Development Of Florfenicol Susceptibility Breakpoint For Actinobacillus Pleuropneumoinae

Actinobacillus pleuropneumoinae（APP）is an important causative agent of veterinary clinical respiratory tract infections,with high morbidity and mortality all year round,and can be transmitted by air and animal contact,seriously endangering the healthy development of the farming industry.Florfenicol（FF）,as a synthetic monofluorinated derivative of methylsulfonamides,is widely used in the treatment of pneumonia caused by respiratory bacteria such as APP in pigs because of its rapid and complete absorption and wide distribution in the tissues.However,the resistance rate of APP to FF has been increasing year by year due to large or irrational clinical use.Susceptibility breakpoint are an important basis for evaluating changes in drug susceptibility and formulating dosing regimens.However,to date,no criteria have been established to determine the resistance of APP to FF in China.In order to protect the clinical potential of FF and slow down the drug resistance problem,it is urgent to establish our own susceptibility breakpoint.In this study,the technical guidelines were referenced based on Clinical and Laboratory Standards Institute（CLSI）and the European Commission Antimicrobial Susceptibility Testing（EUCAST）.Through antibiotic sensitivity testing and data collection,the minimum inhibitory concentration（MIC）distribution of FF was obtained for 182 strains of APP from pigs,and the wild type cut-off was established.The hollow fiber infection model（HFIM）was used to carry out PK/PD study in plasma and pulmonary interstitial fluid to optimize clinical dosage regimen.With the help of population pharmacokinetic（PPK）model of FF,the pharmacodynamic cut-off was established;Develop clinical cut-off by conducting clinical treatment trials of APP with different sensitivity levels;Finally,the susceptibility breakpoint of APP to FF were established.1.Pharmacodynamic study and establishment of the wild-type cut-off of FF against APPThe MIC distribution of 182 strains of APP was obtained through clinical isolation in laboratory and published experimental data.The MIC results were analyzed by using the ECOFFFinder,and the wild type cut-off was established.MIC distribution range of FF to182 strains of APP in pigs is 0.125-16μg/m L.MIC₅₀and MIC₉₀are 1μg/m L and 4μg/m L,respectively.The wild-type cut-off is 8μg/m L.The MIC,mutant prevention concentration（MPC）and post-antibiotic effect（PAE）of the tested strain 121 in TSB broth were determined by broth microdilution method.The MIC of the tested strain 121 is 4μg/m L.The MBC is 8μg/m L.The MPC is 16μg/m L.The MSW is 4-16μg/m L.The PAE produced by exposure to FF for 1 h is 0.071-0.89 h,and the PAE produced by exposure to FF for 2 h is 0.23-1.16 h.2 In vitro PK/PD study and establishment of pharmacodynamic cut-off of FF against APPConstruction of physiologically based pharmacokinetic（PBPK）Model:Using the PBPK model of FF,we obtained the concentration-time curve of FF in plasma and pulmonary interstitial fluid at different doses of 10,20,40 mg/kg/24h and 10,20,40mg/kg/48h.Using Phoenix software to fit the data,and determine the k₁₀=0.050,t_1/2β=13.84h in plasma,k₁₀=0.15,t_1/2β=4.61 h in pulmonary interstitial fluid.Construction of a HFIM:15 m L bacterial fluid of 10⁶CFU/ml was inoculated into the extra-capillary space（ECS）of HFIM,and simulated the PK of different administration schemes in plasma and pulmonary interstitial fluid.The 100μL samples from ECS were counted at 0,1,3,6,9,12,24,and 48 h for PD analysis.The 1.2 m L samples from the ECS were taken at 0,0.25,0.5,0.75,1,2,4,6,8,12,24,36,and 48 h（added two sampling points of 27,30 h for each 24 h administration）for PK analysis.The results showed that in plasma and pulmonary interstitial fluid,only at a dose of 40 mg/kg/24h could the bacteria be completely killed at 30h and 36h,and bacteria did not recovery growth within 48h.Non compartmental simulation and linear regression of PK data showed a good correlation between simulated PK in HFIM and in vivo PK（R²>0.9）.Fitting of PK/PD model:The Sigmaid I_maxmodel in Phoenix software was used to fit the relationship between the