Susceptibility Breakpoint And Resistance Mechanism Of Danofloxacin Against Swine-dervied Escherichia Coli

Escherichia coli(E.coli)is a gram-negative bacterium of Enterobacteriaceae and is found in the gastrointestinal tract of humans and animals.E.coli is a conditional pathogen,and some serotypes are pathogenic,causing serious intestinal infections and systemic infections in livestock and poultry,and causing huge economic losses to the aquaculture industry.E.coli can also cause serious harm to human health and public health through the food chain or the environment.Danofloxacin is a member of the fluoroquinolone antimicrobial agents and used for livestock and poultry.It plays an important role in controlling infectious diseases caused by susceptible bacteria such as E.coli.It was initially recognized that the unique antimicrobial mechanism of fluoroquinolones rarely leads to bacterial resistance,but the number of resistant strains of gram-negative bacteria including E.coli has been increasing since the improper use of danofloxacin for a long time.In addition,there is serious cross-resistance with other members of fluoroquinolones.Therefore,the monitoring of bacterial resistance and the development of a scientific and rational dosage regimen have important guiding significance for the prevention and control of drug-resistant bacteria and the later clinical treatment.The susceptibility breakpoint is an important technical basis for monitoring bacterial resistance.To date,the susceptibility breakpoint of E.coli against danofloxacin has not been established by both the European Committee on Antimicrobial Susceptibility Testing(EUCAST)and the Clinical and Laboratory Standards Institute(CLSI).In addition,there are rare systematic researches on the molecular resistance mechanism of E.coli against danofloxacin at home and abroad.In this study,based on the establishment of the Epidemiological Cutoff Values(ECV)and the pharmacodynamic(PD)cutoff(COPD)of danofloxacin against E.coli,the resistance mechanisms of swine-derived E.coli against fluoroquinolone antimicrobial agents were conducted.The optimal danofloxacin dosage regimen for the treatment of E.coli infection was proposed by the pharmacokinetic(PK)/PD intergration model.The results provide theoretical basis for monitoring of danofloxacin-resistant E.coli and the effective treatment of the infections caused by swine-derived E.coli,which could be sufficient for treatment intestinal infection of E.coli in pig and minimizes possible bacterial resistance.(1)Establishment of the ECV and COPD of danofloxacin against E.coliThe minimum inhibitory concentration(MIC)of danofloxacin against 1233 E.coli strains was determined by microdilution broth according to the guidelines of the CLSI document M07-A9.MIC distribution was performed with software SPSS 22.0 and Epidemiological Cutoff Values(ECV)was established by statistical analysis with using nonlinear least squares regression of Graphpad 6.0.Plasma drug concentration data were used to establish PK model in swine.The COPD was calculated using Monte Carlo simulation with Crystal Ball software.The results show that the MIC distributions of danofloxacin to E.coli range from 0.008 to 128 ?g/m L;the percentage of strains per MIC(0.008,0.016,0.03,0.06,0.125,0.25,0.5,1,2,4,8,16,32,64 and 128 ?g/m L)was 0.73%,3.97%,2.35%,0.73%,3.16%,7.38%,13.22%,10.62%,6.16%,5.43%,7.54%,12.98%,7.62%,8.76% and 9.33%;the MIC50 and MIC90 was respectively 4 and 128 ?g/m L.The MIC distribution are statistically consistent with the normal distribution as both the skewness coefficient(-0.321)and the kurtosis coefficient(-0.731)are ? 1.The ECV was set to 8 ?g/m L by statistical analysis.The probability of danofloxacin attaining an AUC: MIC ratio of at least 125 is 92.25% when MIC=0.03 ?g/m L.Therefore,the COPD was defined at 0.03 ?g/m L.