Joël Doré

Deputy head UMR 1319 Micalis Institute & Scientific Director US 1367 MetaGenoPolis

INRA, Jouy en Josas, France

Gut microbiota for health - lessons of a metagenomic scan -

Gut microbiota for health: lessons of a metagenomic scan Joël Doré, INRA.fr

Microscopic counts >> culture counts : great plate count anomaly

Faecalibacterium prausnitzii

Photos : INRA UEPSD

Bacteroides dorei Escherichia coli

Ruminococcus spp Clostridium difficile in a mouse caecum

Segmented filamentous bacteria anchored in a Peyer’s Patch of mouse intestin

From the romantic « flora »

to the pragmatic ‘microbiota’

100 trillion microorganisms ; 10 times the number of human cells in our body (Savage 1977) ; >150 fold more

genes than in the human genome

Predominantly not yet cultured to date (~70% of dominant species)

Central to Food-Microbiota-Host interactions

(crosstalk between microbiome and human

genome impact immune, neural and endocrine functions)

Mutualistic association & true organ, « protecting our health and well-being »« throughout all stages of our life » ; and amenable to modulations

The human intestinal microbiota

Phylogenetic view : gut bacteria in the ‘family’ tree of life

Eckburg et al. 2005

(11831 séquences ; 391 espèces)

Bacteroidetes

Firmicutes

Actinobacteria

Single gene - 16S rDNA sequence : 3 major phyla among the >50 within currently known bacterial diversity

Classification memo: Domain Phylum Class Order Family Genus species (strain)

Phylogenetics of the human intestinal tract

Sequencing &

phylogenetic profiling

Sequence-based phylogenetics of the dominant human intestinal microbiota was initiated in the mid 1990’s

DNA amplification & extraction of SSUrDNA

Single gene - 16S rDNA sequence based approaches :

• A few dominant phyla • High species diversity • Resistance and resilience = homeostasis • A few prevalent & dominant species = Core microbiota

• colonization is affected by: – Gestational term – Mode of delivery (vaginal delivery or caesarean section) – Maternal nutrition and maternal microbiome – Hygiene of neonatal environment and antibiotic administration – Mode of feeding (breast milk versus bottled milk) and weaning diet

• early colonisation & hygiene hypothesis: exposure to low bacterial diversity in the neonatal period would prevent

or delay maturation of the mucosal immune system and favor aberrant responses to allergens or auto-antigens and onset of associated pathologies

Bach JF. N Engl J Med. 2002;347:911-920 Okada et al Clin Exp Immunol 2010 ; 160:1-9

‘sterile’ in utero, the intestine is colonized at birth

Yatsunenko et al. Nature 2012

microbiome diversity is low in north-Americans compared to Amerindians and Malawians

after the age of 3

Metagenomics of the human intestinal tract

Whole Genome Shotgun

sequencing

The metagenome is made of the combined genomes of all dominant microbes within a given ecosystem

DNA

extraction

Assembly and annotation

Reference gene catalog and gene counts

Initial pilot studies in sequence based metagenomics: Manichanh Gut 2006 => Healthy versus Crohn Gill Science 2006 Kurokawa DNA Res 2008 Manichanh Nucl Acids Res 2008 Qin, Raes et al, Nature 2010

Ashler Mullard. The inside story, Nature 453. May 2008. …

International Human Microbiome Consortium - IHMC : INRA-Paris oct 2005 ; EMBL-Heidelberg nov 2008

S.D. Ehrlich

J. Doré L. Zhao

Humans share a core microbiome, and yet differ at

the level of metagenomic species

On average, each individual carries ~540 000 genes of the initial 3.3 million genes catalog (Qin, Raes et al, Nature 2010)

Similarity:

Core metagenome genes : ~50 % of an individual’s genes are shared by at least 50 % of individuals of the cohort

We are all rather similar!, but not identical!!

Yet, individuality:

Rare genes : genes shared by less than 20 % of individuals = 2.4 million genes

Arumugam, Raes et al, Nature 2011

In an attempt to characterize the ‘average’ human intestinal microbiota, we observed … An organisation of intestinal microbiomes into three assemblages of genes and microbial taxa that were named enterotypes: … les ‘entérotypes’

… order in chaos !?!

