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Background <p>Carbapenem-resistant <i>Citrobacter freundii</i> 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.</p> Objective <p>This study aims to characterize the first IncFII(pBK30683)/IncR plasmid carrying <i>bla</i><sub>KPC-2</sub> in <i>C. freundii</i> ST91, focusing on its transferability, stability, and the role of plasmid lineages in carbapenem resistance dissemination.</p> Methods <p>We investigated a urine-derived ST91 <i>C. freundii</i> 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.</p> Results <p>The 5.64 Mb genome comprised a chromosome and three plasmids. <i>bla</i><sub>KPC-2</sub> was located on an IncFII(pBK30683)/IncR plasmid pMAS5905-KPC carrying a complete conjugation module, the <i>mer</i> operon, and the anti-CRISPR determinant AcrIE9, with the <i>bla</i><sub>KPC-2</sub> embedded in an IS<i>26</i>-bracketed ΔTn<i>6296</i>-like module (IS<i>Kpn27</i>-<i>bla</i><sub>KPC-2</sub>-ΔIS<i>Kpn6</i>). The plasmid transferred to <i>E. coli</i> at 1.93 × 10<sup>–5</sup> 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 <i>bla</i><sub>KPC</sub> (31.82%) and <i>bla</i><sub>OXA-48-like</sub> (30.30%).</p> Conclusion <p>To our knowledge, this is the first characterization of an IncFII(pBK30683)/IncR plasmid carrying <i>bla</i><sub>KPC-2</sub> in a clinical <i>C. freundii</i> 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.</p>

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Characterization of a conjugative IncFII/IncR plasmid harboring blaKPC-2 in a clinical ST91 Citrobacter freundii isolate

  • 期刊论文

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  • 发表日期：

    2026年2月4日

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  • Xiao Liu,
  • Zhen Liu,
  • Zhiwen Sun,
  • Guodong Yan,
  • He Gao,
  • Xuemei Bai,
  • Bike Zhang,
  • Duochun Wang

摘要

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 blaKPC-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. blaKPC-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 blaKPC-2 embedded in an IS26-bracketed ΔTn6296-like module (ISKpn27-blaKPC-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 blaKPC (31.82%) and blaOXA-48-like (30.30%).

Conclusion

To our knowledge, this is the first characterization of an IncFII(pBK30683)/IncR plasmid carrying blaKPC-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.