Antibiotic trapping by plasmid-encoded CMY-2 β-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in Escherichia coli

Antimicrob Agents Chemother. 2013 Aug;57(8):3941-9. doi: 10.1128/AAC.02459-12. Epub 2013 Jun 3.

Abstract

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and 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 either in vitro or in vivo with 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 various E. coli strains, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Anti-Bacterial Agents / therapeutic use
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Cell Membrane Permeability*
  • Cephalosporins / pharmacology
  • Drug Resistance, Multiple, Bacterial
  • Enzyme Activation
  • Escherichia coli / drug effects*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli Infections / drug therapy
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Female
  • Humans
  • Hydrolysis
  • Meropenem
  • Microbial Sensitivity Tests
  • Periplasm / drug effects
  • Plasmids / genetics
  • Plasmids / metabolism*
  • Protein Binding
  • Thienamycins / pharmacology
  • Thienamycins / therapeutic use*
  • Young Adult
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism*

Substances

  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • Cephalosporins
  • Escherichia coli Proteins
  • Thienamycins
  • beta-lactamase CMY-2
  • beta-lactamase CTX-M-15
  • beta-lactamase OXA-2
  • beta-Lactamases
  • nitrocefin
  • Meropenem