Identification of mcr-13 in the Endangered Lynx pardinus, and mcr-1 in Sus scrofa, Expands the Diversity of Plasmid-Mediated Colistin Resistance Determinants of Escherichia coli from Wildlife

Background: Plasmid-mediated colistin resistance threatens the effectiveness of one of the last-line antibiotics for treating multidrug-resistant Gram-negative infections. Although wildlife is increasingly recognized as a reservoir of mobile antimicrobial resistance genes, its role in the emergence and dissemination of novel plasmid-mediated colistin resistance (mcr) genes remains poorly understood. Objectives: This study aimed to characterize two distinct mcr-carrying E. coli isolates identified in Spanish wildlife. Methods: Among 807 faecal samples from healthy wild animals screened, two mcr-positive E. coli isolates were identified. Genomic characterization included whole-genome sequencing, plasmid analysis, and functional assays, followed by heterologous expression and lipid A characterization by Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC-HRMS). Results: Analysis of the two mcr-positive E. coli isolates revealed the first mcr-1-positive isolate from a wild boar (Sus scrofa) in Extremadura, Spain, carrying mcr-1 on a 33.3-kb conjugative IncX4 plasmid, while the isolate recovered from an Iberian lynx harbored mcr-13, a novel plasmid-borne mcr-like gene encoding a putative phosphoethanolamine (pEtN) transferase on a 163-kb non-conjugative plasmid. MCR-13 showed 82% amino acid identity with MCR-10, increased colistin MIC two-fold in a susceptible E. coli host, and mediated phosphoethanolamine modification of lipid A, although to a lower extent than MCR-1. Conclusions: The co-occurrence of established (mcr-1) and a novel mcr-like gene mcr-13, further supports the role of wildlife as a potential reservoir of emerging resistance genes and highlights the value of including wildlife in One Health surveillance programs.