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Why are bacteria relevant to Crohn’s?
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Animal model data suggests the inflammation of CD
results from an abnormal response to the intestinal
flora
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Transgenic animal models do not develop CD if raised in
sterile environment
CD relapses have been associated with gut pathogens
like Campylobacter, Salmonella, Shigella, Yersinia,
pathogenic E. coli
Controversy is whether a specific etiologic agent is
required vs. any general flora
Pathogenesis of other mycobacterial infections (TB,
leprosy, MAC in AIDS) shares features of CD
Infectious agents implicated in IBD
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Diplostreptococci (UC)
Bacteroides necrophorum
Bacteroides fragilis
Pseudomonas maltophilia
Helicobacter hepaticus or pylori
species and,
Shigella
Chlamydia
Listeria
measles
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Wolinella
Coxsakie A, B
Reovirus
Polio virus
Norwalk virus
Influenza
herpes virus
Paramyxovirus
pathogenic E. coli
Is Crohn’s disease due to an infectious etiology?
Ballard et al. IDCP; Nov 2002
Ballard et al. IDCP; Nov 2002
n=393
March
2000
Is Crohn’s Disease caused by MAP?
Ballard et al. IDCP; Nov 2002
n=393
What is MAP?
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M. avium
 subsp paratuberculosis
 subsp lepraemurium
 subsp silvaticum and
 strains not belonging to
any of these species that
possibly form new species
Very slow grower, fastidious, and
intracellular pathogen
Resistant to chlorine
Survives pasteurization
 UK survey 7% of cartons
positive
 Has been detected in human
breast milk of CD patients
 Ubiquitous in water supply, etc
 UK has established guidelines
to eradicate in food supply
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First suggested as a cause of
Crohn’s by Dalziel (described
Crohn’s) in 1913
1986 isolated from Crohn’s patients
(Chiodini et al. J Clin Microbiol.
1984;20:966-971)-spheroplasts
Case report of child w/paraTB
scrofula, then Crohns, which
resolved on rifabutin and
clarithromycin
Mixed results (0-100%) regarding
presence of MAP in tissue samples
from CD patients Sanderson JDet al. Mycobacterium
paratuberculosis DNA in Crohn's disease tissue. Gut. 1992;33:890-896.
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High seroprevalence in CD but
variable results (p35 and p36
antibodies), and recent sero study
inconclusive
What is Johne’s disease ?
chronic inflammatory disease of ruminants (6% of
US cattle), and also primates
 Ileitis with chronic diarrhea and wasting
 Easy to detect MAP
 Not segmental, no
fistula/strictures/abscesses/ulcers
 caused by MAP
 UK 2002 guidelines for eradication (published 8/04)
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Koch’s postulates
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1890 German physician/bacteriologist Robert Koch
Koch's postulates are as follows:
 The organism must be present in every case of a specific disease, but not in healthy
subjects.
 The organism must be isolated from the host with the disease and grown in pure
culture.
 The specific disease must be reproduced when a pure culture of the organism is
inoculated into a healthy susceptible host
 The organism must be recoverable from the experimentally infected host
may not hold if:
 bacteria (M. leprae, R. rickettsiae) cannot be "grown in pure culture" in the
laboratory-but PCR and other molecular techniques allow identification
 no animal model of infection exists
organism may also cause disease if:
 acquired extra virulence factors making it pathogenic
 gains access to deep tissues via trauma, surgery, an IV line, etc
 infects an immunocompromised patient.
Ignores subclinical infection
Many examples of accepted infectious etiologies that do not fit the model
 Cholera
 leprosy
Are they met?
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The bacteria must be present in every case of the disease.
