

Roadmap

-

-Understanding whether changes in

Understanding whether changes in

the human

microbiome

can be

correlated with changes in human

health.

(2)

Bacterial 16S RNA

From (open-access):http://en.wikipedia.org/wiki/16S_ribosomal_RNA. Accessed September 23, 2008.

Examples of Non-Culture

Based Techniques

Mshvildadze, Neu and Mai Nutrition Reviews, 2009

Bacterial 16SrRNA Gene Sequences

From Intestinal

Microbiota

of Animals

Dethlefsen, L et al: Nature. 2007 Oct 18;449(7164):811-8. Dethlefsen, L et al: Nature. 2007 Oct 18;449(7164):811-8.

Site Specific Distribution of Bacterial

Phyla in Healthy Humans

Agenda

•

MicroecologyMicroecologyof the Developing GI Tract.of the Developing GI Tract.

•

Relationship of Intestinal Microbes to Relationship of Intestinal Microbes to

Innate Immune system and

Innate Immune system and Inflammatory Inflammatory pathways in the GI tract

pathways in the GI tract

•

Relationship of microbial ecology the fetus Relationship of microbial ecology the fetus

to intestinal inflammation and prematurity

•

Relationship of Intestinal Microbes to Relationship of Intestinal Microbes to

Protection of the GI tract.

•

The Role of The Role of ProbioticsProbiotics(Live vs. Dead vs. (Live vs. Dead vs.

Components),

Components), PrebioticsPrebioticsand and ““PostbioticsPostbiotics””. .

Don’t mess

with my

Microbiome

(3)

The Human Microbiome Project

Peter J. Turnbaugh, Ruth E. Ley, Micah Hamady, Claire M. Fraser-Liggett, Rob Knight & Jeffrey I. Gordon

Nature 449, 804-810(18 October 2007)

Commensal

Microbes: Beneficial

Effects for the Host

•

• Nutrient metabolism

Nutrient metabolism

•

• Tissue development

Tissue development

•

• Resistance to colonization with

Resistance to colonization with

pathogens

•

• Maintenance of intestinal

Maintenance of intestinal

“

homeostasis

”

Development of Intestinal

Angiogenesis With Microbes

•

Villi from (P14) versus (P28) conventionally raised mice.

•

Capillary networks are stained green (FITC).

Stappenback, Hooper and Gordon,PNAS, 2002

Development of Intestinal

Angiogenesis With Microbes

Stappenback, Hooper and Gordon, PNAS, 2002

HOW DO BACTERIA

PREVENT DAMAGE

TO THE INTESTINAL

TRACT?

(4)

Bacteria and Immune

Activation

Neish, et al

Science, 2000

Tales From the Crypt

-

Paneth Cell

defensins

and

angiogenins

Ganz,T.

Nature Immunology 2003

TLRs

, Microbes and Protection

•

Oral dextran sulfate sodium (DSS) to induce Oral dextran sulfate sodium (DSS) to induce colonic injury

colonic injury

•

Deplete mice of intestinal Deplete mice of intestinal microfloramicroflora

•

Reintroduce Reintroduce commensalscommensalsand commensal and commensal products

products

Wild type mice

MyD88-/-

TLR4-/-

TLR2-/-Key adaptor molecule for TLR signaling

Rakoff-Nahoum S et al. Cell2004

TLR Knockouts and Survival

and Weight Loss

Rakoff-Nahoum S et al. Cell 2004

Could Antibiotics lead to

unforeseen consequences?

(5)

Commensal microflora are required for protection from mortality due to colonic epithelial injury

Depletion of all commensals: increased mortality

Rakoff-Nahoum S et al. Cell 2004

Recognition of commensal ligands required for protection from colon injury

Rakoff-Nahoum S et al. Cell 2004

From Madara J. Building an intestine – architectural contributions of commensal bacteria.

New Engl. J. Med. 2004; 351: 1685-86.

TLR ligands and signaling are crucial for the intestinal surface to protect and repair itself in the face of infectious or inflammatory insult

Implications

•

• Gut epithelium and immune system

Gut epithelium and immune system

do not simply tolerate commensal

microflora

but are dependent on

them

•

• Manipulation of the intestinal

Manipulation of the intestinal

microflora

might lead to major

consequences.