Humans differ at the level of ecological make-up of

their intestinal microbiota

Danes n=85; Illumina

US n=154; 454

Bacteroides Prevotella Ruminococcus

Europeans, Americans,

Asians. n=33

Sanger

Arumugam, Raes et al, Nature 2011

3 enterotypes ; 3 microbial drivers ; 3 ecological settings

Humans studied so far belong to

one of three enterotypes

‘Density plots’ pour ~400

échantillons

Enterotypes appear as densely populated zones within the ecological landscape of all possible compositions. They strongly suggest ecological driving forces. Arumugam et al. Nature 2012

Enterotypes may be regarded as preferred patterns in the ecological landscape of human intestinal microbiome

Data d

en

sity (Fraction

of d

ata close

to a cen

tral po

int )

Each metagenome appears quite stable, even at the finest level of nucleotide sequence at which variants (SNPs) remain over time within a person’s microbiome. Schloissnig et al. Nature 2012

Scheffer, Nature 2001

Total DNA

MetaHIT & iMOMi Databases • 4 - 8 million genes • 6000+ genomes

Gene Catalog • Sub-populations • Client Specific • Environment

Specific

Stool sample

Gene counts profiles

50+ million tags/sample

Sambo-MetaQuant

- Quantitative Metagenomics -

SAMBO samples processing

MetaQuant NGS

Identification quantification

www.microbiome-standards.org/ Cardona et al BMC Microbiol. 2012

Humans differ by species, by enterotypes

and also by gut bacterial gene counts

Low High Gene count

n=277

Known species n=10

Unknown species n=58

Humans intestinal microbiota share large similarities but also differences that permit stratification, with potential applications in personalized / digitized medicine and nutrition

Marker species for low/high gene-count microbiota

Each column is an individual Each row is a gene, 50 are displayed Colors reflect gene abundance

low high

Although it has a genome of its own, the microbiota • exerts unique functionalities, essentially protective, many of which are conserved in humans & complementary to human-gene encoded functions • intimately interacts with food and with human cells, with the immune & neural systems, and organs far beyond the gut (liver, adipose tissue, brain) • is markedly distorted in many immune mediated diseases = dysbiosis • is a great source of biomarkers with use in stratification of disease/risk

Because it has a genome of its own, it may be modulated, with perspective to maintain or restore normobiosis/homeostasis in disease or risk

• in structure and probably even more so in functions • by diet, by functional foods • by full fecal microbiota transplantation (FMT), currently tested in immune disorders (A. Vrieze et al Gastroenterology 2012)

Gut microbiota is an organ of the host !

