In Vitro Study On The Influence Of Different Vancomycin Combinations And Bacterial Density On Mutant Prevention Concentrations Of Vancomycin Against Methicillin-resistant Staphylococcus Epidermidis

ObjectiveTo determine the mutant prevention concentrations (MPCs) of vancomycin alone and in combination with rifampicin, levofloxacin or fosfomycin against methicillin-resistant Staphylococcus epidermidis (MRSE) in vitro and provide theoretical evidence for optimal antimicrobial therapy.Methods15strains of Staphylococcus epidermidis collected in Anhui Center for Surveillance of Bacterial Resistance in2010were randomly chosen and Specific identification of MRSE was conducted using mecA gene sequences, intragenic primer sets for staphylococcus16SrRNA and S. epidermidis species-specific gene sequence.MICs against MRSE isolates were determined by the standard doubling dilution method on M-H agar.Checkerboard synergy testing was conducted for susceptible strains and the fractional inhibitory concentration index (FICI) was calculated to know better the interactivity between vancomycin and its partner.Bacterial suspension of1010CFU/ml was enriched in M-H broth. Mutant prevention concentrations of vancomycin, rifampicin, levofloxacin and fosfomycin individually and in vancomycin combination against susceptible MRSE isolates were determined using doubling-dilution method on M-H agar. The concentrations of levofloxacin (2mg/L) and rifampicin (2or4mg/L) used in combination with vancomycin reflect their mean serum concentrations at steady state in healthy adults and adult pulmonary tuberculosis patients, respectively. The corresponding dosing regimens are500mg po or i.v. once daily for levofloxacin and450or600mg po or i.v. once daily for rifampicin. The concentration of fosfomycin used in combination with vancomycin equals the susceptible breakpoint concentration of staphylococcus to fosfomycin. The concentrations of rifampicin, levofloxacin and fosfomycin used in combination with vancomycin were all below respective MPCs.Cells recovered from the highest antibiotic concentration after72h incubation were passaged twice on drug-free M-H agar before retesing putative mutant for susceptibility. If antibiotic MIC against the cells recovered from the highest antibiotic concentration after72h incubation was below the corresponding resistant breakpoint concentration1010CFU were challenged by higher antibiotic concentration (i.e.,50%×,60%×,70%×,80%×,90%×,100%×MPC) to see whether higher antibiotic MIC against mutant would result.ResultsTotally10strains of MRSE were confirmed.The susceptibility rates of10MRSE isolates to vancomycin, rifampicin, levofloxacin and fosfomycin were100%,100%,50%and90%, respectively.The fractional inhibition concentration indices (FICI) for vancomycin combination with rifampicin, levofloxacin or fosfomycin were≥1.5but≤2,≥1.5but≤2, and>0.5but≤1.5, respectively, implying indifferent interactivity.The MPCs against susceptible MRSE strains were16～32mg/L,>64mg/L,4～16mg/L and≥64mg/L for vancomycin, rifampicin, levofloxacin and fosfomycin, respectively. Against mutants selected for by vancomycin, rifampicin, levofloxacin and fosfomycin individually antibiotics had standard MICs of2-4mg/L for vancomycin,>64mg/L for rifampicin,4～8mg/L for levofloxacin and≥64mg/L for fosfomycin, respectively.The MPCs of vancomycin combination with rifampicin (2or4mg/L), levofloxacin (2mg/L) and fosfomycin (32mg/L) were16～32mg/L,16～32mg/L and1～4mg/L, respectively.ConclusionSingle-step mutation can lead to resistance of MRSE to rifampicin, levofloxacin and fosfomycin rather than non-susceptibility to vancomycin. The MPCs of vancomycin can be greatly reduced when combined with fosfomycin. ObjectiveTo explore the influence of different bacterial density with fixed inoculum size on the mutant prevention concentrations (MPCs) of vancomycin against methicillin-resistant Staphylococcus epidermidis (MRSE).Methods10MRSE isolates collected in Anhui Center for Surveillance of Bacterial Resistance in2010were chosen for the study. Specific identification of MRSE had been conducted previously using mecA gene sequences, intragenic primer sets for staphylococcus16SrRNA and S. epidermidis species-specific gene sequence.Vancomycin MICs against MRSE isolates were determined by the standard doubling dilution method on M-H agar.Bacterial suspension of1010CFU/ml was enriched in M-H broth. Approximately1.2×1010CFU were diluted into200and30ml M-H broth containing known vancomycin concentrations (doubling dilutions) to produce bacterial density of6.0×107and4.0×108CFU/ml, respectively. The lowest drug concentration blocking visible growth in MHB after36h incubation with shaking at37℃was defined as MPC.Cells recovered from the highest vancomycin concentrations after36h incubation (i.e., 1/2MPC) were diluted (to eliminate the carryover effects of vancomycin) and passaged twice in drug-free culture (M-H agar and M-H broth successively) before retesting putative nutants for susceptibility. If vancomycin MICs against the cells recovered from1/2MPC were below the MPCs,1.2×1010CFU were challenged by50%×,60%×,70%×,80%×,90%×, and100%×MPC to see whether higher vancomycin MIC against mutant would result.ResultThe vancimycin MICs against the10MRSE isolates were0.5or1mg/L.The MPCs were2or4mg/L and8or16mg/L when determined by bacterial density of～6×107and～4×108CFU/ml, respectively.For bacterial density of6×107CFU/ml, against cells recovered from1/2MPC vancomycin had MICs identical to the MPCs against the corresponding parental strains. For bacterial density of4×108CFU/ml, against cells recovered from1/2MPC vancomycin had wild-type MICs, but higher vancomycin concentration (i.e.,60%×,70%×,80%×,90%×MPC) can select mutants against which vancomycin MIC was2or4mg/L. For a given parental strain of MRSE, against the least susceptible single-step mutants selected from different density vancomycin had identical susceptibility. In contrast to the parental strains, vancomycin MICs against the single-step mutants usually increased by at least4fold (with the exceptions of2strains).ConclusionVancomycin MPC against MRSE increases with bacterial density, so MPC should be regarded as the actual rather than the theoretically standard low-inoculum MIC. Against the mutants selected from different bacterial density with an inoculum size of1010CFU vancomycin has identical susceptibility.A large enough inoculum size is the premise to select for mutant, but bacterial density decides the position of the mutant selection window.Non-susceptibility of MRSE to vancomycin cannot be acquired through single-step mutation.