(2)Mechanisms of fluoroquinolone resistance in E.coli from swineIn this study,a total of 479 clinical isolates of swine E.coli were selected and all the strains were isolated from seven provinces in China.These strains covering the MIC distribution range(0.0075->128 ?g/m L)of danofloxacin against E.coli were selected for the study of susceptibilty test of E.coli against norfloxacin,ciprofloxacin,ofloxacin and levofloxacin.According to the results of susceptibility test,74 strains are resistant to the four fluoroquinolone agents above were selected for the study of resistance genotype.The mechanism of drug resistance caused by chromosomal mutation of E.coli and plasmid-mediated fluoroquinolones resistance mechanism were systematically analyzed.The results of susceptibility test show that the percentage of strains of resistant to four human fluoroquinolone antimicrobials(norfloxacin,ciprofloxacin,ofloxacin and levofloxacin)was positively correlated with the degree of resistance to danofloxacin and there is no geographical difference in this correlation.The double mutation or single mutation of gyr A,par C,par E,mar R and acr R genes on chromosome of E.coli is one of the main mechanisms leading to E.coli resistance to fluoroquinolones.Among the 74 E.coli clinical isolates,no mutations were found in gyr B.There are 39 strains carried double or single mutations in gyr A and these mutations are S83 L and D87 to N,Y,G,or H.Twenty-one strains carried single or double mutations in par C(S80I and E84K).Two strains carried double par E mutations(I355T and L416F)and carried two gyr A mutations and one par C mutations.A total of 26 strains with mutations in mar R,25 strains of those carried single mutation(G123S)and 1 strain of those carried two mutations(D87N and G123S).Two strains carried mutations in acr R,the mutations were respectively V33 G and S60-.Neither of the two strains carried gyr A mutation.The genes that mainly mediated resistance to fluoroquinolone antimicrobials on the plasmids of E.coli were qnr S,oqx AB and aac(6')-Ib-cr.There are 7 strains carried the qnr S gene,29 strains carried the oqx AB gene,and 9 strains carried the aac(6')-Ib-cr gene.There are no qnr A,qnr B,qnr C,qnr D and qep A genes were detected.The strains carried two gyr A mutations can mediate the high level of resistance to fluoroquinolones.The plasmids of E.coli carried the qnr S,oqx AB and aac(6')-Ib-cr genes increase the resistance to fluoroquinolone antimicrobials such as danofloxacin.(3)PK/PD integration of danofloxacin against E.coli in swineThe number of the strains with an MIC equal to 0.5 ?g/m L were the most according to the MICs distribution of 1233 clinical strains.Therefore,a total of 69 strains(MIC=0.5 ?g/m L)isolated were conveniently selected for serotype determination,and then for the pathogenicity test.An E.coli strain HP501(O158)was selected for subsequent in vitro PD experiments and PK/PD integration model.In this study,ultrafiltration probes were successfully implanted into the ileum of piglets with the aid of anesthetic and then collected the ileum ultrafiltrate.After danofloxacin was intramuscularly administered at a dose of 2.5 mg/kg,blood and ileum ultrafiltrate were collected at different time points and then determined by High Performance Liquid Chromatography.PK parameters for plasma and ileum ultrafiltrate were calculated.The MIC and minimum bactericidal concentration(MBC)of danofloxacin against HP501 in MH broth,plasma and ileum ultrafiltrate were determined according to the methods recommended by CLSI.The mutant prevention concentration(MPC)and post-antimicrobial effect(PAE)of danofloxacin against HP501 in MH broth was also determined.The in vitro and ex vivo time kill curve of danofloxacin against HP501 were determined in MH broth and ileum ultrafiltrate collected at different time points,respectively.The relationship between the AUC24h/MIC,Cmax/MIC and the log10 difference between the initial bacterial count(CFU/m L)and the bacterial count after 24 h of incubation for ultrafiltrate was established.The values of PK/PD parameters(AUC24h/MIC and Cmax/MIC)required