Emergence of Mobilized Tigecycline Resistance Gene Cluster <i>tmexCD1-toprJ1</i> in <i>Raoultella ornithinolytica</i> From a Swine Farm, China

Tigecycline is considered a last-line therapeutic option for treating infections caused by multidrug-resistant (MDR) Gram-negative bacteria. The emergence of <i>tmexCD1-toprJ1</i>, a plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster, poses a growing threat to tigecycline efficacy. While this gene cluster has primarily been identified in <i>Klebsiella pneumoniae</i>, its presence in other species remains poorly characterized. In this study, we investigated the occurrence, genetic features, resistance profile, and virulence potential of <i>tmexCD-toprJ</i>-positive <i>Raoultella ornithinolytica</i> isolates from a swine farm in China. A total of 126 samples were collected from a swine farm and screened for tigecycline-resistant isolates. Species identification was performed using MALDI-TOF MS, while PCR and sequencing were applied to detect <i>tmexCD-toprJ</i>. Antimicrobial susceptibility testing was assessed by agar and broth microdilution methods. Whole-genome sequencing with hybrid assembly provided insights into genetic organization. Conjugation and electroporation experiments were conducted to assess plasmid mobility, and virulence was assessed using the <i>G. mellonella</i> infection model. Six <i>R. ornithinolytica</i> isolates (4.76%) were identified as carrying <i>tmexCD1-toprJ1</i> and exhibited multidrug resistance, including reduced susceptibility to tigecycline (minimal inhibitory concentrations [MICs] 4-16 mg/L). The resistance genes were located on highly similar IncFIB plasmids, which lacked typical conjugative elements. However, IS26 sequences flanking the gene cluster suggested potential for horizontal transfer. Phylogenetic analysis indicated possible clonal dissemination. All isolates carried chromosomally encoded virulence genes, and in vivo assays in <i>G. mellonella</i> revealed moderate to high pathogenicity. These findings expand the ecological distribution of <i>tmexCD1-toprJ1</i> to <i>R. ornithinolytica</i> in livestock environments, underscoring the role of swine farm water systems as potential reservoirs. The coexistence of antimicrobial resistance and virulence determinants highlights the urgent need for strengthened surveillance and containment strategies to prevent further dissemination of tigecycline resistance.