Identification of plasmids by PCR based replicon typing in bacteremic Klebsiella pneumoniae

Background: Klebsiella pneumoniae is a notorious pathogen with plasmid mediated resistance to all classes of antibiotics. It is important to determine the plasmid profile coding for resistance genes. Plasmid profile varies among geographical regions and tracking the types helps in determining the MDR and XDR K. pneumoniae spread especially in hospital setting. Aim of the present study was to determine the plasmid profile and types among bacteraemic K. pneumoniae. Materials and methods: Ninety consecutive K. pneumoniae collected over a period of three months from blood cultures were characterised by PCR for plasmid profile. Inc plasmid types were determined by PCR based replicon typing (PBRT) and carbapenemases were determined by multiplex PCR. For a subset of isolates hybrid assemblies were developed by sequencing with Ion Torrent and MinIon. Results: Overall, PBRT showed 29% of isolates carried four plasmids including IncHI1B, IncFIA, IncFII(K) and IncR. The most common type of plasmid was IncHI1B (93%) followed by IncFII(K) (89%) and IncR (82%). IncFIA was predominant among carbapenem resistant isolates. Almost all plasmids identified in K. pneumoniae were AMR plasmids, except two isolates which had virulence plasmids. IncX3 plasmid observed in this study was previously reported to be self-disseminating. Furthermore, the hybrid genome sequencing revealed complete structural arrangements of plasmids, which would be missed in short-read sequencing. NDM and OXA48-like were co-produced in 59% of the carbapenem resistant isolates. Bla(OXA-232) was present on ColKP3; aac(6')-lb3 and rmtF on IncFIB. Conclusion: Diverse plasmid profile among the successive K. pneumoniae isolates indicates the transfer of resistance genes through different types of plasmids. IncHI1B, IncFIA, IncFIIK and IncR were the prevalent plasmid types. Hybrid assembly revealed bla(OXA-232) was present on ColKP3 unlike global reports of IncL/M. Hybrid assemblies provide better plasmid structure that long and short read assemblies. There was no significant association of beta-lactamases with specific Inc groups in this study.