To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, bla_CTX–M–55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A–:B– plasmid pS13-2, respectively, fused with IncFI:A–:B– plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A–:B– plasmid and IncFI:A–:B– plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.

Background: Klebsiella pneumoniae is an opportunistic zoonotic pathogen that can cause respiratory diseases in chickens. The aim of this study was to investigate the biofilm formation, resistance, and virulence of K. pneumoniae isolates from chickens in Henan Province, China, between 2023 and 2025, and to analyze the associations among these characteristics. Methods: We identified the isolates through blood agar culture, Gram staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Hypermucoviscosity phenotype, biofilm-forming ability, antimicrobial resistance and virulence genes were assessed by the string test, crystal violet assay, disk diffusion and polymerase chain reaction (PCR), respectively. Results: A total of 102 K. pneumoniae isolates were identified. Of these, 81.4% were biofilm-forming strains, and 15.7% exhibited the hypermucoviscosity phenotype. The susceptibility profiles of the K. pneumoniae isolates indicated high resistance to penicillins (>90.0%) and low resistance to carbapenems (<10%), with 88.2% identified as multidrug-resistant. The results of virulence gene detection indicated that luxS (79.4%), mrkD (75.5%), and uge (73.5%) were the most prevalent virulence genes, followed by wabG (49.0%), ybtA (34.3%), and iucA (20.6%). The association analysis indicated that biofilm formation was significant association with multidrug-resistant and the carriage of virulence genes. Compared with non–biofilm–forming isolates, biofilm–forming isolates showed significantly higher resistance to ceftiofur and florfenicol, as well as higher detection rates of luxS, mrkD, and uge (p < 0.05). Conclusions: The data obtained in this study reveal the pathogenic potential and multidrug–resistant characteristics of this pathogen, highlighting the need for surveillance and monitoring.