A One Health Perspective on the Plasmid Backbone Preference and Evolutionary Adaptation of tmexCD-toprJ in Klebsiella spp

Background: Antimicrobial resistance (AMR) poses a critical One Health challenge, linking human, animal, and environmental health through the movement of multidrug-resistant (MDR) bacteria and resistance determinants. The tmexCD-toprJ gene cluster, an efflux pump conferring high-level resistance to tigecycline and eravacycline. However, its plasmid backbone preferences and evolutionary trajectories in Klebsiella spp . remain insufficiently characterized. Methods: This study investigated the plasmid backbone preference and evolutionary characteristics of tmexCD-toprJ -harboring plasmids in Klebsiella spp . using whole-genome sequencing of three clinical strains carrying tmexCD-toprJ collected from 2018 to 2023. Conjugation assays, comparative genomics, and global epidemiological analysis were performed to assess plasmid mobility, genetic context, and evolutionary direction under the One Health framework. Results: All three isolates (K7, K36, and K307) exhibited MDR and harbored major resistance genes, including blaIMP-4, mcr-1.1 , and blaNDM-1 , respectively. The plasmid from K36 was transferable to EC600 (frequency, 10 − 7 ), confirming cross-species mobility. Global database analysis revealed that tmexCD-toprJ -positive Klebsiella spp . isolates (n=92) originated mainly from humans (59.8%), followed by animals (37.0%) and environments (3.3%). Phylogenetic and plasmid analyses the tmexCD1-toprJ1 variant was mainly associated with these hybrid plasmids, frequently co-localizing with sul1, qnrB , and strA/B to form stable “tigecycline–aminoglycoside–sulfonamide” co-resistance modules. In contrast, tmexCD2-toprJ2 was more often inserted into classical resistant plasmids. Conclusion: These findings demonstrate that tmexCD-toprJ has evolved as a highly mobile resistance determinant within Klebsiella spp . disseminating across the human–animal–environment interface via hybrid plasmids and horizontal gene transfer. This underscores the urgent need for integrated One Health surveillance and containment strategies to mitigate plasmid-mediated multidrug resistance and its global public health impact. Keywords: tmexCD-toprJ, Klebsiella spp, one health, horizontal gene transfer, hybrid plasmids