The Study Of Post-antibiotic Effect And Mutant Prevention Concentration Against Multidrug-Resistant Pseudomonas Aeruginosa

Objective:To explore the differences between Amikacin,Isepamicin alone and combined with piperacillin-tazobactam respectively in PAE,MPC,MPC/MIC,MSW against the clinic isolated strains of MRPA in our hospital, based on determining the MICs, PAEs and MPCs in vitro, Providing the laboratory evidence on preventing antibiotics resistance for guiding antibiotic therapy and controlling nosocomial infections.Methods:1 the MICs of Amikacin,Isepamicin against the MRPA were determined by agar plates dilution method : 1.5μl 1.5×10⁷cfu/ml bacterial liquid of the MRPA were plated in M-H agar plates contained various concentrations of Amikacin,Isepamicin,and the MICs were defined as the lowest concentration of the antibiotics that prevented visible growth after16～20h incubation at 35°C.2 MICs of Amikacin combined with piperacillin-tazobactam, Isepamicin combined with piperacillin-tazobactam for the MRPA were determined in cation-supplemented M-H broth using the micro-dilution technique: based on their proven susceptibility to Amikacin, Isepamicin and piperacillin-tazobactam evaluated in the study, the two drugs (respectively 50μl )and samples with an inoculum of 5×10⁵cfu/ml (100μl) were added to series of test wells investigated by checkerboard technique (drug A(B) at the same concentration was combined with drug B(A) in series of concentrations).The combined MICs were defined as combined concentration of the antibiotics at the lowest FIC ratio that prevented visible growth after 16～20h incubation at 35°C. 3 the PAE determinations against the MRPA were evaluated in M-H agar by colony-counting method:cell suspensions of 106～l07cfu/ml in the late logarithmic phase of growth were exposed 2 hours to Amikacin, Isepamicin, piperacillin-tazobactam,Amikacin combined with piperacillin-tazobactam, Isepamicin combined with piperacillin-tazobactam at concentrations of 1/2MIC,1MIC,2MIC,4MIC without shaking,Controls not exposed to the antibiotics were treated similarly. The antibiotics were removed by centrifugation (4000r/min 10 min), washed and centrifuged again, which was repeated in triplicate. Control assays with untreated isolates were performed in the same fashion. At 0, 1, 2, 3, 4, 5 and 6 h of incubation, 0.1mL aliquots were plated in triplicate on pre-reduced nutrition agar and streaked for individual colony counts. All agar plates were incubated aerobically at 35℃for 16～20 h and colony counts determined. The PAE was defined as the time difference (in hours) for antibiotic-treated organisms to increase in number by 1 log10 minus the same determination for non-treated cultures of the same test organism.4 The MPCs of Amikacin, Isepamicin alone and combined with piperacillin-tazobactam against the MRPA were determined by agar plates dilution method: the cells of 3×10¹⁰cfu/ml MRPA were enriched in MH broth .100μl bacterial liquid of MRPA were plated in agar plates containing various concentrations of amikacin, Isepamicin alone or combined with piperacillin-tazobactam.1.2×10¹⁰cfu bacterial liquid of MRPA were plated in each drug concentration and incubated at 35℃for 72 h. The lowest concentrations that no colonies grow on drug-containing agar plates were MPCs.5 To analyze MIC50, MIC₉₀, FIC, PAE50, PAE90, MPC50, MPC₉₀, MPC/MIC(selective index SI) of Amikacin,Isepamicin alone and combined with piperacillin-tazobactam against clinic isolated strains of MRPA and evaluate the influence of Amikacin,Isepamicin combined with piperacillin-tazobactam respectively on PAE,MPC,MPC/MIC MSW against MRPA.Results: 1 The MICs of the tested MRPA against Amikacin, Isepamicin alone and combined with piperacillin-tazobactam respectively are listed in Table 2. MIC50, MIC₉₀values for Amikacin in combined group was 8, 9.6fold lower than in single group, MIC50, MIC₉₀values for Isepamicin in combined group was 16, 16fold lower than in single group, MICmin, MICmax values for Amikacin in combined group was 32, 8fold lower than in single group, MICmin, MICmax values for Isepamicin in combined group was 15, 16fold lower than in single group. The difference of MIC between single drugs and drug combinations not only amikacin but isepamicin were significant, P<0.05. However, no other significant differences were observed between amikacin and isepamicin both single drugs and drug combinations.2 Synergy was defined as a FIC index≤0.5; plus was defined as FIC index>0.5～1;no interaction was defined as FIC index>1～2;and antagonism was defined as FIC index >2. Synergy was demonstrated in all MRPA strains with amikacin combined with piperacillin-tazobactam, isepamicin combined with piperacillin-tazobactam . No statistically significant differences were seen in FIC between the two groups.3 In same drugs but different concentration levels, statistically significant differences in PAEs were found among the four drugs and drug combinations, P<0.05. No statistically significant differences were seen in PAEs of amikacin and isepamicin in the same times MIC. Significant differences in PAEs were demonstrated between single drugs and drug combinations. Considerable differences in PAEs were observed at the same levels of 1/2MICcombined, 1MICcombined, 2MICcombined between two drug combinations, while they are plus effect ; No significant differences was demonstrated at 4MICcombined in their synergy and plus effect.4 The statistically significant differences were observed in SI when amikacin, isepamycin were compared among the four drugs and drug combinations. Considerable decreases in the amikacin MPC₉₀ and isepamicin MPC₉₀ were seen (10.3 fold, 16.1 fold respectively). MPC₉₀/MIC₉₀ in amikacin combined group was decreased from 18.13 to 15.36. MPC₉₀/MIC₉₀ in isepamicin combined group was decreased from 17.07 to 16. Significant difference was shown in the two groups. We divided the MPC results (interpreted according to CLSI methodology) to observe how the strains distribute, and the results were showed significantly differences among the four conditions. Evidently decreases were presented in drug combinations than single drugs respectively.Compared with drug alone, SI were decreased (major), non-changed, increased in combined group.Conclusions:1 The effect of synergy of both amikacin plus piperacillin-tazobactam and isepamicin plus piperacillin-tazobactam against MRPA was observed2 The effect of synergy could be demonstrated at 4MICcombined when amikacin, isepamicin Combined with piperacillin-tazobactam, plus effect at 1/2MICcombined, 1MICcombined, 2MICcombined.3 Combined with piperacillin-tazobactam could decrease the MPC of amikacin , isepamicin alone against MRPA , this decrease is helpful for the concentration of amikacin , isepamicin beyond MPC, and is in advantage to improve the ability of preventing drug resistant development. The decrease effect in MSW was more signifiant in amikacin combined with piperacillin-tazobactam than in isepamicin combined with piperacillin-tazobactam.4 The influence of amikacin, isepamicin combined with piperacillin-tazobactam on MSW of amikacin , isepamicin against MRPA included : narrowed(major),enlarged (single)and non-changed. The narrow MSW is in advantage to improve the ability of reducing the selective enrichment of resistant mutants, while enlarged MSW is reverse.