Engineering of a monitorable expression system to characterize β-lactamase genes in Enterobacteriaceae.

Bacteria are becoming progressively more resistant to available antimicrobials. The increased ease and availability of genome sequencing has made it possible to identify putative, novel antimicrobial resistance (AMR) genes bioinformatically. However, no standardized system is available to phenotypically characterize the ability of novel AMR genes in Enterobacteriaceae to confer resistance and impact bacterial physiology and pathogenicity in relation to expression levels. We previously used plasmid pBAD24, which allows for arabinose-inducible expression of heterologous genes, and Escherichia coli Top10 to characterize mobile colistin resistance genes. Based on the pBAD24 backbone, we constructed a new plasmid (pBAD25) that carries a kanamycin resistance gene (instead of an ampicillin resistance gene). We show that our expression system allows for the characterization of five different blaOXA genes, which differ in their ability to confer susceptibility to β-lactams, detected protein levels, and impact on bacterial growth. We characterized blaOXA-48b, a close relative of blaOXA-48, previously uncharacterized in E. coli, to be phenotypically similar to blaOXA-48, and blaOXA-549, a previously uncharacterized gene of the blaOXA-548 family, as encoding a β-lactamase that is detected intra- but not extracellularly, has moderate growth defects, and decreases susceptibility to carbapenems and ampicillin. Additionally, we found that, in blaOXA expressing strains, (i) levels of intracellular proteins and bacterial growth negatively correlate and (ii) susceptibility to 2nd and 3rd generation cephalosporins and susceptibility to different carbapenems positively correlate. Our results demonstrate that the expression of AMR genes, specifically blaOXA genes, through pBAD25 allows for easy characterization of putative, novel AMR genes.