Effects of iron supplements and iron-containing micronutrient powders on the gut microbiome in Bangladeshi infants: a randomized controlled trial

Anemia is highly prevalent globally, especially in young children in low-income countries, where it often overlaps with a high burden of diarrheal disease. Distribution of iron interventions

(as supplements or iron-containing multiple micronutrient powders, MNPs) is a key anemia reduction strategy. Small studies in Africa indicate iron may reprofile the gut microbiome towards

pathogenic species. We seek to evaluate the safety of iron and MNPs based on their effects on diversity, composition, and function of the gut microbiome in children in rural Bangladesh as

part of a large placebo-controlled randomized controlled trial of iron or MNPs given for 3 months (ACTRN12617000660381). In 923 infants, we evaluate the microbiome before, immediately

following, and nine months after interventions, using 16S rRNA gene sequencing and shotgun metagenomics in a subset. We identify no increase in diarrhea with either treatment. In our primary

analysis, neither iron nor MNPs alter gut microbiome diversity or composition. However, when not adjusting for multiple comparisons, compared to placebo, children receiving iron and MNPs

exhibit reductions in commensal species (e.g., Bifidobacterium, Lactobacillus) and increases in potential pathogens, including Clostridium. These increases are most evident in children with

baseline iron repletion and are further supported by trend-based statistical analyses.

Anemia affects almost 300 million pre-school-aged children worldwide, and is most prevalent in children in low- and middle-income countries (LMICs).1 In 2019, the prevalence of anemia in

children aged 6–59 months was 49.0% in South-East Asia, and 43.1% in Bangladesh.2 Anemia in young children is often accompanied by a concomitant burden of diarrhea in low-income settings.

Diarrhea causes 10% of all child deaths in Asia and Africa,3 precipitated by unsafe water, sanitation and hygiene (WASH) conditions. The World Health Organization (WHO) recommends universal

distribution of iron (either as iron-containing multiple micronutrient powders—MNPs, or iron supplements, e.g., drops) to all young children where anemia is prevalent.4,5 However, the safety

of this approach is uncertain, with large field trials of iron-containing MNPs suggesting an increased risk of diarrhea.6

Trials in sub-Saharan Africa have reported pathogenic reprofiling of the gut microbiome (an increase in pathogenic enterobacteria and a decrease in commensal species) in young children

receiving iron-containing MNPs, associated with evidence of increased intestinal inflammation. This may relate to an increase in colonic iron that facilitates the growth of potentially

pathogenic bacteria at the expense of commensal species, leading to dysbiosis.7 An effect of iron interventions on the microbiome has been observed in studies conducted in sub-Saharan

Africa. A study of Kenyan children receiving iron-containing MNPs reported that iron promoted growth of enteropathogens e.g., Escherichia, Salmonella and Clostridium, and increased

intestinal inflammation.8 A second Kenyan study reported that even low-dose iron pathogenically reprofiled the microbiota and exacerbated intestinal inflammation.9 In both cases, these

conclusions were drawn from statistical analyses of the microbiome that were not adjusted for multiple comparisons, and could represent false discovery (i.e., false positive) findings.

Crucially, there remains limited data evaluating microbiome effects in South Asia, where effects from iron on the clinical endpoint of diarrhea have been observed.6 Iron status (deficiency

or repletion) influences intestinal iron uptake10 and could, therefore, potentially modify the effects of interventions on the microbiome.

Defining the safety of iron interventions is critical, given the ongoing public health recommendation for their distribution.11 There is an urgent need for well-powered, placebo-controlled

clinical trials that evaluate the safety of iron interventions on the gut microbiome using a rigorous study design to examine causal relationships.

To address this, we leverage the BRISC (Benefits and Risks of Iron InterventionS in Children) trial, a large, placebo-controlled double-blind, double-dummy randomized controlled trial

conducted in rural Bangladesh in which infants were randomized to receive three months of daily iron syrup, MNPs or placebo.12 In this large sub-study we present here, we evaluate stool

samples from a subset of BRISC participants at baseline, after three months of intervention, and after a further 9-month post-intervention follow-up. Samples are analyzed using 16S rRNA

amplicon sequencing, with a subset also analyzed with shotgun metagenomic sequencing. This combined methodology enables us to define causal iron-induced gut microbiome reprofiling by

exploring the effects of iron and MNPs compared to placebo on diversity and composition across an unprecedented sample size while also undertaking a high-resolution evaluation of the effect

of iron on species and function.

