Establishment Of Susceptibility Breakpoints For Danofloxacin And Tylosin Against Haemophilus Parasuis In Swine

Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the etiological agent of Glasser’s disease,which is characterized by fibrinous polyserositis,arthritis,meningitis and pneumonia.Danofloxacin is a fluoroquinolones antibiotic,with broad antimicrobial spectrum,strong bactericidal activity,quick absorption,and high bioavailability.It was mainly used for the treatment of pig and cattle,sheep,chicken and other animal respiratory tract infection.Tylosin is a macrolide antibiotic,commonly used for the treatment of porcine respiratory disease.As the abuse of antibiotics and environmental stresses,antimicrobial resistant H.parasuis emerged in different degrees,which brought serious threat to global economy and public health.However,the susceptibility breakpoints for H.parasuis have not been established.We mainly referred the standards of Haemophilus or Actinobacillus pleuropneumoniae established by CLSI in clinical.Therefore,we established the susceptibility breakpoints for H.parasuis according to the process recommend by CLSI and EUCAST,in order to guide clinical medication,and provide scientific data for determination of susceptibility breakpoints.1 Wildtype cutoff(CO_WT)for Danofloxacin and Tylosin against H.parasuis35 H.parasuis strains were isolated from the clinically diseased lung,Combined with 8 strains given by Xiaojuan Xu and 100 strains preserved in laboratory,it was 143 H.parasuis strains in total.The antimicrobial susceptibility of 143 strains was determined by agar dilution method recommended by CLSI.The MIC₅₀for danofloxacin and tylosin against H.parasuis were 4μg/m L and 16μg/m L,the MIC₉₀were 16μg/m L and 32μg/m L,respectively.The CO_WTunder different confidence intervals were obtained by the Ecoffinder software.The CO_WTfor danofloxacin and tylosin against H.parasuis were 16μg/m L and 64μg/m L,respectively.2 Formulating PK-PD cutoff(CO_PD)and dosage regimens for danofloxacin against H.parasuis81 H.parasuis strains including MIC₅₀,MIC₉₀,wildtype cutoff were selected for the serotyping of enterbacterial repetitive intergenic consensus-PCR（ERIC-PCR）.18 strains for serotyping 5 were screened for virulence test in mice.16 g～20 g healthy Balb/c mice were selected and divided into 19 groups.Each strain was a group of 5 Balb/c mice,and each mouse was injected with 0.5 m L 1×10⁹CFU by intraperitoneal injection..Finally,MIC₅₀strain H80 was selected to research on PK-PD experiment.5 different MIC strains H42,H80,H12,H83 and H17 were selected for clinical treatment experiment,The MIC and MBC of H.parasuis H80 in broth and pulmonary epithelial lining fluid（PELF）were determined by broth microdilution method recommended by CLSI.The MIC in broth and PELF were 4μg/m L and 2μg/m L,MBC were 8μg/m L and 4μg/m L,respectively.The antibacterial activity of danofloxacin in PELF is stronger than that of in broth,maybe the PELF contain certain antibodies or white cells or other medium which can enhance the antibacterial activity of danofloxacin.The mutant prevention concentration（MPC）of H.parasuis H80 were 20μg/m L.The post antibiotic effect（PAE）of H.parasuis H80 exposed to different concentrations of danofloxacin incubated with 1 h and 2 h were 0.59 h～2.38 h and 0.86 h～2.95 h,respectively.According to the results of MIC,danofloxacin at different concentrations were incubated with H.parasuis H80.0.1 m L bacterium solution were taken for plate counting at different time points,and drawn the vitro killing curve of danofloxacin against H.parasuis.According to the concentration of danofloxacin at different time points in PELF after administration,the ex-vivo killing curve was drawn by incubating