Accelerated Laboratory Evolution Generates Growth-Costly High-Transfer Variants of an mcr-1-Bearing IncI2 Resistance Plasmid

This study aimed to characterize the plasmid-associated changes and host fitness consequences accompanying experimentally enriched high-transfer variants of the mcr-1-bearing IncI2 plasmid p14E1050.

We established an Accelerated Laboratory Evolution (ALE) system that coupled pMP6-mediated mutagenesis with iterative selection for enhanced conjugation frequency of the mcr-1-bearing p14E1050. Evolved lineages were characterized using conjugation assays, plasmid copy number (PCN) quantification, growth curve measurement, RNA-seq, and whole-genome sequencing (WGS).

Under selection for plasmid transmissibility, the mcr-1-bearing IncI2 plasmid p14E1050 rapidly evolved an enhanced conjugation phenotype, exhibiting a 41.05 ± 14.77-fold increase in conjugation frequency. WGS identified 32 mutations within the plasmid backbone of evolved variants. Increased PCN was associated with elevated conjugation frequency, while transcriptomic analysis of independent high-transfer variants revealed shared host-plasmid transcriptional remodeling, including upregulation of conjugation-related genes and pathways involved in nucleotide metabolism. Notably, enhanced transmissibility was accompanied by reduced host growth performance, indicating a cost to host-plasmid compatibility.

Accelerated laboratory evolution rapidly enriched high-transfer variants of the mcr-1-bearing IncI2 plasmid p14E1050, accompanied by increased PCN and reduced host growth. These findings suggest that high-transfer states can emerge during accelerated plasmid evolution, highlighting the relevance of evolutionary accessibility when evaluating antimicrobial resistance (AMR) plasmid dissemination risk.