Introduction:

The minimum inhibitory concentration (MIC), an in-vitro microbiological test, where the effective inhibition of bacterial growth is measured, can be related to the maximum concentration (Cmax) achieved in plasma, when the antimicrobial is administered. A Cmax/MIC ratio of 8-10 appears to give an effective killing level in vivo for the aminoglycosides. The area under the curve (AUC) of the antimicrobial concentration curve, achieved in plasma over a 24 hour period following its administration, can also be used. The AUC divided by the MIC gives the AUIC (area under the inhibitory curve) and an effective killing ratio is approximately 100-125 hr for the fluoroquinolones.

The purpose of this paper was to examine the relationships between the Cmax, AUC and MIC of tiamulin, a primarily bacteriostatic antibiotic, against a specific isolate of Mycoplasma gallisepticum (MG) and correlate them with results from tiamulin dose-titration studies for the prevention and treatment of airsacculitis or chronic respiratory disease (CRD) in chickens.

Materials and methods:

a. Pharmacodynamics
The MIC of tiamulin for the MG isolate (FS 9) used in the artificial infection studies was 0.0039μg/ml (Drews et al, 1975). The minimum bactericidal concentration (MBC) was not recorded, however work with other strains of MG has shown that as the concentration of tiamulin increases, the killing activity increases, until an MBC can be established (Windsor, 2004 unpublished data) (see Graph 1). The MBC was approximately 16 times the MIC and therefore an MBC of 0.0624μg/ml was used in the subsequent relationship calculations.

b. Pharmacokinetics

An early study by Laber and Schütze (1977) looked at the serum levels of tiamulin in 7-week-old chickens after a gavaged dose of 25mg/kg and 50mg/kg bodyweight (see Graph 2). The tiamulin concentrations in serum were determined using an agar-well microbiological assay technique with Staphylococcus aureus (ATCC 29067) as the test organism. The Cmax after a single gavage dose of tiamulin at 50mg/kg bodyweight was 3.7μg/ml and the AUC 24 hours was calculated at 32.5μg hr/ml. Approximately linear results were achieved with 25mg/kg bodyweight at 1.86μg/ml and 13.72μg hr/ml.

c. Artificial infections studies - dose titration for prevention and treatment of airsacculitis

In a prevention dose-titration study (Laber and Schütze, 1975), tiamulin was administered by gavage at increasing dose rates, to 3-week-old chicks, daily, for three days, starting at the same time as the infection. The birds were infected by an inoculation of MG (FS 9) broth into the left post-thoracic air sac. Necropsy was performed 8 days after infection and the air sacs were scored for lesions and cultured for MG. The criteria for successful treatment were the absence of air sac lesions and MG could not be isolated from the air sacs. As a result, this demonstrated a mycoplasmacidal or eliminatory endpoint for the mycoplasma.

In a treatment dose-titration study (Laber and Schütze, 1975), 3-week old chicks were infected as above, but treatment was administered for 3 days, 7 days after infection. Necropsies were carried out 8 days after the start of treatment as before, and air sacs were scored for lesions and cultured for MG. The criterion for successful treatment was the lack of recovery of MG, as treatment could not completely reverse the lesions of airsacculitis. Similarly, a mycoplasmacidal or eliminatory endpoint could be determined.

Results and calculations:

a. Prevention study
The maximum effective dose (EDmax) of tiamulin given to completely inhibit the recovery of MG was 10mg/kg bodyweight (ED50% was 5.1mg/kg bwt).


Calculations - prevention:

The Cmax and AUC for the dose of 10mg/kg bodyweight can be estimated (E) by dividing the 50mg/kg bodyweight figures of 3.7μg/ml and 32.5μg hr/ml by 5 respectively, to give 0.74μg/ml and 6.5μg hr/ml. These figures were used in the calculations (see Table 1)

MIC (μg/ml)	0.0039	MBC E (μg/ml) (MIC x 16)	0.0624
Cmax @ 10mg/kg E (μg/ml)	0.74		0.74
Cmax/MIC ratio	189.7	Cmax/MBC ratio	11.9
AUC @10mg/kg (μg hr/ml)	6.5		6.5
AUC/MIC (hr) (AUIC)	1667	AUC/MBC (hr)	104

Commonly, the parameters used for successful treatment with bactericidal compounds are: - the aminoglycosides (Cmax/MIC = 8-10) and the fluoroquinolones (AUIC = 100-125hr). These figures are well exceeded by the ratios for tiamulin i.e. 189.7 for Cmax/MIC and 1667 for AUIC. However if the figures are divided by the MBC of tiamulin (MBC/MIC ratio times 16 for MG) then more typical ratios are demonstrated of 11.9 (Cmax/MBC ratio) and 104 (AUC/MBC ratio).

b. Treatment study
The ED50% was assessed as 21.2mg tiamulin/kg bodyweight but the EDmax was not achieved, even at 80mg/kg, where there was a 70% cure (see Graph 4).

