Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in pigs. M. hyopneumoniae infection can lead to considerable economic losses in the pig-breeding industry. Here, this study established a first-order absorption, one-compartment model to study the relationship between the pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. hyopneumoniae in vitro. We simulated different drug concentrations of timicosin in the fluid lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of tilmicosin against M. hyopneumoniae with an inoculum of 106 CFU/mL was 1.6 μg/mL using the microdilution method. Static time–kill curves showed that if the drug concentration >1 MIC, the antibacterial effect showed different degrees of inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log10 CFU/mL, thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was reduced from 3.61 to 5.11 log10 CFU/mL upon incubation for 96 h in a dynamic model with a dose of 40–200 mg, thereby achieving mycoplasmacidal activity. The area under the concentration-time curve over 96 h divided by the MIC (AUC0–96 h/MIC) was the best-fit PK/PD parameters for predicting the antibacterial activity of tilmicosin against M. hyopneumoniae (R2 = 0.99), suggesting that tilmicosin had concentration-dependent activity. The estimated value for AUC0–96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline was 70.55 h and 96.72 h. Four M. hyopneumoniae strains (M1–M4) with reduced sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of these strains to tylosin, erythromycin and lincomycin was also reduced significantly. For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V was found only in resistant M. hyopneumoniae strains (M3, M4). In conclusion, in an in vitro model, the effect of tilmicosin against M. hyopneumoniae was concentration-dependent and had a therapeutic effect. These results will help to design the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and minimize the emergence of resistant bacteria.

= 333 21 Text = 3434 22 Figure counts: 5 2 23 Abstract 24 Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in 25 pigs. M. hyopneumoniae infection can lead to considerable economic losses in the 26 pig-breeding industry. Here, this study established a first-order absorption, 27 one-compartment model to study the relationship between the 28 pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. 29 hyopneumoniae in vitro. We simulated the drug concentration of timicosin in the fluid 30 lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of 31tilmicosin against M. hyopneumoniae with an inoculum of 10 6 CFU/mL was 1.6 32 μg/mL using the microdilution method. Static time-kill curves showed that, if the 33 drug concentration >1 MIC, the antibacterial effect showed different degrees of 34 inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log 10 CFU/mL, 35 thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was 36 reduced from 3.61 to 5.11 log 10 CFU/mL upon incubation for 96 h in a dynamic 37 model with a dose of 40-200 mg, thereby achieving mycoplasmacidal activity. The 38 peak concentration by MIC (C max /MIC) and the area under the concentration-time 39 curve over 96 h divided by the MIC (AUC 0-96 h /MIC) were the best-fit PK/PD 40 parameters for predicting the antibacterial activity of tilmicosin against M. 41 hyopneumoniae (R 2 = 0.99), suggesting that tilmicosin had concentration-dependent 42 activity. The estimated value for C max /MIC and AUC 0-96 h /MIC for 2log 10 (CFU/mL) 43 reduction and 3log 10 (CFU/mL) reduction from baseline was 1.44 and 1.91, and 70.55 44 h and 96.72 h, respectively. Four M. hyopneumoniae strains (M1-M4) with reduced 3 45 sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of 46 these strains to tylosin, erythromycin and lincomycin was also reduced significantly. 47 For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V 48 was found only in resistant M. hyopneumoniae strains (M3, M4). In conclusion, in an 49 in vitro model, the effect of tilmicosin against M. hyopneumoniae was 50 concentration-dependent and had a therapeutic effect. These results will help to design 51 the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and 52 minimize the emergence of resistant bacteria. 53 54 Keywords: Mycoplasma hyopneumoniae, tilmicosin, in vitro dynamic model, 55 Pharmacokinetic, Pharmacodynamic, enzootic pneumonia, resistance. 56 57 1 Introduction 58 Mycoplasma hyopneumoniae is the primary pathogen of mycoplasmal pneumonia in 59 pigs. M. hyopneumoniae is widespread in various regions, and can cause huge 60 economic losses to the pig industry [1]. Infected pigs are the main source of infection. 61 The pathogen can be transmitted directly through air and contact, so the infection rate 62 is extremely high [2]. Once flocks of pigs are infected with M. hyopneumoniae, 63 eradication is difficult because M. hyopneumoniae can tr...