Microbial Ecology of the

Uterus and Fetal GI tract:

Nexus with Innate and

Adaptive Immunity and

Disease

Prematurity

•

• 4 million U.S. births per year

4 million U.S. births per year

•

• 12% (480,000) premature

12% (480,000) premature

•

• 2% (80,000) < 1.5 kg BW (3 lbs)

2% (80,000) < 1.5 kg BW (3 lbs)

(VLBW or very low birthweight)

(6)

Relationship of Amniotic

Infection (without ruptured

membranes) to Prematurity

•

• Quantitative amniotic fluid cultures were

Quantitative amniotic fluid cultures were

performed on 12 patients delivered of

premature infants.

•

• Only 2 of the 12 had premature rupture of

Only 2 of the 12 had premature rupture of

membranes.

•

• Seven of the 10 women with in preterm

Seven of the 10 women with in preterm

labor who had intact membranes had

colony counts greater than 1,000 per ml.

Bobbit, JR: J. Reproductive Medicine, 1977 Goldenberg, RL NEJM, 2000

FERGUSON, JAMES E. II MD*; Clin Obstet Gynecol. 2007 Jun;50(2):454-67 Issue:Volume 50(2), June 2007, pp 454-467

Microbial Identification

Techniques: Culture vs. PCR

DiGiulio DB et al. PLoS one Aug., 2008

PCR and Culture Results with

AF Markers of Infection

DGiulio DB et al. PLoS one Aug., 2008

DiGulio DB PLoS ONE. 2008 Aug 26;3(8):e3056.

Outcomes According to PCR

and Culture Results

(7)

DiGulio DB PLoS ONE. 2008 Aug 26;3(8):e3056.

Gestational Age at Delivery as a

Function of Bacterial

rRNA

Concentration

Fetal Origin of Sustained

“

Fetal

”

Inflammatory

Response

•

Infants born preterm have different Infants born preterm have different

concentrations of many inflammatory markers in concentrations of many inflammatory markers in blood drawn several days after birth compared blood drawn several days after birth compared to infants born at term.

to infants born at term.

•

The degree of association between the levels of The degree of association between the levels of inflammatory markers and preterm birth inflammatory markers and preterm birth correlated with the degree of prematurity. correlated with the degree of prematurity.

•

These results support the hypothesis that the These results support the hypothesis that the fetus is an important source of inflammation and fetus is an important source of inflammation and may play a role in the causes of preterm birth. may play a role in the causes of preterm birth. Skogstrand K et al. Obstetrics and Gynecology, May 2008

This is not a lung! The fetus in late gestation swallows up to 450 ml/day.

The Gut is the Motor that

Drives Systemic

Inflammation and Multiple

Organ Dysfunction!

Barrier Function:loss of

Epithelial Integrity

IL IL--88 INTESTINAL EPITHELIUM S(F)IRS Bacterial or toxin Translocation LOCAL OR DISTAL ORGAN INJURY -NEC -Chronic lung disease -Neuro-developmental delays. -Premature Labor??

Hypothetical Pathways :

Proinflammatory

Cytokines

Leading to IVH and or White

Matter Damage

(8)

Don’t mess

with my

Microbiome

!

Clark RH, et al. Pediatrics 2006;117:1979-87

TOP TEN LIST OF NICU

MEDICATIONS

Antibiotics in Premature

Infants

•

Ampicillin and aminoglycoside used in >90% of Ampicillin and aminoglycoside used in >90% of prematures less than 30 weeks gestation for at prematures less than 30 weeks gestation for at least 2 days:

least 2 days: WHY? WHY? –

–CanCan’’t tell the difference between RDS and t tell the difference between RDS and pneumonia.

pneumonia. –

–Premature delivery might be caused by infection.Premature delivery might be caused by infection. –

–Want to prevent infections because of high Want to prevent infections because of high susceptibility due to immunologic immaturity. susceptibility due to immunologic immaturity. –

–Hardware (ET and OG tubes, umbilical lines, etc.).Hardware (ET and OG tubes, umbilical lines, etc.). –

–Sensitivity of blood cultures suboptimal Sensitivity of blood cultures suboptimal

Delayed Colonization and

“

Unnatural Selection

”

: Why

Worry?

•

• The first colonization of the intestine is

The first colonization of the intestine is

one of the most profound immunological

exposures faced by the newborn infant.

