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

Lianmin Bao,1,* Tongxi Hu,2,3,* Qing Zhang,4 Heping Lin,1 Hanhan Yan,1 Han Gao,1 Xiaojing Liu,1 Zongxiao Shangguan1 1Department of Respiratory and Critical Care Medicine, The Third Affiliated Hospital of Wenzhou Medical University, Ruian, People’s Republic of China; 2School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 4Department of Laboratory Medicine, The Third Affiliated Hospital of Wenzhou Medical University, Ruian, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zongxiao Shangguan, Email [email protected]: Intestinal colonization by multidrug-resistant Escherichia coli provides a silent reservoir for dissemination of last-line resistance. The co-occurrence of blaNDM-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 blaNDM-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 blaNDM-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 blaNDM-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 blaCTX-M-199. Importantly, all three resistance plasmids identified above: the blaNDM-5-carrying IncX3 plasmid in L1771 and the blaNDM-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, blaNDM-5, mcr-1.1, IncX3, IncI2, conjugation