Dietary iron and metal-based growth promoters differentially modulate the gut resistome and Escherichia coli virulome in weaned pigsOA

Background High levels of zinc oxide(ZnO)and copper sulfate are widely used as alternative growth promoters in postweaning pig diet.However,excessive exposure to these metals may drive co-selection for heavy metal(HMR)and antibiotic resistance(AMR).Nursery diets also contain abundant iron to offset the low bioavailability of plantderived iron,yet how dietary iron influence gut dysbiosis and microbial resistance in postweaning pigs remains unclear.This exploratory study examined the effects of dietary iron and metal-based growth promoters on the fecal resistome of postweaning pigs using shotgun metagenomics and whole-genome sequencing(WGS).Methods Fifty weanling pigs were stratified and randomly assigned to five dietary treatments for 24 d.Experimental diets included a control diet(Con)containing 25,139,and 141 mg/kg of Cu,Fe,and Zn,respectively,a low-iron diet(LFe,19 mg Fe/kg),a high-iron diet(HFe,1,219 mg Fe/kg),a high-copper diet(HCu,257 mg Cu/kg),and a high-zinc diet(HZn,2,631 mg Zn/kg,including 2,490 mg Zn/kg from ZnO).All pigs were orally administered with F18 enterotoxigenic Escherichia coli(ETEC)on d 13–16.Metagenome sequencing were performed on d 24 fecal DNA(n=24)to identify HMR genes(BacMet Predicted database)and AMR genes(CARD database).Functional annotation was performed using HUMAnN3.Whole genome sequencing(WGS)was conducted on 120 E.coli isolates from fecal cultures on d 1,12,and 24,and AMR and virulence genes were identified from contig assemblies using ABRicate.Results Dietary metal treatments significantly alteredβ-diversity of HMR genes compared with Con,with HZn differing from both HCu and LFe(P<0.05).Fecal iron levels correlated with sodB(ρ=0.64,P=0.075),an iron-containing superoxide dismutase,while fecal copper levels correlated with pcoC(ρ=0.66,P=0.075),a plasmid-mediated copper resistance gene.Across metagenomes,172 AMR genes were identified,dominated by glycopeptide and tetracycline resistance.While dietary iron had minimal effects on fecal AMR profile,HZn induced the largest shifts in resistome,including increases of ant(9)-la,conferring aminoglycoside resistance on mobile genetic elements,and adeF,encoding a multidrug efflux pump(P<0.05).Functional profiling revealed enrichment of carbohydrate metabolism pathways in HZn group(P<0.05).WGS of E.coli isolates showed distinct AMR profiles under HZn on d 24 and distinct virulence profile under LFe on d 12,exhibiting increased prevalence of exotoxin and T3SS genes(P<0.05).Conclusion Dietary iron restriction enhanced E.coli virulence genes,whereas excessive ZnO induced the most pronounced changes in the gut resistome and microbial metabolism,highlighting a risk for AMR co-selection and marked influence on gut microbiota.