Administration of ferrous sulfate drops has significant effects on the gut microbiota of iron-sufficient infants: a randomised controlled study

Abstract

We read with interest the work by Jaeggi et al 1 and Paganinni et al 2 and commend their efforts. Despite differences in iron concentration, infants’ age and sequencing techniques, both studies demonstrate unfavourable iron effects on gut microbiota with decreased abundance of bifidobacteria and lactobacillus, and increased abundance of pathogenic bacteria in iron-deficient/anaemic Kenyan infants. We have investigated changes in gut microbial composition due to iron fortification or supplementation in healthy, Swedish infants. Iron-sufficient infants at 6 months of age were randomly allocated to receive low-iron-fortified formula (1.2 mg Fe/day; n=24), high-iron-fortified formula (6.6 mg Fe/day; n=24) or no-added-iron formula with liquid ferrous sulfate supplementation (iron drops; 6.6 mg Fe/day; n=24) for 45 days. All participants gave their informed consent before inclusion through parents or legal guardians. Total iron intake was 1.2, 6.4 and 5.7 mg/day (all differences p<0.01) in the low-iron, high-iron and iron-drops group, respectively. Stool samples were collected before and after the intervention. We applied 16S rRNA gene amplicon sequencing of the V3–V4 region to profile the gut microbiome using Illumina MiSeq. We used QIIME3 to assess composition and diversity of gut microbiota and the DESeq2 package4 to investigate differences in relative abundance of gut bacteria among the groups. PICRUSt was used to predict metagenome functional content.5 Vaginally delivered infants (n=53) with paired stool samples were included in the analyses. There were no significant differences in anthropometrics or iron-related biomarkers among the randomisation groups; no adverse effects were reported (diarrhoea, increased rates of infections, other illnesses, etc), and growth was not affected (table 1).6 Table 1 Baseline characteristics of the study participants and anthropometric and biochemical values at the 45-day follow-up. Low-iron formula High-iron formula Fe drops Participants (n) 18 18 17 Girls (n) 7 9 11 Birth weight (g)* 3717±560 3548±425 3800±436 Birth length (cm)* 51.1±2.2 50.2±1.6 51.7±1.7 Age at inclusion (months)* 6.1±0.3 6.1±0.2 6.1±0.3 Baseline Follow-up P values† Baseline Follow-up P values† Baseline Follow-up P values† P values‡ Weight (kg)* 8.3±1.0 9.1±1.1 <0.001 8.0±1.2 8.8±1.1 <0.001 8.4±0.9 9.2±0.9 <0.001 0.49 Length (cm)* 68.4±2.4 71.3±2.7 <0.001 67.4±2.8 69.9±2.6 <0.001 68.2±2.3 71.7±3.9 <0.001 0.26 Hb (g/L)* 111.6±6.0 110.2±9.0 0.71 112.2±7.0 112.9±5.9 0.62 118.0±11.5 112.2±5.8 0.06 0.51 S-Fe (µmol/L)* 9.5±4.2 9.5±4.3 0.66 9.7±3.8 8.7±3.6 0.42 8.8±4.5 9.6±3.6 0.64 0.78 S-transferrin (g/L)* 2.2±0.3 2.4±0.4 0.07 2.2±0.3 2.2±0.3 0.66 2.3±0.4 2.2±0.2 0.70 0.32 S-ferritin (µg/L)§ 89.4±44.7 61.2±32.5 <0.001 72.3±40.7 70.5±47.0 0.81 109.3±85.8 92.2±62.9 0.14 0.17 F-calprotectin (µg/g)¶ 132 (71, 241) 121 (55, 211) NS** 120 (59, 238) 105 (62, 421) NS** 263 (104, 345) 151 (109, 492) NS** NS††** Data are mean/geometric mean±SD or median (25th, 75th percentile). *Mean ±SD. †P values for within-group differences, paired-samples t-test. ‡P values for between-group differences, ANOVA. §Geometric mean ±SD. ¶Median (25th, 75th percentile). **P values for within-group differences, related-samples Wilcoxon signed-rank test. ††P values for between-group difference, independent-samples Kruskal-Wallis test. F, faecal; Hb, haemoglobin; NS, not significant at p=0.05; S, serum. In this study, we confirm findings that consumption of high-iron formula is associated with decreased relative abundance of Bifidobacterium (p<0.001, 60% vs 78%) after only 45 days of intervention, but we did not detect enhanced growth of pathogenic bacteria. However, we were able to partly confirm previous findings regarding abundance of lactobacilli due to iron consumption. We found lower relative abundance of Lactobacillus sp (p<0.007, 8% vs 42%) in infants who received iron drops versus high-iron-formula group. Unexpectedly, we also found higher relative abundance of Lactobacillus sp (p<0.0002, 42% vs 32%) in high-iron compared with low-iron formula group; this result challenges the hypothesis that the mode of iron administration has a direct effect on lactobacilli colonisation in the gut. Furthermore, the iron-drops group had lower abundance of Streptococcus (p<0.0003, 0.2% vs 0.9%) but higher abundance of Clostridium (p<0.05, 25% vs 9%) and Bacteroides (p<0.02, 1.2% vs 0.9%) compared with the high-iron formula group (figure 1). In the present study, all groups received formula with added galacto-oligosaccharides (GOS) at 3.3 g/L. This prebiotic may mitigate adverse effects of iron fortification on gut microbiota,2 but in the iron-drops group, iron was administered apart from the formula meals. Thus, we cannot exclude a possible protective effect of GOS on the gut microbiota of infants in our study. Figure 1 Differences in gut bacterial composition depend on the concentration and administration mode of the consumed iron. In the cladogram, showing the results of the microbiome analysis over time, taxa are grouped on the basis of synapomorphy. The outermost small, white circles represent the 561 OTUs (operational taxonomic units). Differences in gut microbial composition between the high-Fe-formula group versus the low-Fe-formula group over time are presented in the yellow component around the cladogram, where blue bars represent lower relative abundance of bacteria in the high-Fe-formula group compared with the low-Fe-formula group and the red bars represent higher relative abundance in the high-Fe-formula group compared with the low-Fe-formula group, respectively. Differences in gut microbial composition between the high-Fe-formula group versus the Fe-drops group over time are presented in the red component around the cladogram, where the blue bars represent lower relative abundance of bacteria in the high-Fe-formula group and the red bars represent higher relative abundance in the high-Fe-formula group compared with the Fe-drops group, respectively. OTU, operational taxonomic unit. As in the study by Paganinni et al,2 faecal calprotectin did not differ between the groups (table 1), but in our study, it correlated positively with Clostridium difficile in high-iron-formula (rSpearman=0.4, p<0.01) and iron-drops intervention groups (rSpearman=0.48, p<0.004). The bacterial function pathway related to Staphylococcus aureus infection (KEGG module 05150)5 was significantly lower in the iron-drops group compared with the low-iron-formula group (p=0.027). This is a novel finding which suggests that changes in bacterial composition due to administration of iron drops may reduce the protective response of the gut microbiota to bacterial infections. Nevertheless, no effects on the health of the participants were seen due to this. To summarise, in healthy, non-anaemic Swedish infants, consumption of high-iron formula is associated with significantly lower abundance of bifidobacteria compared with low-iron formula, and administration of iron as drops, even in a dose comparable with the daily iron requirement and for a short time, leads to decreased relative abundance of lactobacilli and potentially increases susceptibility to bacterial infection.