In Vitro And Vivo Pharmacodynamics Of Linezolid Combined With Fosfomycin Against MRSA

Objectives:Methicillin-resistant Staphylococcus aureus（MRSA）is one of the common pathogens in hospitals and communities.It is characterized by high morbidity,high mortality and susceptibility to drug resistance,making it impossible for existing antibiotics to meet the demand for treatment of MRSA infections.In this study,the human simulated regimens of linezolid combined with fosfomycin was used in an in vitro PK/PD model and in vivo animal infection model.The purpose of this study was to discuss the combined bactericidal effect of MRSA and the optimization of dosage regimens,so as to provide support for clinical drug administration.Methods:The synergistic effect of linezolid/fosfomycin combination therapy on eight MRSA isolates was evaluated by in vitro susceptibility testing and checkerboard studies.The strain with the best synergistic effect was selected to simulate linezolid600mg iv 0.5h q12h(Ke=0.144h^-1)and fosfomycin 1g iv 0.5h q8h or 2g iv 0.5h q8h(Ke=0.0693h^-1)in an in vitro PK/PD model for single and combination therapy of MRSA high-load inoculation.The total population and the 2,4,8,16×MIC resistant subpopulations were used as pharmacodynamic indexes for dose optimization.Based on the above optimized drug regimen,the in vivo synergistic bactericidal was further studied in murine bacteremia model.Finally,the resistance genes of Linezolid（23S r RNA domain,ribosome protein L3,L4 domain,methyltransferase gene cfr and abc transporter gene optr A）and fosfomycin（Fos B,Mur A,Glp T and Uhp T）were detected after 72h in vitro dynamic time-kill study to explore the effect of combined administration on the mechanism of drug resistance.Results:Eight MRSA strains were all susceptible to linezolid and fosfomycin.The results of the checkerboard method showed that combination therapy had a synergistic effect on 7 strains and 1 strain had an additive effect.The linezolid/fosfomycin combination was synergistic against all isolates in checkerboard studies.The strain NO.8 was selected(MIC_LZD=1 mg/L;MIC_FOF=2 mg/L)for in vitro and in vivo dynamic time-kill study.TheΔlog CFU_0-24of the dose of linezolid 600 mg q12h were-1.18 log₁₀CFU/m L in vitro PK/PD model and bacteremia model,indicating that linezolid monotherapy exerted a sustained bacteriostasis effect against MRSA.Compared with linezolid,fosfomycin monotherapy（1 g q8h or 2 g q8h）produced faster initial killing in the first 8h(0.69-1.28 log₁₀CFU/m L)in the in vitro PK/PD model,followed by steady regrowth.While theΔlog CFU_0-72of linezolid（600 mg q12h）combined with fosfomycin（1 g or 2 g）were-1.42log₁₀and-2.39log₁₀CFU/m L,respectively.In vivo murine bacteremia model,the blood colony number of control mice was 4.22±0.18log₁₀CFU/m L at 0h and gradually increased to 6.23±0.45 log10 CFU/m L.All control mice died at 24 h.However,the mice treated with the linezolid（600mg q12h）,fosfomycin（2g q8h）alone and in combination still survived at 24 h.TheΔlog CFU_0-24of linezolid,fosfomycin alone and combined groups in the blood of mice were-0.90±0.11,0.01±0.05 and-2.06±0.15,respectively.For resistance study,fosfomycin prevented the emergence of 2×MIC mutants resistant to linezolid at 72 h.While linezolid can only reduce the enrichment of 2×,4×,8×and 16×MIC fosfomycin-resistant subpopulations,but not inhibit their emergence.Moreover,combination therapy also inhibited the emergence of fosfomycin resistance genes compared to fosfomycin monotherapy.2×MIC mutants resistant to linezolid carried 23Sr RNA resistance genes in linezolid monotherapy group,while no linezolid-related resistance genes were detected in the combination group.After fosfomycin monotherapy,only 16×MIC fosfomycin-resistant mutants amplified positive fragments and its resistance genes of fosfomywere Mur A,Glp T and Uhp T,while only Uhp T resistance genes were detected after combination therapy.Conclusions:（1）In vitro PK/PD dynamic time-kill study showed that linezolid（600mg q12h）combined with fosfomycin 1 g or 2 g q8h had synergistic bacteriogenic effect on MRSA.（2）In vivo animal experiments suggested that linezolid（600mg）combined with fosfomycin（2g）had better synergistic bactericidal effect than in vitro.（3）Linezolid combined with fosfomycin can inhibit the mutation of MRSA-related resistance genes and the enrichment of resistance subpopulations in vitro,but more in vivo experiments are needed for verification,so as to provide evidence for clinical drug use.