Characterization of tmexCD2-toprJ2-carrying carbapenem-resistant Raoultella ornithinolytica from hospital sewage in Zhejiang Province of China, 2022–2023

• tmexCD2-toprJ2 -positive Raoultella ornithinolytica from hospital wastewater co-harboring bla KPC-2 , bla NDM-1 , and bla IMP-4 reveals dual resistance in Zhejiang, China. • All isolates exhibited multidrug resistance including tigecycline and carbapenems. • Mobile plasmids harboring tmexCD2-toprJ2 facilitate environmental dissemination. • Hospital wastewater may serves as an environmental reservoir of emerging resistance genes. Hospital wastewater serves as a critical source of antimicrobial resistance genes (ARGs), particularly those associated with opportunistic pathogens like Raoultella ornithinolytica . This study aimed to characterize carbapenem-resistant R. ornithinolytica (CR-ROR) isolates carrying the tmexCD2-toprJ2 gene cluster from hospital sewage in Zhejiang province, China. Wastewater samples collected from four hospitals in Zhejiang Province were screened for carbapenem-resistant isolates. Whole-genome sequencing using the Illumina platform, combined with bioinformatic analysis, was performed to investigate phylogenetic relationships, associated ARGs, and the structural features of plasmids in tmexCD2-toprJ2 -positive CR-ROR isolates. Five genetically distantly related tmexCD2-toprJ2 -positive CR-ROR isolates were identified. All exhibited multidrug-resistant (MDR) phenotypes and carried various ARGs, including carbapenemase genes such as bla KPC-2 , bla NDM-1 , and bla IMP-4. Genomic analysis revealed that tmexCD2-toprJ2 was plasmid-borne and frequently flanked by mobile genetic elements (MGEs), suggesting a high risk of horizontal gene transfer. The presence of tmexCD2-toprJ2 -carrying CR-ROR in hospital sewage underscores the role of wastewater as a potential environmental reservoir for clinically resistant genes. Ongoing surveillance of hospital effluents is crucial for tracking the dissemination of high-risk antimicrobial resistance determinants and informing timely public health interventions.