•

• Crosstalk induces gene expression in both

Crosstalk induces gene expression in both

the epithelium and the immune system.

•

• Niches are formed as part of a a

Niches are formed as part of a a

potentially long lasting

biofilm

located

within the luminal

glycocalyx

.

What Does Neonatal Antibiotic

Treatment Do To Gastrointestinal

Tract Development?

•

100 mg/kg/d Clamoxyl (Amoxicillin) compared with 100 mg/kg/d Clamoxyl (Amoxicillin) compared with saline control.

saline control.

•

All bacteria were significantly reduced especially All bacteria were significantly reduced especially Lactobacillus, mainly in colon.

Lactobacillus, mainly in colon.

•

Affymetrix gene microarrays performed.Affymetrix gene microarrays performed.

•

1010--30% of the genes undergoing maturational changes 30% of the genes undergoing maturational changes showed modulation by the antibiotic so that their normal showed modulation by the antibiotic so that their normal pattern of maturation was either accelerated or slowed pattern of maturation was either accelerated or slowed down.

down.

•

MHC genes markedly affectedMHC genes markedly affected——required for required for tolerizationtolerization to luminal antigens.

to luminal antigens.

Schumann, A et al. Physiological Genomics 23:235-245,2005

Intravenous Antibiotics

and NEC

Cotten,C.M.,Pediatric Research 2007

(9)

Persistent Biofilm Formation:

Should we Worry?

_Prematures

Probiotic

_{: Randomized Trials}

Studies in

Schanler, R. Arch. Dis. Child. Fetal Neonatal Ed. 2006;91;395-397

Alternatives to Live

Probiotic

Microbes

•

• Inactivated by heat or UV irradiation.

Inactivated by heat or UV irradiation.

•

• Pre and Post

Pre and Post

-

biotics

•

• Toll receptor agonists

Toll receptor agonists

--LPS (TLRLPS (TLR--4)4) --LTA (TLRLTA (TLR--2)2) --Flagellin (TLRFlagellin (TLR--5)5) --CpGCpGnucleotide (TLRnucleotide (TLR--9)9)

Potential Dark Side of

Prebiotics

•

Promoting growth of Promoting growth of NICU bad bugs. NICU bad bugs.

•

Increased bacterial Increased bacterial translocation translocation

•

Unknown long term Unknown long term effects of early effects of early manipulation of manipulation of microbiome microbiome..

Prebiotics

: Problems in

Premature

•

• Prebiotics on their own can only enhance

Prebiotics on their own can only enhance

the growth of bacteria already present in

the gut.

•

• If health promoting species are not

If health promoting species are not

present to begin with, the prebiotic is

unlikely to be effective.

(10)

Increased Bacterial

Translocation in Rats Fed Using

Pup in Cup

Barrat, et al Pediatric Research E published ahead of Print 2008

Prevention with Mother’s Milk: NEC in

Premature Infants (UK)

Lucas & Cole, Lancet 1990;336:1519-23

Formulas Only (n=236) 24 (10.2%)17 (7.2%)

Formulas plus

Mother’s Milk (n=437) 16 (3.7%) 11 (2.5%) Human Milk (n= 253) 11 (4.3%) 3 (1.2%)

In-Hospital Diet All Cases Confirmed Cases

Breast Milk Microbial Origin:

Maternal Intestine?

Perez, PF. Pediatrics 2007;119;e724-e732

Relation Between Infection,

Antibiotic Usage and Diseases

Bach, JF NEJM 2002

GNP vs. MS, Type 1 diabetes

and Asthma

Bach, JF NEJM 2002

(11)

Th1 and Th2 Diseases

_{HOWEVER, THE INCIDENCE}

OF TH

-

1 PREDOMINANT

DISEASES (AUTOIMMUNE,

E.G. TYPE 1 DIABETES)

AND

TH

-

2 (ALLERGY, ATOPY,

ASTHMA) HAVE BOTH

INCREASED.

CS15

“

Old Friends

”

Hypothesis

Rook, et al. Gut 2005

Are there Long Term

Effects of Intestinal

Microbiome Manipulation?

