Triple Threat: Elucidating Evolution Under Antibiotic, Metal, and Phage Pressure in Escherichia coli

ABSTRACT Antibiotic resistance is a growing challenge in the treatment of bacterial infections, prompting interest in alternative therapies. Understanding how bacterial populations evolve under different selective pressures is important for predicting resistance pathways. This study aimed to investigate the evolutionary responses of E. coli populations to different antimicrobial environments and assess how these conditions influence resistance to phage infection, ampicillin, and gallium nitrate. Populations of EC‐WT and EC‐Phage‐Resistant E. coli were experimentally evolved for 10 days under different selection conditions: ampicillin, gallium nitrate, and a combination of ampicillin and gallium nitrate. Following the evolution period, phage resistance was assessed using spot assays. Growth fitness in the presence of ampicillin and gallium nitrate was measured using assays conducted in 96‐well plates. Whole‐genome sequencing was performed to identify mutations associated with adaptive responses. Several evolved populations developed resistance across multiple treatments regardless of the initial selection condition. Notably, all phage‐resistant mutants exhibited triple resistance to phage infection, ampicillin, and gallium nitrate. Sequencing analyses revealed multiple mutations associated with increased growth fitness under ampicillin and gallium nitrate exposure. These findings demonstrate that E. coli populations can rapidly evolve multidrug resistance under diverse selection pressures. In some populations, epistatic interactions between preexisting phage‐resistance mutations and newly acquired mutations limited growth‐rate fitness, suggesting that such interactions may constrain certain evolutionary pathways of resistance. Examining phage therapy through an evolutionary framework can help identify potential resistance pathways and inform the development of more effective strategies for treating multidrug‐resistant bacterial infections.