Convergence of Carbapenem and Colistin Resistance in Intestinal Escherichia coli ST167/ST2659 Driven by IncX3 blaNDM-5 and IncI2-Associated Mcr-1.1 Mobility

Objective: Intestinal colonization by multidrug-resistant Escherichia coli provides a silent reservoir for dissemination of last-line resistance. The co-occurrence of bla NDM-5 and the plasmid-mediated colistin resistance gene mcr-1.1 is particularly concerning, yet the plasmid architectures and transfer potential of colonizing isolates remain poorly characterized. Methods: Two bla NDM-5 - and mcr-1.1 -positive E. coli isolates were recovered from fecal samples during colonization screening were investigated (L1771 and L2386). Antimicrobial susceptibility was determined by MIC testing. Short- and long-read sequencing was used to achieve plasmid-level resolution. S1-nuclease PFGE and Southern blot hybridization were performed to localize resistance genes. Conjugation assays were conducted to evaluate plasmid transferability. Results: Both isolates were resistant to colistin (MIC 4 mg/L) and carbapenems (L1771: imipenem 32 mg/L, meropenem 32 mg/L; L2386: imipenem 16 mg/L, meropenem 16 mg/L). L1771 contained a bla NDM-5 -carrying IncX3 plasmid (~46 kb), whereas mcr-1.1 was located on the chromosome. In contrast, plasmid-resolved analysis of L2386 revealed a high-risk configuration involving two distinct resistance plasmids: a bla NDM-5 -carrying IncX3 plasmid (~48 kb) and an mcr-1.1 -carrying IncI2 plasmids (~65 kb). Together, these plasmids formed a multi-plasmid “last-line resistance package” linking carbapenem and colistin resistance within a single isolate. The IncI2 mcr-1.1 plasmid p2803-MCR co-carried bla CTX-M-199 . Importantly, all three resistance plasmids identified above: the bla NDM-5 -carrying IncX3 plasmid in L1771 and the bla NDM-5 -carrying IncX3 plasmid together with the mcr-1.1 -carrying IncI2 plasmid in L2386 were transferable by conjugation, supporting strong potential for horizontal spread and co-selection. Conclusion: This study provides plasmid-resolved and functionally validated evidence that gut-colonizing E. coli can assemble a fully conjugative, multi-plasmid resistance package linking carbapenem and colistin resistance, including an IncI2 mcr-1.1 platform with potential broad-host-range dissemination. These findings underscore the need for surveillance that resolves plasmid backbones and transferability, not only resistance gene presence. Keywords: Escherichia coli , intestinal colonization, bla NDM-5 , mcr-1.1 , IncX3, IncI2, conjugation