Impact of iron, GOS and LF on Kenyan infant gut microbiota in vitro
Iron fortification in infants living in rural areas of low- and middle-income countries has been associated with microbial gut dysbiosis and higher rates of infection. Modified micronutrient supplements that favour a healthy gut microbiota while improving iron absorption are under investigation. The addition of prebiotic galacto-oligosaccharides (GOS) in combination with iron-sequestering bovine lactoferrin (bLF) was shown to increase iron absorption in Kenyan infants in vivo. However, the direct effect of GOS and bLF treatment on the Kenyan infant gut microbiota during iron fortification must be assessed. In this study, we investigated the impact of GOS and bLF alone and combined on the gut microbiota of infants living in a rural area of Kenya during iron supplementation in vitro.  Kenyan infant fecal microbiota were cultivated in anaerobic batch fermentations and in the continuous PolyFermS model designed to closely mimic the Kenyan infant colon and diet (milk and maize porridge). First, different doses of iron, GOS and bLF were screened during batch fermentations in 24-well plates inoculated with three different infant microbiota. Next, the effect of iron, GOS and bLF on microbiota community structure was investigated using two Kenyan infant continuous intestinal PolyFermS model. Finally, PolyFermS culture supernatants were assessed on barrier integrity, enterotoxigenic E. coli (ETEC) infection and inflammatory response using a transwell co-culture of epithelial and immune cells.  A dose-dependent increase in SCFA production, Bifidobacterium and Lactobacillus/Leuconostoc/Pediococcus (LLP) was detected during 24 h batch fermentation of three donor microbiota with iron supplementation with GOS alone and combined with bLF. Iron with GOS alone and combined with bLF at concentrations mimicking the in vivo dose supplementation also strongly promoted SCFA production, Bifidobacterium and LLP during continuous fermentation of two donor microbiota in the PolyFermS model, while potential harmful taxa, including C. difficile and C. perfringens, additionally were inhibited. In all donor microbiota, supplementation of iron alone and combined with bLF did not have detectable effects on microbiota composition and metabolic activity. Cell barrier integrity increased upon exposure to PolyFermS culture supernatant from one of the two microbiota which was treated with GOS combined with bLF during iron supplementation. ETEC infection was not impacted by culture supernatants, while infection-induced cell cytotoxicity was decreased by supernatant from one microbiota treated with GOS alone and combined with bLF. Further, the pro-inflammatory response was decreased with PolyFermS culture supernatant from one microbiota treated with iron and GOS alone and combined with bLF and with culture supernatant from the second microbiota treated with iron and bLF alone.  In conclusion, addition of GOS alone and combined with bLF promoted SCFA production and beneficial gut microbes, and inhibited outgrowth of enteropathogens during iron supplementation in two independent Kenyan infant PolyFermS models. Further, we observed strengthening of the epithelial barrier and decrease in cell death and pro-inflammatory response during ETEC infection with PolyFermS culture supernatants from microbiota treated with GOS alone and with bLF and with bLF alone during iron supplementation. However, the effects were donor dependent. Overall, the beneficial effects of GOS combined with bLF were similar compared to GOS alone but superior compared to bLF alone during iron supplementation in vitro.