An IS26-assembled three-module chimeric plasmid co-localizing bla NDM-1 and bla KPC-2 undergoes post-transfer rearrangement in Klebsiella pneumoniae.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a severe global public health threat, driven largely by plasmid-borne carbapenemase genes such as bla NDM-1 and bla KPC-2. Although CRKP strains co-producing multiple carbapenemases are increasingly reported, the co-localization of these genes on a single plasmid remains rare, and the evolutionary dynamics and stability of such hybrid resistance plasmids are poorly understood. METHODS: In this study, we characterized a highly imipenem-resistant clinical ST11 CRKP isolate (W61) by combining whole-genome sequencing, conjugation assays, structural variation (SV) analysis, S1-PFGE, Southern blotting, and antibiotic susceptibility testing. RESULTS: The isolate harbored a novel 224-kbp chimeric plasmid (pW61_1) and exhibited an imipenem minimum inhibitory concentration (MIC) of 512 μg/mL. Plasmid pW61_1 was composed of three modules fused through IS26-mediated cointegration: module 1 (IncFIB), module 2 (IncFII, bla KPC-2 +), and module 3 (IncN2, bla NDM-1 +). Under laboratory conditions, W61 maintained a high carbapenem-resistance retention rate in its native host even after 10 days of serial passage without antibiotic exposure. In addition, pW61_1 retained high structural stability after 10 days of serial passage under high imipenem concentrations. In the K. pneumoniae recipient, pW61_1 rearranged into smaller, resistance-retaining SVs, including SV-D3 (bla NDM-1 +) and SV-D4 (bla KPC-2 +), primarily through IS26-driven replication-associated excision. In the Escherichia coli J53 recipient, only small rearranged derivatives of pW61_1 were retained, each carrying one or both carbapenemase genes. DISCUSSION: Our findings demonstrate that the tri-modular plasmid is transferable and undergoes post-transfer rearrangement under laboratory conditions. We hypothesize that this tri-modular plasmid may play an important role in the dissemination of carbapenemase genes, and therefore continued surveillance of this plasmid and its rearranged derivatives is warranted.