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It is widely known that integrative and conjugative elements (ICEs) play an important role in the transmission of resistance genes and other exogenous genes. The present study aimed to characterize the three novel ICEs including ICEGpa76, ICEGpa44, and ICEGpa11, from Glaesserella parasuis. The ICEs from G. parasuis strains d76, Z44, and XP11 were predicted and identified by whole-genome sequencing (WGS) analysis, ICEfinder, and PCR. Characterization of G. parasuis strains carrying ICEs were determined by conjugation assay, antimicrobial susceptibility testing, WGS, phylogenetic analysis, and comparative sequence analysis.The WGS results showed that three ICEs from G. parasuis have a common genetic backbone belonging to characteristics ofthe ICEHpa1 family. The sequence comparison showed that the ICEHpa1 family has five hot spots (HSs) determined by IS6, IS110, and IS256. Moreover, two variable regions (VRs), VR1 and VR2 were determined by multidrug resistance genes and the rearrangement hotspot (rhs) family, respectively. VR1 consists of multidrug resistance genes, ISApl1s, and other accessory genes, while VR2 is composed of IS4, rhs family, transposase, and hypothetical protein genes. Conjugation experiments and MICs revealed that three ICEs could be transferred to G. parasuis strain IV52, indicating these three ICEs could be transmitted horizontally among G. parasuis strains. Additionally, the difference in resistance genes from ICEs might be due to the insertion function of the ISApl1s in VR1, and the rhs family in VR2 might evolve andthen be stably inherited in G. parasuis. These results further elucidated the transmission mechanism of exogenous genes in G. parasuis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism., Methods: We employed whole-genome sequencing to decipher the genetic architecture of pS130-4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests., Results: pS130-4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both bla KPC-2 and hypervirulence genes. Our conjugation experiments established that pS130-4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10 -5 and 10 -4 . Remarkably, pS130-4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130-4's virulence remained intact postconjugation., Conclusion: A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)