with H.parasuis H80.The bactericidal effect in vitro and ex-vivo increased with the increase of concentration,shown significant concentration dependence.The final selected PK-PD parameter was AUC/MIC.A total of 6 healthy weaned piglets（about 20 kg）were experimented to establish pharmacokinetic models in plasma and lung.The dosage regimens were 2.5 mg/kg by intramuscular injection.After administration,the plasma was collected at 0.08 h,0.16 h,0.25 h,0.5 h,0.75 h,1 h,1.5 h,2 h,3 h,4 h,6 h,8 h,10 h,12 h,24 h,36 h and 48 h,the PELF was collected by electron microscopy at 0.5 h,1 h,1.5 h,2 h,3 h,4 h,6 h,8 h,10 h,12 h,24 h,36 h and 48 h.The concentration of danofloxacin in plasma and PELF was detected according to the established High Performance Liquid Chromatography（HPLC）method.The two compartment model in Winnonlin software was used to simulate pharmacokinetic parameters of danofloxacin in plasma and PELF.In plasma,the peak time(T_max)was 0.23±0.07 h,the peak concentration(C_max)was 0.67±0.01μg/m L,the area under the concentration-time curve（AUC）was 4.47±0.51 h·μg/m L;in PELF,T_maxwas 1.61±0.15 h,C_maxwas 3.67±0.25μg/m L,AUC was 24.28±2.70 h·μg/m L.According to the results of the ex-vivo bactericidal curve,the(AUC₂₄/MIC)_exof danofloxacin under different concentrations was calculated by Sigmoid E_maxequation E=E_max-((E_max-E₀)×C^N)/(C^N+EC^N₅₀) simulation.The values of(AUC₂₄/MIC)_ex at E=0,-3 and -4（inhibition,sterilization and eradication）were 12.73,28.68 and 44.38,respectively.The(AUC₂₄/MIC)_exat E=-3 was the pharmacodynamic target.The probability of target attainment（PTA）at different MIC was simulated by the Monte Carlo analysis.The CO_PDfor danofloxacin against H.parasuis in PELF and plasma was 0.5μg/m L and 0.125μg/m L,respectively.The PK-PD data and MIC₅₀of clinical strain were plugged into the equation Dose=(MIC×(AUC₂₄/MIC)_ex)/fu×CL/F,the dosage under different bactericidal effect（inhibition,sterilization and eradication）were 4.58 mg/kg,10.32 mg/kg and 15.97 mg/kg.3 Establishment of clinical cutoff for danofloxacin against H.parasuisIn this trail,66 healthy weaned piglets（about 20 kg）were divided into 11 groups,the experimental groups were challenged with the screening 5 different MICs strains H42,H80,H12,of H83 and H17 by intranasal inoculation,1×10¹⁰CFU,two times a day.The dosage regimens were recommended by PK-PD regimen,10.32 mg/kg,two times a day.After challenging,the relevent symptoms of pigs were observed.Then the probability of cure（POC）was obtained and analyzed.Different analysis methods were used to analyze clinical cutoff according to the relationship between POC and MIC distribution obtained from this experiment.Firstly,by eye-view analysis,when POC is equal to 90%,the clinical cutoff should be located at 0.125μg/m L～1μg/m L;the window of clinical cutoff located at 0.125μg/m L～4μg/m L recommended by the‘Windo W’approach;nonlinear regression analysis shows that when the POC is 90%,MIC is 0.428μg/m L,the recommended clinical critical value that is 0.428μg/m L;lastly,the clinical cutoff was less than 0.56μg/m L by CART（Classification and regression tree）analysis.There were differences between these results obtained by different analysis method.Combined with the distribution of MIC in the experiment,we chosen the highest MIC in the range from the measured MIC.Therefore the clinical cutoff for danofloxacin against H.parasuis was0.25μg/m L.In all experiments,the wildtype cutoff was 16μg/m L,the PK-PD cutoff was 0.5μg/m L,and the cilinical cutoff was 0.25μg/m L.The three cutoffs were analyzed by a method recommended by CLSI,these three cutoffs accorded with the formulation of CO_WT>CO_PD>CO_CL,the susceptibility breakpoints for Danofloxacin against H.parasuis was 16μg/m L.