Establishment Of Susceptibility Breakpoints For Danofloxacin And Apramycin Against Escherichia Coli In Chickens

For poultry,Escherichia coli(E.coli)can cause intestinal infection through colonization in the intestine,but also can transfer to the parenteral tract to cause extraintestinal infection.Chickens mainly infected at 3 to 7 weeks of age,and the main symptoms of E.coli infection are pericarditis,perihepatitis,peritonitis,ophthalmitis and so on.It has brought huge economic losses to the chicken industry.Danofloxacin(fluoroquinolones)and apramycin(aminoglycosides)are two broad-spectrum antibiotics for veterinary purposes,which are used to prevent and treat infections caused by Gram negative(G-)bacteria(such as E.coli)in chickens,pigs and so on.At present,due to the long-term non-standard use of antibiotics,the drug resistance of E.coli is becoming more and more serious.However,the criteria for determining the resistance of these two drugs to E.coli have not been established at home and abroad.The purpose of this study is to establish the susceptibility breakpoints of danofloxacin and apramycin,so as to provide a theoretical basis for standardizing the clinical application of these two drugs in veterinary medicine in China.(1)Determination of wildtype cutoff(COWT)for danofloxacin and apramycin against E.coliIn this study,1412 strains of E.coli from chickens were collected and identified from large-scale chicken farms in China.The minimal inhibitory concentration(MIC)of danofloxacin and apramycin were measured by broth dilution method.The MIC distribution of danofloxacin in the range of 0.125 μg/mL to 64 μg/mL showed a bimodal distribution.The peak values were 0.5μg/mL and 8 μg/mL,respectively.When the MIC was 8 pg/mL,215 strains of bacteria accounted for 15.7%,and the distribution was the largest.The MIC50 and MIC90 of danofloxacin against chicken E.coli were 4 μg/mL and 64 μg/mL respectively.However,apramycin showed a single peak distribution in the range of 2 μg/mL to 256 μg/mL.The peak value was MIC=8 μg/mL.There were 828 strains of bacteria in this area,accounting for 60.7%of the total.The MIC50 and MIC90 of apramycin against E.coli were 8 and 16μg/mL respectively.The MIC values of the two drugs were converted to Log2MIC.The critical values of wild type of danofloxacin and apramycin were 4 g/mL and 16 g/mL respectively by non-linear regression analysis,NORMINV and NORMDIST methods.(2)Analysis of drug resistance of E.coli to danofloxacin and apramycinE.coli with different MIC values were selected and the correlation between resistance genes and MIC values was detected by polymerase chain reaction(PCR).The results showed that in E.coli,the positive rates of oqxAB,aac(Ib-cr),qnrA,qnrB,qnrS and qepA genes increased to some extent with the increase of MIC value,while the positive rates of qnrC and qnrD were 0.Similarly,the positive rate of apramycin-resistant gene aac(3)-Ⅳ and npmA was also MIC-dependent.Twenty-four strains of highly drug-resistant bacteria to danofloxacin and 16 strains of highly drug-resistant bacteria to apramycin were further screened by broth microdilution method.The susceptibility of these two strains to 17 commonly used antibiotics of Gram-negative bacteria was tested by disk diffusion method.The results showed that both strains had multidrug resistance,with the lowest resistance of 10 drugs and the highest resistance of 15 to 16 drugs.It was also found that all danofloxacin-resistant strains showed 100%resistance to amoxicillin,ampicillin,nalidixic acid,enrofloxacin,ciprofloxacin,doxycycline,tetracycline and trimethoprim,but were relatively sensitive to amikacin and fosfomycin.All apramycin-resistant strains showed 100%resistance to amoxicillin,ampicillin,gentamicin,doxycycline,tetracycline,trimethoprim and florfenicol.Interestingly,all apramycin-resistant strains are resistant to gentamicin,but relatively sensitive to amikacin and spectinomycin.