Galacto-oligosaccharides alone and combined with lactoferrin impact the Kenyan infant gut microbiota and epithelial barrier integrity during iron supplementation in vitro

Abstract

Aim: Iron supplementation to African weaning infants was associated with increased enteropathogen levels. While cohort studies demonstrated that specific prebiotics inhibit enteropathogens during iron supplementation, their mechanisms remain elusive. Here, we investigated the in vitro impact of galacto-oligosaccharides (GOS) and iron-sequestering bovine lactoferrin (bLF) alone and combined on the gut microbiota of Kenyan infants during low-dose iron supplementation. Methods: Different doses of iron, GOS, and bLF were first screened during batch fermentations (n = 3), and the effect of these factors was studied on microbiota community structure and activity in the new Kenyan infant continuous intestinal PolyFermS model. The impact of different fermentation treatments on barrier integrity, enterotoxigenic Escherichia coli (ETEC) infection, and inflammatory response was assessed using a transwell co-culture of epithelial and immune cells. Results: A dose-dependent increase in short-chain fatty acid (SCFA) production, Bifidobacterium and Lactobacillus/Leuconostoc/Pediococcus (LLP) growth was detected with GOS alone and combined with bLF during iron supplementation in batches. This was confirmed in the continuous PolyFermS model, which also showed a treatment-induced inhibition of opportunistic pathogens C. difficile and C. perfringens. In all tests, supplementation of iron alone and combined with bLF did not have a significant effect on microbiota composition and activity. We observed a strengthening of the epithelial barrier and a decrease in cell death and pro-inflammatory response during ETEC infection with microbiota fermentation supernatants from iron + GOS, iron + bLF, and iron + GOS + bLF treatments compared to iron alone. Conclusion: Overall, beneficial effects on infant gut microbiota were shown using advanced in vitro models for GOS alone and combined with bLF during low-dose iron supplementation.

Keywords: Gut microbiome; ex vivo models; iron fortification; micronutrient; prebiotic; weaning infant.

Figure 1

Overview of batch fermentation and PolyFermS experiments. IR with immobilized Kenyan infant fecal microbiota and connected PolyFermS second-stage treatment reactors (TR1-6). Each fecal microbiota was immobilized and cultivated separately. (A) IR microbiota of donors 1 to 3 were used for batch fermentations in 24-well plates, and (B) IR of donors 4 and 5 were connected to TRs. IR: Inoculum reactors; TRs: test reactors; qPCR: quantitative PCR; HPLC: high-performance liquid chromatography; T: temperature; RT: retention time; STAB: stabilization period; TREAT: treatment period.

Figure 2

Overview of mammalian cell model setup using a transwell system to assess the impact of differently treated PolyFermS supernatant on the epithelial barrier, pathogen infection, and inflammatory response. TEER: Transepithelial electrical resistance; MOI: multiplicity of infection; LDH: lactate dehydrogenase; ETEC: enterotoxigenic Escherichia coli; NFκB: nuclear factor kappa B; SEAP: secreted embryonic alkaline phosphatase.

Figure 3

Quantification of beneficial and potential pathogenic taxa before and after treatment with iron, GOS, and bLF in PolyFermS. Mean ± SD of log₁₀ bacteria/mL effluent is shown for the last three days of stabilization (STAB) and the last three days of treatment (TREAT) of (A) two experimental periods of donor microbiota 4 and (B) three experimental periods of donor microbiota 5. Significant differences between STAB and TREAT in the corresponding periods are indicated. ^*P < 0.05, ^**P < 0.01. Enterobacteriaceae and C. difficile were also detected in donor 5 [Supplementary Figure 5]. GOS: Galacto-oligosaccharides; bLF: bovine lactoferrin.

Figure 4

PCoA of binary and weighted Jaccard distance metrics of before and after treatment with iron, GOS and bLF in PolyFermS. The last three days of stabilization and treatment of two and three experimental periods are shown for (A) donors 4 and (B) 5, respectively. The top 8 genera associated with the community composition are plotted as vectors in weighted Jaccard. PCoA: Principal coordinate analysis; GOS: galacto-oligosaccharides; bLF: bovine lactoferrin.

Figure 5

Differential abundance analysis (DESeq2) at genus level after treatment with iron and GOS with/without bLF in PolyFermS. Barplots show log₂-fold changes of genera significantly (P < 0.05) different in relative abundance between the last three days of iron co-supplementation with GOS and/or bLF and the last three days of supplementation with iron alone for all experimental periods in (A) donor 4 and (B) donor 5 microbiota. GOS: Galacto-oligosaccharides; bLF: bovine lactoferrin.

Figure 6

Quantification of total and intermediate metabolites and SCFA after treatment with iron and GOS with/without bLF in PolyFermS. Mean ± SD of metabolite concentration is shown for the last three days of stabilization (STAB) and the last three days of treatment (TREAT) of (A) two experimental periods of donor 4 and (B) three experimental periods of donor 5. Significant differences between STAB and TREAT in the corresponding periods are indicated. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. SCFA: Short-chain fatty acid; GOS: galacto-oligosaccharides; bLF: bovine lactoferrin.

Figure 7

Effect of PolyFermS supernatant treatments on epithelial barrier, infection and inflammation in a Caco-2/HT29-MTX/THP-1 Blue cell co-culture model. Barrier integrity was assessed by TEER measurement after 24-h exposure to 20% of microbiota supernatant of (A) donor 4 and (B) donor 5. CFU of adhered and invaded ETEC and cell cytotoxicity were assessed by plating and LDH release, respectively, after 3 h of infection in the presence of 20% microbiota supernatant. NFκB activation in THP1-Blue cells was assessed after 21 h of further incubation following infection. Mean ± SD is shown, n = 3 independent cell passages. TEER: Transepithelial electrical resistance; CFU: colony forming units; ETEC: enterotoxigenic Escherichia coli; LDH: lactate dehydrogenase; NFκB: nuclear factor kappa B.