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1 carbapenem antibacterial activity and resistance mechanisms two recombinant Escherichia coli AcrAB efflux pump building

Author: HanHui
Tutor: YouXueFu
School: Beijing Union Medical College
Course: Microbial and Biochemical Pharmacy
Keywords: clinical isolated strains Drug resistance Carbapenemase Escherichia coli AcrAB efflux pump
CLC: R446.5
Type: Master's thesis
Year: 2011
Downloads: 119
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Abstract


Carbapenem antibiotics are strongly effective drugs for clinical infections. But in recent years the number of carbapenem resistant strains is increasing. The emergence and spread of carbapenemases, especially the transferable carbapenemases have brought great challenges to the clinical treatment of infectious diseases.This study investigated the sensitivity and resistance to carbapenems in clinical isolated strains from Beijing hospital and their carbapenemase genotype.352 clinical isolates of eight species were collected from Beijing hospital in 2007-2009. Their MIC to meropenem, imipenem/cilastatin, panipenem and faropenem were done by the serial two-fold agar dilution method. The sensitivity and resistance of these clinical strains were studied according to the MIC results. Meropenem, imipenem/ cilastatin, panipenem and faropenem are effective to most clinical isolates, with the sensitivity rates were 82.1%,85.5%,82.1%and 83.2%, respectively. While the resistance rates were 12.8%,12.8%,12.8%and 12.5%, respectively. Among the eight species, Acinetobacter baumannii and Pseudomonas aeruginosa were significantly more resistant than other species.Acinetobacter baumannii and Pseudomonas aeruginosa strains were chosen for PCR screeing to find the carbapenemase genes. One from fourteen Pseudomonas aeruginosa strains was identified carrying VIM-2 gene, while seventeen Acinetobacter baumannii strains were all identified carrying OXA-23 gene. The VIM-2 and OXA-23 genes were cloned and ligated with pET30a vecter. Then the expressing plasmids were transformed into Escherichia coli BL21 strain. The VIM-2 and OXA-23 protein expression systems were successfully constructed. And the activities to meropenem and imipenem of purified VIM-2 and OXA-23 enzymes were determined by the hydrolysis test. The clonal strain of NDM-1 gene was constructed using the genomic DNA of ATCC Klebsiella pneumoniae BAA2146 as the template. Efflux pump is one of the most essential mechanisms of multiple drug resistance in Escherichia coli. Among all of the efflux pumps of Escherichia coli, AcrAB-To1C pump has the broadest substrate spectrum and highest efflux power. It can pump out many kinds of antibiotic, such as tetracyclines, chloramphenicol, quinolones, macrolides,β-lactams and rifampicin.In this study, we amplified acrA-acrB genes from the genome of K12 Escherichia coli and constructed the expression plasmid pET30a-acrAB. Finally, the expression plasmid was transformed into the BL21(DE3) strain which had been knocked out acrAB and acrEF genes, got the YH216 strain efficiently expressing AcrAB pump.MIC assay of different classes of antibiotics showed that the MIC in YH216 was much higher than in the original strain and negative control strain. After the induction of IPTG, the MIC of YH216 was higher than before. These results demonstrated that AcrAB was assembled correctly and had expressive activity. Furthermore, this expressive activity can be induced by IPTG. When treated with proten pump inhibitor CCCP, the MIC of YH216 was similar to that of negative control strain. This further validated the activity of AcrAB pump.

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