Potential dissemination of IncHI2/IncHI2A plasmids carrying <i>mcr</i> -9.4 complex transposon in chicken-derived <i>Enterobacter hormaechei</i>

The escalating global prevalence of antimicrobial resistance(AMR) represents a critical public health challenge, particularly concerning the compromised efficacy of polymyxins-essential therapeutic agents against carbapenem-resistant Gram-negative pathogens. This crisis is exacerbated by the plasmid-mediated horizontal gene transfer mechanism, which facilitates the inter-reservoir dissemination of resistance determinants across anthropogenic, zoogenic, and environmental microbiomes. This study investigated a multidrug-resistant <i>Enterobacter hormaechei</i> strain GS32 isolated from a deceased 180-day-old laying hen. Antimicrobial susceptibility testing, whole-genome sequencing, and comparative genomics were employed to analyze resistance profiles, plasmid architecture, and genetic mobility. Conjugation assays assessed plasmid transferability. Results revealed <i>E. hormaechei</i> GS32 harbored a 255 kb IncHI2/IncHI2A plasmid carrying <i>mcr-</i>9.4(pGS32-1) within a conserved transposon (IS<i>1R-qseB/qseC-wbuC-mcr-</i>9.4-IS<i>903B</i>) alongside 14 additional resistance genes [e.g., <i>tet</i>(D), <i>mph</i>(A), and <i>sul2</i>] and heavy metal resistance determinants. The pGS32-1 demonstrated high similarity to those in <i>Salmonella</i> spp. and <i>Citrobacter freundii</i>, suggesting cross-species transmission. Conjugation to EC600 occurred efficiently (frequency: [7.92 ± 0.75] × 10^-²). To our knowledge, the present study provides the first evidence of the presence of an IncHI2 carrying <i>mcr</i>-9.4 in <i>E. hormaechei</i> isolated from poultry. The pGS32-1 was frequently found in <i>Enterobacter</i> sp. (including <i>E. hormaechei</i> and <i>Enterobacter cloaca</i>e), <i>Salmonella</i> sp., and other bacteria such as <i>C. freundii</i> and <i>Leclercia adecarboxylata</i>, indicating the cross-species transmission capability of IncHI2 plasmids, highlighting its role in disseminating polymyxin resistance across ecological niches. These findings underscore the urgent need for enhanced antimicrobial resistance surveillance in livestock and stricter antibiotic stewardship to mitigate the emergence of a multidrug-resistant pathogen under the One Health framework.IMPORTANCEPolymyxin, as the last-line therapeutic agent against carbapenem-resistant Gram-negative bacterial infections, is facing increasing clinical challenges due to the emergence of novel resistance mechanisms. In this study, a strain of <i>Enterobacter hormaechei</i> GS32 harboring an IncHI2/IncHI2A-type plasmid (pGS32-1) was isolated from deceased laying hens. This plasmid carries a multidrug resistance gene cluster, including <i>mcr</i>-9.4, and exhibits high-efficiency conjugative transfer capability. The <i>mcr</i>-9.4 gene is located within a conserved transposon structure (IS<i>1R-qseB</i>/<i>qseC-wbuC-mcr</i>-9.4-IS<i>903B</i>), colocalized with other resistance genes on the plasmid, suggesting its potential integration as a more complex transposon substructure into this plasmid type. Previous studies have demonstrated that IncHI2-type plasmids are predominantly distributed among <i>Enterobacteriaceae</i> species such as <i>Klebsiella pneumoniae</i> and <i>Salmonella</i> spp. Notably, pGS32-1 exhibits high homology with plasmids identified in <i>Salmonella</i> spp. and <i>Citrobacter freundii</i>, indicating the cross-species transmission potential of IncHI2/IncHI2A-type plasmids and their role in expanding the reservoir of resistance genes.