Metagenomic signatures of dysbiosis

in immune mediated diseases

We identify bacterial genes & genomes specific of the microbiome of patients

BMI

inflammatory bowel diseases and obesity

d = 0.1

Canonical weights

d = 0.1

Dorea.f ormicigenerans

Bacteroides.v ulgatus.ATCC.8482

Bacteroides.sp..9_1_42FAA

Faecalibacterium.prausnitzii.SL3.3

Roseburia.intestinalis.M50.1

Bacteroides.unif ormis

Bacteroides.sp..2_1_7

Coprococcus.comes.SL7.1

Clostridium.sp.SS2.1

unknown.sp.SS3.4

Eubacterium.rectale.M104.1

Dorea.longicatena

Bacteriodes.xy lanisolv ens.XB1A

Collinsella.aerof aciens

Alistipes.putredinis Bacteroides.sp..4_3_47FAA

Bacteroides.sp..D4

Bacteroides.dorei

Ruminococcus.sp.SR1.5

Bacteroides.sp..2_2_4

Bif idobacterium.longum.subsp..inf antis.CCUG.52486

Eubacterium.hallii

Bacteroides.ov atus Parabacteroides.merdae

Bif idobacterium.adolescentis

Ruminococcus.torques.L2.14

Bacteroides.thetaiotaomicron.VPI.5482

Bacteroides.sp..D1

Parabacteroides.distasonis.ATCC.8503

Eubacterium.siraeum.70.3

Ruminococcus.obeum.A2.162

Ruminococcus.bromii.L2.63 Bif idobacterium.bif idum.NCIMB.41171

Bacteroides.caccae

Bacteroides.eggerthii Streptococcus.thermophilus.LMD.9 Bacteroides.stercoris

Bacteroides.coprocola

Prev otella.copri

Bacteroides.f ragilis.3_1_12

Clostridium.bolteae Eubacterium.v entriosum

Eubacterium.bif orme

Bacteroides.f inegoldii

Bacteroides.plebeius Clostridium.bartlettii

Escherichia.coli.O157.H7.str..EC4115

Holdemania.f ilif ormis

Clostridium.sp.M62.1

Ruminococcus.gnav us

Ruminococcus.lactaris

Bacteroides.capillosus Clostridium.sp.L2.50

Subdoligranulum.v ariabile

Desulf ov ibrio.piger.ATCC29098

Catenibacterium.mitsuokai

Bif idobacterium.pseudocatenulatum

Clostridium.leptum

Parabacteroides.johnsonii

Methanobrev ibacter.smithii.DSM2375

Anaerotruncus.colihominis

Bif idobacterium.animalis.subsp..lactis.AD011

Bacteroides.cellulosily ticus

Bif idobacterium.catenulatum Clostridium.sy mbiosum

Gordonibacter.pamelaeae.gen.nov .sp.Nov

Akkermansia.muciniphila.ATCC.BAA.835

Mitsuokella.multacida Clostridium.nexile Blautia.hy drogenotrophica

Coprococcus.eutactus

Clostridium.asparagif orme

Blautia.hansenii

Anaerostipes.caccae

Bacteroides.intestinalis

Clostridium.spirof orme

Bacteroides.pectinophilus

Bacteroides.coprophilus

Streptococcus.gordonii.str..Challis.substr..CH1

Clostridium.ramosum

Megamonas.hy permegale.ART12.1

Streptococcus.pneumoniae.Hungary 19A.6

Eubacterium.dolichum

Lactobacillus.delbrueckii.subsp..bulgaricus.ATCC.11842

Clostridium.scindens

Mollicutes.bacterium.D7

Buty riv ibrio.crossotus

Enterococcus.f aecalis.TX0104

Haemophilus.inf luenzae.86.028NP

Enterobacter.cancerogenus

Lactobacillus.sakei.subsp..sakei.23K Lactobacillus.saliv arius.ATCC.11741

Bif idobacterium.brev e

Bif idobacterium.dentium Collinsella.stercoris

Streptococcus.inf antarius

Streptococcus.mutans.UA159

Lactobacillus.acidophilus.NCFM Fusobacterium.nucleatum.subsp..nucleatum.ATCC.25586 Lactobacillus.gasseri.ATCC.33323

Lactobacillus.f ermentum.IFO.3956

Proteus.mirabilis.HI4320

Lactobacillus.casei.BL23

Helicobacter.pullorum.MIT.98.5489 Bif idobacterium.angulatum

Klebsiella.pneumoniae.342

Collinsella.intestinalis

Escherichia.f ergusonii.ATCC.35469 Actinomy ces.odontoly ticus

Lactococcus.lactis.subsp..cremoris.MG1363

Streptococcus.sanguinis.SK36

Campy lobacter.hominis.ATCC.BAA.381

Pediococcus.pentosaceus.ATCC.25745

Clostridium.methy lpentosum

Bry antella.f ormatexigens

Buty riv ibrio.f ibrisolv ens.16.4

Citrobacter.sp..30_2

Methanosphaera.stadtmanae.DSM.3091

Clostridium.hy lemonae

Clostridium.sp..7_2_43FAA

Enterococcus.f aecalis.TX1332

Porphy romonas.gingiv alis.ATCC.33277

Pseudomonas.aeruginosa.LESB58

Citrobacter.sp

Enterobacter.sp..638

Desulf ov ibrio.v ulgaris.str...Miy azaki.F.

Haemophilus.parasuis.SH0165

Pasteurella.multocida.subsp..multocida.str..Pm70

Leuconostoc.mesenteroides.subsp..mesenteroides.ATCC.8293

Citrobacter.koseri.ATCC.BAA.895

Clostridium.perf ringens.ATCC.13124

Campy lobacter.concisus.13826

Enterococcus.sp.7L76 Trophery ma.whipplei.str..Twist

Candidatus.Sulcia.muelleri.GWSS Clostridium.dif f icile.630

Lactobacillus.hilgardii.ATCC.8290

Lactobacillus.reuteri.SD2112

Salmonella.enterica.subsp..enterica.serov ar.Heidelberg.str..SL476

Finegoldia.magna.ATCC.29328

Streptococcus.suis.05ZYH33

Thermoanaerobacter.sp..X514

Actinobacillus.pleuropneumoniae.serov ar.7.str..AP76

Anaerof ustis.stercorihominis Clostridium.phy tof ermentans.ISDg

Proteus.penneri

Streptococcus.py ogenes.MGAS10750 Lactobacillus.ultunensis.DSM.16047 Lactobacillus.helv eticus.DPC.4571