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The bacteria must be isolated from the host with the disease and grown in
pure culture
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First isolated in 1984 (Chiodini)
Has been isolated from Crohn’s patients, and recently from blood (?less
chance of environmental contamination)
The specific disease must be reproduced when a pure culture of the
bacteria is inoculated into a healthy susceptible host
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Essentially yes for MAP in Johne’s disease
Not established for CD, although most recent papers have shown higher
correlation
Yes but not repeatedly
The bacteria must be recoverable from the experimentally infected host
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Yes for Johne’s (strains of human origin)
Table 1 Summary of the frequency of polymerase chain reaction studies for the presence or absence of Mycobacterium paratuberculosis in Crohn's disease (CD), ulcerative colitis (UC), and non-inflamm
Reference
Target
Non-IBD
UC
CD
Rosenburg et al 19918
Wu et al 19919
Sanderson et al 199210
Dell'Isola et al 199411
Lisby et al 199412
Fidler et al 199413
Suenaga et al 199514
Not stated, not IS900
Pan mycobacterial sequences
IS900
IS900
IS900
IS900. Also detected non M paratuberculosis sequences
IS900
GroEL
IS900
IS900
16S rRNA
Culture
IS900
IS900 mRNA
IS900
Mycobacteria
M paratuberculosis
M paratuberculosis 16S rRNA
IS900
MP2
IS900
IS900
16S rRNA any mycobacteria
IS900
IS900
IS900
16S rRNA
IS1110 element and probed 16S rRNA
IS900
0/6 (0%)
nd
5/40 (12.5%)
7/24 (29%)
3/28 (11%)
0/20 (0%)
14/16 (87.5%)
14/16 (87.5%)
1/26 (4%)
0/15 (0%)
0/1 (0%)
0/23 (0%)
4/35 (11%)
0/2 (0%)
0/11 (0%)
13/23 (57%)
0/23 (0%)
0/22 (0%)
0/21 (0%)
0/21 (0%)
0/12 (0%)
0/11 (0%)
2/20 (10%)
0/20 (0%)
0/3 (0%)
0/13 (0%)
0/11 (0%)
nd
0/12 (0%)
nd
nd
1/23 (4.3%)
1/5 (20%)
2/10 (20%)
0/10 (0%)
11/18 (61%)
11/18 (61%)
0/49 (0%)
2/15 (13%)
nd
nd
nd
4/4 (100%)
nd
6/13 (46%)
0/13 (0%)
0/10 (0%)
0/6 (0%)
0/6 (0%)
nd
0/5 (0%)
1/27 (4%)
0/27 (0%)
0/14 (0%)
0/4 (0%)
nd
1/1 (100%)
nd
0/21 (0%)
0/20 (0%)
26/40 (65%)
13/18 (72%)
11/24 (46%)
4/31 (13%)
10/10 (100%)
10/10 (100%)
0/68 (0%)
2/9 (22%)
0/4 (0%)
0/23 (0%)
10/26 (38%)
8/8 (100%)
0/23 (0%)
17/36 (47%)
0/36 (0%)
0/31 (0%)
0/10 (0%)
0/10 (0%)
0/7 (0%)
1/21 (5%)
2/47 (4%)
0/47 (0%)
0/30 (0%)
0/13 (0%)
3/11 (27%)
7/20 (35%)
0/3 (0%)
Rowbotham et al 199515
Murray et al 199516
Kreuzpaintner et al 199517
Erasmus et al 199518
Mishina et al 199619
Frank and Cook 199620
Dumonceau et al 199621
Dumonceau et al 199722
Al-Shamali et al 199723
Riggio et al 199724
Clarkston et al 199825
Cellier et al 199826
Chiba et al 199827
Kanazawa et al 199928
Tiveljung et al 199929
*Ikonomopoulos et al 200030
daggerGibson et al 200031
* M paratuberculosis sequences in sarcoidosis were also found. dagger 0/21 from orofacial granulomatosis.
nd, not done.
