Investigation of Antimicrobial Action against Mycoplasma mycoides subsp. mycoides SC

John Mitchell1, Declan McKeever1, Quintin McKellar2. 1Royal Veterinary College, Hatfield, UK, 2University of Hertfordshire, Hatfield, UK.

Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) is the causative agent of Contagious Bovine Pleuropneumonia (CBPP), an economically important disease of cattle in sub-Saharan Africa (Thomson, 2005). Current control relies on public sector deployment of live attenuated vaccines; however, these are of limited efficacy (Rweyemamu et al., 1995) and, as an alternative, farmers are using antimicrobials to treat their animals. Without any information on appropriate dosing, such use may result in poor clinical outcomes and development of resistance (McKellar et al., 2004). To date, studies on antimicrobials for the treatment of CBPP have been few; as an initial step to selecting a drug and defining a dosage schedule using a pharmacokinetic-pharmacodynamic approach, the effector kinetics of three antimicrobials against MmmSC, namely oxytetracycline (OTC), danofloxacin and tulathromycin, were determined in artificial medium (Mycoplasma Experience, Reigate, UK) and adult bovine serum (Sigma-Aldrich, Poole, UK).

Minimal inhibitory concentrations (MIC) were established for these antimicrobials against MmmSC strain B237, a virulent Kenyan isolate, using a macrodilution technique and subsequently time-kill curves at various multiples of the MIC were constructed over a 24h period in both matrices. Pharmacodynamic analysis was performed using Phoenix WinNonlin 6.2 software (Pharsight, Mountain View, CA, US) and data were fitted to the sigmoid Emax model, E = E0 + (EmaxCeN)/(EC50N+CeN), where E is the antimycoplasmal effect (change in log10 cfu.mL-1 after 24h compared to the initial titre), E0 is the difference in log10 cfu.mL-1 after 24h compared to the initial titre when no antimicrobial is present, Emax is the maximum antimycoplasmal effect, EC50 is the AUC (area under curve):MIC ratio for an antimicrobial that gives rise to 50% of the maximal response, Ce is the AUC:MIC ratio of the antimicrobial in the effect compartment (i.e. artificial medium or serum) and N is the Hill coefficient, which reflects the slope of the relationship between antimycoplasmal effect and AUC:MIC. Where appropriate, AUC:MIC ratios were obtained for mycoplasmastatic (E = 0) and mycoplasmacidal (E = -3, 99.9% reduction in cfu.mL-1) activity of antimicrobials, and for virtual mycoplasmal elimination (E = -4, 99.99% reduction in cfu.mL-1).

MICs and pharmacodynamic analysis for OTC, danofloxacin and tulathromycin against MmmSC B237 in artificial medium and serum are given in the table 1. In artificial medium, while oxytetracycline was mycoplasmastatic, both danofloxacin and tulathromycin showed mycoplasmacidal activity. In serum, a similar picture was observed for OTC and danofloxacin, albeit with higher MIC values, which may, at least in part, be a result of plasma protein binding of these drugs. Interestingly, the MIC for tulathromycin was considerably lower in serum than artificial medium; however, the antimycoplasmal effect of this drug was also lower.

In conclusion, all three antimicrobials demonstrated efficacy against MmmSC in vitro but to differing extents. In serum, OTC and tulathromycin were bacteriostatic and therefore would require help from the host’s immune system to clear infection in vivo. Danofloxacin was bactericidal, relying on the immune system to a lesser degree but unable to bring about virtual mycoplasmal eradication. The study also highlights the importance of determining MICs and effector kinetics in biological matrices as these can vary widely from data obtained in artificial media. The next step is to generate time-kill curves using ex vivo samples, thus allowing for both matrix effects and the possibility that parent drug and active metabolites exert combined effects.

Table 1: MICs for OTC, danofloxacin and tulathromycin against MmmSC B237 in artificial medium (inoculum size 107 cfu.mL-1) and adult bovine serum (inoculum size 106 cfu.mL-1), and pharmacodynamic analysis of data obtained from in vitro time-kill studies. MS, mycoplasmastatic; MC, mycoplasmacidal; VME, virtual mycoplasmal elimination.

McKellar QA, Sanchez Bruni SF, Jones DG (2004). Pharmacokinetic/pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine. J Vet Pharmacol Ther 27(6): 503-514.

Rweyemamu MM, Litamoi J, Palya V, Sylla D (1995). Contagious bovine pleuropneumonia vaccines: the need for improvements. Rev Sci Tech Oie 14(3): 593-601.

Thomson GR (2005). Contagious bovine pleuropneumonia and poverty: A strategy for addressing the effects of the disease in sub-Saharan Africa. Research report, DFID Animal Health Programme, Centre for Tropical Veterinary Medicine, University of Edinburgh, UK.