Resolving plasmid-encoded carbapenem resistance dynamics and reservoirs in a hospital setting through nanopore sequencing.

The growing resistance of Enterobacterales to last-resort antibiotics such as carbapenems puts a significant burden on healthcare systems, also due to plasmids driving a rapid spread of carbapenem resistance. We here evaluate the use of long-read nanopore sequencing to investigate carbapenem resistance dynamics and the role of plasmid transfers and environmental reservoirs in the hospital setting. Over 13 months, routine clinical diagnostics identified recurring isolates of carbapenem-resistant Citrobacter species carrying Klebsiella pneumoniae carbapenemases (KPCs) and/or OXA-48-like carbapenemases from patient screening and hospital drain samples. While routine diagnostic approaches provided limited insights into the carbapenem resistance dynamics, we show that near-complete de novo assembly of chromosomes and plasmids by long-read nanopore sequencing allowed for high-resolution strain identification, plasmid profiling, and antibiotic resistance gene detection. Notably, genomically nearly indistinguishable Citrobacter freundii of the high-risk sequence type ST91 genomes were recovered from screening samples collected in the same hospital room 1 year apart. We further provide evidence of a KPC-2-encoding IncN plasmid that is likely to have spread across bacterial species and between patient and drain isolates, which emphasizes the role of contaminated drains in the persistence and dissemination of antimicrobial resistance within the hospital environment. Overall, this study demonstrates the value of long-read nanopore sequencing for uncovering the complex dynamics of carbapenem resistance spread and persistence in the hospital setting and its potential implications for infection prevention and control.