Role of AmpG in the resistance to β-lactam agents, including cephalosporins and carbapenems: candidate for a novel antimicrobial target

Abstract

Background: A complex cascade of genes, enzymes, and transcription factors regulates AmpC β-lactamase overexpression. We investigated the network of AmpC β-lactamase overexpression in Klebsiella aerogenes and identified the role of AmpG in resistance to β-lactam agents, including cephalosporins and carbapenems.

Methods: A transposon mutant library was created for carbapenem-resistant K. aerogenes YMC2008-M09-943034 (KE-Y1) to screen for candidates with increased susceptibility to carbapenems, which identified the susceptible mutant derivatives KE-Y3 and KE-Y6. All the strains were subjected to highly contiguous de novo assemblies using PacBio sequencing to investigate the loss of resistance due to transposon insertion. Complementation and knock-out experiments using lambda Red-mediated homologous recombinase and CRISPR-Cas9 were performed to confirm the role of gene of interest.

Results: In-depth analysis of KE-Y3 and KE-Y6 revealed the insertion of a transposon at six positions in each strain, at which truncation of the AmpG permease gene was common in both. The disruption of the AmpG permease leads to carbapenem susceptibility, which was further confirmed by complementation. We generated an AmpG permease gene knockout using lambda Red-mediated recombineering in K. aerogenes KE-Y1 and a CRISPR-Cas9-mediated gene knockout in multidrug-resistant Klebsiella pneumoniae-YMC/2013/D to confer carbapenem susceptibility.

Conclusions: These findings suggest that inhibition of the AmpG is a potential strategy to increase the efficacy of β-lactam agents against Klebsiella aerogenes.