| Background and ObjectivesIn recent years,the increasing prevalence of carbapenem-resistant K.pneumoniae strains poses a serious threat to patients.In order to solve the problem of limited treatment options,the re-evaluation of some old agents are awaking.Fosfomycin,as one of the old neglected agents,has the merits of broad spectrum,better pharmacokinetics and safe characters.Moreover,it is highly effective in the treatment of multidrug-resistant(MDR)microorganisms.However,the treatment effect of fosfomycin monotherapy is not clear and it alone shows the rapid selection of resistant variants.Therefore,it is of great value to clear how to promote rational utilization of fosfomycin against MDR bacterial infections.The aim of this study was to examine the in vitro synergistic antibacterial effects of fosfomycin in combination with imipenem,ertapenem,tigecycline,colistin,or amikacin against KPC-producing K.pneumoniae.Then we evaluated the bactericidal effect of the optimal dosage regimen at different dose using dynamic model with simulating human pharmacokinetics in vitro,providing therapeutic advice for the clinical selection of antibiotics.Methods1.In the first section,a total of 170 consecutive nonduplicate clinical K.pneumoniae isolates with exclusion of sputum samples were collected from two hospitals in Beijing and Zhejiang province.Carbapenemase-producing isolates were identified by modified Hodge test and the positive strains were included in this study.Carbapenemase genes were detected by PCR and antimicrobial susceptibilities to 24 antimicrobial agents were determined.In addition,Multilocus Sequence Typing(MLST)was performed on all strains.2.In the second section,the chequerboard method were used to detect synergism between fosfomycin and imipenem,ertapenem,tigecycline,colistin,or amikacin against 136 isolates.Then bacteriacidal activity of fosfomycin alone and combination with other five antibiotics was evaluated by time-kill assays.At last,the Pharmacokinetics Auto Simulation System 400(PASS-400)system was employed to simulate different dosing regimens.Pharmacodynamic parameters were calculated to access the antibacterial effect.3.In the third section,we combined time-kill assays with genomic analysis.Samples were collected at different concentrations and time points during the time-kill assays and sequenced the whole genomes.The possible mechanisms of fosfomycin sensitivity variation were identified by comparative genomic analysis.Results1.One hundred and thirty-six KPC-producing K.pneumoniae strains were positive in modified Hodge test.One isolate was coexistence of blaKPC-2 and blaIMP-4,another was carrying both blaKPC-2 and blaIMP-8.According to the results of antimicrobial susceptibilities test,there were 83 multidrug-resistant strains and 53 extensively drug-resistant strains.A total of 18 ST types were identified and 6 novel ST types were submitted to pasteur database.The most prevalent was ST11(79.4%),followed by ST 15(6.6%),and others ST types rate below 1.5%,indicating clonal spread in the two hospitals.2.The chequerboard method revealed that five drug combinations had a significant additive effect against the 136 KPC-producing K.pneumoniae strains.Fosfomycin plus carbapenem(fosfomycin/imipenem and fosfomycin/ertapenem)showed the largest synergistic effect(against 15.44%and 22.06%of isolates,respectively).And higher in vitro synergistic activity was observed against fosfomycin-susceptible isolates(20.69%and 34.48%,respectively)than against-resistant isolates.Fosfomycin showed a concentration-dependent bactericidal activity in vitro.Neither fosfomycin(at 1×MIC)nor tigecycline(at 1 mg/1 and 1×MIC)showed an antibacterial effect.Likewise,imipenem or ertapenem(at 1×MIC)alone caused a 1 log 10 reduction in CFU/mL at 2 h.Colistin caused a 3 log 10 reduction in CFU/mL during the first hour of the experiment,although there was re-growth after 4 h.Amikacin(at 1 xMIC)alone showed a bactericidal effect against this isolate at 12 h.The combination of fosfomycin/colistin and fosfomycin/amikacin showed synergistic activity and the bactericidal activity enhancement was dependent on the concentration of fosfomycin.PASS-400 studies showed the killing effect of fosfomycin/colistin and fosfomycin/amikacin combination therapies were much more pronounced than monotherapies against sensitive strains.The area between the control growth and antibacterial killing curves(IE)of fosfomycin(8g,every 8 hours)plus amikacin(15 mg/kg,once-daily)and fosfomycin(8g,every 8 hours)plus colistin(2.5mg/kg,every 12 hours)were higher(>170 and>200 LogCFU/mL·h-1,respectively)than that of monotherapies against sensitive strains.3.The differential expression was only less than 5%at different concentrations and time points by comparative genomic analysis.Antibiotic resistance genes were analyzed and found one add antibiotic inactivation enzyme at 4×MIC at 4 hour and 8×MIC at 12 hour.In the isolates collected from 32xMIC concentration of fosfomycin at 6 hour and 12 hour,a short fragment of the uhpT gene was found upstream of the uhpT gene.ConclusionsIn conclusion,with the analysis of KPC-producing K.pneumoniae isolated from two hospitals in Beijing and Zhejiang province,we found clonal spread in two hospitals and all isolates were multidrug-resistant.Fosfomycin showed a concentration-dependent bactericidal activity against KPC-producing K.pneumoniae and the UhpT transporter played an important role in the change of susceptibility to fosfomycin.Fosfomycin combined with imipenem,ertapenem,tigecycline,colistin,or amikacin were much more pronounced than monotherapies.The combination of fosfomycin(8g,every 8 hours)plus colistin(2.5mg/kg,every 12 hours)and fosfomycin(8g,every 8 hours)plus amikacin(15 mg/kg,once-daily)were effective on maximizing bacterial killing. |
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