Detection of MAP DNA in Tissue by FISH Using CSLM and Nested PCR
Romero et al, IBD: Volume 11(2), February 2005, pp 116-125
Serologic evidence for MAP in CD
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Highly variable
Bernstein et al, JCM: Mar 2004; 42 (3) 1129-35
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Population based trial in Manitoba (high Crohn’s rate)
ELISA to MAP
>900 subjects, 283 w/CD
No correlation between MAP seropositivity and CD, among siblings, UC
pts, CD pts, normal controls
No epidemiologic/demographic correlation with high likelihood of MAP
exposure
Most recent data
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Bull et al. Detection and verification of Mycobacterium avium subsp
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Naser et al (Lancet 2004;364:1039-44)-blasted by editorials
paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals
with and without Crohn's disease. J Clin Microbiol. 2003;41:2915-2923.
 92% of intestinal biopsies + for MAP (vs. 26%)
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Small (n=28 w/CD, 15 controls, some w/other bowel disease)
Isolated MAP from blood of 50% of CD, 22% of UC and none of non-IBD pts
No other organisms isolated (so not just leaky mucosa)
But 20% of non-IBD pts (n=15) had MAP DNA in blood
sequenced strains
Sechi LA - Am J Gastroenterol - 01-JUL-2005; 100(7): 1529-36
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Survey of patients having ileocolonoscopy in Sardinia
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83% of CD had MAP by PCR vs 10% of controls (OR= 43)
MAP was isolated in 63% vs 10%
Initial form isolated was Z-N negative, then reverted to the Z-N positive
form.
Antibiotics in Crohn’s
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Variable data for metronidazole,
although overall appears to have
activity in Crohn’s, but poorly
tolerated
Case reports in 1980’s reported
dramatic improvement in CD when
pts rx for concomitant TB
MAP is resistant to many anti-TB
agents, and macrolides are the most
active agent
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Lowest MICs
High intracellular concentrations
At least 17 negative trials, but
none included macrolides
Small open label study w.clari alone
reported dramatic benefit
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N=25
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4 trials with clarithromycin show
benefit
 Sample sizes <60
 Follow-up range 12-54 mo
 Triple drug therapy
 Enpoints variable (CR vs
improved scores)
 All open label
single controlled trial (Goodgame
et al)
 N=30, randomized
 Only 12mo f/u and 3mo rx
 No benefit
For
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NOD2 defect predisposes to infection with intracellular bacterial
infections and is associated with CD [Girardin SE et al. Trends Immunol. 2003;24:652].
MAP RNA has been detected in a large proportion of CD
patients, indicating viability
organism has been cultivated from stool, intestinal tissue, and
peripheral blood in patients with Crohn disease [Bull TJ et al. J Clin Microbiol.
2003;41:2915].
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Clinical trials suggest benefit of effective combination therapy,
albeit mostly uncontrolled small trials. Duration of rx and
follow-up may have been inadequate.
Negative Ziehl-Neelson staining may reflect pathogenic non-cell
wall forms. Confirmed by culture data.
Against
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Finding of MAP DNA may reflect non-viable MAP coincidentally passing
through the GI tract
Isolation from blood may reflect only increased intestinal permeability as a
result of CD
Johne’s is more similar to MAC wasting syndrome than UC
Data from seroprevalence studies has been highly variable
If MAC is cause, then why don’t immunosuppressants exacerbate the
infection?
