Joint Application Of Antibiotics Reduce Mutant Selection Windows Of The Antibacterial Agents Against Bacterium

BackgroundAntimicrobial resistance is a complex problem that is likely to require attention at many levels. Issues concerning dosing are addressed by the mutant selection window hypothesis. This hypothesis maintains that drug-resistant mutant subpopulations present prior to initiation of antimicrobial treatment are enriched and amplified during therapy when antimicrobial concentrations fall within a specific range (the mutant selection window). The upper boundary of the mutant selection window is the MIC of the least drug-susceptible mutant subpopulation, a value called the mutant prevention concentration (MPC). Closing or narrow MSW would help to avoid occurrence of drug resistance. When two antibacterial agents with different mechanisms of action and higher than their own MIC were used jointly, the bacteria required two drug resistance mutations at the same time in order to close MSW and control resistance of bacterium effectively.ObjectiveTo explore whether two antibiotics combined will reduce the mutant selection window and control resistance of bacterium effectively in vitro and in the animal vivo. We also observed the mechanism of resistance for the accumulated and increased drug-resistant bacteria in the MSW.Method1. In vitro test:We selected three kinds of standard strains which were pseudomonas aeruginosa (PA), staphylococcus aureus(SA) and Escherichia coli(ECO) to experiment.1.1 PA:Choose ceftazidime(CAZ), amikacin(AMK) and ciprofloxacin(CIP) as the experimental antibiotics. Determine the minimal inhibitory concentration (MIC) of CIP,AMK,CAZ alone by agar plates dilution method, and the combined MIC was determined by checkerboard method. The MPC both sole and combined were determined by the method of agar plates dilution method, and then the value of SI (MPC/MIC) will be acquired. According to the degree that SI drops to determine whether reduce the MSW.1.2 SA:Determine the MIC and MPC of vancomycin to SA by E-test and agar plates dilution method. Use checkerboard method and agar plates dilution method to determine the MIC and MPC of vancomycin to SA after combining leofloxacin and rifamycin, so as to observe the value of SI.1.3 ECO:Choose CIP and AMK as the experimental antibiotics. Determine the MIC of CIP and AMK alone by agar plates dilution method, and the combined MIC was determined by checkerboard method. The MPC both sole and combined were determined by the method of agar plates dilution method, and then the value of SI will be acquired.2. Mechanism of resistance:We selected the standard strain of PA, and observed the role of active efflux mechanism in the mechanism of resistance for the accumulated and increased drug-resistant PA in the MSW.2.1 E-test was used to determine the MIC of four single uses which were MEM, CAZ, AMK and CIP.2.2 Checkerboard test was employed to determine the MIC of joint use between anti-PA agents and efflux pump inhibitors (EPI). We selected five kinds of EPI which were Reserpine, Omeprazole, Azithromycin, CCCP and PAβN.2.3 Agar plate double dilution was adapted to detect MPC for single use of antibacterial agents and their joint use. After their accordant SI were calculated, the capacity to reduce drug-resistant mutants was determined based on the decrease in SI. Meanwhile, different SI were tested before and after MEM, CAZ, CIP used with AMK respectively and AMK used with CIP jointly. Then different capacity to prevent drug resistance mutations of PA was compared between joint use antibacterial agents and with EPI, so as to observe the role of active efflux mechanism in the mechanism of resistance for the accumulated and increased drug-resistant PA in the MSW.3. Animal experiment:We observed that whether CIP combined tobramycin will reduce the MSW of CIP to PA and control the resistance in rabbit.3.1 Construct the rabbit tissue cage model.3.2 The pharmacokinetic parameters in rabbit of CIP were determined by high performance liquid chromatography.3.3 Grouping:Fifty-five rabbits were randomly divided into eleven groups by random number table. One group was used physiological saline. Five groups were used CIP single; other five groups were applied CIP combined tobramycin.1010cfu/ml concentrations of ATCC27853 were injected into the rabbit tissue cages.3.4 Dosage regimen:design the dosage regimen according to the pharmacokinetic parameters in rabbit of CIP. Inject into the edges of rabbit ears for 3 days and 10 times a day. The dosing interval was 2 hours. Both single and combined groups, the steady state concentration of CIP reached 1MIC,2MIC,4MIC,8MIC and 16MIC respectively. The dosage regimen of tobramycin according to the reference, and made its steady state concentration reached 2-4MIC. Three days after we extracted the tissue juices, then diluted the tissue juices and coated on agar plate, so as to observe the growing condition of mutants.Result1. In vitro test:1.1 PA:The sole SI:CIP(16),AMk(16),CAZ(>32). The two drugs combined SI: ciprofloxacin-amikacin(CIP-8;AMK-4);ciprofloxacin-ceftazidime(CIP-4;CAZ-8);ceft azidime-amikacin(AMK-8;CAZ-4).1.2 SA:The MIC and MPC of vancomycin to SA were 1.0μg/mL and 51.2μg/mL. The SI were 51.2. The MPC and SI of vancomycin combined with rifamycin and leofloxacin were 8.0μg/mL,16; 12.8μg/mL,25.6.1.3 ECO:The sole SI of two antibiotics were 16(CIP) and 32(AMK). When two antibiotics combined, if the concentrations of AMK reach 2MIC and 2MIC concentration of CIP can restrain the mutants. After these two antibiotics combined, the value of SI come to be 8and 8.2. Mechanism of resistanceThe SI for four single antibacterial agents of MEM, CAZ, AMK and CIP were>32, >32,16,16; for MEM, CAZ, AMK, CIP combining with Reserpine were 16,16,16 and 8 respectively; combining with Omeprazole were 16,32,16 and 8 respectively; combining with Azithromycin were 8,16,16 and 8 respectively; combining with CCCP were 8,8,16 and 8 respectively; combining with PAβN were 8,16,16 and 8 respectively. The SI for MEM, CAZ and CIP combining with AMK were 4,4 and 8 respectively. When Amikacin was combined with Ciprofloxacin, its SI was 4.3. Animal experiment3.1 Single groups:There were mutants grew on agar plates of 1MIC,2MIC, 4MIC and 8MIC groups, and the colony number dropped off along with the raise of concentration of CIP. When the concentration of CIP reached 16 MIC, none of mutants was aroused.3.2 Combined groups:There were mutants grew on agar plates of 1MIC and 2MIC groups. There was no mutant on agar plates of 4MIC,8MIC and 16MIC groups.ConclusionBoth in vitro and vivo, two antibiotics combined will reduce the MSW of bacterium, so as to control the resistance effectively. Although joint application of antibacterial agents and EPI could narrow MSW of some antibacterial agents, it had an insignificant effect, which was far weaker than joint use with another antibacterial agent. This showed that, for the accumulated and increased drug-resistant bacteria in the MSW, neither their active efflux system was activated extensively nor new efflux system was generated under the pressure of antibacterial agents with concentration higher than MIC.