Lactobacillus.johnsonii.NCC.533 Lactobacillus.paracasei.subsp..paracasei.ATCC.25302

Anaerococcus.hy drogenalis Bif idobacterium.gallicum

Enterobacter.sakazakii.ATCC.BAA.894

Staphy lococcus.saprophy ticus.subsp..saprophy ticus.ATCC.15305

Canonical weights

Variables

Dorea.f ormicigenerans

Bacteroides.v ulgatus.ATCC.8482

Bacteroides.sp..9_1_42FAA

Faecalibacterium.prausnitzii.SL3.3

Roseburia.intestinalis.M50.1

Bacteroides.unif ormis

Bacteroides.sp..2_1_7

Coprococcus.comes.SL7.1

Clostridium.sp.SS2.1

unknown.sp.SS3.4

Eubacterium.rectale.M104.1

Dorea.longicatena

Bacteriodes.xy lanisolv ens.XB1A

Collinsella.aerof aciens

Alistipes.putredinis Bacteroides.sp..4_3_47FAA

Bacteroides.sp..D4

Bacteroides.dorei

Ruminococcus.sp.SR1.5

Bacteroides.sp..2_2_4 Bif idobacterium.longum.subsp..inf antis.CCUG.52486

Eubacterium.hallii

Bacteroides.ov atus Parabacteroides.merdae Bif idobacterium.adolescentis

Ruminococcus.torques.L2.14

Bacteroides.thetaiotaomicron.VPI.5482

Bacteroides.sp..D1

Parabacteroides.distasonis.ATCC.8503

Eubacterium.siraeum.70.3

Ruminococcus.obeum.A2.162

Ruminococcus.bromii.L2.63 Bif idobacterium.bif idum.NCIMB.41171

Bacteroides.caccae

Bacteroides.eggerthii Streptococcus.thermophilus.LMD.9

Bacteroides.stercoris

Bacteroides.coprocola

Prev otella.copri

Bacteroides.f ragilis.3_1_12

Clostridium.bolteae Eubacterium.v entriosum

Eubacterium.bif orme

Bacteroides.f inegoldii

Bacteroides.plebeius Clostridium.bartlettii

Escherichia.coli.O157.H7.str..EC4115

Holdemania.f ilif ormis

Clostridium.sp.M62.1

Ruminococcus.gnav us

Ruminococcus.lactaris

Bacteroides.capillosus Clostridium.sp.L2.50

Subdoligranulum.v ariabile

Desulf ov ibrio.piger.ATCC29098 Catenibacterium.mitsuokai

Bif idobacterium.pseudocatenulatum

Clostridium.leptum

Parabacteroides.johnsonii

Methanobrev ibacter.smithii.DSM2375

Anaerotruncus.colihominis

Bif idobacterium.animalis.subsp..lactis.AD011

Bacteroides.cellulosily ticus

Bif idobacterium.catenulatum Clostridium.sy mbiosum Gordonibacter.pamelaeae.gen.nov .sp.Nov

Akkermansia.muciniphila.ATCC.BAA.835

Mitsuokella.multacida Clostridium.nexile

Blautia.hy drogenotrophica

Coprococcus.eutactus

Clostridium.asparagif orme

Blautia.hansenii Anaerostipes.caccae

Bacteroides.intestinalis

Clostridium.spirof orme

Bacteroides.pectinophilus

Bacteroides.coprophilus

Streptococcus.gordonii.str..Challis.substr..CH1

Clostridium.ramosum

Megamonas.hy permegale.ART12.1

Streptococcus.pneumoniae.Hungary 19A.6

Eubacterium.dolichum

Lactobacillus.delbrueckii.subsp..bulgaricus.ATCC.11842

Clostridium.scindens

Mollicutes.bacterium.D7

Buty riv ibrio.crossotus

Enterococcus.f aecalis.TX0104

Haemophilus.inf luenzae.86.028NP

Enterobacter.cancerogenus Lactobacillus.sakei.subsp..sakei.23K Lactobacillus.saliv arius.ATCC.11741

Bif idobacterium.brev e

Bif idobacterium.dentium Collinsella.stercoris

Streptococcus.inf antarius

Streptococcus.mutans.UA159

Lactobacillus.acidophilus.NCFM Fusobacterium.nucleatum.subsp..nucleatum.ATCC.25586 Lactobacillus.gasseri.ATCC.33323

