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

Resistance to beta-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 beta-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 beta-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.