biostudies-literatureUnknown13(1)Boyd-Vorsah S<h4>Background and objectives</h4>Copper is an essential micronutrient and a widely used antimicrobial, yet its widespread application may accelerate microbial resistance. We investigated how long-term copper (II) sulfate (CuSO₄) exposure drives genetic and phenotypic changes in <i>Escherichia coli</i>, focusing on survival, resistance mechanisms, and antibiotic cross-resistance.<h4>Methodology</h4>Fifty <i>E. coli</i> populations were evolved for 55 days under progressively increasing CuSO₄ concentrations. Whole-genome sequencing (WGS) identified genetic adaptations, while phenotypic changes were assessed using minimum inhibitory concentration (MIC) and fitness assays across CuSO₄ and antibiotic gradients.<h4>Results</h4>CuSO₄ imposed strong selective pressure, with only 16% of populations surviving prolonged exposure. Survivors exhibited up to eight-fold increases in CuSO₄ resistance, though some reverted to ancestral resistance levels when selective pressure was removed. Fitness assays showed that CuSO₄-selected populations maintained significantly higher fitness in high CuSO₄ environments than controls and ancestors (<i>P</i> < .001). WGS revealed diverse mutations in stress-response and metal-tolerance genes (<i>cusA</i>, <i>acrB</i>, <i>corA</i>, <i>fur</i>, and <i>ybhA</i>) without a single resistance signature. Although antibiotic cross-resistance was not observed, some CuSO₄-selected populations displayed elevated MICs for levofloxacin, colistin, trimethoprim, fosfomycin, and meropenem. Similar trends in controls suggest that additional factors, such as adaptation to laboratory media, also contribute to resistance.<h4>Conclusions and implications</h4>CuSO₄ exerts strong and variable selective pressure on <i>E. coli</i> populations, promoting diverse resistance pathways through distinct genetic and physiological mechanisms. While some CuSO₄-selected strains exhibited increased antibiotic resistance, trends in controls highlight the complexity of resistance evolution. These findings emphasize the need to monitor copper-driven antimicrobial resistance.Evolution, medicine, and public health176-187https://www.ebi.ac.uk/biostudies/studies/S-EPMC12409786biostudies-literatureSurvival, resistance, and fitness dynamics of &lt;i&gt;Escherichia coli&lt;/i&gt; populations after prolonged exposure to copper.PMC12409786Bui BYeh PJTorres Ortiz ABoyd-Vorsah SPulido SfalseSurvival, resistance, and fitness dynamics of &lt;i&gt;Escherichia coli&lt;/i&gt; populations after prolonged exposure to copper.<h4>Background and objectives</h4>Copper is an essential micronutrient and a widely used antimicrobial, yet its widespread application may accelerate microbial resistance. We investigated how long-term copper (II) sulfate (CuSO₄) exposure drives genetic and phenotypic changes in <i>Escherichia coli</i>, focusing on survival, resistance mechanisms, and antibiotic cross-resistance.<h4>Methodology</h4>Fifty <i>E. coli</i> populations were evolved for 55 days under progressively increasing CuSO₄ concentrations. Whole-genome sequencing (WGS) identified genetic adaptations, while phenotypic changes were assessed using minimum inhibitory concentration (MIC) and fitness assays across CuSO₄ and antibiotic gradients.<h4>Results</h4>CuSO₄ imposed strong selective pressure, with only 16% of populations surviving prolonged exposure. Survivors exhibited up to eight-fold increases in CuSO₄ resistance, though some reverted to ancestral resistance levels when selective pressure was removed. Fitness assays showed that CuSO₄-selected populations maintained significantly higher fitness in high CuSO₄ environments than controls and ancestors (<i>P</i> < .001). WGS revealed diverse mutations in stress-response and metal-tolerance genes (<i>cusA</i>, <i>acrB</i>, <i>corA</i>, <i>fur</i>, and <i>ybhA</i>) without a single resistance signature. Although antibiotic cross-resistance was not observed, some CuSO₄-selected populations displayed elevated MICs for levofloxacin, colistin, trimethoprim, fosfomycin, and meropenem. Similar trends in controls suggest that additional factors, such as adaptation to laboratory media, also contribute to resistance.<h4>Conclusions and implications</h4>CuSO₄ exerts strong and variable selective pressure on <i>E. coli</i> populations, promoting diverse resistance pathways through distinct genetic and physiological mechanisms. While some CuSO₄-selected strains exhibited increased antibiotic resistance, trends in controls highlight the complexity of resistance evolution. These findings emphasize the need to monitor copper-driven antimicrobial resistance.2025-01-01T00:00:00Z20252026-06-03T02:53:42.404Z2026-04-23T03:12:59.516ZS-EPMC124097864091763410.1093/emph/eoaf015