Le Microbiote Intestinal

Joël Doré INRA, MetaGenoPolis, Jouy-en-Josas, FRANCE

Vendredi 4 avril 2014 - Institut Pasteur - Paris

19ème Colloque sur le

Contrôle Epidémiologique des Maladies Infectieuses

Microbiota acquisition and early development

Microbiota composition – a metagenomics view

Dysbiosis – towards risk biomarkers

Microbiota functions – towards new bioactives

Le microbiote intestinal:

Microbiota acquisition and early development

• Intestinal colonization is affected by : – Gestational term

– Mode of delivery (vaginal or Cesarean)

– Higene of neonatal environment

– Maternal microbiomes and maternal nutrition

– Food & feeding mode (breast milk vs formula ; weaning)

• Early colonisation and hygene hypothesis: early exposure to low microbial diversity would prevent or delay

maturation of the mucosal immune system and thereby promote aberrant responses to allergens or autoantigens and development of associated diseases

Bach JF. N Engl J Med. 2002;347:911-920

Okada et al Clin Exp Immunol 2010 ; 160:1-9

Dominguez 2010

Is species richness affected by long term changes?

Yatsunenko et al. Nature 2012

Species richness

Sp

ecie

s r

ich

ne

ss

Impact, over generations, of :

• Nutritional transition?

• Repeated antibiotherapy?

• …

Microbiota composition – a metagenomics view

Whole Genome

shotgun sequencing

The metagenome, made of the combined genomes

of all dominant microbes within a given ecosystem

DNA

extraction

Assembly and annotation

Reference gene catalog

and gene counts

Reference gene catalogs: 3.3 million bacterial genes/ 124 subjects 10 million bacterial genes/1267 subjects

500,000 bacterial genes/individual

Comparable gene catalog for Europeans, Americans, Japanese, Chinese

50% bacterial genes of each microbiome shared by >50% surrounding individuals: metagenomic core

Qin et al, Nature 2010

Arumugam et al, Nature 2011

57 Common species

Human microbiomes differ

by gut bacterial genes, species and enterotypes

‘Density plots’ for ~400 individuals

- ecological landscape -

Da

ta d

ensity

(Fra

ction

of d

ata

close to

a cen

tral p

oin

t )

Scheffer, Nature 2001

Bacteroides

Prevotella

Ruminococcus

Human microbiomes differ

by gut bacterial gene counts

Low High Gene count

n=277

Each column is an individual

Each row is a gene, 50 are displayed

Colors reflect gene abundance

less more

Known

species

n=10

Unknown

MGS

n=58

Low gene count (low bacterial richness)

individuals (23%) have less healthy

metabolic & inflammatory traits

Microbiome: source of biomarkers for stratification and monitoring

Frailty in seniors Van Tongeren et al., 2005 Crohn’s Disease Seksik et al., 2003; Sokol et al., 2006, 2008, 2009 Ulcerative colitis Sokol et al., 2008; Martinez et al., 2008 Pouchitis Lim et al., 2009, Kühbacher et al., 2006 Obesity Ley et al., 2007; Kalliomäki et al., 2008 Type-2 diabetes Cani and Delzenne, 2009 Type-1 diabetes Dessein et al., 2009; Wen et al., 2008 Cœliac disease Nadal et al., 2007; Collado et al., 2009 Allergy Kirjavainen et al., 2002; Björkstén, 2009 Autism Finegold et al., 2002; Paracho et al., 2005 Cancer colorectal Mai et al., 2007; Scanlan et al., 2008 Cardiovascular Wang et al. 2011 others….

Chronic immune diseases

associated with dysbiosis of the microbiome

Prevention = risk detection + risk alleviation

Metagenomic signatures

of dysbiosis

Diagnostic genes and genomes are 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)

Host genetic markers from

GWAS AUC=0.6

Microbiome biomarkers are more

discriminant than clinical assessment and

the human genome in Type 2 diabetes

AUC = 0.84

ROC analysis :

Metagenomic signatures of risk :

diversity is a key stratifier?

