Study On MPC Of Levofloxacin Combined With Fosfomycin Against Staphylococcus Aureus And Study On PK/PD Parameters Of Levofloxacin Predict The Emergence Of Resistant Mutants In Rabbit Infection Model

【Objective】To investigate the in vitro effects of levofloxacin combined with fosfomycin upon 30 clinical isolates of Staphylococcus aureus (15 methicillin resistant and 15 methicillin sensitive Staphylococcus aureus). To investigate the in vitro bactericidal activity of levofloxacin alone and combined with fosfomycin against 3 strains MSSA and 1 stain MRSA. To evaluate the mutant prevention concentrations of levofloxacin alone and in combination with fosfomycin against staphylococcus aureaes. To predict the emergence of resistant mutant in rabbit tissue cage infection model using the PK/PD parameters.【METHOD】(1) A checkerboard method that adhered to the recommendations of Clinical Laboratory Standards Institute(CLSI) was applied to assess the synergism effect between levofloxacin and fosfomycin, the Fraction Inhibitory Concentration Index(FICI) was calculated according to the results. (2) Three MSSA strains and one MRSA from clinical specimens were used in this study. Time-kill curve method was carried out with different concentration of the two drugs both alone and in combination. (3)MPC of the two drugs against S.aureaes was determined by agar dilution method,The mutantal frequency of levofloxacin and fosfomycin were calculated, (4) Two S.aureaes strains which have same MIC and different MPC to levofloxacin were enriched in broth and infected the rabbit tissue cage. Infected rabbits were treated orally with various doses of levofloxacin for 5 days, lml tissue fluid was drew from the tissue cage after drug administration 2,4,6,8,10 hours. The concentrations of levofloxacin was determined by High Performance Liquid Chromatographic method. The PK/PD parameters were calculated. Both bacterial susceptibility and viable bacteria inside the tissue cage were monitored.【RESULT】(1) The FICI of MSSA less than 0.5,from 0.5 to 4, more than 4 were 40%, 60%,0 respectively. The FICI of MRSA less than 0.5,from 0.5 to 4, more than 4 were 73.8,26.2,0 respectively. (2)Levoloxacin alone showed a concentration-dependent time-kill curve against three strains of MSSA, fosfomycin showed a nonconcentration-dependent time-kill curve. Neither levofloxacin nor fosfomycin alone inhibited bacterial growth at 1/4 MIC; but the combination of this two drugs at 1/4 the respective MIC showed a synergistic bactericidal effect, a 2～3 log10 decrease with respect to the most active antibiotic alone. (3) The MPC/MIC ratios of levofloxacin alone for 10 strains of MSSA and 10 srains MRSA were 4～64,4～16 respectively,The MPC/MIC ratios of the combination were 2～8,1～8 respectively, the ratios for the combination decreased 2-8 fold and drug mutantal frequencies were also lower than the single application of levofloxacin for 10～102. (4) There were 11 and 13 rabbits who had isolated resistant mutants in the two group. When AUC/MPC and Cmax/MPC were above 21.4 and 1.7 in SA99 group, and when above 18.2,1.12 in SA6648 group, completed eradication occurred and no decrease in susceptibility was observed. On the other hand, when AUC/MPC and Cmax/MPC were below the above values, the susceptibility decreased.T>MPC and Tmsw did not show any correlation with the emergence of resistance. The AUC/MIC and Cmax/MIC were less predictive than the AUC/MPC and Cmax/MPC to predict the emergence of resistance. There were no resistance emergence when the whole serum concentration was below the MIC. Target genes mutations within grlA (ser80-phe, Glu84→Lys) and mutations within gyrA (Ser84→Leu,Asp73→Gly) were associated with levofloxacin resistance. AUC/MPC and Cmax/MPC were more stable and more correct than AUC/MIC and Cmax/MIC to predict the resistance of S.aureaes.【CONCLUSION】In vitro the combination of levofloxacin and fosfomycin presented synergistic and indifferent action.There was no antagonistic action. The combination of subinhibitory concentrations of levofloxacin and fosfomycin presented synergism,exerting a bactericidal effect. The combination also has a bactericidal effect against MRSA. Combination use of antimicrobial agents can narrow the Mutant Selection Window and reduce the MPC/MIC ratios. The emergence of resistant strains was decreased. A MPC-based threshold (for example, AUC24/MPC) is more suitable than a MIC-based parameters (for example, AUC24/MIC) as a target parameter for blocking resistant subpopulation proliferation.