Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 β-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem

Abstract

While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC β-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase. Bla _CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like β-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

Keywords: CMY-2-like; E. coli; OmpC; OmpF; antimicrobial resistance; liquid chromatography-mass spectrometry; meropenem; parallel reaction monitoring.

FIGURE 1

In vitro exposure to increasing meropenem concentrations and selection of mutants.

FIGURE 2

Analysis of the presence of CMY-2-like, cAmpC, and the porins OmpC and OmpF in mutant isolates of different E. coli strains selected under increasing meropenem concentrations using LC-MS/MS and Western blotting. On the Y-axis the meropenem minimum inhibitory concentration and mass spectrometry signal intensity of each endogenous peptide divided by signal intensity of each stable isotope labeled (SIL) peptide. When a peptide was not detected in a mutant isolate, the corresponding symbol was left out. All detected peptides were shown even when ratios to SIL approached zero. Molecular weight standard proteins are indicated (in kDa) at the left of the blots. Strains Top10F’-p2761 and Top10F’-pmr42 were used in the Western blot analysis as positive controls for CMY-2 and strain 187 as a positive control for cAmpC. (A–F) Represent the different clinical strains referred to throughout the “Results” section.