Abstract
BACKGROUND: Carbapenem-resistant Citrobacter freundii is increasingly reported and recognized as an opportunistic host of mobile carbapenemase-encoding elements in healthcare-associated settings. However, lineage-associated plasmid vehicles and their transfer-related features in this species remain incompletely characterized. OBJECTIVE: This study aims to characterize the first IncFII(pBK30683)/IncR plasmid carrying bla(KPC-2) in C. freundii ST91, focusing on its transferability, stability, and the role of plasmid lineages in carbapenem resistance dissemination. METHODS: We investigated a urine-derived ST91 C. freundii isolate (MAS5905) through antimicrobial susceptibility testing, whole-genome sequencing, conjugation, 20-day plasmid stability, and growth curve assays. Comparative genomics included plasmid phylogeny of pMAS5905-KPC-like sequences and ST91 phylogenomics. RESULTS: The 5.64 Mb genome comprised a chromosome and three plasmids. bla(KPC-2) was located on an IncFII(pBK30683)/IncR plasmid pMAS5905-KPC carrying a complete conjugation module, the mer operon, and the anti-CRISPR determinant AcrIE9, with the bla(KPC-2) embedded in an IS26-bracketed ΔTn6296-like module (ISKpn27-bla(KPC-2)-ΔISKpn6). The plasmid transferred to E. coli at 1.93 × 10(–5) was retained at 100% over 20 days, and imposed a significant fitness cost. Phenotypically, MAS5905 was resistant to multiple β-lactams (including ertapenem and meropenem) and fluoroquinolones, while remaining susceptible to cefepime, ceftazidime, trimethoprim-sulfamethoxazole, aminoglycosides, tetracyclines, and nitrofurantoin. Plasmid comparisons resolved six IncFII/IncR clades with a conserved backbone and broad host range. ST91 phylogenomics revealed multiple clades and, in the analyzed public dataset, a high carbapenemase gene prevalence (75.76%), dominated by bla(KPC) (31.82%) and bla(OXA-48-like) (30.30%). CONCLUSION: To our knowledge, this is the first characterization of an IncFII(pBK30683)/IncR plasmid carrying bla(KPC-2) in a clinical C. freundii ST91 isolate. The findings highlight plasmid lineages as drivers of carbapenem resistance dissemination and underscore the need for plasmid-focused genomic surveillance, particularly for monitoring transferable resistance vehicles across clinically relevant hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-026-04795-1.