Probiotics

and eczema

•

KaplanKaplan--Meier curves for children without eczema at the Meier curves for children without eczema at the ages of 2, 4, and 7 years in

ages of 2, 4, and 7 years in LactobacillusLactobacillusGG (GG (LGG;LGG;n = n = 64) and placebo (n = 68) groups;

64) and placebo (n = 68) groups; PP= .008 by log= .008 by log--rank rank test.

test.

Kalliomäki M, Journal of Allergy and Clinical Immunology

Volume 119, Issue 4, April 2007, Pages 1019-1021

Probiotics

: Late Complications

•

LGG administered to mothers prior to delivery and then LGG administered to mothers prior to delivery and then to the infants shortly after delivery: decreased atopic to the infants shortly after delivery: decreased atopic dermatitis in the group receiving LGG, but more of dermatitis in the group receiving LGG, but more of allergic rhinitis and asthma in the

allergic rhinitis and asthma in the LactobacillusLactobacillusGG group GG group at 7 years

at 7 yearsa_._.

•

At 2 years of age, LGG didnAt 2 years of age, LGG didn’’t result in differences in t result in differences in atopic dermatitis, but there was a statistically significant atopic dermatitis, but there was a statistically significant increase in wheezing bronchitis (26% vs. 9%) in the increase in wheezing bronchitis (26% vs. 9%) in the LGG

LGG--treated grouptreated groupb_._.

•

At the age of 12 months, the rate of sensitization to At the age of 12 months, the rate of sensitization to common allergens was significantly higher in the common allergens was significantly higher in the probiotic

probioticgroupgroupc_._.

a.

a. KalliomakiKalliomakiM.etM.etal. J Allergy al. J Allergy ClinClinImmunolImmunol2007 Apr.;119(4):10192007 Apr.;119(4):1019--21.21. b.

b. Kopp MV, et al Pediatrics. 2008 Apr.;121(4):e850Kopp MV, et al Pediatrics. 2008 Apr.;121(4):e850--66

c.

(12)

Slide 62

CS15

Should this read the incidence of...has increased rather than the increase in....has increased

(13)

Don’t mess

with my

Microbiome

!

Take Home Messages

•

There is a close relationship between intestinal There is a close relationship between intestinal microbes and development of the GI tract, immunity,

microbes and development of the GI tract, immunity,

and other systems.

•

Many microbes cannot readily be detected by usual Many microbes cannot readily be detected by usual culture based techniques.

culture based techniques.

•

Microbes in amniotic fluid are related to premature Microbes in amniotic fluid are related to premature delivery via an inflammatory response.

delivery via an inflammatory response.

•

We still have a lot to learn about intestinal microbes We still have a lot to learn about intestinal microbes and their interactions with the developing host to

and their interactions with the developing host to

manipulate them safely, especially in select groups,

e.g. premature infants .

Intestinal Microflora

The Developing Intestinal

The Developing Intestinal

Microbial Ecosystem:

Microbial Ecosystem:

Relationship to Subsequent

Relationship to Subsequent

Health and Disease

Health and Disease

Josef Neu, M.D.

Josef Neu, M.D.

University of Florida

University of Florida

[email protected]

[email protected]

Extra! Scientists Discover a

Extra! Scientists Discover a

New Organ in Humans

New Organ in Humans

!

!

Intestinal Microflora

Intestinal Microflora

The

The

Microbiome

Microbiome

•

•

Microbiome

Microbiome

–

–

The Microbiome is the

The Microbiome is the

full collection of microbes (bacteria,

full collection of microbes (bacteria,

fungi, viruses, etc.) that naturally

fungi, viruses, etc.) that naturally

exist within the human body.

exist within the human body.

•

•

Goal of NIH

Goal of NIH

Microbiome

Microbiome

Roadmap

Roadmap

-

-Understanding whether changes in

Understanding whether changes in

the human

the human

microbiome

microbiome

can be

can be

correlated with changes in human

correlated with changes in human

health.

health.

Bacterial 16S RNA

Bacterial 16S RNA

Examples of Non-Culture

Based Techniques

Bacterial 16SrRNA Gene Sequences

Bacterial 16SrRNA Gene Sequences

From Intestinal

From Intestinal

Microbiota

Microbiota

of Animals

of Animals

Site Specific Distribution of Bacterial

Site Specific Distribution of Bacterial

Phyla in Healthy Humans

Phyla in Healthy Humans

Agenda

Agenda