PK/PD parameter of FF and the effect.The best PK/PD parameter was used to predict the efficacy of FF,as well as the PK/PD parameter values required to achieve different antibacterial effects,and developed a reasonable dosage regimen.After fitting with the sigmoid I_maxmodel,the optimal PK/PD parameter was determined to be AUC_0-24/MIC.The corresponding PK/PD parameter values for achieving different antibacterial effects（bacteriostasis,sterilization,eradication）in plasma were 7.24,22.41,and 48.83 h,respectively,and 8.23,23.95,and 38.36 h in pulmonary interstitial fluid,respectively.The doses for prevention,treatment,and clearance in plasma were 8.48,26.23,and 57.16 mg/kg b.w.,respectively,and the doses in pulmonary interstitial fluid were 5.43,23.95,and 38.36 mg/kg b.w.,respectively.The final clinical treatment dosage regimen is 25mg/kg b.w.,once a day,for two consecutive days.Construction of a PPK model:Collect clinical PK data of 17 groups of pigs injected intramuscularly with FF to construct a PPK model.Through Loglike,-2（LL）,AIC,and BIC evaluations,it was determined that the covariate（weight）was not highly correlated with the model,and a non covariate model was selected as the final PPK model.Goodness of fit（GOF）analysis,visual predictive check（VPC）,and bootstrap testing were performed on the model,and the results showed that the final model had good accuracy and stability.Establishment of pharmacodynamic cut-off:The PPK model was used to simulate the PK curve of 1000 pigs after 96 hours of administration,obtain PK/PD target values under different MICs when the probability of target attachment（PTA）=90%,and finally determined the pharmacodynamic cut-off in plasma is 1μg/m L.The crystal ball software was used to simulate the data of 10000 pigs in Monte Carlo.The PTA under different MICs was calculated in plasma and pulmonary interstitial fluid based on the pharmacodynamic target value（E=-3）,and the maximum MIC was obtained with the PTA higher than 90%,which is the pharmacodynamic cut-off.The pharmacodynamic cut-off in plasma and pulmonary interstitial fluid obtained by Monte Carlo simulation are 2 and 4μg/m L,respectively.3.Establishment of clinical cut-off and susceptibility breakpoint for FF against APPEstablishment of clinical cut-off:36 piglets were randomly divided into 1 control group and 5 experimental treatment groups,with 6 piglets in each group.Pathogenic clinical strains 068,BW25,121,APP7,and APP7-1 were selected（MIC=0.5μg/m L,1μg/m L,4μg/m L,8μg/m L,16μg/m L）to challenge piglets.After the onset of piglets,FF 20mg/kg b.w was injected intramuscularly,once every two days,twice in a row.The cure rate and mortality rate of each group of piglets were counted.The clinical cut-off was determined by using Windo W,CART algorithm and nonlinear regression analysis.The results show that the cure rates of different experimental groups were 100%,100%,83.3%,83.3%and 66.7%respectively.Combining the results of Windo W,CART algorithm and nonlinear regression analysis,the clinical cut-off was determined by 4μg/m L.Formulation of susceptibility breakpoint:Refer to the guidelines for the formulation of susceptibility breakpoint for CLSI and EUCAST in the field of human medicine,and the values of wild type cut-off,pharmacodynamic cut-off,and clinical cut-off were compared to determine the susceptibility breakpoint.When the three critical values are equal,the value is determined as the final susceptibility breakpoint.When the three critical values are not equal,a flow chart is developed based on the CLSI susceptibility breakpoint to determine the final susceptibility breakpoint.The final susceptibil ity breakpoint of APP to FF was 4μg/m L.By constructing the PBPK model and HFIM,the PK and PD interactions of FF against APP under dynamic conditions in vitro were investigated to develop reasonable dosing regimens applicable to different clinical conditions,to provide a more realistic response to the therapeutic situation in target animals after dosing,and to establishsusceptibility breakpoint in line with China’s national conditions by constructing the PPK model to delay the drug resistance problem and to protect the clinical use potential of FF.