Objective: Iron-containing fortified foods and supplements are important tools to reduce the consequences of iron deficiency, including anemia. However, unabsorbed iron from these interventions may perturb gastrointestinal integrity and the gut microbiome by promoting pathogen growth and proliferating antimicrobial resistance genes (ARGs). We aimed to evaluate the effect of household use of multiple micronutrient-fortified bouillon cubes on the gut resistomes of Ghanaian children aged 2-5 years.

Methods: This study is nested in a double-blind randomized controlled trial where households of enrolled children (n=690) received biweekly bouillon cube rations fortified with either:1) multiple micronutrients––vitamins A: 200 μg retinol/g, B12:1.2 μg/g, B9: 80 μg folic acid/g; zinc: 3 mg/g, iodine: 30 μg/g, and iron: 4 mg/g or 2) iodine only (control) for 38 weeks. Stool samples were collected into nucleic acid preservation tubes at baseline and week 35 from a randomly selected subgroup (n=136) and subjected to shotgun metagenomic sequencing. Resistomes were profiled via the Comprehensive Antibiotic Resistance Database and microbial species were identified using MetaPhlAn 4. Microbial determinants of ARGs were determined using correlational analyses (Compositionality Corrected by Renormalizaion and Permutation). Difference in differences (DiD) mixed models estimated intervention effects on microbial taxa, ARG richness (number of unique ARGs) and normalized ARG abundance (copies per million), including specifications by individual ARGs, antibiotic drug class, and resistance mechanism assessed via MaAsLin 3.

Results: Children (mean age at enrollment = 40 months) consumed ~1.5-1.9 g bouillon/d, and household intervention adherence was >99%. Biweekly prevalence of caregiver-reported antibiotic use and diarrhea was low. The intervention did not affect normalized ARG abundance over time (geometric mean ratio DiD: 0.99; 95% CI: 0.91–1.08; p=0.78). The control group had a 9% reduction in ARG richness relative to the intervention group over time (incidence rate ratio DiD: 0.91 95% CI: 0.80–0.92; p < 0.01). No intervention effects emerged within ARG drug classes or resistance mechanisms (q > 0.05). Sixteen ARGs showed nominal changes over time (p < 0.05), but none remained significant after adjustments for multiple comparisons (q > 0.05). Escherichia coli was the primary microbial contributor of ARGs in fecal samples but was not differentially abundant between the control and intervention groups over time.
Conclusion: Multiple micronutrient-fortified bouillon cubes had no meaningful effects on gut resistomes of children in this context, suggesting minimal risk of ARG-mediated gastrointestinal or infectious morbidity.