The role of extended spectrum beta lactamases encoding genes in carbapenem resistance in escherichia coli and klebsiella pneumoniae at the American University of Beirut Medical Center -

Abstract

Background: Carbapenem resistance is continuously emerging worldwide, in extended spectrum beta lactamase (ESBL) and non-ESBL producing Enterobacteriaceae. Mechanisms of carbapenem resistance have been shown in a previous study done at the Department of Experimental Pathology, Immunology and Microbiology, to be due to the effect of carbapenemases in ESBL and non-ESBL producing Escherichia coli and Klebsiella pneumoniae. However, carbapenem resistance, has also been observed in this study in ESBL producing E. coli and K. pneumoniae lacking carbapenemases encoding genes, but having outer membrane porin (OMP) impermeabilities and-or efflux pump (EP) activity. To that purpose, we attempted in this project, to assess by electroporation, 1) the effect of the ESBLs CTXM-15 and TEM-1, encoded by bla- CTXM-15 and bla- TEM-1genes, and 2) the effect of the carbapenemases OXA-48 and NDM-1, encoded by bla -OXA-48 and bla -NDM-1 genes, singly and in combination, on carbapenem resistance in electrocompetent cells, lacking OMP impermeabilities and- or EP activity, in order to determine the sole effect of these enzymes on carbapenem resistance, without the effect of intrinsic mechanisms of resistance in the bacterial cells. Methods: Seven previously stored ESBL and non ESBL producing and carbapenem resistant K. pneumoniae and E. coli isolates harboring a selection of ESBL and -or carbapenemases encoding genes, bla-CTX-M-15, bla-TEM-1, bla-NDM-1 and bla-OXA-48 were selected in this project. Three control carbapenem resistant isolates lacking ESBL and-or carbapenemases encoding genes, but having OMP impermeabilities and -or EP activity, were also previously stored and used in this project as well. Pulsed Field Gel Electrophoresis (PFGE) analysis was performed. Extracts of total and plasmid DNA were subjected to Polymerase Chain Reaction (PCR) amplification of all encoding genes. Electroporation experiments utilizing E.coli GC-10 electrocompetent recipient cells were done. Antimicrobial susceptibility and PCR amplification were pe