(3)Establishment of PK/PD cutoff(COPD)for danofloxacin against E.coliThe chicken model of E.coli infection was established.After single oral administration of 5 mg of danofloxacin,blood samples and intestinal fluids of four intestinal segments(duodenum,jejunum,ileum and cecum)were collected from healthy and infected chickens at different time points.And the drug concentration was measured by high performance liquid chromatography fluorescence detection.Population pharmacokinetics of four intestines fitted a two-compartment model.The absorption rate(Ka),clearance rate(CL)and distribution volume(V)of healthy chickens from duodenum,jejunum to ileum declined in turn.Compared with healthy chickens,the CL of danofloxacin in infected chickens decreased from duodenum,jejunum to ileum,and were lower than the corresponding intestinal segments of healthy chickens,Ka in duodenum and ileum decreased significantly,and V in jejunum and ileum increased.F in ileum was higher in healthy group and infected group.The growth curve of E.coli 078 in MH broth,ileal blood and plasma showed the same trend.The minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)of danofloxacin against E.coli 078 in broth,plasma and four intestinal fluids were determined by microbroth dilution method.The results showed that the MICs of danofloxacin in MH broth and plasma were both 0.016 μg/mL.The MIC values in four intestinal fluids were higher than those in plasma and MH broth.The MIC values in jejunal fluid were the highest(6.4 g/mL),while that in ileal fluid were the lowest(0.64 μg/mL).The time-killing curves of danofloxacin against E.coli 078 in vitro and ex-vivo(ileum and duodenum)were plotted by plate dumping counting method.Bactericidal activity of danofloxacin in vitro and ex vivo showed concentration dependence,so PK/PD parameter AUC/MIC was selected to formulate COPD.Based on the pharmacokinetic(PK)and pharmacodynamic(PD)characteristics of danofloxacin in plasma and four intestinal segments,the COPD of danofloxacin were finally established using data from infected chicken ileum contents.The PK data of danofloxacin in plasma and ileum were analyzed by Winnonlin software.The average time to peak(Tmax),concentration to peak(Cmax)and area under the time curve(AUC24)of danofloxacin in infected chicken plasma were 1 h,0.38 μg/mL and 2.06 h μg/mL,respectively.Tmax,Cmax and AUC in ileum of danofloxacin were 2.7 h,18.95 μg/mL and 261.78 h μg/mL,respectively.Then the Sigmoid Emax model in Winnonlin software was used to simulate the PK/PD data of ileum.When E=-3 was selected,the AUC/MIC value was the pharmacodynamic target.The pharmacodynamic targets of danofloxacin in ileum were 421.45.Finally,the PK/PD data of danofloxacin in ileum were simulated by Crystal ball software.The probability of AUC/MIC reaching pharmacodynamic targets under different MIC values was obtained.The maximum MIC with probability of target attainment(PTA)greater than 90%was selected as the COPD.The COPD of danofloxacin in ileum for E.coli were 0.54 g/mL.(4)Formulating of Clinical cutoff(COCL)for danofloxacin against E.coliIn this study,clinical treatment of danofloxacin in chickens included infection group,infection treatment group and a blank control group,with 5 chickens in each group.Five pathogenic E.coli strains with different MIC values were screened by polymerase chain reaction(PCR)and infection test in chickens.Each infection group corresponds to three infection treatment groups.The treatment regimen is 5 mg/kg,2 times/day for 3 consecutive days;10 mg/kg,1 time/day for 3 consecutive days and 20 mg/kg,1 time/day for 3 consecutive days,respectively.The results showed that there was no significant difference in the cure rate among the three treatments.Therefore,5 mg/kg treatment results were selected to establish the COCL.Firstly,the "WindoW"method recommended by EUCAST was used.Resultly,the COCL Of danofloxacin for E.coli ranged from 0.5 to 16 μg/mL.Then,MIC values and treatment results of clinical strains were then processed by CART classification tree regression analysis,When POC equals 90%,the COCL>2.25μg/mL.Therefore,4 ug/mL was recommended as COCL of danofloxacin against E.coli.In conclusion,COWT=COCL=4μg/mL,COPD is 0.54 μg/mL of danofloxacin in this study.Therefore,the susceptibility breakpoint of danofloxacin was 4 μg/mL.and the COWT of apramycin was 16 μg/mL,and its COPD and COCL need to be further studied and determined.