Lactobacillus.f ermentum.IFO.3956

Proteus.mirabilis.HI4320

Lactobacillus.casei.BL23 Helicobacter.pullorum.MIT.98.5489 Bif idobacterium.angulatum

Klebsiella.pneumoniae.342

Collinsella.intestinalis

Escherichia.f ergusonii.ATCC.35469 Actinomy ces.odontoly ticus

Lactococcus.lactis.subsp..cremoris.MG1363

Streptococcus.sanguinis.SK36

Campy lobacter.hominis.ATCC.BAA.381

Pediococcus.pentosaceus.ATCC.25745

Clostridium.methy lpentosum

Bry antella.f ormatexigens

Buty riv ibrio.f ibrisolv ens.16.4

Citrobacter.sp..30_2

Methanosphaera.stadtmanae.DSM.3091

Clostridium.hy lemonae

Clostridium.sp..7_2_43FAA

Enterococcus.f aecalis.TX1332

Porphy romonas.gingiv alis.ATCC.33277

Pseudomonas.aeruginosa.LESB58

Citrobacter.sp

Enterobacter.sp..638

Desulf ov ibrio.v ulgaris.str...Miy azaki.F.

Haemophilus.parasuis.SH0165 Pasteurella.multocida.subsp..multocida.str..Pm70

Leuconostoc.mesenteroides.subsp..mesenteroides.ATCC.8293

Citrobacter.koseri.ATCC.BAA.895

Clostridium.perf ringens.ATCC.13124

Campy lobacter.concisus.13826

Enterococcus.sp.7L76 Trophery ma.whipplei.str..Twist

Candidatus.Sulcia.muelleri.GWSS Clostridium.dif f icile.630

Lactobacillus.hilgardii.ATCC.8290

Lactobacillus.reuteri.SD2112

Salmonella.enterica.subsp..enterica.serov ar.Heidelberg.str..SL476

Finegoldia.magna.ATCC.29328

Streptococcus.suis.05ZYH33

Thermoanaerobacter.sp..X514

Actinobacillus.pleuropneumoniae.serov ar.7.str..AP76

Anaerof ustis.stercorihominis Clostridium.phy tof ermentans.ISDg

Proteus.penneri

Streptococcus.py ogenes.MGAS10750 Lactobacillus.ultunensis.DSM.16047 Lactobacillus.helv eticus.DPC.4571 Lactobacillus.johnsonii.NCC.533 Lactobacillus.paracasei.subsp..paracasei.ATCC.25302

Anaerococcus.hy drogenalis Bif idobacterium.gallicum

Enterobacter.sakazakii.ATCC.BAA.894

Staphy lococcus.saprophy ticus.subsp..saprophy ticus.ATCC.15305

Variables

02

46

Eigenvalues

d = 5

Scores and classes

N:N

Y:CD

Y:UC

Axis1 Axis2

Axis3

Inertia axes

d = 2

Classes

N:N

Y:CD

Y:UC

p-value: 0.031

Crohn Patients

UC Patients

Healthy Controls

Guarner (HUVH, Barcelona) Wang Jun (BGI, Shenzen) Ehrlich, Lepage, Tap (INRA)

Pedersen (SDC, Copenhagen) Wang Jun (BGI, Shenzen) Ehrlich (INRA, Paris)

M0 surgical resection

M6 colonoscopy

Still in remission

or

Endoscopic relapse

20 patients with active CD, requiring ileo-caecal resection :

FISH analysis of biopsies

•Eub338 (Eubactia)

• Bac303 (Bacteroides-Prevotella)

• Ent1458 (Enterobacteria)

• Erec482 (Clostridium coccoides)

• Lab158 (Lactobacillus-Enterococcus)

• Bif164 (Bifidobacterium)

• Fprau645 (Faecalibacterium prausnitzii)

Faecalibacterium prausnitzii is associated with protection from endoscopic inflammation relapse 6 months after surgery. … It’s a bacterial signature of high gene count microbiota …

Harry Sokol, Philippe Langella et al.

PNAS 2008

Mucosal Dysbiosis in Crohn’s Disease

F. prausnitzii at M0 (p=0.027)

3.3% Remission at M6 0.3% Relapse at M6

Gut microbial dysbiosis in Crohn’s Disease beyond Faecalibacterium prausnitzii

Reference F. prausnitzii Other species under-represented

under-represented

Sokol et al, PNAS 2008 yes Not explored

Willing et al. 2009 yes Subdoligranulum sp, Roseburia sp.