 In trials where organism was isolated, finding did not correlate with
immunosuppression
 Increased MAP detection not reports with infliximab and other
immunosuppressants
MAP has been isolated from normals and UC patients and may represent
epiphenomenon
No epidemiological evidence that CD is associated with exposure to
ruminents
Future
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Results of Australian RPCT of 2 years’ rx of 214 pts with
clari/rifabutin/clofazimine pending
Complete genome of MAP just published (Proc Natl Acad Sci U
S A - 30-AUG-2005; 102(35): 12344-9)
RPCT’s with long term follow-up using drugs known to be active
against MAP
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Better case definitions and more uniform therapeutic endpoints
More basic science
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?Australian study
Pre-exposure histology, cultures and PCR then post-exposure
Immunogenetics
Better epidemiology (?trace a strain from cow to human)
Antibiotics in Crohn’s
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32. Thomas GA, Swift GL, Green JT, et al. Controlled trial of antituberculous chemotherapy in Crohn's disease: a five year follow
up study. Gut. 1998;42:497-500. Bibliographic Links [Context Link]
33. Prantera C, Kohn A, Mangiarotti R, et al. Antimycobacterial therapy in Crohn's disease: results of a controlled, double-blind
trial with a multiple antibiotic regimen. Am J Gastroenterol. 1994;89:513-518. Bibliographic Links [Context Link]
34. Rutgeerts P, Geboes K, Vantrappen G, et al. Rifabutin and ethambutol do not help recurrent Crohn's disease in the
neoterminal ileum. J Clin Gastroenterol. 1992;15:24-28. Bibliographic Links [Context Link]
35. Hampson SJ, Parker MC, Saverymuttu SH, et al. Quadruple antimycobacterial chemotherapy in Crohn's disease: results at 9
months of a pilot study in 20 patients. Aliment Pharmacol Ther. 1989;3:343-352. Bibliographic Links [Context Link]
36. Shafran I, Kugler L, El-Zaatari FA, et al. Open clinical trial of rifabutin and clarithromycin therapy in Crohn's disease. Dig
Liver Dis. 2002;34:22-28. Bibliographic Links [Context Link]
37. Hermon-Taylor J. Treatment with drugs active against Mycobacterium avium subspecies paratuberculosis can heal Crohn's
disease: more evidence for a neglected public health tragedy. Dig Liver Dis. 2002;34:9-12. [Context Link]
38. Borody TJ, Leis S, Warren EF, et al. Treatment of severe Crohn's disease using antimycobacterial triple therapy: approaching a
cure? Dig Liver Dis. 2002;34:29-38. [Context Link]
39. Hulten K, Almashhrawi A, El-Zaatari FA, et al. Antibacterial therapy for Crohn's disease: a review emphasizing therapy
directed against mycobacteria. Dig Dis Sci. 2000;45:445-456. Bibliographic Links [Context Link]
40. Borgaonkar MR, MacIntosh DG, Fardy JM. A meta-analysis of antimycobacterial therapy for Crohn's disease. Am J
Gastroenterol. 2000;95:725-729.
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?since then
?4 studies
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Hermon-Taylor J, Bull TJ, Sheridan JM, et al.
Causation of Crohn's disease by Mycobacterium
avium subspecies paratuberculosis. Can J
Gastroenterol. 2000;14:521-539. Bibliographic
Links [Context Link]
Hermon-Taylor J. Protagonist: Mycobacterium
avium subspecies paratuberculosis is a cause of
Crohn's disease. Gut. 2001;49:755-756.
Summary of MAP and Crohn’s
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The NOD2 genetic defect predisposes to infection with intracellular bacterial
infections and is associated with Crohn disease [Girardin SE et al. Trends Immunol.
2003;24:652].
The DNA of M. avium ss paratuberculosis has been detected by PCR in 90% of cases,
by in situ hybridization in 70% of cases, and by RT-PCR in up to 100% of cases [Sechi
LA et al. J Clin Microbiol. 2001;39:4514; Mishina D et al. Proc Natl Acad Sci USA.
1996;93:9816].
This organism has been cultivated from stool, intestinal tissue, and peripheral blood in
patients with Crohn disease [Bull TJ et al. J Clin Microbiol. 2003;41:2915].
Serologic tests support this association, but results have been variable [Collins MT et
al. J Clin Microbiol. 2000;38:4373].
Multiple antibiotic trials with Crohn disease have been negative including those using
antituberculous agents. However, treatment of this pathogen requires macrolides, and
since 1997, 4 trials with macrolides have shown complete clinical remission in up to
66% [Greenstein RJ. Lancet Infect Dis. 2003;3:507].
This organism causes a similar, but not at all identical, disease in cattle characterized by
granulomatous inflammation of the intestine [Mortensen HJ. Dairy Sci. 2004;87: 2108].