Genomic evolution and stability of the mcr-1-harboring IncI2 plasmid in the presence and absence of colistin

Abstract Background The emergence of plasmid-mediated colistin resistance, primarily driven by the mcr-1 gene, represents a major global health threat. IncI2 plasmids, one of the leading carriers of mcr-1, have been frequently recovered from clinical and agricultural settings. However, their persistence in the absence of antibiotic pressure and adaptive responses to colistin exposure remain poorly understood. Methods We conducted 60-day laboratory evolution experiments using Escherichia coli C600 carrying the mcr-1-harboring IncI2 plasmid pBD110 under three colistin concentrations (0, 2, and 4 mg/L). Stability was evaluated using polymerase chain reaction (PCR). Bacterial fitness was assessed using growth curve analysis and competition assays. Antimicrobial susceptibility was determined by the broth microdilution method. Conjugation potential was examined using conjugation experiments. Genomic alterations were investigated using whole-genome sequencing combined with bioinformatic analysis. Results pBD110 was stably maintained for 120 passages under all conditions, with no significant loss observed in the absence of colistin. Under strong selection (4 mg/L), plasmid abundance increased, whereas moderate pressure (2 mg/L) led to fitness costs and reduced plasmid copy number. Whole-genome sequencing revealed distinct adaptive strategies: plasmids under non-selective conditions accumulated mutations in conjugation-related genes, enhancing transfer frequency, whereas those under colistin exposure retained structural stability but acquired shufflon inversions that impaired conjugation. Host genomes accumulated numerous chromosomal mutations, particularly in metabolic and stress response pathways, to compensate for resistance-associated burdens. Conclusions IncI2 plasmids exhibit dual evolutionary strategies. In the absence of colistin, they optimized horizontal transfer, whereas under selective pressure, they prioritized the stability and vertical inheritance of mcr-1. These findings provide new insights into the persistence and dissemination of colistin resistance and highlight evolutionary trade-offs that shape plasmid-host coadaptation.