Novel Substitutes of Phthalate Esters (PAEs) Promote the Propagation of Antibiotic Resistance Genes via Ferroptosis: Implication for the Environmental Safety Evaluation of PAE Substitutes

Abstract

The horizontal transfer of antibiotic resistance genes (ARGs) has become a major threat to global public health. Recent studies have found that ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation and glutathione depletion, may play a critical role in the dissemination of ARGs among environmental microbes. Here, we demonstrated for the first time that phthalate esters (PAEs) and their substitutes significantly enhanced plasmid conjugation by triggering ferroptosis-related pathways. Classical ferroptosis-associated responses, including the hyperpolarization of the cell membrane potential, elevated production of reactive oxygen species, and heightened membrane permeability, were observed under the stress of PAEs or their substitutes. Through integrated transcriptomic and metabolomic analyses, we revealed that these compounds triggered iron dysregulation via the upregulation of iron acquisition and storage pathways while suppressing DNA replication, concurrently causing oxidative damage that stimulated the plasmid conjugation. Molecular docking simulations revealed that PAEs and their substitutes competitively disrupted the functionality of ferric uptake regulator (Fur) protein, a master controller of intracellular iron homeostasis, with superior binding affinity than its natural ligand Fe²⁺. Integrated metagenomic sequencing and homology analyses demonstrated the conservation of Fur protein across biofilm microbiota and functional implications in iron homeostasis. Structural analysis based on the characteristic molecular fingerprints of chemicals pinpointed aliphatic chains as the crucial structure responsible for enhancing ARG propagation between bacteria. Our findings uncovered a mechanism by which PAEs and their substitutes exacerbated ARG dissemination through ferroptosis-mediated conjugation, providing crucial insights for environmental risk assessment and resistance mitigation strategies.