MICs and MBCs of Tiamulin against
Actinobacillus pleuropneumoniae (APP)
in broth and 100% Serum
Andrew Pridmore1, David Burch2 and Peter Lees3
1
Don Whitley Scientific Ltd, Shipley, West Yorkshire, BD177SE, UK;
2
3
Octagon Services Ltd, Old Windsor, Berks, SL42NR, UK;
Royal Veterinary College, North Mymms, Hatfield, Herts, AL97TA, UK
ABSTRACT
Attempts to integrate the pharmacokinetics of
tiamulin (Denagard® – Novartis) in plasma with
tiamulin MICs against Actinobacillus
pleuropneumoniae (App) have proven ineffective,
despite good clinical and microbiological responses
to treatment. The purpose of this study was to
determine MICs and MBCs of tiamulin against App
and whether these would be consistently reduced
by culturing the organism in 100% swine serum.
Tiamulin activity was measured against 19 UK field
isolates of App (collected 2003-2009) and type strain
(ATCC 27090). Broth microdilution MIC/MBC tests were
performed in accordance with CLSI guideline M31-A3,
using ‘Veterinary Fastidious Medium’ [supplemented
Mueller-Hinton (MH) broth, pH 7.3] without serum or in
100% swine serum.
For precision, a modified overlapping doubling dilution
series was used (tiamulin concentration range 0.3 to 72
µg/ml). MIC/MBC plates were incubated in 5% CO2 for 24-
Attempts to integrate the pharmacokinetics (PK) of
tiamulin (Denagard® - Novartis Animal Health Inc.)
concentrations in plasma with the pharmacodynamics
(PD) measured as Minimum Inhibitory Concentration
(MIC) against Actinobacillus pleuropneumoniae (App),
using standardized MIC determinations, have been
unsuccessful and led to considerations that lung Ref.1
or leucocyte concentrationsRef.2 might play a significant role,
as estimated intracellular concentrations were 8.5µg/g but
only 0.47µg/ml in plasma (ratio 18:1).
The MIC and MBC results for 19 field isolates and the reference strain were determined following growth in VFM.
Only 3 field isolates and the reference strain grew in 100% serum and there was no reduction in MIC.
A
B
C
D
E
F
G
H
Results in
culture medium (VFM)
This is considered unjustifiable by some authorities Ref.3
because the infectious agent resides outside the cell in the
extracellular compartment. Moreover, serum/plasma
concentrations are better established for pharmacokinetic
integration for most antibiotics.
12
14
12
It was the purpose of this study to determine whether the
MIC and Minimum Bactericidal Concentration (MBC) of
tiamulin against App would be consistently reduced by
culturing the organism in 100% swine serum in
comparison with those achieved in serum-free culture
medium.
2
8
M
AT3 E R4 I A 5L S 6A N7D M
E T9 H 10
O D 11
S
A
14
DWC 10418
12
14
DWC 10423
18
7
9
10
14
14
20
12
32
12
14
10
24
Broth microdilution MIC tests were performed using an
overlapping
double-dilution series of tests with tiamulin
E
(range 0.3 to 72 µg/ml) in accordance with CLSI guideline
F
M31-A3, using Veterinary Fastidious Medium (VFM) G
MHB,
5% lysed horse blood, yeast extract & yeast
concentrate
supplement, without serum (exact CLSI
H
method) and in 100% swine serum. MIC plates were
incubated in 5% CO2 for 24-48h.
2
3
4
5
A
72
64
56
48
B
36
32
28
C
18
16
D
9
6
7
DWC 10429
DWC 10950
MIC
DWC 10953
MBC
MIC Breakpoint
Resistance ≥ 16.0 µg/ml
DWC 18792
12
DWC 18822
12
DWC 18824
12
DWC 18826
12
DWC 18829
28
36
18
D
DWC 10427
DWC 15145
36
36
Tiamulin
activity was measured against 19 field isolates
B
(collected 2003 – 2009) and one type strain (ATCC 27090)
ofCApp.
56
DWC 15140
18
12
32
DWC 10417
20
A preliminary study
with serum 50% : MHB 50%
indicated that there was a reduction in geometric mean
MICs of seven App isolates by 6.6 fold and 2 isolates’
MICs were reduced by 32 fold in the presence of serum.
24
24
DWC 10413
12
Ref.6
APP STRAIN
Results in
100% Swine Serum
ATCC 27090
18
However, in an artificial infection study, an App isolate with
a tiamulin MIC of 4µg/ml was effectively treated, both
clinically and bacteriologically, with 180 ppm tiamulin in the
drinking water. Studies using 2.5% serum in the culture
media Ref.4 and 100% serum Ref.5 reduced the MIC values for
tulathromycin against a variety of bacterial respiratory
pathogens but in particular Pasteurella multocida by eight
and fifty times, respectively and against App Ref.4 by thirtytwo times.
1
The mean MBC/MIC ratio for tiamulin against App was
only 1.74: 1, although tiamulin is classed as a
bacteriostatic drug. Only 3 of the isolates and the
reference strain grew in 100% swine serum and
their MICs were not lower than those determined in MH
broth.
R E S U LT S
INTRODUCTION
1
48h. MBC was reported as the lowest concentration
producing a 99.9% (1000 fold) reduction in bacterial
density in the sub-cultured well contents, relative to the
positive control well.
10
10
16
18
14
24
24
DWC 20758
DWC 20759
24
DWC 6903
24
12
28
DWC 6908
24
8
9
10
11
12
40
72
64
56
48
40
MIC (µg/ml)
Results
MBC (µg/ml)
24
20
36
32
28
24
20
14
12
10
18
16
14
12
10
8
7
6
5
9
8
7
6
5
E 4.5
4
3.5
3
2.5
4.5
4
3.5
3
2.5
F 2.25
12
14
7-18
12.3
1
MIC/MBC 50%
MIC/MBC 90%
Range
MIC/MBC mean
MIC/MBC ratio
18
36
9-36
21.4
1.74
2
1.75
1.5
1.25
2.25
2
1.75
1.5
1.25
G 1.13
1
0.88
0.75
0.62
1.13
1
0.88
0.75
0.62
H 0.56
0.5
0.44
0.38
0.31
0.56
0.5
0.44
0.38
0.31
MIC and MBC results of tiamulin
against App grown in VFM.
DISCUSSION
Overlapping dilution series employed for MIC/MBC determination.
The MBC of tiamulin against each isolate was reported as
the lowest concentration producing a 99.9% (1000 fold)
reduction in bacterial density in the sub-cultured well
contents relative to the positive control well.
Growth of App isolates was poor in 100% serum but the MICs were not reduced as previously observed with 50% serum.
The MICs results of the isolates grown in VFM were in accordance with previous surveys. The mean MBC was only
1.74 times the MIC for tiamulin against App, which is surprising as it is considered primarily a bacteriostatic antibiotic.
This is in accordance with its clinical response Ref.1 but does not clarify the relationship between plasma concentrations and
MIC/MBC.
References:
1. Burch & Klein (2008) Proc IPVS Congress, 2, 494
2. Nielsen & Szancer (1998) Proc IPVS Congress, 3, 241
3. Mouton et al (2008) J Antimicrob Chemother, 61, 235-237
4. Godinho et al (2005) Vet Therapeutics, 6, 2, 113-121
5. Illambas et al (2008) Proc AAVM Conference, 93
6. Burch et al, (2009) J Vet Pharmacol Therap, (Suppl 1), 68-69
This poster was first presented at the Fifth International Conference on Antimicrobial Agents in Veterinary Medicine (AAVM), May 2010