Linezolid Combined With Fosfomycin Against Enterococcus Using In Vitro Pharmacokinetic/Pharmacodynamic Model

Objective:The rapid spread of antibiotic resistance among enterococcus has prompted considerable interest in determining the dosage regimen of linezolid combined with fosfomycin.Different co-administration regimens have different effects on the development of resistance and the killing effect of enterococcus.Therefore,this experiment simulates the bactericidal effect of different drug delivery regimens of two drug combinations on enterococcus through in vitro PK/PD model,and reveals the interaction mechanism between drugs and bacteria from a quantitative perspective through a semi-mechanical PK/PD mathematical model,which provides simulation tools and basis for dose optimization of the combined drug delivery.Methods:The minimum inhibitory concentrations of linezolid and fosfomycin against eight hospital-isolated enterococcus were determined according to the agar dilution method,and whether the combination of the two drugs had a synergistic effect was evaluated by the checkerboard assay and in vitro static bactericidal assay.In the in vitro PK/PD model,linezolid simulated steady-state dosing concentrations（4,8,12 mg/L）at the standard dose of 600 mg q12h,and fosfomycin simulated the dosing regimen of 4g,6g,8g,iv 0.5 h,q 8h in the clinic,and finally the above single-drug regimen was combined.Based on the above bactericidal experimental data,NONMEN software was used to establish a semi-mechanical PK/PD mathematical model to obtain parameters such as bacterial growth rate,maximum bactericidal rate and half of the drug concentration when the maximum bactericidal rate was reached,the mathematical model is then judged to be successful by fitting a goodness-of-fit plot and visual prediction checking（VPC）.Finally,the success of the extrapolation experiments of the mathematical model was verified as a simulation tool to further predict the efficacy of single drugs and combinations.Results:Among the 8 strains of enterococcus,2 strains were resistant to linezolid(MIC_LZD≥8 mg/L)and 1 strain was resistant to fosfomycin(MIC_FOF≥256 mg/L).The results of the checkerboard method showed that linezolid combined with fosfomycin had a synergistic effect on 7 strains of enterococcus and 1 strain had an additive effect,no antagonistic effect was observed.In the in vitro static bactericidal assay,both linezolid and fosfomycin alone had no bactericidal effect,while theΔlog CFU_0-24of the combined administration were-2.18±0.08,-2.13±0.09 and-2.29±0.13 against three strains of enterococcus,respectively.The results of in vitro PK/PD model showed that fosfomycin alone was resistant to all drugs at 24 h.The best bactericidal dosing regimen of linezolid was 12 mg/L,withΔlog CFU_0-24of-0.98 and-1.09 for the 2clinical isolates,respectively.The results of co-administration showed that theΔlog CFU_0-24of linezolid（8 mg/L）combined with fosfomycin（6 g,iv 0.5h,q 8h）on three clinical isolates and the standard strain ATCC 29212 were-3.39,-2.24,-2.26 and-2.31,respectively.TheΔlog CFU_0-24of linezolid（12 mg/L）combined with fosfomycin（8 g,iv 0.5h,q 8h）on the 2 clinical isolates were-3.00,-3.07.In the semi-mechanical PK/PD model building and validation,the goodness-of-fit plots showed that the predicted and measured values were basically close,and the visual prediction check（VPC）plots indicated that the 95%confidence interval（CI）of the model predictions covered almost all the observed data,indicating successful model building.Extrapolation experiments of the mathematical model revealed a consistent decreasing trend between predicted and measured values of linezolid（12 mg/L）combined with fosfomycin（8 g,iv 0.5h,q 8h）on the standard strain ATCC 29212,with the maximum difference between them controlled between 1 log₁₀CFU/m L,confirming the value of the model as a tool to predict the bactericidal effect of newly administered doses.In the NONMEN software,prediction of new dosing regimens revealed that linezolid（≥16mg/L）combined with fosfomycin（6 g or 10 g,iv 0.5h,q8h）could provide a 4 log₁₀CFU/m L reduction at 24h relative to the initial bacterial load for 2 clinical isolates.Conclusions:（1）Checkerboard method,in vitro static time-kill experiments showed that linezolid combined with fosfomycin can play a synergistic bactericidal effect within the achievable concentration range in human;（2）For sensitive or resistant enterococcus,the results of in vitro dynamic time-kill experiments recommended that the dosing regimen of linezolid（8 or 12 mg/L）combined with fosfomycin（6 g or 8 g iv 0.5h q 8h）can play a synergistic bactericidal and bactericidal effect;（3）The in vitro semi-mechanical PK/PD model can adequately characterize the bactericidal profile and has good external validation results,which can be used as a simulation tool for future dose optimization.The model prediction results indicated that in the co-administration regimen,the concentration of linezolid was increased above 16 mg/L to provide a better synergistic bactericidal effect,but more in vivo experiments are needed for validation.