Study On In Vitro Antimicrobial Activity And Mutant Prevention Concentration Of Tigecycline Against Multidrug-resistant Acinetobacter Baumannii

Objective: To determine the minimal inhibitory concentration (MIC) of tigecyclineagainst multidrug-resistant Acinetobacter baumannii (MDR-AB), and to assess theclinical efficacy of tigecycline at different doses in treatment of MDR-AB pneumoniawith a PK/PD model using Monte-Carlo stimulation. To investigate the in vitro effectsof tigecycline combination usage against MDR-AB and to evaluate the effect of protonpump inhibitors (PPIs) on in vitro antimicrobial activity of tigecycline against severalcommon species of clinical pathogens. Based on the theory of MSW, MPCs oftigecycline alone and combination usage was to be measured, and to make a primaryresearch on the mechanisms of tigecycline resistance.Method:(1) The MICs of tigecycline against135MDR-AB strains were determined bythe standard two-fold agar dilution method. Pharmacodynamic exposures, measured asthe ratio of steady-state total drug area under the curve to MIC (AUC/MIC), weremodelled using Monte-Carlo simulation which incorporates pharmacokinetic data oftigecycline from patients with pneumonia, and the cumulative fraction of response (CFR)was calculated according to preset threshold of achieving a level of microbiologicefficacy.(2) A checkerboard method that adhered to the recommendations of ClinicalLaboratory Standards Institute (CLSI) was applied to assess the synergism effect oftigecycline combination usage, and the Fraction Inhibitory Concentration Index (FICI)was calculated according to the results.(3) The MICs of tigecycline alone and combinedwith PPIs against6common species of clinical pathogens were determined by thestandard two-fold agar dilution method, and Time-kill curve method was carried outwith tigecycline alone and combined with different concentrations of PPIs.(4) TheMICs and mutant prevention concentration (MPC) of tigecycline alone and combinedwith colistin E or sulbactam were measured through the two-fold agar dilution method, and then selection indexes (SI) were calculated.(5) The MICs of tigecycline alone andcombined with CCCP against resistant mutants selected during the process of testing theMPC were determined by two-fold agar dilution method, and RT-PCR was used toassess the relative quantitative expression of Ade B and Ade J efflux pump genes.Result:(1) Among135MDR-AB strains,97%isolates displayed susceptibility and3%were intermediate to tigecycline. According to Monte-Carlo simulation, A CFR of61.62%was predicted for tigecycline at current dosage (50mg q12h) and dosage increased to100mg q12h in this simulation can approach the clinical goal.(2) By combination usage,the MIC50of tigecyline against MDR-AB decreased dramatically. Tigecycline/colistincombination displayed that FICI≤0.5,0.5<FICI<1, FICI=1,1<FICI≤2, FICI>2were4.3%,20%,11.4%,64.3%and0%, respectively. Tigecycline/sulbactam combinationshowed that FICI≤0.5,0.5<FICI<1, FICI=1,1<FICI≤2, FICI>2were10%,54.3%,25.7%,10%and0%, respectively.(3) Compared with tigecycline alone, an addition of5-10mg/L PPIs could increase the MICs of tigecycline by0to2times and the additionof50mg/L PPIs could increase the MICs of tigecycline by4to>128times. Thetime-kill curves of tigecycline combined with PPIs were above those of tigecyclinealone, indicating that bacterial colony counts increased compared with tigecycline alone.(4) The MPC range of tigecycline alone was4-32mg/L and the SI range was4-64. Withan addition of colistin at4mg/L, the MPCs decreased2-4times and the MPCsdecreased4-8times at8mg/L; with an addition of sulbactam at32mg/L, the MPCsdecreased2-8times and the MPCs decreased4-64times at64mg/L,.(5)With anaddition of CCCP, the primary strains showed no MIC decreases. Except for the AB10mutant strain, the MICs of the other four mutant strains decreased16-128folds with anaddition of CCCP and the relative quantitative expression of AdeB efflux pump genesincreased at least2folds. No increase of the relative quantitative expression of AdeJefflux pump genes was detected in this study.Conclusion:(1) Despite tigecycline presented higher susceptibility against MDR-ABisolates, the probability of target attainment evaluated by Monte-Carlo simulation wasnot optimal at current dosage.(2) Tigecycline/colistin combination displayed indifference in most tested isolates, whereas the tigecycline/sulbactam combinationshowed synergistic and partial synergistic activity in most isolates.(3) In vitrosusceptibility of tigecycline can be influenced by an addition of PPIs in the test medium.(4) The MPCs of tigecyline, colistin and sulbactam were quite high and the MSWs werewide, indicating the easy selection of drug-resistant mutant strains; combination usagecould enhance their mutant prevention ability effectively.(5) Decreased susceptibility totigecycline in MDR-AB is associated with the overexpression of the AdeABC multidrugefflux pump.