Impacts of dietary copper on the swine gut microbiome and antibiotic resistome

Restrictions on antibiotic growth promoters have prompted livestock producers to use alternative growth promoters, and dietary copper (Cu) supplementation is currently being widely used in pig production. However, elevated doses of dietary Cu constitute a risk for co-selection of antibiotic resistance and the risk may depend on the type of Cu-based feed additives being used. We here report the first controlled experiment investigating the impact of two contrasting Cu-based feed additives on the overall swine gut microbiome and antibiotic resistome. DNA was extracted from fecal samples (n = 96) collected at four time points during 116 days from 120 pigs allotted to three dietary treatments: control, divalent copper sulfate (CuSO₄; 250 μg Cu g^-1 feed), and monovalent copper oxide (Cu₂O; 250 μg Cu g^-1 feed). Bacterial community composition, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) were assessed, and bioavailable Cu ([Cu]_bio) was determined using whole-cell bacterial bioreporters. Cu supplementation to feed increased total Cu concentrations ([Cu]_total) and [Cu]_bio in feces 8-10 fold and at least 670-1000 fold, respectively, but with no significant differences between the two Cu sources. The swine gut microbiome harbored highly abundant and diverse ARGs and MGEs irrespective of the treatments throughout the experiment. Microbiomes differed significantly between pig growth stages and tended to converge over time, but only minor changes in the bacterial community composition and resistome could be linked to Cu supplementation. A significant correlation between bacterial community composition (i.e., bacterial taxa present) and ARG prevalence patterns were observed by Procrustes analysis. Overall, results of the experiment did not provide evidence for Cu-induced co-selection of ARGs or MGEs even at a Cu concentration level exceeding the maximal permitted level for pig diets in the EU (25 to 150 μg Cu g^-1 feed depending on pig age).