Relevance of bacteriain the pathogenesis of IBD

Philippe Marteau, MD, PhDMedico-surgical Departmentof Digestive Diseases,Lariboisière hospital, Paris

Pathogenesis of IBD

immunologiques

Geneticbackground

Environment

Immune response

Intestinal Intestinal floraflora

Relevance of bacteria in IBD

• Lesions in IBD predominate in areas of highest bacterial exposure

• Failure of induction of colitis in germ free animals

• Colitis can be transferred with T cells reactive to bacterial antigens

• Loss of tolerance to the flora in patients with IBD

Relevance of bacteria in IBD

• The flora influences epithelial and immune cells

• Receptors for bacterial signals

• Polymorphisms of some of them (NOD2, TLR4) associated with a higher risk of IBD

• Defensins & Crohn’s disease

• Lesions in IBD predominate in areas of highest bacterial exposure

• Failure of induction of colitis in germ free animals

• Colitis can be transferred with T cells reactive to bacterial antigens

• Loss of tolerance to the flora in patients with IBD

Relevance of bacteria in IBD

• Role of the faecal stream ?

• Efficacy of antibiotics ?

• Antibodies against micro-organisms. What do they mean ?

• Effectiveness of probiotics in models and clinical situations ?

• Does the composition of the faecal and mucosal flora differ between patients with IBD & controls ?

Bacteria & POUCHITIS

• Dysbiosis

• Effectiveness of metronidazole and ciprofloxacinin acute pouchitis (RCTs)

• Efficacy of some probiotics (VSL#3, 3RCTs) to prevent recurrence of chronic relapsing pouchitisand first episode

Gionchetti P et al. Gastroenterology 2000;119:305-9

n=40 VSL#3 (3g x 2/d) vs placebo 9 n=40 VSL#3 (3g x 2/d) vs placebo 9 monthsmonths

Maintenance Maintenance ofof remissionremission ofof relapsingrelapsingpouchitispouchitis withwith thethe probioticprobiotic VSL#3 VSL#3

VSL#3VSL#3

PlaceboPlacebo

• Pouchitis is rare when IAA is performed for other reasons than IBD

• Bacteria are not « THE » (only) « CAUSE »

• … pouchitis is not due to « A PATHOGEN »

Think different

• Pouchitis is rare when IAA is performed for other reasons than IBD

• Bacteria are not « THE » (only) « CAUSE »

• … pouchitis is not due to « A PATHOGEN »

Bacteria in other IBD ?

• Dysbiosis ?

• Effectiveness of antibiotics ?

• Efficacy of probiotics ?

Bacteria & Postoperativerecurrence of ileal Crohn’s disease

• Dysbiosis

• Fecal stream diversion (very few subjects)

• Effectiveness of imidazole antibiotics (RCTs)

• No (clear) efficacy of probiotics so far

Faecal stream diversion in Crohn’s disease

Stomy Anastomosis Whole faecal stream Filtered faecal stream

Imidazole antibiotics & postoperative recurrence of ileal CD

Rutgeerts et al. Gastroenterology 1995

60 patients curative ileal resection

metronidazole (20 mg/kg/d) 3 months vs placebo

Recurrence at 12wks lesions 52% vs 75% (P = 0.09)

severe lesions 13% vs 43% (P = 0.02)

at 1 year clinical 4% vs 25%

Imidazole antibiotics & postoperative recurrence of ileal CD

Rutgeerts et al. Gastroenterology 2005

80 patients curative ileal resection

Ornidazole (1g/d) 1 year vs placebo

Recurrence

1 year clinical 7.9% vs 37.5% (P= 0.0046)

1 year lesions 53.6% vs 79% (P = 0.037)

Antimicrobial effect of imidazoles ? … not studied

Immunomodulating and anti-oxydant properties

% endosc. relapse grade > 1

PLACEBOLA1

Prevention of postoperative recurrence of Crohn’s D.

Double blind placebo controlled trial

98 subjects operated for CD

Lactobacillus johnsonii LA1 (4 109 cfu/d) or placebo 6 months

Marteau et al.. Gut 2006

49% 64%

05

1015202530354045

Grade 0 Grade 1 Grade 2 Grade 3 Grade 4

%

LA1

Placebo

C D

A : Listeria monocytogenes; B : measles

C : Mycobacterium paratuberculosis; D : Saccharomyces cerevisiae

AB

Crohn’s disease and pathogens ? : conflicting results

Naser S et al. Lancet 2004;127:412

MycobacteriumMycobacterium aviumavium paratuberculosisparatuberculosisD

étec

tion o

fM

AP

46%50%

45%

22% 20%

0%0%

20%

40%

60%

80%

100%

Crohn n=28 UC n=9 Controls n=15

intestine Blood

Sechi L et al. Am J Gastroenterol 2005;100:1529–1536

Sardinia 1.6 million people - 3.5 million sheep and 100,000 cattle

Johne’s disease and Mycobacterium avium subspeciesparatuberculosis infection are endemic

Intestinal biopsies in CD and controls

CD Controls pPCR + 83.3% 10.3 % 0.000001Culture + 63.3% 10.3 % 0.00001

Ulcerative colitis

• Dysbiosis

• Efficacy of some probiotics to prevent recurrence(best evidence= E. coli Nissle 1917 vs 5-ASA…)

• No Effectiveness of antibiotics (RCTs)

Conclusions

No bacterium fulfiling the Koch’s criteria and « causing » IBD

The « clinical relevance of bacteria » is

• established in pouchitis

• possible in postoperative recurrence of CD

• a good track for researchers in other situations

Think different

•

Is it possible to discover new pathogens ?

