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9/6/2010
1
Group 2:
GUT MICROBIOTA AND
DISEASE
Castelldefels
August 29, 2010
GUT MICROBIOTA
AND DISEASE
Francisco Guarner, Chair University Hospital Vall d'Hebron Spain
James Versalovic, Co-Chair Texas Children's Hospital USA
John Bienenstock McMaster Brain-Body Institute Canada
Dusko Ehrlich INRA France
Inna Eiberger Merck Selbstmedikation GmbH Germany
Kirsty Hunter Nottingham Trent University UK
Annett Klinder University of Reading UK
Krishna Madduri The Dow Chemical Company USA
Chays Manichanh University Hospital Vall d’Hebron Spain
Peggy Martini Kraft Foods USA
Ravi Menon General Mills Inc USA
Alicia Murcia University Hospital Vall d´Hebron Spain
Arthur Ouwehand Danisco Finland
Junjie Qin Beijing Genomics Institute China
Yehuda Ringel University of North Carolina USA
Ian Rowland University of Reading UK
Saskia van Hemert Winclove Netherlands
Johan van Hylckama Vlieg Danone Research France
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GUT MICROBIOTA AND DISEASECastelldefels 2010
Aims:
• To define traits/characteristics/structure of a
“normal” gut microbiota
• To review clinical conditions associated with
dysbiosis. Is dysbiosis a pathogenetic factor of
disease?
Dusko Ehrlich:
Definition of a normal structure of the human gut microbiota
Jim Versalovic:
The intestinal microbiota in children
Junjie Qin:
Microbiota in Chinese Type II Diabetes patients and control individuals
Udi Ringel:
Microbiota in IBS (human studies)
John Bienenstock:
Microbiota and IBS (animal studies)
Ian Rowland:
Microbiota in colon cancer
Francisco Guarner:
Microbiota in IBD
Kirsty Hunter:
Gut microbiota in physical stress
Chaysavanh Manichanh:
Transplantation of gut microbiota in experimental models
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The « healthy » intestinal microbiome
Can we define health? A question for doctors!
Health not black and white. There is a range. Arthur C. Ouwehand
Health microbiome may also not be black and white - can’t we expect that there is a range?
Ehrlich, ISAPP 2010
The Human Gut Metagenome
# of genes
Non-redundant gene set 3,299,822
Common (>50% of individuals) 294,110
Rare (<20% of individuals) 2,375,655
Average gene set per individual 590,384
Qin et al, Nature 2010
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The landscape of « healthy » intestinal
microbiome
What is the form of the landscape?
- a continuum – any combination of microbial
species is possible
- discrete states – some microbial community
structures are favoured
Ehrlich, ISAPP 2010
The Distal Gut Microbiomes of Healthy Children
Differ from Healthy Adults
• Healthy
Children
• Healthy
Adults
V1V3 regions only (1 replicate/sample)
11 samples from healthy adults
(18-40 yo) sequenced
by HGSC at Baylor.
29 samples from healthy children
(7-12 yo) sequenced by HGSC
at Baylor
454 16S metagenomic sequencing
14-16K reads per sample
Versalovic et al, 2010
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Gene catalog update
884,577 novel genes assembled from Chinese samples
124 EU samples
73 CN samples
Contribution to gene catalog Contribution to sequencing read
Qin et al, 2010
Intestinal Microbiota and IBS
• Only few studies
=> Results are not consistentand sometimes conflicting
• Mostly small studies
• Considerable limitations (lack of information on subtypes, use of antibiotics, hospitalized patients)
Intestinal Microbiota and IBSIntestinal Microbiota and IBS
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• Significantly lower levels oflactobacillus ssp. in D-IBSvs. C-IBS
Malinen et al, Am J Gastroenterol 2005
qPCR analysis
• Significantly higher levelsof veillonella spp. in C-IBSvs. controls
• RT-PCR of 20 bacterialgroups covering 300different species
Intestinal Microbiota and IBSIntestinal Microbiota and IBS
• The observations seen on theIBS subtype analyses did nothold on the mixed group (pooled samples) analysis.
Malinen et al, Am J Gastroenterol 2005
• Results were reported by genomic DNA in 1g of wet weight fecal samples
qPCR analysis
=> Results may depend on theIBS subtype.
Intestinal Microbiota and IBSIntestinal Microbiota and IBS
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We need
Clinical implications
Conclusions and Personal Perspective
• High quality studies to better characterize the intestinal microbiota in well definedpatients with IBS.
• More studies to help determine the reasons for the observed alteration in themicrobiota (cause or effect?).
• More studies on the host-microbiota interaction to help determine the mechanismsby which the intestinal microbiota affect GI function and functional GI symptoms.
• The current data together with recent studies indicating beneficial effects of manipulation of the intestinal microbiota by prebiotics, probiotics, and antibiotics,provide the rational for targeting intestinal bacteria in the treatment of IBS.
• The enthusiasm for the use of this approaches in IBS has outpaced the scientificevidence.
