IS 26 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 IS 26 -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, IS 26 also underpinned amplification of a 47 kb multiple drug resistance (MDR) region encompassing nine antibiotic resistance genes and six IS 26 insertion sequences. Tandem-repeat analysis and experimental validation demonstrated that bla KPC-2 amplification was indeed mediated by IS 26 , 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 IS 26 -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. IMPORTANCE Klebsiella pneumoniae carbapenemases (KPCs) are powerful β -lactamases that enable Gram-negative pathogens to destroy clinically important carbapenems in antibiotic therapies. In particular, KPC-2 is difficult to detect due to a lack of instrument sensitivity in regular laboratory screens, which leads to misdiagnosis and poor treatment outcomes. It remains unclear how bla KPC-2 rapidly induces exceedingly high-level resistance against carbapenems following the challenges of sublethal antibiotics. Here, we demonstrated that, under sublethal doses of antibiotics, insertion sequence IS 26 mediated rapid amplification of multiple resistance determinants, including bla KPC-2 and a multiple drug resistance (MDR) region, which was accompanied by intricate genetic rearrangement.