The PK/PD Study Of Colistin Combined With Enrofloxacin On E. Coli Infected Chickens

Colistin,also known as Polymyxin E,is a peptide antibiotic with strong antibacterial activity against Escherichia coli（E.coli）.In the early 1980s,with the increasing reports of strong nephrotoxicity and neurotoxicity of colistin,colistin was gradual ly abandoned.But after 2000,multi-drug resistant super bacteria（Multi-Drug Resistant Gram-Negative Bacteria,MDR-GNB）appeared worldwide,and the emergence of MDR-GNB made the clinic face the dilemma of no drugs available.As MDR-GNB spreads the infection from time to time,people have to reassess and use the previously abandoned colistin.Recently,domestic scholars have discovered the colistin resistance gene（mcr-1）in enterobacteria with multi-drug resistance.Based on safety considerations,the Ministry of Agriculture of my country prohibits the use of colistin as a feed additive.But it needs to be emphasized that,compared with other drugs,colistin is still one of the most effective drugs for the treatment of MDR-GNB bacterial infections,so the research on colistin still cannot stop.In response to the problem of bacterial resistance caused by mcr-1,colistin combination is one of the strategies to overcome this problem.Enrofloxacin,as a third-generation fluoroquinolone antibiotic,has a good th erapeutic effect on diseases caused by E.coli.In addition,there have not been too many reports on the combined effects of colistin and enrofloxacin.This article aims to study the effects of colistin and enrofloxacin alone or in combination on E.coli containing mcr-1 through the Pharmacokinetics/Pharmacodynamics（PK/PD）model,and provide for follow-up research More experimental support.The E.coli strain SZ31 containing the mcr-1 gene isolated from diseased chickens is stored in our laboratory.SZ31 is the target strain for this study.（1）In vitro single and combined antibacterial test of colistin and enrofloxacin on SZ31The Minimum Inhibitory Concentration（MIC）of colistin and enrofloxacin on SZ31 was determined by the micro broth method.The results showed that the MIC of colicin and enrofloxacin was 4μg/m L,and the MIC of enrofloxacin was 64μg/m L.The checkerboard dilution method was used to determine the Fractional Inhibitory Concentration Index（FICI）of these two drugs against SZ31,and the measured FICI value was 0.504.According to the criteria for determining the antibacterial index,0.5<FICI<0.75 is a partial synergistic effect.It can be seen that enrofloxacin and colistin have a partial synergistic antibacterial effect.Finally,in vitro minimum Bactericidal Concentration（MBC）and Mutation Preventive Concentration（MPC）determination tests were carried out.The MBC of colistin and enrofloxacin were 4μg/m L and 64μg/m L,respectively.The MPC is 6.4μg/m L and 51.2μg/m L,respectively.（2）Use SZ31 strain to build chicken infection E.coli modelThree virulence genes,pho A,omp A and fim C,were detected in SZ31 by PCR.The 1-day-old Laihang chicks were purchased and fed to 7-days old,and 10⁸ CFU/m L SZ31 bacterial solution was intraperitoneally injected to construct a chicken infection model of E.coli.Observed by clinical symptoms 24 hours later,the chickens were depressed,with messy coats,bloodshot feces and no obvious signs of improvement.Through pathological dissection,a large amoun t of yellow fibrous exudate can be seen in the abdominal cavity.A large number of pink colonies with neat edges and uniform size can be seen after the blood Mac Conkey plate is coated.The results showed that the model of chicken infected with E.coli was successful.（3）PK/PD study of colistin and enrofloxacin in chickensHigh Performance Liquid Chromatography（HPLC）was used to determine the pharmacokinetics（PK）data of colistin and enrofloxacin in chickens,and Winnonlin 5.2.1 software was used for analysis.The PK parameters of colistin are as follows:peak time(T_max)is 1.5～2 h,elimination half-life(T_1/2)is 2.32～2.97 h,peak concentration(C_max)is 0.57～4.17μg/m L,drug concentration-The area under the time curve(AUC_last or AUC_0-24h)is 2.32～20.45μg×h/m L;the pharmacokinetic parameters of enrofloxacin are 2 h,T_1/2 are 2.78～3.3 h,and C_max are 1.07～2.18μg/m L,AUC_0-24h is 6.67～13.3μg×h/m L.The results showed that there was no significant difference in the PK data of colistin and enrofloxacin for single and combined use（p>0.05）.The PK/PD parameters(AUC_0-24h/MIC)and the antibacterial effect were fitted by the Sigmoid Emax model.The results showed that the two had a good correlation when used alone or in combination,with R² being 0.9542 and 0.9735,respectively.Pharmacodynamics（PD）studies have found that colistin and enrofloxacin alone have no obvious antibacterial effect on SZ31,and the combination of the two has better antibacterial effect.In summary,this study evaluated the antibacterial effects of colistin and enrofloxacin through an in vitro combined antibacterial test,and found that the combination of these two drugs can significantly improve the antibacterial effect of SZ31.The antibacterial effects of colistin and enrofloxacin in vivo were studied through the PK/PD model,and the relationship between the antibacterial effect and the dosage of the drug was evaluated,which provided a reference for solving the problem of bacterial resistance.