Study On The Pharmacokinetic-pharmacodynamic (PK-PD) Model Of Tilmicosin Against Mycoplasma Gallisepticum In Vitro

Tilmicosin has a fairly broad spectrum of efficacy and has been used as an ideal medicine for clinical treatment of mycoplasmosis.This study determined the in vitro antibacterial activity of tilmicosin against M.gallisepticum,and established an in vitro PK-PD model.One-compartment open model with first order absorption of pharmacokinetics for tilmicosin was successfully established.It was proposed that this model which simulated different clinical doses will be used to provide reference for designing dosage regimen.This study investigated the antibacterial activity of tilmicosin against M.gallisepticum strain S6.The minimal inhibitory concentration（MIC）values of tilmicosin with an inoculum of 10⁵ and 10⁷ CFU/mL were 0.01 and 0.02μg/mL by using Micro-dilution method.The MIC values for an inoculum of 10⁵,10⁷ and 10⁹ CFU by the agar dilution method were 0.02,0.04 and 0.16μg/mL,respectively.The results of static time-killing curves with constant drug concentration（0⁶4 MIC）showed that the amount of M.gallisepticum reduced to the limit of detection after 36 h when the concentration exceed 1 MIC.With the increase of MIC multiple,the kill rate increased but the rate of increase slowed down.The maximum kill rate was 0.53 h^-1.This experiment was based on accurately simulating the drug concentration of lung tissue.According to the clinically recommended dose,7 different dose groups（1,2.5,5,7.5,10,5 and 20 mg）were designed in vitro dynamic model.The amount and susceptibility of M.gallisepticum were detected through application of this model.In dynamic time-killing studies,maximum antimycoplasmal effect was observed in all dose groups after 5 days.Tilmicosin produced a maximum antimycoplasmal effect of 6.51Log₁₀CFU/mL reduction.The correlation coefficients between AUC_24h/MIC,C_max/MIC,%T>MIC and antibacterial effect were 0.87,0.88 and 0.49,respectively,by analyzing of the inhibitory Sigmoid Emax model.The results showed that C_max/MIC was the best PK-PD parameter for predicting the antimicrobial activity of tilmicosin against M.gallisepticum.Magnitude of C_max/MIC and AUC_24h/MIC predicted for 1log reduction were 2.17 and 62.58 h.Tilmicosin showed concentration-dependent activity.A total of 7 M.gallisepticum strains with decreased susceptibility to tilmicosin were isolated from 7 dose groups.The MIC values of strain M3,M4,M5 were significantly higher than strain S6 for tilmicosin.In addition,in vitro susceptibility for tylosin,erythromycin,doxycycline,amikacin against these strains was also determined by the micro-dilution method.7 strains of M.gallisepticum acquired resistance to erythromycin as well as to tylosin.However,no change of susceptibility for amikacin and doxycycline in these strains was observed.Gene mutation analysis was performed on the basis of annotation single nucleotide polymorphism（SNP）using strain S6 genome as the reference.Resistant mutation was not found in rplD and rplV,genes encoding ribosomal proteins L4 and L22 for strain M3,M4 and M5.No mutation was detected in domain II and V of the 23S rrnA gene for strain M4.For strain M5,G495T mutation occurred in domain II of the 23S rrnA gene,but domain V of the 23S rrnA gene remained unchanged.For Strain M3,the T854A mutation in domain II of the 23S rrnB gene and the G2799A mutation in domain V of the 23S rrnB gene were associated with resistance.These mutations have not been reported previously.