Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

Abstract

ABSTRACTA liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS)Escherichia colistrain, but during admission, a carbapenem-resistant (CR)E. colistrain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CRE. colilacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CRE. colipossessedblaCTX-M-15andblaOXA-1. The CRE. colistrain additionally harboredblaCMY-2and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation eitherin vitroorin vivowith meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into variousE. colistrains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of “trapping” by CMY-2 β-lactamase plays a role in carbapenem resistance.