Screening Of Novel Anti - Drug - Resistant Gram - Negative Bacteria

Over the past decades, antibiotic resistance in Gram-negative bacilli has been increasingly serious, especially reflects in the carbapenem-resistant Enterobacteriaceae, which includes Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter aerogenes, and the carbapenem-resistant non-fermenters, like Acinetobacter baumannii and Pseudomonas aeruginosa. Among these, resistance in Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa is the most serious, and it is very difficult to treat infections due to these bacteria.Developing potentiating agents of antimicrobial drugs based on the resistant mechanism which can enable noneffective antibiotics regain activity is an important way to overcome the problem of Gram-negative bacilli resistant. Therefore, in this research, New Delhi metallo-β-lactamase 1(NDM-1) expressed by "superbugs" and the major efflux pump of Pseudomonas aeruginosa MexAB-OprM were chosen for establishing screening model to screen inhibitors, then validate and evaluate the inhibitors, in order to obtain potentiating agents of anti-G-bacillus drugs having promising clinical application.In the first part of this study, we cloned and expressed Klebsiella pneumoniae ATCC BAA-2146 NDM-1, purified its protein using Ni2+ affinity chromatography, acquiring relatively pure and soluble enzyme having natural activity. The high-throughput screening model was built based on the enzyme activity assays using meropenem and nitrocefin as substrate, optimized in the aspects of substrate concentration, enzyme concentration and DMSO. Also the feasibility of optimized model were evaluated. 76,800 compounds were screened using this model, of which 16 compounds appear inhibitory activity against NDM-1,4 of them capable of reversing the tolerance of bacterium expressing NDM-1. The compound IMB-XW2 can increase the sensitivity of E.coli expressing NDM-1 to meropenem and ampicillin 8 folds at 4 ug/ml, without having activity against other G+ and G- pathogenic microorganisms. Then through the study of enzyme kinetics, we elucidated IMB-XW2 was a competitive inhibitor whose inhibition constant is 0.22 uM and ICso is 0.69 uM. By adopting fluorescence quenching,we found its action mode was static quenching. In addition, the liver and kidney cells toxicity of IMB-XW2 were evaluated, to HepG2 and Vero its TCso was 5.078 μg/ml and 6.430 μg/ml respectively, showing a relatively strong cytotoxicity. The ADMET and toxicological properties were also forecasted, which provided reference for further optimization. This inhibitor were hopeful to be a novel anti-drug resistant gram-negative bacteria drug or lead compound.In the second part of this study, we constructed strains specific expressing MexAB-OprM and preliminary screened 76,800 compounds using the high-throughput screening model of MexAB-OprM, acquiring 147 active compounds with a screening positive rate of 0.94%.Then after comparing the MIC of acquired compounds with 40 μg/ml tetracycline hydrochloride combined with the compounds, we obtained 4 inhibitors. Furthermore, we estimated the inhibitory activities by determining the MIC of inhibitors combined with multiple antibiotics,discovering that under the concentration of 10μg/ml, IMB-P4 can improved the sensitivity of the strains specific expressing MexAB-OprM of Pseudomonas aeruginosa to various antibiotics 4-16 times. Antibacterial activity assay showed that IMB-P4 had no activity against other G+ and G-pathogenic microorganisms. Also, we evaluated the liver and kidney toxicity of IMB-P4, to HepG2 and Vero its TCso was 4.551 μg/ml and 46.620 μg/ml, respectively. The IMB-P4 is expected to be a novel drug targeting efflux pump or lead compound.