Qin et al, Nature 2010 yes yes

Kang et al, IBD 2010 yes Ruminococcus sp, Bacteroides group.

Mondot et al, IBD 2010 yes Subdoligranulum sp, Ruminococcus sp. Oscillibacter sp, Bifidobacterium sp,..

Joossens et al. 2011 yes Ruminococcus sp, Bifidobacterium sp,..

For review: Legage et al Gut 2012

Many are bacterial signatures of high gene count microbiota …

High relapse rate: > 1 per year Low relapse rate: < 1 per year

Below 108 copies per g: OR 2.29 (1.07-4.90) for relapsing condition (p<0.05)

Below 3 copies per 1000: OR 3.13 (1.40-6.96) for frequent relapsing condition (p<0.01)

Faecalibacterium prausnitzii in UC:

associated with Relapse Rate

p<0.05 vs. High *

Varela, Manichanh et al, APT 2013

105

106

107

108

109

High Low

Co

pie

sF

p /

g s

too

l

* *

0

2

4

6

High Low

Co

pie

sF

p / 1

00

0 B

acte

ria

RELATIVE ABUNDANCE CONCENTRATION IN FECAL SAMPLES

relapse rate relapse rate

… It’s a bacterial signature of high gene count microbiota …

Recovery of the F. prausnitzii population after relapse was associated with maintenance of clinical remission

Low Gene-counts in UC:

associated with higher Relapse Rate

associated to time since last relapse: associated to relapse frequency: most frequent relapses in low gene counts N

ulb

er

of

gen

es

pe

r d

om

inan

t m

etag

en

om

e

T0 (baseline) T1 (6 sem.) T2 (12 sem.)

2 weeks run-in Probiotic versus placebo consumption

HUVH, Barcelona, Guarner et al.; Danone, Derrien et al.

Randomized Double-Blind Placebo-Controlled Trial Microbiome stability computed based on quantitative metagenomic profiling

High gene-count microbiome patients

Placebo Probiotic controls

p=0.06

All patients

p=0.01

Mic

rob

iota

sta

bili

ty

Microbiome diversity permits stratification in responders/non-responders.

p=0.06

Placebo Probiotic

p=0.01

Mic

rob

iota

sta

bili

ty

Low Gene-counts in UC: predictive of non-response

to microbiota stabilization by a probiotic

12 MGS AUC = 0.84 Linear additive model

Metagenomic species show a good discrimination power between obese and lean, in contrast to human genome

ROCs for individual MGS

Etude de population de 154 jeunes Danois

False positive

True positive

Intestinal Microbiota and Obesity in human

n= 154 Danes

18 obesity risk loci AUC = 0.58

False positive rate

n = 6,510 middle-aged Danes

Andreasen et al. Diabetes 2010

Le chatelier et al, Nature, 2013

∆ 230 K genes, 35% ∆ 240 K genes, 40%

High gene count n=68

High gene count n=23

Low gene count n=31

Low gene count n=13

Le chatelier et al, Nature, 2013; Cottillard et al, Nature, 2013

Micro Obes

The low gene count individuals display increased adiposity, insulin resistance, dyslipidaemia, and inflammation

Obese people differ by gut bacterial gene counts,

and species

Each column is an individual Each row is a set of 50 gene per species Colors reflect gene abundance

low high

Le chatelier et al, Nature, 2013; Cottillard et al, Nature, 2013

6 weeks hyper low caloric diet

Low gene count patients

High gene count patients

Low

High

Although partly corrected by calory-restriction, a low gene count of the microbiota predicts a lesser response in terms of weight loss, insulin resistance and correction of inflammatory tone

intervention stabilization

intervention : low fat, high protein and low glycemic index carbohydrates with a large variety of fibers from fruits and vegetables. 1200-1500 Kcal

Time (weeks)

Low Gene-counts in obesity: predictive of a poor

response to nutritional intervention

Cotillard et al, Nature 2013

Functional and phylogenetic shifts in the LGC microbiome

Le chatelier et al, Nature, 2013

in Spanish UC patients (F Guarner, Barcelona): • diversity is consistently lower in patients microbiota

• Lower gene count predicts higher relapse rate of chronic acute phases

in Danish obese patients (O Pederson, Copenhagen): • indicates higher weight gain over time • higher inflammatory context and biomarkers of risk of comorbidities

in French obese patients (K Clément, Paris): • higher inflammatory context and biomarkers of risk of comorbidities • Low gene count predicts worst response to calory-restricted diet in terms of weight loss, improvement of inflammatory tone, biology and adiposity.

