IS26 Veers Genomic Plasticity and Genetic Rearrangement toward Carbapenem Hyperresistance under Sublethal Antibiotics

Multidrug-resistant Gram-negative carriers of Klebsiella pneumoniae carbapenemases (KPCs) often subvert antibiotic therapy due to inadequate sensitivity in laboratory detection. Although unstable gene amplification has been recognized to crucially contribute to underestimation or misestimation of antimicrobial resistance in clinical isolates, the precise mechanisms underlying carbapenem resistance driven by amplification of bla(KPC-2) remain obscure. Here, we reported that IS26-mediated amplification of bla(KPC-2) rapidly and robustly gave rise to carbapenem hyperresistant phenotypes in an Escherichia coli clinical strain following sublethal meropenem or tobramycin preexposure. Intriguingly, IS26 also underpinned amplification of a 47 kb multiple drug resistance (MDR) region encompassing nine antibiotic resistance genes and six IS26 insertion sequences. Tandem-repeat analysis and experimental validation demonstrated that bla(KPC-2) amplification was indeed mediated by IS26, which was further experimentally shown to involve intricate genetic rearrangement. Such gene amplification arose dynamically under antibiotic stress and subsided upon antibiotic withdrawal. Instead of reducing the amplification of the IS26-flanked MDR region, drug combinations in vitro exacerbated it. Our study, thus, provides valuable insights into how dynamic gene amplification processes can precipitously transform resistance status and complicate diagnosis.