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.

Dimard E Foudraine,John Hays,Nikolaos Strepis,Agnes A Wattel,Jan Tommassen,Marian T Ten Kate,Annelies Verbon,Wil H F Goessens,Camiel N M Aarents,Theo M Luider,Corné H W Klaassen,Lennard J M Dekker,Ria Van Boxtel

doi:10.3389/fmicb.2022.793738

Dimard E Foudraine, John Hays + Show 11 more

Open Access

https://doi.org/10.3389/fmicb.2022.793738

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Journal: Frontiers in microbiology	Publication Date: Feb 28, 2022
Citations: 3	License type: CC BY 4.0

Affiliation: Erasmus MC, Utrecht University

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. BlaCMY–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.

Highlights

There are several antibiotic classes known, of which β-lactams are the most diverse and most frequently prescribed (Pandey and Cascella, 2020). β-lactam antibiotics can be categorized into several sub-classes, including the broad-spectrum carbapenem class
We developed a single targeted LCMS/MS assay for the detection and quantification of the E. coli porins OmpC and OmpF, as well as CMY2-like and cAmpC β-lactamases
Peptides were selected for the detection of CMY-2-like, cAmpC, OmpC and OmpF using LC-MS/MS (Table 1)

Summary

INTRODUCTION

There are several antibiotic classes known, of which β-lactams are the most diverse and most frequently prescribed (Pandey and Cascella, 2020). β-lactam antibiotics can be categorized into several sub-classes, including the broad-spectrum carbapenem class. No reliable phenotypic assays are currently available to detect the absence of OmpC and OmpF. This information is important, as isolates which lack one or both of these main porins can rapidly become resistant to multiple antibiotics (including carbapenems) after antibiotic exposure (Pages et al, 2008; Goessens et al, 2013; van Boxtel et al, 2017; Vergalli et al, 2020). We developed a single targeted LCMS/MS assay for the detection and (relative) quantification of the E. coli porins OmpC and OmpF, as well as CMY2-like and cAmpC β-lactamases. We used the developed assay to analyze how the abundance of the porins and β-lactamases correlated to resistance, and in addition, we applied whole-genome sequencing (WGS) to place our findings within the context of the underlying genetic mechanisms

MATERIALS AND METHODS

RESULTS

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DISCUSSION

DATA AVAILABILITY STATEMENT