ObjectivesThe study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producingKlebsiella pneumoniaeclinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out.MethodsPlasmids were isolated from 10 clinical isolates ofKlebsiella pneumoniae, and from a corresponding set ofEscherichia colitransconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. ForE. colitransconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results.ResultsAll carbapenemase-producingK. pneumoniaeisolates (carrying eitherblaNDM-1, or/andblaOXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of theblaNDM-1andblaESBL-plasmids identified in this study were previously described. MostblaNDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange betweenK. pneumoniaeisolates of this collection. The majority of NGS-based AMR profiles from theE. colitransconjugants correlated well with both NGS-based predicted and conventional AST results.ConclusionThis study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.