A nutritional link to antimicrobial resistance: iron scarcity promotes plasmid co-selection in Escherichia coli

Multidrug-resistant (MDR) Escherichia coli represents a larger fraction of the E. coli population in preweaned dairy calf feces compared to older postweaned calves and adult cows. Previous work demonstrated that the MDR genotype is strongly associated with iron-acquisition genes (iucABCD-iutA and sitABCD), which are primarily carried on IncFIB plasmids that frequently co-encode these systems with antimicrobial-resistance genes in bovine E. coli. We hypothesized that iron limitation in the preweaned calf gut favors E. coli carrying these iron-acquisition systems, thereby indirectly selecting for antimicrobial resistance. To test this, we quantified growth by isothermal microcalorimetry in bovine cecal medium and used RT-qPCR to measure expression of iron-acquisition genes under iron-replete and iron-limited conditions. Under iron-replete conditions, plasmid carriage imposed a negligible fitness cost. Under iron limitation, IncFIB-positive strains generated up to 3.5-fold more cumulative heat and sustained metabolic activity 6-10 h longer than their plasmid-negative counterparts. This advantage corresponded to strong siderophore gene upregulation, with iucA expression increased by up to 38.4-fold. Multivariate analysis further distinguished plasmid-bearing from plasmid-free groups under iron stress. The results of this study demonstrate that plasmid maintenance costs are negligible in rich medium, and they are fully offset by the significant growth advantage conferred by iron-acquisition systems in iron-limited growth conditions. These findings suggest that gut iron availability contributes to the maintenance of antimicrobial resistance in young calves and highlight nutrition-based strategies as potential interventions to reduce antimicrobial resistance in these animals.IMPORTANCEThe preweaned calf gut is an iron-poor environment that favors Escherichia coli carrying IncFIB plasmids encoding both siderophore systems and antimicrobial resistance genes. Our findings show that these plasmids convert a potential liability, the cost of extra DNA, into a fitness advantage under iron limitation. This nutritional-genetic interaction explains why multidrug-resistant strains persist in young calves and suggests that dietary iron management could help reduce antimicrobial resistance in livestock, with implications for food safety and public health.