Clonal outbreak of NDM-1-producing Enterobacter hormaechei belonging to high-risk international clone ST78 with the coexistence of tmexCD2-toprJ2 and mcr-9 in China

•China has seen as emergence of Enterobacter hormaechei strains producing NDM-1 co-harbouring tmexCD2-toprJ2 and mcr-9.•This study reports a clonal outbreak of tmexCD2-toprJ2 and mcr-9-positive carbapenem-resistant E. hormaechei belonging to high-risk international clone ST78 between 2018 and 2021 in hospitalised patients.•The integrases and IS elements were also drivers of the horizontal transmission of tmexCD2-toprJ2, blaNDM–1 and mcr-9.•There is a silent spread and rapid evolution of mcr-9-carrying E. hormaechei worldwide. The emergence of carbapenem-resistant Enterobacter cloacae complex (CR-ECC) has posed significant global challenges to the clinical treatment of healthcare-associated infections. This study reports the clonal outbreak of NDM-1-producing Enterobacter hormaechei (E. hormaechei) with the coexistence of tmexCD2-toprJ2 and mcr-9 in China. During the outbreak (January 2018 – December 2021), 15 non-repetitive multidrug-resistant E. hormaechei strains were obtained from 13 patients in a tertiary hospital. Antimicrobial susceptibility testing, plasmid stability, plasmid conjugation, plasmid fitness evaluation, colistin induction, whole-genome sequencing, and bioinformatics analysis were performed. A phylogenetic tree was constructed based on single nucleotide polymorphisms of core genomes to illustrate the evolutionary dynamics of mcr-9-carrying E. hormaechei strains worldwide. The 15 E. hormaechei strains belonged to the high-risk international clone ST78 and co-harboured tmexCD2-toprJ2 and blaNDM-1, of which 12 E. hormaechei strains carried the mcr-9 gene. Whole-genome sequencing analysis revealed that tmexCD2-toprJ2 and blaNDM-1 coexisted on the IncFIB/IncFII-type plasmid, which could be transferred to Escherichia coli J53 by conjugation and had a significant effect on host fitness. The mcr-9 gene was located between two insertion sequences, IS903B and IS1R, but lacked the two-component system regulatory gene qseBC, which might be the reason for all mcr-9-positive E. hormaechei strains remaining susceptible to colistin. The expression of mcr-9 was not inducible in strains confirmed by colistin induction assays. Phylogenetic analysis illustrated the silent spread and rapid evolution of mcr-9-carrying E. hormaechei worldwide. This study enriched the epidemiological and genomic characterisation of the coexistence of tmexCD2-toprJ2 and mcr-9 in ST78 CR-ECC isolates and demonstrated that they could prolong clonal dissemination in a tertiary hosp