Establishment Of Susceptibility Breakpoint Standard For Avilamycin Against Clostridium Perfringens In Chicken

Clostridium perfringens is the main etiologies of necrotic enteritis in chicken,they could cause digestive tract symptoms such as diarrhea,hemorrhagic stool and intestinal mucosal injury in young chickens,and even cause high fatality rate around50%,and brings serious economic losses to the breeding industry.Avilamycin has the advantages of low residue,fast metabolism and weak toxicity,and also has good efficacy in preventing and treating bacterial digestive tract diseases in animals,especially for C.perfringens from chickens.Under the strict restriction of using growth-promoting antibiotics in European Union,how to apply avilamycin in the treatment and control of C.perfringens infection in chickens has become the key point of rational drug use and drug resistance control.Clinical breakpoint is the standard for judging bacterial sensitive or resistance to antimicrobial drugs,and is also the basis for predicting clinical therapeutic effect and designing rational drug delivery schemes.However there is no relevant report about the breakpoint for avilamycin against C.perfringens in chicken at home and abroad.This thesis was to establish clinical breakpoint of C.perfringens against avilamycin,which referred the values of wild-type cutoff,PK/PD cutoff,clinical cutoff according to the Clinical and Laboratory Standards Institute（CLSI）and European Committee on Antimicrobial Susceptibility Testing（EUCAST）.This will also provide a theoretical basis for guiding the study of drug resistance standard in China and the rational use of avilamycin in clinical.1.Establishment wild-type cutoff for avilamycin against C.perfringens in chicken628 chicken intestinal samples were provided from 5 provinces in Hubei and Hunan etc.236 strains was suspected as C.perfringens on TSC medium,128 strains of C.perfringens type A were identified by PCR.The Minimum inhibitory concentration（MIC）of 128 strains were determined by agar dilution method of CLSI M11-A7,The MICs showed a single peak normal distribution and ranged from0.015μg/m L to 4μg/m L,and MIC₅₀ and MIC₉₀ were 0.06μg/m L and 0.5μg/m L.The MICs of 95%,99%and 99.9%confidence interval were 0.25μg/m L,0.5μg/m L and 1μg/m L according to the Ecoffinder software.In this study,the MIC value of0.25μg/m L corresponding to 95%confidence interval was defined as the epidemiological cut-off values（ECOFFs）or CO_WT for avilamycin against C.perfringens in chicken.2.Establishment PK/PD cutoff for avilamycin against C.perfringens in chickenPD study in-vitro and ex-vivo:C.perfringens SX10 was determined from the virulence test of clinical isolates.The MIC,minimum antibiotic concentration（MBC）,mutant prevention concentration（MPC）,post-antibiotic effect（PAE）and killing curves of the strain for avilamycin were tested during the research,which result showed the MIC,MBC and MPC of avilamycin against SX10 were 0.25μg/m L,0.5μg/m L and 3.2μg/m L,the mutation selection window（MSW）range was from 0.25μg/m L to 3.2μg/m L in vitro,the MIC,MBC in ex vivo were 0.25μg/m L,0.5μg/m L.Combined 1×MIC,2×MIC and 4×MIC avilamycin with SX10,the PAE produced by combined exposure for 1 h was 0.33 h,0.71 and 1.28 h,for 2 h was 0.52 h,1.19 h and1.67 h.The in-vitro and ex-vivo killing curves showed that the antibacterial effect of avilamycin against C.perfringens was significantly concentration dependent.PK study:130 Sanhuang broilers aged 3-4 weeks were divided into two groups randomly,65 in each group.One group was infected artificially with 5m L of 10¹⁰CFU/m L of SX10 bacterial solution liquid artificially for 3 days to establish a model of chicken necrotic enteritis,and the other group was healthy model.Took the plasma and ileum contents from both healthy and diseased group in every 0.5 h,0.75 h,1 h,2h,3 h,4 h,5 h,6 h,8 h,12 h,24 h,36 h after administration,and determined the avilamycin in plasma and ileum contents by High Performance Liquid Chromatography（HPLC）,every time point 5 chickens.The result of HPLC showed that avilamycin in plasma was not detected,in the ileum was contented.Analyzed the PK data in the ileum contents of healthy and diseased chickens by Winnonlin software,the Peak time(T_max)were both 1 h,the peak concentration(C_max)were 15.89μg/m L and 23.43μg/m L,the area under concentration-time killing curve of AUC_all were46.03 h*μg/m L and 56.16 h*μg/m L.PK/PD modeling:Combined ex-vivo PD data and PK data,determined AUC/MIC was the PK/PD parameter,analyzed the Sigmoid Emax model in Winnonlin software:when E=0,-3 and-4,the PK/PD parameters of healthy group was 25.69 h,29.71 h,31.31 h,the diseased group was 36.64 h,46.10 h and 49.96 h.Finally,the parameter of 46.10 h in the disease group when E=-3 was selected as the pharmacodynamic target,and the PK/PD cutoff for avilamycin against C.perfringens(CO_PD)was 1μg/m L by Monte Carlo simulation.3.Establishment of clinical cutoff for avilamycin against C.perfringens in chicken165 broilers aged 3～4 weeks were divided into 5 groups randomly,and infected virulence strains into the groups with MIC 0.015μg/m L,0.06μg/m L,0.25μg/m L,0.5μg/m L,4μg/m L to establish 5 disease models,30 each.After the successfully attack,each model group was randomly divided into positive treatment sub-group and negative non-treatment control sub-group,15 each.In addition,use 15 chickens in blank control groups as comparison.Scored the clinical symptoms of each group,counted the number of death and bacteriological prognosis,and analyzed the probability of cure（POC）of different MIC strains,which final result showed that the mortality of blank control group was 0,the negative non-treatment group death rate was higher than 20%.In positive treatment group,the smaller the MIC,the lower the clinical symptom score,the better the bacteriological prognosis,and the higher the cure rate.The clinical cutoff(CO_CL)was determined to be 0.5μg/m L by analyzing the relationship between MIC and POC.4.Establishment of breakpoint for avilamycin against C.perfringens in chickenReferred the tree structure of breakpoint in CLSI,and combined the above three cutoff values,determined CO_PD>CO_CL>CO_WT,and the breakpoint of chicken C.perfringens to avilamycin was 0.5μg/m L.The breakpoint established in this study can be used as a reference threshold to determine whether C.perfringens from chickens is sensitive or resistant to avilamycin,and provide a scientific and rational drug regimen for the prevention,treatment and eradication of C.perfringens from chickens.This will be a great significance to control the drug resistance of C.perfringens and to regulate the effective use of avilamycin.