Both ionic and nanoparticle iron have been proposed as materials to control multidrug-resistant (MDR) bacteria. However, the potential bacteria to evolve resistance to nanoparticle bacteria remains unexplored. To this end, experimental evolution was utilized to produce five magnetite nanoparticle-resistant (FeNP_1–5) populations of Escherichia coli. The control populations were not exposed to magnetite nanoparticles. The 24-h growth of these replicates was evaluated in the presence of increasing concentrations magnetite NPs as well as other ionic metals (gallium III, iron II, iron III, and silver I) and antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). Scanning electron microscopy was utilized to determine cell size and shape in response to magnetite nanoparticle selection. Whole genome sequencing was carried out to determine if any genomic changes resulted from magnetite nanoparticle resistance. After 25 days of selection, magnetite resistance was evident in the FeNP treatment. The FeNP populations also showed a highly significantly (p < 0.0001) greater 24-h growth as measured by optical density in metals (Fe (II), Fe (III), Ga (III), Ag, and Cu II) as well as antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). The FeNP-resistant populations also showed a significantly greater cell length compared to controls (p < 0.001). Genomic analysis of FeNP identified both polymorphisms and hard selective sweeps in the RNA polymerase genes rpoA, rpoB, and rpoC. Collectively, our results show that E. coli can rapidly evolve resistance to magnetite nanoparticles and that this result is correlated resistances to other metals and antibiotics. There were also changes in cell morphology resulting from adaptation to magnetite NPs. Thus, the various applications of magnetite nanoparticles could result in unanticipated changes in resistance to both metal and antibiotics.

The rise in antimicrobial resistant bacteria have prompted the need for antibiotic alternatives. To address this problem, significant attention has been given to the antimicrobial use and novel applications of copper. As novel applications of antimicrobial copper increase, it is important to investigate how bacteria may adapt to copper over time. Here, we used experimental evolution with re-sequencing (EER-seq) and RNA-sequencing to study the evolution of copper resistance in Escherichia coli. Subsequently, we tested whether copper resistance led to rifampicin, chloramphenicol, bacitracin, and/or sulfonamide resistance. Our results demonstrate that E. coli is capable of rapidly evolving resistance to CuSO₄ after 37 days of selection. We also identified multiple de novo mutations and differential gene expression patterns associated with copper, most notably those mutations identified in the cpx gene. Furthermore, we found that the copper resistant bacteria had decreased sensitivity when compared to the ancestors in the presence of chloramphenicol, bacitracin, and sulfonamide. Our data suggest that the selection of copper resistance may inhibit growth in the antimicrobials tested, resulting in evolutionary trade-offs. The results of our study may have important implications as we consider the antimicrobial use of copper and how bacteria may respond to increased use over time.