Knockout of Targeted Plasmid-Borne β-Lactamase Genes in an Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile

Abstract

The critical situation regarding antibiotic resistance requires a more in-depth effort for understanding underlying mechanisms involved in antibiotic resistance, beyond just detecting resistance genes. The methodology presented in this work provides a framework for knocking out selected resistance factors, to study the adjustments of the bacterium in response to a particular antibiotic stress, elucidating the genetic response and proteins that are mobilized. Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne β-lactamase genes ( bla OXA-1 , bla TEM-1 , and bla CTX-M15 ) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted β-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the bla CTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of bla OXA-1 and bla TEM-1 affected fewer proteins’ expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss. IMPORTANCE The critical situation regarding antibiotic resistance requires a more in-depth effort for understanding underlying mechanisms involved in antibiotic resistance, beyond just detecting resistance genes. The methodology presented in this work provides a framework for knocking out selected resistance factors, to study the adjustments of the bacterium in response to a particular antibiotic stress, elucidating the genetic response and proteins that are mobilized. The protocol uses MS-based determination of the proteins that are expressed in response to an antibiotic, enabling the selection of strong candidates representing putative resistance factors or mechanisms and providing a basis for future studies to understand their implications in antibiotic resistance. This allows us to better understand how the cell responds to the presence of the antibiotic when a specific gene is lost and, consequently, identify alternative targets for possible future treatment development.