Abstract
Background/Objective: The rise of multidrug-resistant bacteria underscores the urgent need for alternative antimicrobial strategies. Metal-based compounds and bacteriophage (phage) therapy have emerged as promising candidates, but the evolutionary trade-offs associated with these selective pressures and their combination remain poorly understood. This study aimed to investigate how prior exposure to T4 phage influences Escherichia coli B's subsequent adaptation to iron (III) stress and to assess the resulting phenotypic and genomic signatures of dual resistance. Method: In this study, we performed experimental evolution using Escherichia coli B to investigate adaptive responses under four conditions: control (LB broth), T4 phage-only, iron (III) sulfate-only, and sequential phage followed by iron (III) exposure. Each treatment consisted of ten independently evolved populations (biological replicates), all derived from a common ancestral strain and passaged daily for 35 days. Phage resistance evolved rapidly, with complete resistance observed within 24 h of exposure. Results: In contrast, iron-selected populations evolved tolerance to high iron concentrations (1000-1750 mg/L) over time at a cost to resistance in other metals (gallium and iron (II) and antibiotics (tetracycline). Notably, prior phage exposure altered these outcomes: phage/iron-selected populations retained phage resistance and iron tolerance but showed diminished resistance to iron (II) and distinct antibiotic sensitivity profiles. Whole-genome sequencing revealed stressor-specific adaptations: large deletions in phage receptor-related genes (waaA and waaG) under phage pressure, and selective sweeps in iron-adapted populations affecting regulatory and membrane-associated genes (qseB, basR, aroK, fieF, rseB, and cpxP). Conclusions: These results demonstrate that the sequence of environmental stressors significantly shapes phenotypic and genetic resistance outcomes. Our findings highlight the importance of fitness epistasis and historical contingency in microbial adaptation, with implications for the design of evolution-informed combination therapies.