Antimicrobial susceptibility of
Mycobacterium avium subsp.
paratuberculosis (MAP)
M.Y. Krishnan, E.J.B. Manning, M.T. Collins*
University of Wisconsin-Madison
MAP causes paratuberculosis (Johne’s disease)
IBD of animals
Estimated at 22% in 1996
“…..results suggest that at least one-fourth of
U.S. dairy operations may have a relatively high
percentage of infected cows in their herds.”
Johne’s disease – Crohn’s disease
Similar Clinical Presentation
Onset @ sexual maturity
Diarrhea
Weight loss
Abdominal pain
Chronic
Debilitating
Progressive
Johne’s disease – Crohn’s disease
Similar Gross Pathology
Thickened
Thickened
Corrugated
Corrugated
Non-ulcerated
Non-ulcerated
JD
CD
Johne’s disease – Crohn’s disease
Similar Histopathology
Diffuse
Granulomatous
Giant cells
Dilated lymphatics
JD
CD
“The association of MAP and Crohn’s disease, based on PCR or ELISA
testing, is well established and we doubt that important further
insights may be gained from additional case-control studies….”
September, 2007
How
How can
can humans
humans be
be exposed
exposed to
to MAP?
MAP?
Pasteurization
Does Not Kill 100% of MAP
† MAP: most heat-resistant
Mycobacterium sp. (lab studies):
„ U.S. & U.K. & Germany
† MAP: can be cultured from
retail milk:
„ 1.8% in U.K. (AEM May 2002)
„ 1.6% in Czech Rep. (AEM March 2005)
„ 2.8% in U.S. (J. Food. Protect. May 2005)
Cows With Paratuberculosis
are Culled & Consumed
† Cows pass antemortem
veterinary inspection.
† Cows pass postmortem
veterinary inspection.
† Infected cows
frequently have
disseminated MAP
infections.
Test-and-cull
Test-and-cull JD
JD control
control programs
programs on
on farms
farms
are
are essentially
essentially test-and-feed-to-the-public.
test-and-feed-to-the-public.
Not all MAP-infected cows are thin.
IDEXX ELISA S/P = 2.0 = Strong-positive
Colorado
ColoradoState
StateUniversity:
University:
53%
53% of
of cull
cull dairy
dairy cows
cows had
had disseminated
disseminated MAP
MAP infections.
infections.
(beyond
(beyondthe
thegut
gutand
andassociated
associatedlymph
lymphnodes)
nodes)
57%
57% such
such MAP
MAP-infected
-infected cull
cull dairy
dairy cows
cows were
were clinically
clinically normal.
normal.
Antognoli
Antognolietetal.
al.Veterinary
VeterinaryMicrobiology
Microbiology127:300,
127:300,2008
2008
Colorado
ColoradoState
StateUniversity:
University:
53%
53% of
of cull
cull dairy
dairy cows
cows had
had disseminated
disseminated MAP
MAP infections.
infections.
(beyond
(beyondthe
thegut
gutand
andassociated
associatedlymph
lymphnodes)
nodes)
57%
57% such
such MAP
MAP-infected
-infected cull
cull dairy
dairy cows
cows were
were clinically
clinically normal.
normal.
Antognoli
Antognolietetal.
al.Veterinary
VeterinaryMicrobiology
Microbiology127:300,
127:300,2008
2008
Americans consume >50 lb.
of ground beef/person/yr.
30% of ground beef is
from cull dairy cows
Vehicles
Source
Susceptible
host
Spectrum of Human + Mycobacteria Outcomes
Mycobacterial factors:
Strain / virulence
Exposure dose
Exposure frequency
Human factors:
NOD2 defect
NRAMP-1
Age of exposure
M. tuberculosis
Subclinical
M
Latent
Skin test-pos
Clinical
M. leprae
Subclinical
Tuberculoid
Skin test-pos
Intermediate
Lepromatous
M. paratuberculosis
IBS
Ulcerative
colitis
Intermediate
Crohn’s
disease
CD-severe
perforating
Study Aim:
Establish the critical concentrations of
antibiotics necessary to inhibit or kill MAP.
Critical concentration definitions:
MIC = minimal inhibitory concentration
(lowest drug concentration necessary to stop MAP growth)
MBC – minimum bactericidal concentration
(lowest drug concentration necessary to kill MAP)
Methods – General / Nontechnical
† Mix various concentrations of
antibiotics with MAP and
observe culture for growth or
killing of MAP.
„
Antimicrobial susceptibility testing (AST)
† Culture system+measurement
„ MGIT ParaTB medium &
„ MGIT 960 instrument.
MGIT = Mycobacterial Growth Indicator Tube
Novel Aspects of Study
† First extensive study on human-origin MAP
† First study to use MGIT system for MAP AST
„ Results correlated with 2 other AST methods
„ MIC and MBC values determined
† First proof of drug stability in MGIT ParaTB
Medium
† First report of interactions of 6-MP (anti-
inflammatory drug commonly used for treatment of CD)
with multiple antibiotics
† First test of novel ethambutol derivatives
from Sequella® vs MAP
Results: Conventional Drugs
† MGIT AST methods agree with standard methods
† Considerable MAP strain drug susceptibility variability
† Susceptibility pattern similar to MAC, although most
drugs tested vs MAP do not have MICs reported for MAC
† Most potent drugs
1. Azithromycin & Clarithromycin
2. Ciprofloxacin
3. Amikacin & Rifampin
† Drugs with no effect on MAP
„ Isoniazid = anti-tuberculosis drug
„ Dapsone = anti-leprosy drug
† Combinations of 2 or 3 of the most effective antibiotics
showed neither synergistic nor antagonistic activity
Antibiotic + 6-MP Interactions
† 6-MP inhibited but did not kill 6 of 9
human MAP strains
† 6-MP was synergistic with multiple
antibiotics (improve antibiotic action)
† 6-MP was not antagonistic with any
antibiotics (did not interfere with antibiotic)
Results: Sequella® Compounds
† 5 of 10 compounds showed anti-MAP
activity
† Compound SQ641 (capuramycin
analogue) had anti-MAP potency
equal to or greater than conventional
drugs (MIC 0.125 – 2.0 µg/mL)
Importance
† Provides direction for drug selection:
„ Ex vivo trials (infected macrophages)
„ Animal treatment trials
„ Human clinical trials
† Standardizes methods for in vitro
susceptibility testing of MAP
† Supports studies searching for new
anti-MAP therapeutics