Pharmacokinetic/Pharmacodynamic (PK-PD) Profiles Of Antibacterial Agents Against Mycoplasma Gallisepticum

Mycoplasma gallisepticum is one of the most important pathogens that cause chronic respiratory disease（CRD）in chicken.Increased embryo mortality,as well as reduced weight gain efficiency and egg production were found in infected chickens,which can lead to considerable economic losses in poultry industry.Although several antimicrobial agents are currently recommended for the treatment and prevention of M.gallisepticum infection,such as tetracyclines,macrolides,quinolones,and pleuromutilins,the development of antimicrobial resistance has been contributed by the long-time overuse and misuse of antibiotics.It is difficult to cultivate M.gallisepticum for the reason that it is minute in size and completely lacks a bacterial cell wall;and it should be cultivated on specially formulated media.Accordingly,there were few investigations of M.gallisepticum.The ’mutant selection window’ （MSW）hypothesis postulates that a drug concentration zone exists in which resistant mutants are selectively amplified.Firstly,the perferred type of animal serum to support M.gallisepticum growth on agar plates has been selected.Mutant selection window（MSW）of danofloxacin,doxycycline,tilmicosin,tylvalosin and valnemulin against M.gallisepticum strain S6 has been determined in vitro.Killing of M.gallisepticum strain S6 by these antimicrobials was also studied by incubating M.gallisepticum into medium containing the compounds at the minimal concentration that inhibits colony formation by 99%（MIC99）and the mutant prevention concentration（MPC）.Based on the morphoLogy and colony numbers of M.gallisepticum on agar plates,the four kinds of serum in the order of the applicability for culturing M.gallisepticum were swine serum>horse serum>bovine serum>mixed serum.The MPC/MIC₉₉ values for each agent were as follows:danofloxacin>tilmicosin>tylvalosin>doxycycline>valnemulin.MPC generated more rapid and greater magnitude killing than MIC99 against M.gallisepticum.Under exposure of 10⁵¹0⁹ CFU/mL at MPC drug levels,valnemulin had the slowest rate of reduction in viable organisms and danofloxacin had the highest rate of reduction.This study investigated the antibacterial activity of doxycycline against M.gallisepticum strain S6株.In static time-killing studies with constant antibiotic concentrations（0 to 64MIC）,M.gallisepticum colonies were quantified and kill rates were calculated to estimate the drug effect.An in vitro dynamic model（the drug concentrations are fluctuant）was also established and two half-lives of 6.78 h and 12 h were simulated.The samples were collected for drug concentration determination and viable counting of M.gallisepticum.In static time-killing studies,doxycycline produced a maximum antimycoplasmal effect of 5.62 Log₁₀CFU/mL reduction and the maximum kill rate was 0.11 h^-1.In the in vitro dynamic model,doxycycline had a mycoplasmacidal activity in the two regimens,and the maximum antimycoplasmal effects were 4.1（6.78 h）and 4.75（12 h）Log₁₀ CFU/mL reduction,respectively.Furthermore,the cumulative percentage of time over a 48 h period that the drug concentration exceeds the MIC（%T>MIC）was the pharmacokinetic-pharmacodynamic（PK-PD）index that best correlated with antimicrobial efficacy（R²=0.986,compared with0.897 for the peak level divided by the MIC and 0.953 for the area under the concentration-time curve over 48h divided by the MIC）.The estimated%T>MIC values for 0 Log₁₀CFU/mL reduction,2 Log₁₀ CFU/mL reduction and 3 Log₁₀ CFU/mL reduction were 32.48%,45.68%and 54.36%,respectively,during 48 h treatment period of doxycycline.In conclusion,doxycycline shows excellent effectiveness and time-dependent characteristics against M.gallisepticum strain S6 in vitro.In the present study,danofloxacin concentrations were simulated below the MIC₉₉（the drug concentration required to inhibit colony formation by 99%）,between the MIC₉₉ and MPC（the mutant prevention concentration）,and above the MPC in an in vitro dynamic model.The relationship between danofloxacin pharmacokinetics and pharmacodynamics（PK-PD）parameters and development of resistance for M.gallisepticum were explored based on different dosing regimens.Danofloxacin concentration,counts of viable cell and susceptibility were determined during the experiment.The mutations in gyrA,gyrB,parC and parE as well as efflux pumps were examined.M.gallisepticum lost danofloxacin susceptibility when drug concentrations were between the lower boundaries of the mutant selection window.For PK-PD parameters,there were mutant selection enrichment when 0.37h<AUC_{24 h}/MPC<14.47 h.The single mutation in gyrA（Ser83→Arg）was found in all mutants,while double mutations in gyrA and parC（Ala64→Ser）were observed only in the mutant with the highest MIC.In addition,no change of susceptibility in the mutants was observed in the presence of reserpine and carbonyl cyanide 3-chlorophenylhydrazone（CCCP）.This suggested that efflux pump were most likely not acting on the resistant mutants strains.An in vivo M.gallisepticum infection model was established.Danofloxacin was orally administrated to the infected chickens once daily for 3 days.Not only the concentration of danofloxacin in plasma and lung tissues were analyzed,but also the counting of viable cell and susceptibility changes in air sac and lung tissues were determined.Point mutations in gyrA,gyrB,parC and parE of the selected resistant mutant strains were examined.In addition,the susceptibility of enrofloxacin,ofloxacin,levofloxacin,gatifloxacin and norfloxacin against these mutant strains were also determined.The PK profiles indicated that danofloxacin concentrations in lung tissues were higher than plasma.Mycoplasmacidal activity was found when M.gallisepticum was exposed to danofloxacin at the dose group above 2.5 mg/kg.The ratios of AUC24 h/MIC（the area under the concentration-time curve over 24 h divided by the MIC）for 2 Log₁₀ CFU/mL and 3 Log₁₀ CFU/mL reduction were31.97 and 97.98 L·h/kg,respectively.Substitutions of Ser83→Arg or Glu87→Gly in gyrA;Glu84→Lys in parC were observed in the resistant mutant strains that selected from the dose group of 1 and 2.5 mg/kg.MICs of danofloxacin,enrofloxacin,ofloxacin,levofloxacin,gatifloxacin and norfloxacin against the resistance mutant strains with a single mutation in position-83 were higher than that with a single mutation in position-87.These findings suggested that danofloxacin may be therapeutically effective to treat M.gallisepticum infection in chickens if administered at a dosage of 5.5 mg/kg once daily for 3 days.