Between September 2018 and February 2019, we collected 923 baseline stool samples, 796 samples at the subsequent post-intervention time point 13 weeks later, and 578 samples at the

post-follow-up time point (Fig. 1A, Supplementary Fig. 1). Overall, a sample was provided at baseline from 84% of participants approached for this sub-study, and of these, 86% provided a

sample at midline and 63% at endline. Baseline characteristics of the cohort are summarized in Table 1. 16S rRNA amplicon sequencing was performed on 923 samples at baseline, 796 at midline,

and 578 at endline; a subset of 319, 320 and 315 samples also underwent shotgun metagenomic sequencing at these time points, respectively (Fig. 1B). Baseline characteristics of the shotgun

metagenomic sub-cohort are summarized in Table S1(Supplementary Material).

A BRISC trial schema showing assessment and sampling time points. B Flow diagram outlining stool samples for 16S rRNA and shotgun metagenomic sequencing. C Stacked bar plot presenting

taxonomic composition of baseline samples at study entry (i.e., baseline) based on relative abundance of the top five genera overall. Each bar represents a sample, with relative abundance on

the y-axis. Samples are grouped by trial arm (16S rRNA data). Taxonomic boxplots showing relative abundance of the four most abundant bacterial phyla by sampling time point (D) 16S rRNA

data and (E). shotgun metagenomic data. Boxplot center lines denote median value, with bounds of box indicating 25–75th percentiles. Whisker lines encompass 1.5 x interquartile range from

above the upper and below the lower quartiles. Data points outside whiskers are outliers. (n = 900 at baseline, n = 790 at post-intervention and n = 574 at post-follow-up time points for

(D), and n = 316 at baseline, n = 316 at post-intervention and n = 312 at post-follow-up time points for E). F Heatmap showing relative differences in abundance of genes relating to

microbiome functional profile by sampling time point across all trial arms, with baseline as reference. Relative abundance data underwent centered log ratio normalization/transformation in a

general linear model analysis in MaAsLin2, with data for differentially abundant functional pathways including coefficient (approximating log2-fold change), standard deviation and

FDR-adjusted p value. Only the 10 features exhibiting the greatest relative change in abundance (increase – in red – or decrease – in blue – from baseline) are shown. Abundance is expressed

as −log10(FDR-adjusted p value) * sign(log2-fold change) as per the heatmap calculation used by MaAsLin2. (n = 255 at baseline, n = 231 at post-intervention and n = 312 at post-follow-up

time points) (Shotgun metagenomic data). A, B created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license

(https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Source data for Fig. 1C–F are provided in the Source Data file.

The BRISC trial found that across all participants, neither iron nor MNPs significantly increased parent-reported days with diarrhea (incidence rate ratio (IRR) 1.13 [95% CI 0.89–1.42] for

iron versus placebo, IRR 1.17 [0.93–1.48] for MNPs versus placebo).12 Among children in the microbiome sub-study, again, neither iron nor MNPs statistically significantly increased

parent-reported days with diarrhea (IRR 1.43 [0.89–2.27] for iron versus placebo, IRR 1.24 [0.77–1.98] for MNPs versus placebo).

For the gut microbiome analysis we first examined baseline taxonomic profiles and changes with age. The top five genera detected at baseline are presented in Fig. 1C (source data in

Supplementary Material). No significant baseline differences were seen between arms at the genus or species levels. We then examined changes to the microbiome as children grew from 8 to 20

months of age. Using 16S and shotgun metagenomics, we observed reductions in Actinomycetota and Pseudomonadota phyla and increased Bacteroidota over this 12-month timeframe (Fig. 1D, E).

Compared to baseline, many key functional pathways altered as children reached the 3-month post-intervention period (11 months of age); 155 pathways were differentially abundant compared to

baseline (91 increased and 64 reduced). At 9 months follow-up (age 20 months), 349 pathways were differentially abundant (113 up and 236 down) compared to baseline. For both comparisons the

pathway with the greatest increase was dTDP-L-rhamnose biosynthesis I (log2-fold change 0.001, SD 0.0004, adj. p-value 0.003 at 11 months; log2-fold change 0.006, SD 0.0004, adj. p