Study On The Pharmacokinetic-pharmacodynamic(PK/PD) Model Of Danofloxacin Against Mycoplasma Hyopneumoniae In Vitro

Mycoplasma hyopneumoniae is the main pathogen that causes Mycoplasma hyopneumoniae pneumonia,which is widespread in the world,causing significant economic losses to the pig industry.Danofloxacin has a broad spectrum of antibacterial activity and is often used clinically as an ideal drug for the treatment of Mycoplasma hyopneumoniae.By successfully establishing an in vitro model and simulating different drug concentrations of danofloxacin in the lungs,it is hoped to provide data support for danofloxacin in the treatment of porcine mycoplasma infection.In this paper,the in vitro antibacterial activity of dafloxacin against Mycoplasma hyopneumoniae was determined.The MIC values of 10⁵,10⁶and 10⁷CFU/m L bacterial counts measured by microdilution were 0.03125μg/m L,0.0625μg/m L and 0.125μg/m L.The MIC values for an inoculum of 10⁵,10⁶and 10⁷CFU/m L by the agar dilution method were 0.0625,0.125 and 0.25μg/m L.The results of the static bactericidal curve at 0 to 64MIC indicate that the concentration of the drug in the concentration group higher than 2MIC can reduce the mycoplasma hyopneumoniae strain below the detection limit after 48hours,and the 64 MIC group drops below the detection line after only 12 hours.With the increase of the MIC multiple,the sterilization rate is also increasing.By using the Sigmoid E_maxmodel,the sterilization rate and drug concentration are fitted over time.The best fit is0 to 12 hours.R²is 0.994.The maximum sterilization rate is 0.32 h^-1.In this study,seven different doses（0.1,0.5,0.8,1.25,2.5,3.5,and 5μg/m L）were designed in the in vitro dynamic model based on the clinically recommended drug concentration in the lungs.The effect of danofloxacin on Mycoplasma hyopneumoniae and its sensitivity to Mycoplasma hyopneumoniae.In the in vitro dynamic model,the bacterial volume of the 0.1 and 0.5μg/m L dose groups decreased by 0.94 Log₁₀CFU/m L and 1.82Log₁₀CFU/m L,respectively,showing bacteriostatic effects,and the bacterial dose of the other dose groups decreased to more than 3 Log₁₀CFU/m L,show bactericidal effect,the maximum bacterial volume decreased by 5.20 Log₁₀CFU/m L.According to the analysis results of the inhibited Sigmoid E_maxmodel,the correlations between AUC_24h/MIC,C_max/MIC,%T>MIC and antibacterial effect were 0.933,0.887 and 0.667,respectively.The antimicrobial effect of danofloxacin is most related to AUC_24h/MIC,and it is not far from C_max/MIC.The results show that danofloxacin is a concentration-dependent antibiotic against Mycoplasma hyopneumoniae,and AUC_24h/MIC is the most important factor affecting antibacterial effect.The seven groups with doses of 0.5,0.8,1.25 and 2.5μg/m L were screened for reducing the sensitivity of M.hyopneumoniae pneumoniae strains,and their MICs were measured by the microdilution method,of which three were 0.5,1.25 and 2.5μg/m L.The MIC value of the strain increased significantly,and the increase degree was 8 times,16times,and 32 times,respectively.At the same time,its sensitivity to common quinolones such as enrofloxacin and ciprofloxacin was measured,and all of them caused a decrease in sensitivity.Measured the sensitivity to different antibacterial drugs such as doxycycline,womniculin,telmicoxin,and tylosin,and found that the MIC values of all strains with reduced sensitivity to the antibiotics such as doxycycline and standard strain no change compared.Using the ATCC25934 standard strain as the reference genome,by measuring the mutations in the gyr A,gyr B,par C and par E genes of the drug-resistant strains,it was found that the amino acid substitutions at position 83 relative to E.coli gyr A were detected in the 0.5 and 1.25μg/m L dose groups（Ala83→Val）,and the higher-dose 2.5μg/m L dose group not only detected amino acid substitutions at position 83 relative to E.coli gyr A（Ala83→Val）,but also detected amino acid substitutions at position 64 relative to E.coli Par C.（Asp64→Asn）,which reflects that the MIC value of drug-resistant strains may be related to the number of mutation sites.This study investigated in vitro growth curves of M.hyopneumoniae strains resistant to danofloxacin and standard M.hyopneumoniae strains.During the in vitro cultivation of four strains of resistant bacteria,the generation times of the four strains of M1,M2,M3,and M4 were 13.92 h,11.95 h,13.33 h and 13.68 h,which were higher than the standard strain of 11.93 h.And the highest bacterial volume is less than the standard strain.This reflects the growth in vitro in a similar growth environment in vitro,and the growth of drug-resistant strains in vitro does not occupy a significant advantage.