Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli

Abstract

Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying bla_CMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The bla_CMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations.

Keywords: AmpC; CMY-2; Escherichia coli; carbapenem resistance; plasmid-mediated resistance; porins.

FIG 1

Expression of porins in isolate R2761 and derivatives. Outer membrane proteins were either separated by SDS-PAGE and stained with Coomassie brilliant blue (A) or analyzed by Western blotting using a porin-specific polyclonal antiserum (B). Lanes 1, E. coli K-12 strain TOP10F′, which is used as a reference for the major outer membrane proteins; lanes 2, wild-type isolate R2761; lanes 3 to 8, R2761 derivatives mr20, mr26, mr35, mr38, mr41, and mr42, respectively; lanes M, molecular weight marker proteins. The positions of the major outer membrane proteins in the K-12 strain are indicated on the left and the molecular masses of the marker proteins on the right. Only the relevant parts of the gel and blot are shown.

FIG 2

Expression of CMY-2 β-lactamase in isolate R2761 and derivatives. (A) Zymogram on which periplasmic fractions of strain R2761 (lane 1) and its derivatives mr20, mr26, mr35, mr38, mr41, and mr42 (lanes 2 to 7, respectively) were analyzed using nitrocefin as a β-lactamase substrate. Lane M contains molecular weight marker proteins, and their molecular masses are indicated on the left. Two bands with β-lactamase activity were detected, and their apparent molecular masses are indicated at the right. (B) Western blot of periplasmic fractions of R2761 and its derivatives. Lanes are loaded in the same order as in panel A. The blot was probed with an antiserum raised against CMY-2 β-lactamase. Only the relevant parts of the zymogram and the blot are shown.

FIG 3

Overproduction of CMY-2 results from an increased plasmid copy number. (A) Plasmids from strain R2761 and its derivatives and from clinical isolates EC-8 and EC-9 were isolated from transformants of E. coli K-12 strain TOP10F′ and were digested with EcoRI, and the fragments were analyzed on 0.6% agarose gels. Lanes 1 to 4, plasmids from strains R2761 and its derivatives mr20, mr26, and mr42, respectively; lanes 5 and 6, plasmids from strains EC-8 and EC-9, respectively. Each lane contains the plasmid yield from 8 × 10⁸ bacterial cells. The lane marked M shows a molecular marker; sizes are indicated on the left in kilobase pairs. (B) Nucleotide sequences of the hairpin loop region of the inc genes containing single nucleotide substitutions in the plasmids from mr42, EC-8, and EC-9 (indicated in red). The inverted repeats forming the stem of the stem-loop structure are indicated by arrows.