Establishment Of Susceptibility Breakpoints Standards For Avilamycin And Apramycin Against Clostridium Perfringens

Clostridium perfringens can cause gastrointestinal diseases such as necrotizing enterocolitis in swine,leading to severe diarrhea in swine,high morbidity and mortality,and causing huge economic losses to the global aquaculture industry.As animal specific drugs,avilamycin and apramycin have a good therapeutic effect on digestive diseases in livestock and poultry.And it is not easy to produce drug residues,and its toxicity is weak It has extremely high safety and further promotes its application in veterinary clinics However,the unreasonable use of drugs in the raise livestock industry in China will inevitably lead to the emergence of drug resistance.The monitoring of drug resistance is necessary and urgent.The monitoring of drug resistance requires standards.At present,China does not have relevant standards.We usually reference to CLSI.Therefore,in order to monitor the occurrence of drug resistance,analyze the current status and development law of drug resistance,and guide clinical drug use more scientifically.This study refers to the method developed in the CLSI to establish a wild-type cutoff、PK/PD cutoff and clinical cutoff in accordance with China’s national conditions.Provide scientific data for the establishment of susceptibility breakpoints standards1 Formulation of wild-type cutoff for avilamycin and apramycin against Clostridium perfringensBased on multiple PCR,120 positive control strains were identified isolated from clinical.The MIC values of apramycin and avilamycin for Clostridium perfringens in swine were determined using the agar dilution method recommended by CLSI.The MIC50 and MIC90 of apramycin were both 256 μg/mL.The MIC50 and MIC90 of avilamycin were 0.06 μg/mL and 128 μg/mL,respectivelyThe measured MIC data was substituted into the J Turnidge Ecoffinder software model for simulation analysis.It was concluded that the wild-type cutoff of apramycin is 1024 μg/mL,while the wild-type cutof of avilamycin is 0.25 μg/mL2 COPD study of avilamycin against Clostridium perfringensThe strains with strong pathogenicity were screened by the mouse virulence test.The strong pathogenic strains HS42 near COWT were selected.The micro broth dilution method was used to determine the concentration of avilamycin in the broth and the ileum contentss of the swine.The minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)were determined by applying the Clostridium perfringens broth concentrated to 1010 CFU/mL on agar plates containing avilamycin to determine the concentration of antimutagenicity.The PAE effect of avilamycin on Clostridium perfringens was determined by drug removal method.The ileum contentss collected with different concentrations of avilamycin and different time points were used.In vitro and ex vivo bactericidal curves were tested.The results showed that MIC in broth and ileum contentss were both 0.25 μg/mL,MBC in broth and ileum contentss were both 0.5 μg/mL.The MPC of avilamycin against Clostridium perfringens is 3.1 μg/mL,which indicated that the drug resistance mutation window of avilamycin was narrowly taught,probably because it was rarely treated in Clostridium perfringens infections,so there are fewer resistant bacteria.Exposure of Clostridium perfringens to different concentrations of avilamycin for 1 h and 2 h resulted in induced PAE of 0.38 h to 1.35 h and 0.54 h to 1.73 h,respectively.In vitro and in vivo bactericidal curve test results showed that the type of antibacterial effect of avilamycin on Clostridium perfringens was concentration-dependent.Six head 20 kg weaned piglets were used in the experiment to construct an ileal fistula model and used to establish a plasma pharmacokinetic model and a pharmacokinetic model of the digestive tract.The dose was 4 mg/kg.The administration method was gavage.After administration,intestinal contents and plasma were collected at 0.5 h,1 h,2 h,3 h,4 h,5 h,6 h,8 h,10 h,12 h,24 h,36 h,and 48 h,drug concentration in ileum contents and plasma was detected at different time points according to established HPLC method.Experiments showed that the drug concentration of avilamycin in the plasma was below the detection limit.The data obtained was simulated using the first-order absorption two-compartment model in the Winnonlin software.The Tmax of avilamycin in the ileum contents was 4 h,Cmax was 146.3 ± 13.41 μg/mL,and AUC was 428.62±14.23 h·μg/mL.Using the Sigmoid Emax equation for the(AUC24h/MIC)ex value in the half-life bactericidal curve test and Clostridium perfringens concentration The changes were fitted to the values and the PK/PD parameters(AUC24h/MIC)ex parameters were calculated to be 21.60 h,36.15 h,and 53.24 h when E=0,-3,and-4,respectively.The pharmacodynamic target of the COPD is 36.15 h when the value of E=-3 is selected,and Monte Carlo simulation is used to simulate the data of 10,000 swines,and the(AUC24h/MIC)ex compliance rate under different MIC values is obtained,and the compliance rate is obtained.The maximum MIC at or above 90%is the PK/PD cutoff,which is the PK/PD cutoff value of avilamycin against Clostridium perfringens was 8 μg/mL.Combined with the dose formula,the daily doses for the prevention,treatment and eradication of Clostridium perfringens in swine were 0.08 mg/kg,0.12 mg/kg,and 0.2 mg/kg against the tested strains.Accoding to the clinical renconmmended dosage(4 mg/kg),analysis of the reasons for the difference,the clinical recommended dosage is not specifically for the dose of Clostridium perfringens,but for swine in general diarrheal diseases,due to the high concentration of avilamycin in the intestine,Clostridium perfringens is also sensitive to it,so a small amount of avilamycin can achieve the desired bactericidal effect.The daily dose was converted into 3.2 mg/kg,4.8 mg/kg,and 8 mg/kg feed additive.3 Study on clinical cutoff of avilamycin against Clostridium perfringens in swineSixty-six 15 kg～20 kg piglets were randomly divided into 11 groups,each group with six pigs:five healthy control groups and no infection;five negative control groups,infected with 5 different MICs strains and no administration;five experitmental groups with 5 strains of different MICs and administered with avilamycin.Symptoms were scored for each group,and the number of live colonies was counted to evaluate clinical cure or failure and the cure rate was calculated.Analyze the relationship between cure rate and MIC.At present,there is no clear and unified method to establish the clinical cutoff.Therefore,POC and MIC are analyzed by three widely-reported methods.The "WindoW"method was used to calculate the MaxDiff and CAR,and the corresponding MIC range was 0.06 μg/mL～2μg/mL.The non-linear regression was used to fit the POC and MIC curves to obtain the estimated curve model and the POC value was brought in.The corresponding MIC was found to be 0.22 μg/mL;the clinical critical value was greater than 0.16 μg/mL using the CART regression tree analysis.After comprehensive analysis of the above methods,the clinical critical value was finally determined to be close to the MIC of 0.16 μg/mL and 0.22 μg/mL.That is 0.25 μg/mL.According to the flow chart developed by the CLSI publication,the experiment is in line with COWT=COCL,that is,the final susceptibility breakpoint was 0.25 μg/mL.