FGID - Bacteriological and Luminal Factors
Ringel, ISAPP 2010
Kamiya et al: Gut 2006 55 191
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Effect of lactobacillus reuteri on HR response to CRD (80 mm Hg)
92
94
96
98
100
102
rest 10s 20s 30s 40s 50s 60s post 1 post 2
time
% r
esti
ng
hea
rt r
ate broth
lactobacillus
Kamiya et al: Gut 2006 55 191
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GF vs CV animalsGF vs CV animals
�� In CV rats tumour formation more rapid In CV rats tumour formation more rapid and more [malignant] and more [malignant] tumourstumours [Reddy 1974][Reddy 1974]
�� CV rats develop more & larger CV rats develop more & larger tumourstumours[[VanucciVanucci 2008]2008]
�� TcellTcell receptorreceptor--/p53/p53-- mice. CV mice higher mice. CV mice higher tumour incidence (70%) than GF (0%) tumour incidence (70%) than GF (0%) [[KadoKado2001]2001]
�� HFA rats had 2x more DNA adducts than HFA rats had 2x more DNA adducts than CV rats CV rats [[RumneyRumney 1993]1993]
Bacteria & colorectal cancer-2Bacteria & colorectal cancer-2
Molecular methods:Molecular methods:
��CRC risk (CRC risk (vsvs healthy controls) associated withhealthy controls) associated with
�� ��DiversityDiversity of C of C leptumleptum, , C.coccoidesC.coccoides subgroups subgroups (no effect on (no effect on BactBact fragilisfragilis group) group) (Scanlon et al Environ (Scanlon et al Environ MicrobiolMicrobiol 10:1382; 2008; DGGE)10:1382; 2008; DGGE)
�� ��ErecErec; ; ��FprauFprau (4x) ((4x) (nn--Bu producers)Bu producers) ��EntEntfaecalisfaecalis((BalamuruganBalamurugan et al J Gastro et al J Gastro HepatolHepatol 23, 1298, 2008; RT PCR)23, 1298, 2008; RT PCR)
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Summary and conclusionsSummary and conclusions
�� Gut Gut microfloramicroflora is implicated in is implicated in aetiology of colorectal canceraetiology of colorectal cancer
�� Gut microbial products potentially Gut microbial products potentially involve NOC, sulphide, sec BA, involve NOC, sulphide, sec BA, ammonia, phenols, SCFAammonia, phenols, SCFA
�� Modification of gut Modification of gut microfloramicroflora by by propro-- and preand pre--bioticsbiotics alters canceralters cancer--associated events in humansassociated events in humans
Rowland, ISAPP 2010
GUT MICROBIOTA AND DISEASECastelldefels 2010
PATHOGENESIS OF IBD
Asquith & Powrie, J Exp Med 2010
Non-pathogenic Intestinal Microbia
Dysregulated
Immune Response
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Reduced Microbial Gene Diversity in IBD
Qin et al, Nature 2010
IBD CONCLUSIONSExpression of IBD may depend on 3 conditions:
• Genetic susceptibility
• Reduced diversity and instability of the intestinal ecosystem
• Dysbiosis
Imbalance between aggressive vs. protective bacteria associated with clinical relapse
Guarner, ISAPP 2010
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Exercise and immunodepression – the response to psychophysical stress
• Prolonged strenuous exercise results in:
– Increased circulating cortisol
– Postexercise neutrophilia, reduced responsiveness to LPS
– Marked decrease in circulating NK cells and cytotoxic capacity
– Reduced suppressor T cell number, complement, mucosal IgA
– Decreased expression of Toll-like receptors 1, 2, and 4 on monocytes
A heavy schedule of training and competition can lead to transient immune impairment in athletes
Nieman, IJSM, 1994
Hunter, ISAPP 2010
What do we know? Research in athletes to date is limited and inconclusive
• Clancy et al, B. J. Sports Med. 2006
– Fatigued athletes showed sig. reduced secretion of IFN – γ from T cells v healthy athletes. Fatigued athletes who consumed L. acidophilus for a month had levels comparable with healthy athletes
• Kekkonen et al, Int. J. Sport Nut. Ex. Metab. 2007
– Marathon runners consumed either L. rhamnosus GG or placebo for 3 months training then undertook a marathon. No difference in respiratory illness or GI symptoms during three month training. Shorter duration of GI symptoms for 2 weeks post marathon with placebo (no difference in GI episodes)
• Cox et al, B. J. Sports Med., 2008
– 20 elite athletes, crossover trial, L. fermentum VRI-003 v placebo
– No days respiratory illness – 30 during probiotic tmt v 72 during placebo, illness severity sig lower during probiotic tmt (p=0.06)
• Prebiotics – nothing to date
Hunter, ISAPP 2010
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EXPERIMENTAL DESIGN
• Fecal samples, 16S - V4 - pyrosequencing (>2000 sequences/sample) and real time PCR
• Sequences were analyzed with the QIIME pipeline, which providestaxonomic and phylogenetic profiles and comparisons of the 59 samples
Manichanh et al, Genome Research 2010
CLUSTERING BY TREATMENTPrincipal coordinates analysis (PCoA)
Manichanh et al, Genome Research 2010
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GUT MICROBIOTA
AND DISEASE
CONCLUSIONS:
• Too early to define characteristics/structure of a
“healthy” gut microbiota.
• Age related differences confirmed by
metagenomics.
• Microbial signatures associated with disease are
being identified by IHMC projects.
• Prospective studies needed to provide
information about cause-effect relationships.
• Interventions to change gut microbiota may
become relevant to prevent risk of chronic disorders
(Type 2 Diabetes, IBD, IBS, Atopy, Cancer).
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