Prognostic genes and genomes are biomarkers of risks of comorbidities

Low gene count individuals (low bacterial richness, 23%) have less

healthy prognosis

In obesity (O. Pedersen & K. Clément), with:

• less healthy metabolic & inflammatory traits

• higher weight gain over the past 10 years

• more biomarkers of risk of aggravation and comorbidities

• worse response to a calories-restricted diet

In Ulcerative Colitis (F. Guarner), with:

• higher relapse rate of chronic acute phases

• non-responders to probiotic-induced microbiota stabilization

Note: Low bacterial gene count microbiomes are enriched in Bacteroides

enterotype and often lack core-species of dominant firmicutes.

A low gene count microbiome predicts a

worse response to nutritional intervention

intervention stabilization

intervention

1200-1500 Kcal

: low fat, high protein diet, rich in low glycemic index sugars ;

in diverse plant-based dietary fibres.

A low diversity microbiome is predictive of a worse response to calorie-restriction ; especially obesity-associated inflammation

appauvri

enrichi

appauvri

enrichi

Microbiota functions – towards new bioactives

The metagenome ; a window to unexplored microbiota

functions – acting at interfaces :

• Metabolism (food-microbe)

• Mucosal barrier integrity & beyond (cell-microbe) Mucus ; Tight junctions ; Trophicity of epithelium and cell renewal

Immuno-modulation ; Dendritic cells, Paneth cells and defensins

Gut-brain crosstalk

• Barrier against pathogens – colonization prevention

(microbe-microbe)

Clones genes enzymes..

Large homogenous DNA

library

Metagenomic DNA

Bacterial fraction

Recombinant clones

Fosmid

Vectors

Cloning

DNA extraction

- vitamin production (Riboflavin)

- plant polysaccharides degradation (Tasse GenomRes 2010)

- beta-glucuronidase activity (Gloux et al PNAS 2010)

Microbiota functions – food-microbe

Large homogenous metagenomic DNA library

Metagenomic DNA

Bacterial fraction

Recombinant clones

Fosmid Vectors

Cloning

DNA extraction

Metagenomic screening of bacteria-cell crosstalk

9 metagenomic libraries : 340 000 clones

clones genes molecules..

Human Reporter-cell-lines

Cultured strains & Cultured strain DNA library

Microbiota functions – microbe-host

Collab. with Maria Rescigno et al, IEO - Milan

CTRL EPI300 F4

Tissue with control medium control Ecoli clone F4

without

Salmonella

Glue

Sealed cylinder luminal compartment

Tissue specimen

Organ culture inset

Tsilingiri 2012

Bioactive metagenomic clone F4 protects against

Salmonella (FB62)-induced tissue destruction

FB62 EPI300 + FB62 F4 + FB62

with

Salmonella

FB62

Microbiota functions – microbe-microbe

This RumC region is part of a 220 kb ICE ; a transferable “colonisation island”

Fons and col. Crost 2010, 2011; Pujols 2011

In vivo elimination of Clostridium perfringens

by Ruminococcus gnavus E1

Faecal dilution Days post inoculation L

og

10

(CF

U / g

of

fec

es

)

0

2

4

6

8

10

0 2 4 6 8

R. gnavus

C. perfringens

Passive

transit

marker

rumC1 rumC2 rumC3 rumC4 rumC5

rumMC1 rumKC rumRC rumIC1 rumTC rumMC2 rumEC rumIC2 rumFC rumGC rumPC

rumYC

Microbiota as a neglected organ

Relevant to the push for personalized and digital medicine

Relevant for health, preventive nutrition and medical applications Predict responders / non-responders (to nutritional supplements or

therapeutics) Predict relative risk of disease onset in healthy subjects Predict risk of aggravation and co-morbidities in patients

Signatures to assist in diagnosis/prognosis and clinical management of patients

Biomarkers to provide rationale targets and strategies for microbiota modulation

Bioactives with potential for translation in the clinic

INRA Jouy-en-Josas

Christel Béra-Maillet

Hervé Blottière

Catherine Juste

Nicolas Lapaque

Marion Leclerc

Patricia Lepage

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, Peer Borck (EMBL Heidelberg), Francisco Guarner (Val d’Hebron

Hospital Barcelona), Oluf Pedersen (SDC Copenhagen)

and EU-MetaHIT Consortium

Karine Clément, (INSERM U972, CR des Cordeliers) Denis Le Paslier & Eric Pelletier,

(CEA-Genoscope)

and ANR MicroObese consortium

Micro Obes

Merci de votre attention

http://www.gutmicrobiotaforhealth.com/

Michel Fons

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