Microbiome diversity is a key stratifier : A low gene count (low species richness) microbiome

may predict less healthy outcome

Micro Obes

no correlation positive correlation negative correlation

ho

st t

ran

scri

pts

bacterial genera

quantitative data spearman rank correlation -0.5<r<0.5 false discovery rate ≤ 5%

Dysbiosis/loss of diversity (richness) => also loss of host-microbiome crosstalk? Microbiome-transcriptome correlation analysis in genetically identical humans:

Lepage & Häsler et al., Gastroenterol 2011 Stefan Schreiber Lab

healthy individuals

UC-affected discordant twins

healthy individuals

UC-affected discordant twins

UC-unaffected discordant twins

Loss of correlation

Genetic effect

Non-genetic effect

no correlation positive correlation negative correlation

Lepage & Häsler et al., Gastroenterol 2011 Stefan Schreiber Lab

ho

st t

ran

scri

pts

bacterial genera

Why bother ? Dysbiosis/loss of diversity => loss of host-microbiome crosstalk? Microbiome-transcriptome correlation analysis in genetically identical humans:

Dysbiosis of the Gut Microbiota

Low grade inflammation

Stressors

Stressors

Dysbiosis in chronic immune diseases:

Altered intestinal ecology altered host physiology vicious circle

Genetic Predisposition Environment, diet, life-style

Environment, diet, life-style

chicken or egg ; does it matter?

… with fascinating questions of intestinal ecology

the vicious circle should be tackled and broken …

by a combined modulation of: microbiota and inflammation !

& crosstalk

Kong et al, Am J Clin Nutr, 2013 Vrieze et al, Gy, 2012

Microbiota remodeling may be associated with

a resolution of insulin resistance – 2 examples

Fecal Microbiota Transplantation in T2D patients

Bariatric Surgery in morbid obesity : gut microbiota & crosstalk modulation

Increased diversity & restored crosstalk

As opposed to pathogens - host interactions, the cross-talk mechanisms with the commensal microbiota

are poorly understood

How to study these interactions when 70 to 80 % of the commensals are not yet cultured ?

Functional metagenomics

Which are the genes (and from which bacterial species) that are responsible for interactions with the host

and what are their role ? (Inflammatory or anti-inflammatory effects ? …)

Causal agents, contributors, consequence ? Play a role in chronicity ?

Mechanisms ?

Bacterial Fraction

Metagenomic DNA

Functional Metagenomic - 1

Selection

Cloning in E. coli

Epi FOS-5

Picking

Metagenomic Library

Gloux et al., AEM, 2007 Lakhdari et al., PLoS one, 2010

Functional Metagenomic - 2

Human Intestinal Epithelial reporter cells (Luciferase)

Identification of • clones • genes • molecules

modulating key functions in IECs

30 bioactive clones • Immunity (NF-kB) • Cell Proliferation • Metabolism (PPAR g)

Metagenomic Library HTS

9 Metagenomic libraries = 340 000 clones

Gloux et al., AEM, 2007 Lakhdari et al., PLoS one, 2010

Commensal bacteria develop functional crosstalk

with human cells (epithelial, immune cells, beyond..)

Modulation of immune functions

- NF-kB & AP1 pathways, TSLP, …

Modulation of epithelial cell turnover

- AP1, …

Modulation of cellular metabolism

- PPAR gamma, Fiaf, …

20 screens developed ; >50,000 clones or strains screened ;

~ 30 bioactive clones and strains identified Lakhdari 2010, 2011, Madi 2010, Gloux 2011, Kaci 2011, Nepelska 2012, Santos Rocha

2012, Cultrone 2013

Transcription factors Genes of interest

Growth of metagenomic clones (DO 600nm)

Immunomodulatory metagenomic clones on NF-kB

stimulators

inhibitors

NF-k

B a

ctiv

ity

Growth of metagenomic clones

Lakhdari et al, PLoS One 2010

Activators of immune defenses

Inhibitors, anti-inflammatory

Metagenomic Clones

Control E. coli

Stimulates NF-kB, AP1 pathways & TSLP in HT-29, and PIgR & TSLP in Caco-2

Sequence related to Firmicutes (C. Leptum, F. prausnitzii) Secreted factor: trypsin sensitive, heat resistant, 2-3 kDa Key genes for bioactivity encode ABC transporters Bioactivity is MyD88 independent hence TLR independent

