Comparison Of Etimicin In Combination With Meropenem Or Piperacillin/ Tazobactam Against Pseudomonas Aeruginosa

Objective Recent years, with the extensive use of broad-spectrum antimicrobial agents, the resistant rate of Pseudomonas aeruginosa(PA) was gradually increased. The comparison of aminoglycesides combined with carbapenems, β-lactams or others had been studied by some researchers before, but the researches just studied for the minimum inhibitory concentration(MIC) of drugs without the mutant prevention concentration(MPC). The main contents of MSW theory included: When the concentration of drug < MIC,it won’t get treatment. When the concentration > MPC, the strains would continue to alive with mutated 2 times and more. When the concentration was in the range of MSW, it could achieve a good treatment, but the mutants would present as well. In order to solve the above-mentioned problems, the comparison of etimicin in combination with meropenem or piperacillin/ tazobactam against PA has been studied in our studies, according to the theory of mutant selection window(MSW) and some references. And to achieve the following objectives: 1.To determine MIC and MPC of PA used etimicin(ETM) combined with meropenem(MEM) or piperacillin/ tazobactam(TZP), so as to discuss the change of MSW in vitro. And to determine reasonable combined therapy schemes by evaluating the activity of ETM combined with MEM and TZP respectively.2.To study mechanism of ETM treated with MEM or TZP resistance in PA. To reveal the mechanism of drug resistance at the genetic level to provide a scientific theory basis for safe and effective combination treatments in our works, the development of new drug and so on.Methods 1.The MIC was determined by checkerboard microdilution assay, and the fractional inhibitory concentration index was calculated according to MIC. And drew the time-killing carves by counting the colonies. 2.The MPC was admitted by agar plate dilution assay, which inoculated ≥ 1010 CFU ? m L-1 clinical isolates enriched in broth onto a series of drug-containing agar plates, and the selection index was calculated. 3.The resistant genes of PA for aminoglycoside, carbapenem and β-lactamases agents were determined by PCR and DNA sequence analyses.Results 1.The MIC of the standard strains treated with ETM, MEM or TZP alone was 1μg?m L-1, 0.5μg?m L-1, and 1/0.125μg?m L-1 respectively. Compared with the single treatment, the MIC and MPC reduced evidently when ETM combined with MEM or TZP, and there was synergistic effect. 2.The MICs range of ETM, MEM and TZP alone were 4~64μg?m L-1, 0.5 ~ 16μg ? m L-1, 8/1 ~ 32/4μg ? m L-1 respectively. The synergies of 37 multi-drug resistance pseudomonas aeruginosa(MDRP) strains was 43.24%, 37.84%, additive effect was 32.43%, 35.14%, respectively, with no antagonistic effect after ETM+MEM or ETM+TZP. The selection index of MDRP isolates treated with ETM, MEM or TZP were 8~32, 8~32 and 8~32. The selection index of ETM reduced to 1~8 when treated with ETM+MEM or ETM+TZP, and the SI of MEM or TZP reduced to 1~8 and 1~16, compared with the single treatment. 3.The result showed that 43.2% of 37 strains of MDRP carried 2 or more genes. The genes of ATCC27853 mutants selected by TZP contained TEM gene. For the MDRP mutants treated by ETM, MEM, TZP, ETM+MEM and ETM+TZP, respectively, the strains carried 2 or more genes rates were 62.2%, 64.9%, 3.0%, 45.9%, 48.6%. 4. Compared with Gen Bank: L25666.1, M29695.1, DQ842025.1, JN188366.1 and AF074954.1, the rates of 100% homologous carried aac(6’)-Ib, aac(6’)-II, bla IMP-1, TEM and SHV genes were 81.5%(66/81), 86.9%(73/84), 87.7%(50/57), 90.2%(74/82) and 95.3%(1/64), respectively, by gene sequencing. The new mutant subtypes of resistant strains were present. There were 3 kinds mutantype for mutants carried aac(6’)-Ib gene, and for mutants carried aac(6’)-Ib aac(6’)-II, bla IMP-1, TEM and SHV, there were 2 kinds of mutantype respectively.Conclusions There was synergistic/addictive effect against PA strains after ETM combined with MEM or TZP. These results suggest that the combination treatment of ETM+MEM and ETM+TZP against PA might narrow the MSW, so as to prevent the generation of drug-resistant mutants. The mutant rate of 37 MDRP could be reduced when treated with ETM+MEM or ETM+TZP. The results showed that gene mutations were present for some strains. But compared with single therapy, combination therapy could produced a lower incidence of mutations. In short, it is very important to determine reasonable combined therapy schemes and provide the laboratory evidence for preventing the bacterial resistance.