Calculations - treatment:
The Cmax and AUC for the dose of 80mg/kg bodyweight can be estimated by multiplying the 50mg/kg bodyweight figures of 3.7μg/ml and 32.5μg hr/ml by 1.6 respectively, to give 5.92μg/ml and 52μg hr/ml

MIC (μg/ml)	0.0039	MBC E (μg/ml) (MIC x 16)	0.0624
Cmax @ 80mg/kg E (μg/ml)	5.92		5.92
Cmax/MIC ratio	1518	Cmax/MBC ratio	95
AUC @80mg/kg (μg hr/ml)	52		52
AUC/MIC (hr) (AUIC)	13333	AUC/MBC (hr)	833

The Cmax/MIC for tiamulin is 1518, which is vastly in excess of the standard of 10 for bactericidal antibiotics. Even the Cmax/MBC ratio is very high at 95. Similarly the AUIC is very high at 13,333 and the AUC/MBC is also high at 833, nearly 8 times the prevention level. This suggests that other factors are coming into play in this model.

Discussion and conclusions:
From the prevention study, where treatment is given soon after infection, a bactericidal/elimination effect can be achieved against MG at 10mg tiamulin/kg bodyweight when given by gavage. By examining the pharmacodynamic effect of the antibiotic and its pharmacokinetic characteristics, it can be seen that, when the Cmax or AUC is divided by the MBC rather than the MIC, a similar relationship to bactericidal antimicrobials, such as aminoglycosides and fluoroquinolones, can be achieved. The MICs for bactericidal compounds is normally very similar to their MBCs (MBC/MIC ratio 1-2) unlike bacteriostatic compounds.

From the treatment study, it is clear that sufficient damage to the air sacs is caused, during the 7 day untreated exposure, to stop total resolution of lesions once treatment is commenced. It was also evident that high levels of tiamulin (8 times the prevention level) could not eliminate the infectious agent, even though levels well in excess of the MBC were achieved. When air sacs are infected there is inflammation and production of exudate and other debris, which presumably inhibit the antibiotic penetration and destruction of the organism (see Photos 1 & 2) (Toutain et al, 2002). A major consideration of MG infections is the frequent secondary invasion of the respiratory tract by bacteria, especially Escherichia coli (see Photo 3), which causes a septicaemia, inflammation and enlargement of several organs, such as the liver and spleen and a generalized peritonitis, perihepatitis and pericarditis.

From this work, it can be seen that the early treatment/prevention of mycoplasmal infections, before major damage has been caused, is vital to the successful treatment of infections, caused by these organisms and the achievement of a bacteriological cure. Once an infection is well established, a bacteriological cure may not be achievable and surviving mycoplasma may be put into a ‘window’ of selective pressure (Drlica, 2003) and thereby be stimulated to mutate and develop resistance. Fortunately, this is usually a slow process with mycoplasma because of their relatively slow growth but resistance to some antimicrobials has been demonstrated (Burch and Valks, 2001) following prolonged exposure and usage over many years.

Overall, tiamulin is a highly effective antimycoplasmal antibiotic and can cause complete bacteriological cure or elimination, similar to bactericidal antimicrobials, when plasma levels in excess of the MBC are reached, especially in the early stages of infection.

References:

Burch, D.G.S. and Valks, M. (2001) Comparison of minimal inhibitory concentrations (MIC) against chicken mycoplasma of tiamulin and other antimicrobials and their concentrations in the blood. Proceedings of the 12th World Veterinary Poultry Congress, Cairo, Egypt, p322

Drews, J., Georgopoulos, A., Laber, G., Schuetze, E. And Unger, J. (1975) Antimicrobial activities of 81.723 hfu, a new pleuromutilin derivative. Antimicrobial Agents and Chemotherapy, 7, 5, 507-516

Drlica, K. (2003) The mutant selection window and antimicrobial resistance. Journal of Antimicrobial Chemotherapy, 52, 11-17

Laber, G, and Schütze, E. (1975) In-vivo efficacy of 81.723 hfu, a new pleuromutilin derivative against experimentally induced airsacculitis in chicks and turkey poults. Antimicrobial Agents and Chemotherapy, 7, 5, 517-521

Laber, G. and Schütze, E. (1977) Blood level studies in chickens, turkey poults and swine with tiamulin, a new antibiotic. The Journal of Antibiotics, 30, 12, 1119-1122

Toutain, P.L., Del Castillo, J.R.E. and Bousquet-Melou, A. (2002) The pharmacokineticpharmacodynamic approach to a rational dosage regimen for antibiotics. Research in Veterinary Science, 73, 105-114

Toutain, P.L. (2003) Pharmacokinetics/pharmacodynamics integration in dosage regimen optimization for veterinary medicine. Journal of Veterinary Pharmacology and Therapeutics, 26 (Supplement 1), 1-18

Windsor, H. (2004) Report to Novartis 'Determination of the minimal mycoplasmacidal concentration (MMC) of tiamulin against two Mycoplasma synoviae and two M. gallisepticum strains.'

October 2009