Ex Vivo Pharmacokinetic/Pharmacodynamic Relationship Of Cefquinome Against Cow Mastitis Due To Escherichia Coli

Cow mastitis is a common disease that harms the dairy livestock farming.It has a high incidence and is easy to be repeated infection,which brings serious economic losses to the dairy livestock farming.As reported,majority of mastitis is of bacterial origin and just five species of bacteria include Escherichia coli,Streptococcus uberis,Staphylococcus aureus,Streptococcus dysgalactiae and Streptococcus agalactiae,account for almost 90%of all diagnoses.As an 'environmental pathogen',Escherichia coli is always increasing the incidence of cow mastitis.Currently,the treatment of cow mastitis is mainly based on antibiotics,but with the widespread use of antibiotics,bacteria generally develop resistance to a variety of antimicrobial drugs and it brings the problem to the clinical treatment.Cefquinome as fourth-generation cephalosporin developed for use in veterinary medicine,has been used in the treatment of clinical cow mastitis in recent years and has a good therapeutic effect.In order to slow down the resistance of the Escherichia coli to cefquinome,and to protect and maintain the effectiveness of cefquinome.We carried out the ex vivo PK/PD modeling of cefquinome against Escherichia coli in cows.The aim of this study were to obtain the PK/PD parameters and provide the experimental basis for the rational use of cefquinome in clinic.1.Determining the wild-type cutoff and pharmacodynamic study of Escherichia coli to cefquinomeIn order to know the sensitivity of cefquinome to Escherichia coli from clinical and determine the wide-type cutoff.A recommended agar dilution method for the determination of drug sensitivity of cefquinome to 1073 strains of the Escherichia coli.The wide-type cutoff was determined by ECOFFinder software.The MIC distribution of cefquinome to 1073 strains of Escherichia coli was between 0.015?>64 ?g/mL,MIC50 and MIC90 was 0.03 ?g/mL and 4?g/mL,the wild type cut-off was 0.125 ?g/mL.Then,in order to compare the difference of the antibacterial activity about cefquinome against Escherichia coli in MH broth and milk,in vitro pharmacodynamics were studied.The MIC was determined by microdilution method,and ranges of cefquinome against six Escherichia coli from clinical were 0.03?0.125 ?g/mL in MH broth and 0.06?0.25 ?g/mL in milk.It indicates that the protein binding rate of cefquinome in milk is low and can be ignored.The MBC and MPC ranges of cefquinome were 0.06?0.25 ?g/mL and 0.075?0.6 ?g/mL,it has narrow MSW and is insusceptible to mutation.The PAE of cefquinome against Escherichia coli in both MH broth and milk were 0.040?0.746 h and 0.010?0.570 h.It instructe that cefquinome with short PAE.The time-killing curves in MH broth or milk showed that when the initial bacterial concentration was 106 CFU/mL,2×MIC reached the bactericidal effect.When the initial bacterial concentration was 108 CFU/mL,32×MIC reached the bactericidal effect.The amount of bacteria has a great influence on the antibacterial effect of cefquinome.2.Pharmacokinetics of cefquinome in milk of healthy cowsThree healthy Holstein cows were selected.According to the current clinical recommended dose,each breast IMM administration with 75mg cefquinome,and the drug was administered three times at 12 hours intervals.Milk samples were collected at different time points.The drug concentration was determined by HPLC-MS/MS,and the PK analyses were performed using a non-compartmental model provided in the WinNonlin software.PK results showed that,after the first and third administration,AUC0-12h and AUC0-48h were 15.53±5.64 mg·h/mL and 58.46±48.91 mg·h/mL.Cmax were 1.80±0.64 mg/mL and 1.54±0.23 mg/mL.Tmaxwere 1.71±1.18 h and 1.20±2.23 h.T1/2?were 27.84±43.54 h.MRT were 38.74±44.88 h.The results showed that cefquinome reached high concentration rapidly and maintain for a longer time in the milk of healthy cows.3.Ex vivo PK/PD modeling of cefquinome against Escherichia coliMilk samples containing different concentrations of cefquinome were cultured for 24h with different initial amounts of Escherichia coli,and the change amount of bacteria was determined by counting.After the pharmacokinetic parameters were obtained,the optimal PK/PD parameters were selected to construct the ex vivo PK/PD model of cefquinome to the Escherichia coli.The relationship between AUC0-24h/MIC value and bacterial logarithmic variation in the ex vivo time-killing curve test was fitted by Sigmoid Emax model equation.It had good fitting degree and coefficient of association were 0.9080,0.9171,0.8388,0.7946.The AUC0-24h/MIC value of bacteriostatic,bactericidal and clearance effect was determined.The results show that,the AUC0-24h/MIC value of clearance effect to 106 CFU/mL E.coli ATCC 25922 was 347352.44;the AUC0-24h/MIC value of clearance effect to 106 CFU/mL E.coli GM10-1 was 138820.89;the AUC0-24/MIC value of clearance effect to 108 CFU/mL E.coli ATCC 25922 was 404570.75;and to 108 CFU/mL E.coli GM10-1 was 187866.73.In clinical treatment,the appropriate PK/PD parameters can be selected according to the course of mastitis to give play to the ideal therapeutic effect of cefquinome.