Granulobacter bethesdensis

Identification of a novel G-negative rod from a patient with CGDDemonstration of• specific immune reaction by high titer antibody, • ability to cause similar disease when introduced into CGD, but notwild-type mice• recovery of the organism from lesions in the mice… fulfilling of Koch’s postulates for a new pathogen

1

Ruminococcus obeum102c10 -1 clone-HuCB25

129c10 -4 clones-Ruminococcus productus

Clostridium coccoidesClostridium celerecrescens

56c10 -1 clone-Clostridium nexile

HuCC4369c10 -1 clone-

Ruminococcus gnavus46c10 -6 clones-

Eubacterium formicigeneransAdhufec 420Faecalibacterium prausnitzii

Ruminococcus flavefaciensClostridium leptum

32c10 -1 clone-Eubacterium desmolans

Adhufec 296Selenomonas sp. AF385495

Pectinatus frisingensis43c10 -30 clones-

Veillonella ratiMegasphaera elsdenii16c10 -1 clone-

Adhufec 395Phascolarctobacterium faecium137c10 -1 clone-

8c10-1 clone-Streptococcus parasanguinisStreptococcus pneumoniae

Streptococcus salivarius57c10 -3 clones-Pediococcus pentosaceus111c10 -2 clones-

Herbaspirillum seropedicaeOxalobacter formigenesSutterella wadsworthensis

118c10 -3 clones-Escherichia coli62c10 -6 clones-

97c10 -2 clones-Adhufec 153Bacteroides uniformis

Bacteroides vulgatusAdhufec 27124c10 -26 clones-

Adhufec 303Bacteroides stercoris128c10 -3 clones-

Adhufec 12.2540c10 -1 clone-

Bacteroides distasonis68c10 -4 clones-

100

100

100

100

100

100

100

100

92.9

97.8

89.7

71.9

52.4

83.5

93.589.8

95.6100

100

100

100

100

99.998.1

100

100

100

100

88.4

99.1

41.3

100

10098.1

99.9

100

100

99.7

Adhufec : adult human feces clone

GroupeC. coccoides (13)

GroupeC. leptum (1)

GroupeSporomusa (32)

Groupe Streptococcus-Enterococcus (6)

Groupe Sutterella (3)

Groupe desEntérobactéries (6)

Groupe Bacteroides (36)

Gram + Gram - Microflore intestinale dominante de la patiente 1

Total: 97 clones

Molecular inventory of the faecal flora of 4 patients with CD Mangin I et al. FEMS Microbiol Ecol 2004

Numerous clones belonged to species unusual in dominance : Pectinatus, Sutterella,Fusobacterium, Verrucomicrobium,Clostridium disporicum, C.glycolicum, C. ramosum, C. innocuum, C. perfringensThe only molecular species (OTU) shared by all 4 patients

… and at unusually high rate of occurrence (i.e. 7-29% of cloned sequences) : Bacteroides vulgatus

Adding new microbes in IBD…lessons from animal models

• Mono-association of IL10-/-mice with microbes

•• Helicobacter hepaticus,candida albicans, L. lactis, Clostridiumsordelii : no colitis

Kim et al. Gastroenterology 2005;128:891-906

Adding new microbes in IBD…lessons from animal models

• Mono-association of IL10-/-mice with microbes

•• Helicobacter hepaticus,candida albicans, L. lactis, Clostridiumsordelii : no colitis

• E. coli & E. faecalis colitis withdifferent phenotype

E. coliEnterococcusfaecalis

Kim et al. Gastroenterology 2005;128:891-906

Adding new microbes in IBD…lessons from animal models

• Mono-association of HLAB27 TG rats vs IL10-/-mice with the same microbes

• Induction of colitis depends on the host

Escherichia coli

Kim et al. Gastroenterology 2005Rath et al. Infect Immun 1999

Bacteroides vulgatus

Dysbiosis in IBD

• Enteroadherent E. coli in ileal lesions of CD

• Higher N° of bacteria in the mucosa

• Instability of the fecal and mucosal flora in IBD

• Presence of high numbers of unusual bacteria in IBD

• Reduction of biodiversity (discussed)

• Reduction of firmicutes and of their diversity

FaecalFaecal floraflora in in variousvarious colitidiescolitidies andand healthyhealthy subjectssubjects(Sokol et (Sokol et alal. 2005). 2005)

0 ,00%

20 ,00%

40 ,00%

60 ,00%

80 ,00%

100 ,00%

120 ,00%

Erec482/E

UB

Clep/EUB

Bac303/EUB

Bif164/E

UB

Ato291/E

UB

Enter/E

UB

Add itivit y

Gram+

Gram -

C rohn 's D is eas eU lc era t ive c o lit isIn fec t ious c o lit isC ontro l

Bacteria in the mucosaSwidsinki et al. Gastroenterology 2002;122:44-54

Perc

ent o

f pat

i ent

s

Concetration ofmucosal bacteria

Bacteria in the mucosaSwidsinki et al. J Clin Microbiol 2005

CD IBS

YellowYellow bacteriabacteria = = BacteroidesBacteroides((higherhigher concentration in CD concentration in CD identifiedidentified by FISH))by FISH))

Conclusion

• Bacteria play a role in models of IBD and in pouchitis

• Several molecules associated with IBD are bacterialreceptors or « modulators »… (TLRs, NOD, defensins)

• The flora(s) of patients with IBD differs from that ofcontrols and is unstable

• Bacterial concentrations or metabolites may play a role

• It is thus « relevant » to work on intestinal ecology