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 per cent 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 per cent 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 five per cent carbon dioxide for 24 to 48 hours. MBC was reported as the lowest concentration producing a 99.9 per cent (1000-fold) reduction in bacterial density in the sub-cultured well contents, relative to the positive control well.

The mean MBC/MIC ratio for tiamulin against App was only 1.74:1, although tiamulin is classed as a bacteriostatic drug. Only three of the isolates and the reference strain grew in 100 per cent swine serum and their MICs were not lower than those determined in MH broth.

Introduction

Attempts to integrate the pharmacokinetics (PK) of tiamulin concentrations in plasma with the pharmacodynamics (PD) measured as Minimum Inhibitory Concentration (MIC) against Actinobacillus pleuropneumoniae (App), using standardised MIC determinations, have been unsuccessful and led to considerations that lung¹ or leucocyte concentrations² might play a significant role, as estimated intracellular concentrations were 8.5µg/g but only 0.47µg/ml in plasma (ratio 18:1).

This is considered unjustifiable by some authorities³ 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.

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 per cent serum in the culture media⁴ and 100 per cent serum⁵ reduced the MIC values for tulathromycin against a variety of bacterial respiratory pathogens but in particular Pasteurella multocida by eight and 50 times, respectively, and against App⁴ by 32 times.

A preliminary study⁶ with serum 50 per cent : MHB 50 per cent indicated that there was a reduction in geometric mean MICs of seven App isolates by 6.6 fold and two isolates’ MICs were reduced by 32-fold in the presence of serum.

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 per cent swine serum in comparison with those achieved in serum-free culture medium.

Materials and Methods

Tiamulin activity was measured against 19 field isolates (collected 2003 – 2009) and one type strain (ATCC 27090) of App.

Broth microdilution MIC tests were performed using an overlapping double-dilution series of tests with tiamulin (range 0.3 to 72 µg/ml) in accordance with CLSI guideline M31-A3, using Veterinary Fastidious Medium (VFM)-MHB, 5 per cent lysed horse blood, yeast extract & yeast concentrate supplement, without serum (exact CLSI method) and in 100 per cent swine serum. MIC plates were incubated in five per cent carbon dioxide for 24 to 48 hours.

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 per cent (1,000- fold) reduction in bacterial density in the sub-cultured well contents relative to the positive control well.

Results

The MIC and MBC results for 19 field isolates and the reference strain were determined following growth in VFM. Only three field isolates and the reference strain grew in 100 per cent serum and there was no reduction in MIC.

MIC and MBC results of tiamulin against App grown in VFM

Discussion

Growth of App isolates was poor in 100 per cent serum but the MICs were not reduced as previously observed with 50 per cent 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 response¹ 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.

Further Reading

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