to achieving the three levels(bacteriostatic action,bactericidal action and bacterial elimination)of antibacterial effect of danofloxacin in ileum ultrafiltrate.The dose was calculated and the dosage regimen was optimized.Twelve healthy castrated cross-bred piglets were intragastrically administered with 50 m L 1010 CFU/m L E.coli(HP501)daily for 3 consecutive days to establish an E.coli infection model.All the infected piglets were conveniently divided into two groups.The test group(n=6)were intramuscularly administrated with the recommended dose regimen(2.42 mg/kg every 12 h for 3 d)and the control group(n=6)was intramuscularly administered by standard dosing regimen(2.5 mg/kg every 24 h for 3 d).As a result,the Tmax of plasma and ileum ultrafiltrate was 1.1 h and 6 h after single intramuscular injection of danofloxacin at a dose of 2.5 mg/kg.The mean Cmax of the ileum is 13.59 times than that of plasma.The elimination half-life(T1/2?)in the ileum ultrafiltrate(6.84 h)was longer than those in plasma(4.94 h).All the MIC of danofloxacin in MH broth,plasma and ileum ultrafiltrate against E.coli HP501 was 0.5 ?g/m L and its corresponding MBC values were 0.5,1,1 ?g/m L.The MPC of danofloxacin in MH broth was 4 ?g/m L.The time of PAE was positively correlated with the concentration of the drug and the time of exposure of E.coli to drug solution.Both in vitro and ex vivo time kill curves indicate that the mechanism of danofloxacin against E.coli is concentration-dependent,which indicated that the PK/PD parameter most relevant to the antimicrobial effect of danofloxacin is AUC/MIC or Cmax/MIC.According to the PK data in vivo and the MIC ex vivo,the mean AUC/MIC and AUC/MPC were 170.64 h and 21.33 h.The time for which the concentration in ultrafiltrate remained above the MIC and MBC were 29.76 h and 19.32 h,respectively.The mean Cmax/MIC ratio and Cmax/MPC were 13.32 and 1.67,respectively.The calculated mean AUC24h/MIC for ileum ultrafiltrate that produced bacteriostasis,bactericidal activity,and elimination of bacteria were 99.85,155.57,and 218.02 h,respectively.The calculated mean Cmax/MIC for ileum ultrafiltrate that produced bacteriostasis,bactericidal activity,and elimination of bacteria were 4.14,6.49,and 9.05,respectively.Three different doses(1.49 mg/kg,2.42 mg/kg and 3.24 mg/kg)were calculated respectively based on AUC24h/MIC ratio for bacteriostatic,bactericidal and bacterial eradication.The dosage regimen of 2.42 mg/kg danofloxacin after every 12 h treatment for 3 days was the best after simulating different dosage regimens with Mlxplore software.The results of dosage validation indicate that the piglets were treated with 2.42 mg/kg danofloxacin after every 12 h for 3 days that could be sufficient for the treatment of intestinal infection of E.coli.In summary,The COPD(0.03 ?g/m L)was much lower than the ECV(8 ?g/m L)established in our study,implying that the lower COPD in our study may be due to the lower dose of drug administration to piglets.Therefore,the ECV(MIC? 8 ?g/m L)was finally selected as the optimum danofloxacin susceptibility breakpoint for swine E.coli.Danofloxacin-resistant E.coli has significant cross-resistance to other four fluoroquinolone antimicrobial agents(norfloxacin,ciprofloxacin,ofloxacin and levofloxacin)commonly used in human medicine.The resistance mechanisms of swine-derived E.coli to fluoroquinolones is caused by chromosomal mutations(gyr A,par C,par E,mar R and acr R genes double-site or single-site mutation)and caused by the genes(qnr S,oqx AB and aac(6')-Ib-cr)on plasmids that mediated fluoroquinolone resistance.The calculated mean AUC24h/MIC for ileum ultrafiltrate that produced bacteriostasis,bactericidal activity,and elimination of bacteria were 99.85,155.57,and 218.02 h,respectively.The optimal dosage regimen was 2.42 mg/kg danofloxacin after every 12 h for 3 days,which could be sufficient for the treatment of intestinal infection of E.coli and minimizes possible bacterial resistance.