The Effect of Oral Iron Supplementation on Gut Microbial Composition: a Secondary Analysis of a Double-Blind, Randomized Controlled Trial among Cambodian Women of Reproductive Age

The World Health Organization recommends untargeted iron supplementation for women of reproductive age (WRA) in countries where anemia prevalence is greater than 40%, such as Cambodia. Iron supplements, however, often have poor bioavailability, so the majority remains unabsorbed in the colon. The gut houses many iron-dependent bacterial enteropathogens; thus, providing iron to individuals may be more harmful than helpful. We examined the effects of two oral iron supplements with differing bioavailability on the gut microbiomes in Cambodian WRA. This study is a secondary analysis of a double-blind, randomized controlled trial of oral iron supplementation in Cambodian WRA. For 12 weeks, participants received ferrous sulfate, ferrous bisglycinate, or placebo. Participants provided stool samples at baseline and 12 weeks. A subset of stool samples (n = 172), representing the three groups, were randomly selected for gut microbial analysis by 16S rRNA gene sequencing and targeted real-time PCR (qPCR). At baseline, 1% of women had iron-deficiency anemia. The most abundant gut phyla were Bacteroidota (45.7%) and Firmicutes (42.1%). Iron supplementation did not alter gut microbial diversity. Ferrous bisglycinate increased the relative abundance of Enterobacteriaceae, and there was a trend towards an increase in the relative abundance of Escherichia-Shigella. qPCR detected an increase in the enteropathogenic Escherichia coli (EPEC) virulence gene, bfpA, in the group that received ferrous sulfate. Thus, iron supplementation did not affect overall gut bacterial diversity in predominantly iron-replete Cambodian WRA, however, evidence does suggest an increase in relative abundance within the broad family Enterobacteriaceae associated with ferrous bisglycinate use. IMPORTANCE To the best of our knowledge, this is the first published study to characterize the effects of oral iron supplementation on the gut microbiomes of Cambodian WRA. Our study found that iron supplementation with ferrous bisglycinate increases the relative abundance of Enterobacteriaceae, which is a family of bacteria that includes many Gram-negative enteric pathogens like Salmonella, Shigella, and Escherichia coli. Using qPCR for additional analysis, we were able to detect genes associated with enteropathogenic E. coli, a type of diarrheagenic E. coli known to be present around the world, including water systems in Cambodia. The current WHO guidelines recommend blanket (untargeted) iron supplementation for Cambodian WRA despite a lack of studies in this population examining iron’s effect on the gut microbiome. This study can facilitate future research that may inform evidence-based global practice and policy.