| Actinobacillus pleuropneumoniae,formerly known as Haemophilus pleuropneumoniae,is prone to induce pleuropneumoniae in different age of pigs and cause significant mortality.With the abuse of domestic veterinary antibiotics,the therapeutic effect of some drugshas been greatly reduced,so we need to slow the increase of drug resistance.Ceftiofur is the first cephalosporin antibiotic used in animals.It is effective for Gram-negative bacteria and Gram-positive bacteria and has good clinical therapeutic effect for animal respiratory bacteria.Therefore,we need to carry out surveillance measures for Actinobacillus pleuropneumoniae to reduce the resistance of Actinobacillus pleuropneumoniae to ceftiofur.By establishing a criterion for determining the resistance of Actinobacillus pleuropneumoniae to ceftiofur can effectively monitor drug resistance.In this study,we determined the sensitivity of ceftiofur against 135 strains of Actinobacillus pleuropneumoniae to establish the wild type cut-off,and detect the antibacterial effect of ceftiofur on pathogenic bacteria isolated from clinic in vitro and in ex-vivo.Studying the pharmacokinetics of ceftiofur in pigs establish PK-PD model to formulate the best dosage regimen and establish the pharmacodynamics cut-off.Researching the clinical therapeutic effect of ceftiofur against Actinobacillus pleuropneumoniae associated with different MIC pathogenic strains to establish clinical cut-off and set up ceftiofur resistance standard against Actinobacillus pleuropneumoniae,so as to provide reasonable and scientific basis to monitor drug resistance of Actinobacillus pleuropneumoniae.1 Establishing of the wild-type cutoff and pharmacodynamic study of ceftiofur against Actinobacillus pleuropneumoniaeA recommended agar dilution method had been used for the determination of drug sensitivity of ceftiofur against 135 strains of the Actinobacillus pleuropneumoniae.The minimum inhibitory concentration(MIC)was analyzed by ECOFFinder software.The minimal inhibitory concentration and minimum bactericidal concentration(MBC)of ceftiofur against the Actinobacillus pleuropneumoniae BW39 strain in TSB broth medium and piglet serum were determined by micro broth dilution method.At the same time,the mutant prevention concentration(MPC)and the post-antibiotic effect(PAE)of ceftiofur against BW39 strain were determined in vitro.The in vitro and ex-vivo time-killing curves were carried out in TSB and serum containing different concentrations of ceftiofur.The MIC distribution of ceftiofur to 135 strains of Actinobacillus pleuropneumoniae was between 0.0075-4 μg/mL,MIC50 and MIC90 was 0.015 μg/mL and 0.5 μg/mL,the wild type cut-off was 0.125μg/mL,the MIC of ceftiofur against BW39 was 0.5 μg/mL and 1μg/mL in vitro and ex vivo,respectively,and MBCs were both 2 μg/mL.The MPC of ceftiofur to Actinobacillus pleuropneumoniae was 1.6 μg/mL,the mutant selection window(MSW)was 1-1.6 μg/mL.The ceftiofur exerted a PAE range from 0.33-0.66 h after 1h eaposure to the Actinobacillus pleuropneumoniae and 0.73-1.17 h after 2h exposure.The results of in vitro and ex vivo time-killing curve test showed that the antibacterial effect of ceftiofur to APP BW39 strain was time-dependent.2 Pharmacokinetics of ceftiofur in serum of pigs and establishing the pharmacodynamic cutoffTwelve healthy bi-crossbreeding pigs,each weighting about 15 kg,were randomLy divided into two groups,with 6 pigs in each group.Each pig in healthy group and the disease group received ceftiofur hydrochloride injection at a dose of 5 mg/kg·b.w by intramuscular injection of neck.Then plasma were collected at different time points after administration,and the drug concentration was detected by high performance liquid chromatography(HPLC).The best dose regimen was formulated by fitting the ex vivo PK-PD model and the dose calculation formula.The Monte Carlo simulation method was used to simulate the pharmacokinetic data,and the pharmacodynamics cutoff was obtained.After the pharmacokinetic parameters were obtained,the optimal PK-PD parameters were selected to construct the ex vivo PK-PD model of ceftiofur against Actinobacillus pleuropneumoniae.The relationship between AUC/MIC value and bacterial logarithmic reduction was fitted by Sigmoid Emax model equation.The AUC/MIC value of bacteriostatic,bactericidal and clearance effect in the healthy group was 45.73、63.83 and 69.04 h and in disease group was 38.94、47.23 and 49.16 h,respectively.The doses of prevention,treatment and radical treatment by the dose calculation formula were 2.2,3.0,3.3 mg/kg b.w in healthy group,and were 1.9,2.2 and 2.3 mg/kg b.w in disease group,respectively.The mean and standard deviation of AUC were simulated by Monte Carlo simulation,and the pharmacodynamic cutoff of ceftiofur against Actinobacillus pleuropneumoniae was 2 μg/mL.3 The development of clinical cutoff and susceptibility breakpoint for ceftiofur against Actinobacillus pleuropneumoniaeThirty-six piglets were randomLy divided into 6 groups,one group was control group and the rest five groups were experimental groups,each group was six piglets.5 MIC strains of the Actinobacillus pleuropneumoniae with strong pathogenicity were selected to establish an artificial infection piglet disease model.The clinical cutoff was established by using the mathematical statistics algorithm WindoW and CART model.The susceptibility breakpoint of ceftiofur to the Actinobacillus pleuropneumoniae was established by susceptibility breakpoint decision tree.The Actinobacillus pleuropneumoniae BW9,BW29,BW39,QG17 and 18119 strains with MIC of 0.015 μg/mL,0.125 μg/mL,0.5 μg/mL,2 μg/mL and 4 μg/mL,were selected to challenge the experimental group.Then the infected piglets were treated with ceftiofur hrydrocloride injection with 3mg/kg b.w once a day for three days.The cure rates of ceftiofur to 5 MIC strains were 100%,83%,83%,67%and 50%,respectively.The distribution range of the clinical cutoff value was 0.5 μg/mL-1.25 μg/mL by the WindoW and CART algorithm analysis model.The clinical cutoff value of ceftiofur to Actinobacillus pleuropneumoniae was 0.5-1.25μg/mL by WindoW and CART method.The value of the wild type cutoff,the pharmacodynamics cutoff and the clinical cutoff were integrated,and thefinal susceptibility breakpoint was 0.5-1.25 μg/mL.The standard procedures published by the United States Association for Clinical and Laboratory Standards(CLSI)and the European Commission Antimicrobial Susceptibility Testing(EUCAST)were refered to establish susceptibility breakpoint of ceftiofur to Actinobacillus pleuropneumoniae.The aim is to provide scientific theoretical basis for the drug resistance monitoring of Actinobacillus pleuropneumoniae and protect and maintain the efficacy of ceftiofur in clinical treatment. |
