The Mutation Selection Window And Pharmacokinetic/pharmacodynamic Integration Of Several Antibacterial Drugs Against Mycoplasma Hyopneumoinae

Mycoplasma hyopneumoniae is the main pathogen that causes Mycoplasma pneumonia in pigs.The in-depth study of this pathogenic bacteria is of great significance for clinical guidance.In view of this,on the basis of exploring the in vitro pharmacodynamics and mutation selection window（MSW）of M.hyopneumoniae in this experiment,we also conducted an in vitro dynamic model and an in vivo PK/PD model study.The aims of this study are to provide objective data and theoretical support for the clinical treatment of mycoplasma pneumonia,and recommend reasonable drug administration to prevent the emergence of drug-resistant bacteria.In this study,we first investigated the MSW of doxycycline,tulathromycin,danofloxacin,tiamulin,and valnemulin for treating M.hyopneumoniae strain（ATCC 25934）to determine the likelihood of promoting resistance with continued use of these antibiotics.Minimum inhibitory concentration（MIC）values against M.hyopneumoniae were determined for each antimicrobial agent ranging from 10⁵ colony-forming units（CFU）/m L to 10⁹ CFU/m L based on microdilution broth mothod and agar plate methods.MIC values ranged from 0.001 to 0.3125μg/m L based on the microdilution broth method,and from0.008 to 1.0μg/m L based on the agar plate method.The minimal concentration inhibiting colony formation by 99%(MIC₉₉)and the mutant prevention concentration（MPC）were determined by the agar plate method with three inoculum sizes.MPC values ranged from0.0016 to 80μg/m L.MPC/MIC₉₉ values were ordered by tulathromycin>doxycycline>danofloxacin>tiamulin>valnemulin.Antimicrobial killing was determined based on MIC₉₉ and MPC values for all five agents.MPC achieved better bactericidal action than MIC₉₉.Based on pharmacodynamic analyses,danofloxacin,tulathromycin,and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.We established an in vitro dynamic model to explore the relationship between the pharmacokinetic and pharmacodynamic（PK/PD）parameters of tiamulin against M.hyopneumoniae.Static time–killing curves showed that mycoplasmacidal activity(reduced3.0 log₁₀CFU/m L)was achieved during 48 h when the drug concentration was 8 MIC,and with a maximum kill rate of 0.072/h.In dynamic time–killing studies,only the dose-fractionated regimen achieved mycoplasmacidal activity when drug concentration was1.44 and 1.92 mg/L.The duration of post antibiotic effect（PAE）at 1×MIC was 6.27±0.11h,and prolonged as the concentration of tiamulin increased.The cumulative percentage of time over a 48-h period that the drug concentration exceeds the MIC（%T>MIC）was the best PK/PD parameter to predict the antimicrobial activity of tiamulin against M.hyopneumoniae（R²=0.98）.The effect of tiamulin on M.hyopneumoniae was time-dependent and prolonged PAE activity.When the amount of M.hyopneumoniae decreased by 2 and 3 Log₁₀CFU/m L,the corresponding%T>MIC values were 52.99%and96.74%,respectively.The genome of strain ATCC 25934 was used as a reference,and gene-mutation analysis was done.Two strains of M.hyopneumoniae（M1,M2）had acquired resistance to tiamulin as well as to valnemulin,tylosin and amikacin.For strains M1 and M2,a A2058C mutation occurred in domain V of 23S r RNA.Two strains of tiamulin-resistant strains（M1,M2）were simultaneously less sensitive to valnemulin,tylosin and amikacin.Low dose administration of tiamulin is not safe and may lead to selective enrichment of resistant strains.We also determined the relationship between the PK/PD parameters of tulathromycin and M.hyopneumoniae in the in vitro dynamic model.Static time–killing curves showed that the sterilization effect increased with the increase of the drug concentration,and the sterilization effect was reached at 4 MIC.The results of the dynamic model study showed that the best PK/PD fitting parameter was AUC_0-96h/MIC（R²=0.987）.The effect of tulathromycin on M.hyopneumoniae was concentration-dependent.When the amount of M.hyopneumoniae decreased by 2 Log₁₀CFU/m L and 3 Log₁₀CFU/m L,the corresponding AUC_0-96h/MIC values were 354 h and 1956.62 h,respectively.The genome of strain ATCC25934 was used as a reference,and gene-mutation analysis was done.A2058G mutation in the V region of the 23S r RNA gene was found in M1～M6 strains.When the dosage of tulathromycin fall within the mutation selection window,it was easy to induce resistance of M.hyopneumoniae to tulathromycin.Quantitative detection of M.hyopneumoniae in bacterial solution and lung tissue samples was achieved by establishing the real-time PCR method.This method has high specificity and repeatability.Moreover,the logarithm of the amount of M.hyopneumoniae and the Cq value of real-time PCR was good in the range of 3×10² CFU/m L to 3×10⁸CFU/m L（R²=0.9664）,and the minimum detection limit was 3×10² CFU/m L.Quantitative PCR can be used to detect the changes of M.hyopneumoniae in infected piglets before and after administration.A model of M.hyopneumoniae infection in piglets was established to investigate the relationship between tulathromycin and PK/PD synchronization model of M.hyopneumoniae in vivo.The piglets in the infected group were given a single injection of2.5,5,7.5,10,and 15 mg/kg of tulathromycin in the neck.After administration,lung tissue was collected at the time of collection,and the drug concentration of tulathromycin in the lungs and the changes in the amount of M.hyopneumoniae were detected.The in vitro static bactericidal curve showed that when the drug concentration was over 8 MIC,the bactericidal effect was achieved within 60 h,and the bacterial quantity decreased by 4.08～4.12 Log₁₀CFU/m L.The relationship between PK/PD parameters(AUC_0-96h/MIC,%T>MIC and C_max/MIC)and antibacterial effect was fitted using the Inhibitory Sigmoid Emax model in Win Nolin software.The AUC_{0-96 h}/MIC values corresponding to the 0,3,and 4Log₁₀ CFU/Lung reductions were calculated to be 247.03,3220.72 and 10168.88 h,respectively;the corresponding C_max/MIC values were 0.55,19.22 and 50.60,respectively.The results showed that the antibacterial effect of tiamulin on M.hyopneumoniae was concentration-dependent.It was concluded that a single dose of 5.6 mg/kg.bw was effective in the treatment of mycoplasma pneumonia in pigs.