Dietary copper-driven colonic dysbiosis mediates oxidative stress and butyrate deficiency to facilitate the spread of resistome in pigs

Copper-induced transmission of antimicrobial resistance has been well documented in livestock farming environments, but the in vivo mechanisms driving fecal resistome development remain unclear. Here, 120 mg/kg CuSO₄ and copper-peptide were supplemented to piglets, and the fecal resistome development was first analyzed by metagenomic sequencing. In this study, dietary CuSO₄ drove abundant and diverse ARGs and MRGs. Following CuSO₄ deprivation, ARGs and copper resistance exhibited a persistent promotion, whereas most MRGs rapidly declined. The resistance development was characterized by abundant MGEs. This phenomenon expanded the multiple-antibiotic resistance reservoir in fecal community, which was preferentially harbored by pathogens. Furthermore, dietary CuSO₄ disturbed colonic homeostasis, characterized by impaired epithelial integrity and reduced butyrate-producing bacteria abundance, which coincided with an oxidative stress environment and increased prevalence of multiple-resistant pathogens, such as Escherichia coli and Enterococcus spp. In vitro validation further supported these associations, showing that butyrate supplementation and hypoxic conditions alleviated Cu²⁺-induced ROS generation and reduced the frequency of ARGs conjugative transfer. Overall, this study suggests that dietary inorganic copper may contribute to microbial disturbances linked to oxidative stress and potentially facilitate antimicrobial resistance transmission among pathogens, highlighting organic copper as a sustainable alternative for mitigating resistance risks in farmed animals.