Also stimulates IL-8 secretion

M.Nepelska

Clone 5A LAB F4 (from Healthy library)

Transposon insertions are in ABC transporters (18 KO/200 mutants)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

EZ-TN5 EZ-TN5

control

PMA

LB

Epi F

46A

5

D5F

4t

0

500

1000

1500

2000

2500

PMA

LB

Epi

F4

*

6A5

D5F4t

*control

***

Il-8

(p

g/m

l)

Key genes for bioactivity encode ABC transporters

contr

olep

iF4

A5

TNF

Il-1

LPS

0.0

0.5

1.0

1.5 ***

OD

(600)

THP-1 THP-1 MyD88-/-

Bioactivity is MyD88 independent hence TLR independent

Tsilingiri et al Gut 2012

Collab. with Maria Rescigno et al, IEO - Milan

Glue

Sealed cylinder luminal compartment

Tissue specimen

Organ culture inset

New experimental approach to study the properties

of probiotics and bioactive metagenomic clones

Bioactive metagenomic clone F4 protects against

Salmonella (FB62)-induced tissue destruction

Collab. with Maria Rescigno et al, IEO - Milan

CTRL EPI300 F4

Tissue with control medium control E.coli clone F4 without Salmonella

FB62 EPI300 + FB62 F4 + FB62

with Salmonella FB62

Glue

Sealed cylinder luminal compartment

Tissue specimen

Organ culture inset

Tsilingiri 2012

human tissues

Key messages :

1) Reduced microbial diversity (species richness) is a robust indicator of altered intestinal ecology and physiology

2) Altered intestinal ecology associated with immune-mediated disease conditions may correspond to alternative stable states

3) Whether cause or consequence, altered intestinal ecology may contribute to the maintenance of chronic conditions

with altered crosstalk between the gut and the microbiota

4) A dietary intake of diverse plant fibers may promote microbiota diversification

5) Non-empirical interventions to restore normobiosis and healthy crosstalk will require a thorough understanding of gut ecology…

6) Functional metagenomics, a new window into microbe-cell crosstalk

7) Microbiome-based stratification appears promissing --/--

Stratification based on microbiome - future perspectives

Relevant to the push for personalized and digital medicine

Relevant for health, preventive nutrition and medical applications Prediction of responders / non-responders Prediction of relative risk of disease onset in healthy subjects Prediction of risk of aggravation and co-morbidities in patients

Useful to assist in diagnosis/prognosis, in prescription and clinical management of patients

Useful to provide rationale targets and strategies for microbiota modulation

Blottière, De Vos, Ehrlich and Doré, COMICR, 2013

Full and Complete understanding of Human Physiology

INRA Jouy-en-Josas Christel Béra-Maillet Hervé Blottière Marion Leclerc Patricia Lepage Catherine Juste Nicolas Lapaque Tomas de Wouters Antonella Cultrone Malgorsata Nepelska Elsa Jacouton ChenHong Zhang Julien Tap Stanislas Mondot Omar Lakhdari

European Community & ANR-France

Philippe Seksik Harry Sokol Philippe Marteau

S Dusko Ehrlich, Jean Weissenbach (Genoscope, Evry), Wang Jun (BGI, Shenzhen), Peer Borck (EMBL Heidelberg), Francisco Guarner (Val d’Hebron Hospital Barcelona), Oluf Pedersen (SDC Copenhagen), Maria Rescigno (IEO Milan), Liping Zhao (Shanghai JiaoTong University), Jim Versalovic (Baylor College of Medicine, Houston), Baghi Singh (Western Ontario, London) and EU-MetaHIT and IHMS Consortia

Karine Clément (INSERM U972, CR des Cordeliers), Denis Le Paslier & Eric Pelletier, (CEA-Genoscope), Liping Zhao (Shanghai JiaoTong University) and ANR MicroObese consortium

Philippe Langella and col. Bruno Pot Corinne grangette and col.

Micro Obes

Merci de votre attention

http:// www.gutmicrobiotaforhealth.com/

A PLATFORM OF EXCELLENCE DEDICATED TO

QUANTITATIVE AND FUNCTIONAL METAGENOMICS,

FUNDED BY FRENCH GOVERNMENT’S FUTURES INVESTMENTS

Muchas gracias

& please, come visit our new

web-platform !

www.gutmicrobiotaforhealth.com/