Studies For Decreasing Mutant Prevention Concentration Of Staphylococcus Aureus Against Vancomycin Alone And In Combination With Other Antibiotics In Vitro

Objective:To study the comparison of vancomycin (VAN) separately combined with levofloxacin(LVX), rifampincin (RFP) and fosfomycin (FOM) for decreasing mutant preventionconcentration (MPC) of Staphylococcus aureus ATCC29213in vitro.To evaluate the mutant prevention concentrations of vancomycin alone and incombination with rifampicin and fosfomycin against Methicillin-resistantStaphylococcus aureus (MRSA) in vitro, which direct rational applications of availableantibiotics in clinic practice and provide evidence for new strategies for restricting thedevelopment of resistance.Meterials and Methods:Staphylococcus aureus ATCC29213was saved in An Hui Antimicrobial ResistanceInvestigation Center.10clinical isolates of Staphylococcus aureus were collected in thecenter from2009to2010.M-H agar dilution method was used to determine minimum inhibitory concentration(MIC) of VAN、LVX、RFP and FOM alone against Staphylococcus aureus ATCC29213 and10clinical isolates of MRSA.The mutant prevention concentration (MPC) of each agent and combination of themwere tested by the M-H agar dilution method. The Staphylococcus aureus strainATCC29213was enriched the concentration to1010colony forming units per milliliterin broth. High-density cultures were prepared from overnight cultures grown in MHagar medium followed of incubation with shaking at37℃. Inoculateddrugs-impregnated plates were incubated for72h, and the MPC was recorded as thelowest concentration completely inhibiting bacterial growth at this time．The fraction of bacteria recovered curve was traced by colony counting method.The cells of1010colony forming units per milliliter coccus were enriched in broth.Mutant prevention concentrations of the above drugs for10strains of MRSA weredetected with agar dilution method, and value of selectivity index and drug-resistantfrequencies were calculated.Results:MIC of VAN, LVX, RFP and FOM for Staphylococcus aureus strain ATCC29213were1μg/ml,0.125μg/ml,0.008μg/ml,2μg/ml; MPC were6μg/ml,4μg/ml,2μg/ml,>32μg/ml,64μg/ml; and MSW(MPC/MIC) were64,16,>4000,32; MPC(μg/ml) ofeach alone in combination were: VAN+LVX (MPCVAN–4, MPCLVX–0.125),VAN+RFP(MPCVAN–4,MPCRFP–0.008), VAN+FOM(MPCVAN-1, MPCFOM-2).VAN combined with LVX and RFP can decrease to MPCVANused alone1/16times,VAN combined with FOM can decrease to MPCVANused alone1/64times. FOMcombined with the concentration of4.8μg/ml (8MIC×60%) of VAN can decrease MPCFOSto2μg/ml and narrow MSW of FOM used alone1/32times. VAN combinedwith the concentration of28.8μg/ml (16MIC×90%) of FOM can decrease MPCVANto1μg/ml and narrow MSW of VAN used alone1/64times.The fraction of bacteria recovered curve showed that the MSW of FOM combinedwith1MICVAN was narrower than that of FOM used alone; The fraction of bacteriarecovered curve also showed that the MSW of VAN combined with1MICFOM wasnarrower than that of VAN used alone.The selectivity index of vancomycin alone for10strains of MRSA were16~64, andselectivity index of the combination application with rifampicin and fosfomycin forMRSA were2~16and1~8respectively, the selectivity index for the combinationapplication decreased2to32-fold, drug-resistant frequencies were also significantlylower than the single application of vancomycin. The differences all had significance.(P<0.01)Conclusion:Compared with each one alone, the combination of VAN separately with other threeantibiotics can obviously decrease MPCVAN. The combination of VAN and FOM wasmore evidently in dropping MPC and narrowing MSW of VAN.Different concentrations of combination of FOM and VAN can obviously decrease theirMPC. The most dramatically scheme can drop MPC equal to MIC. The combination ofthese two drugs can narrow or even close their MSW and prevent the drug-resistantmutants. Combinations use of antimicrobial agents can decrease the mutant preventionconcentration for10strains of MRSA, and narrow the mutant selection window, so theemergences of resistant strains are decreased.