Emergence of a conjugative hybrid plasmid co-harboring mcr-1.1, mcr-3.39, and tet(X4) in Escherichia coli from wastewater in Seoul: A potential threat to last-line antibiotics.

The emergence of plasmid-mediated antimicrobial resistance to last-line antibiotics such as colistin and tigecycline poses an escalating public health threat. In this study, we identified an Escherichia coli strain (WWCOL-256) isolated from municipal wastewater in Seoul, South Korea, co-harboring the mcr-1.1, mcr-3.39, and tet(X4) genes. Genomic analysis revealed that these resistance genes were located on a 237.9-kb IncFIA(HI1)-HI1A-HI1B(R27) hybrid plasmid (pCOL256-1), with the tet(X4) gene embedded within an ISVsa3-mediated transposon [∆ISVsa3-estT-tet(X4)-ISVsa3] and mcr-1.1 associated with the ISApl1-mcr-1.1-pap2 structure. The mcr-3.39 gene (∆TnAs2-mcr-3.39-dgkA-ISKpn40) was likely acquired from a co-resident IncFII plasmid (pCOL256-2). Functional assays demonstrated that the plasmid pCOL256-1 can be transferred via conjugation and remains stable without antibiotic selection, underscoring its potential to disseminate colistin and tigecycline resistance. Although global surveillance has detected the frequent co-occurrence of the mcr and tet(X) genes, their co-localization on the same plasmid remains rare. To the best of our knowledge, this is the first report of a single plasmid co-harboring the mcr-1, mcr-3.39, and tet(X4) genes, highlighting the urgent need for enhanced environmental genomic surveillance to prevent the spread of multidrug resistance determinants.