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

References

[1] Gedfie S,Melku M.Prevalence and associated factors of iron deficiency and iron deficiency anemia among under-5 children: a systematic review and meta-analysis.Glob Pediatr Health2022;9:2333794X221110860 PMCID:PMC9272181

[2] Giorgetti A,Nyilima S.The effects of 2’-fucosyllactose and lacto-N-neotetraose, galacto-oligosaccharides, and maternal human milk oligosaccharide profile on iron absorption in Kenyan infants.Am J Clin Nutr2023;117:64-72

[3] Paganini D,Kortman GAM.Prebiotic galacto-oligosaccharides mitigate the adverse effects of iron fortification on the gut microbiome: a randomised controlled study in Kenyan infants.Gut2017;66:1956-67

[4] De-Regil LM,Peña-Rosas JP.Point-of-use fortification of foods with micronutrient powders containing iron in children of preschool and school-age.Cochrane Database Syst Rev2017;11:CD009666 PMCID:PMC6486284

[5] WHO. WHO guideline: use of multiple micronutrient powders for point-of-use fortification of foods consumed by infants and young children aged 6-23 months and children aged 2-12 years. 2016.

[6] WHO. Nutritional anaemias: tools for effective prevention and control. 2017. Available from: https://www.who.int/publications/i/item/9789241513067. [Last accessed on 11 Dec 2024]

[7] Puga AM,Montero-Bravo A,Partearroyo T.Iron supplementation at the crossroads of nutrition and gut microbiota: the state of the art.Nutrients2022;14:1926 PMCID:PMC9102039

[8] Tang M,Hendricks AE.Iron in micronutrient powder promotes an unfavorable gut microbiota in Kenyan infants.Nutrients2017;9:776 PMCID:PMC5537890

[9] Jaeggi T,Moretti D.Iron fortification adversely affects the gut microbiome, increases pathogen abundance and induces intestinal inflammation in Kenyan infants.Gut2015;64:731-42

[10] Popovic A,Wang PW.Micronutrient supplements can promote disruptive protozoan and fungal communities in the developing infant gut.Nat Commun2021;12:6729 PMCID:PMC8602372

[11] Mikulic N,Stoffel NU.Prebiotics increase iron absorption and reduce the adverse effects of iron on the gut microbiome and inflammation: a randomized controlled trial using iron stable isotopes in Kenyan infants.Am J Clin Nutr2024;119:456-69 PMCID:PMC10884607

[12] Rai D,Zhuang W,Boettcher J.Longitudinal changes in lactoferrin concentrations in human milk: a global systematic review.Crit Rev Food Sci Nutr2014;54:1539-47

[13] Donker AE,Swinkels DW.The critical roles of iron during the journey from fetus to adolescent: developmental aspects of iron homeostasis.Blood Rev2021;50:100866

[14] Oda H,Yamauchi K.Lactoferrin and bifidobacteria.Biometals2014;27:915-22

[15] Gruden Š.Diverse mechanisms of antimicrobial activities of lactoferrins, lactoferricins, and other lactoferrin-derived peptides.Int J Mol Sci2021;22:11264 PMCID:PMC8541349

[16] Siqueiros-Cendón T,Iglesias-Figueroa BF,Salazar-Martínez J.Immunomodulatory effects of lactoferrin.Acta Pharmacol Sin2014;35:557-66 PMCID:PMC4814036

[17] Chen K,Li H.Effect of bovine lactoferrin from iron-fortified formulas on diarrhea and respiratory tract infections of weaned infants in a randomized controlled trial.Nutrition2016;32:222-7

[18] Motoki N,Tsukahara T.Effects of lactoferrin-fortified formula on acute gastrointestinal symptoms in children aged 12-32 months: a randomized, double-blind, placebo-controlled trial.Front Pediatr2020;8:233 PMCID:PMC7249745

[19] Mikulic N,Mwasi E.Iron absorption is greater from apo-lactoferrin and is similar between holo-lactoferrin and ferrous sulfate: stable iron isotope studies in Kenyan infants.J Nutr2020;150:3200-7

[20] ClinicalTrials.gov. Prebiotic GOS and lactoferrin with iron supplements. Available from: https://clinicaltrials.gov/study/NCT03866837. [Last accessed on 11 Dec 2024]

[21] Vogt JA.Fecal acetate is inversely related to acetate absorption from the human rectum and distal colon.J Nutr2003;133:3145-8

[22] Isenring J,Geirnaert A.In vitro human gut microbiota fermentation models: opportunities, challenges, and pitfalls.Microbiome Res Rep2023;2:2 PMCID:PMC10688811

[23] Zihler Berner A,Dostal A.Novel polyfermentor intestinal model (PolyFermS) for controlled ecological studies: validation and effect of pH.PLoS One2013;8:e77772 PMCID:PMC3813750

[24] Doo EH,Schwab C.Effect of dietary nucleosides and yeast extracts on composition and metabolic activity of infant gut microbiota in PolyFermS colonic fermentation models.FEMS Microbiol Ecol2017;93:fix088

[25] Dostal A,Chassard C,O’Mahony L.Salmonella adhesion, invasion and cellular immune responses are differentially affected by iron concentrations in a combined in vitro gut fermentation-cell model.PLoS One2014;9:e93549 PMCID:PMC3968171

[26] Rachmühl C,Cabrera PM.Long-term continuous cultivation of Kenyan infant fecal microbiota using the host adapted PolyFermS model.Sci Rep2023;13:20563 PMCID:PMC10667343

[27] Cabrera P, Rachmühl C, Derrien M, Bourdet-Sicard R, Lacroix C, Geirnaert A. Comparative prebiotic potential of galacto- and fructo-oligosaccharides, native inulin, and acacia gum in Kenyan infant gut microbiota during iron supplementation.ISME Commun2024;4:ycae033 PMCID:PMC11107946

[28] Poeker SA,Berchtold L.Understanding the prebiotic potential of different dietary fibers using an in vitro continuous adult fermentation model (PolyFermS).Sci Rep2018;8:4318 PMCID:PMC5847601

[29] Hernandez RJ,Guire KE.Capacity of the colon in children.AJR Am J Roentgenol1979;133:683-4

[30] Heinemann M,Lütgehetmann M.Polymicrobial enteric infections in African infants with diarrhoea-results from a longitudinal prospective case-control study.Clin Microbiol Infect2021;27:1792-8

[31] Mikulic N,Paganini D.Consumption of a single dose of prebiotic galacto-oligosaccharides does not enhance iron absorption from micronutrient powders in Kenyan infants: a stable iron isotope study.J Nutr2021;151:1205-12

[32] Tondeur MC,Christofides AL.Determination of iron absorption from intrinsically labeled microencapsulated ferrous fumarate (sprinkles) in infants with different iron and hematologic status by using a dual-stable-isotope method.Am J Clin Nutr2004;80:1436-44

[33] Walczyk T,Zavaleta N.Stable isotope labels as a tool to determine the iron absorption by Peruvian school children from a breakfast meal.Fresenius J Anal Chem1997;359:445-9

[34] Stoddard SF,Hein R,Schmidt TM.rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and archaea and a new foundation for future development.Nucleic Acids Res2015;43:D593-8 PMCID:PMC4383981

[35] Callahan BJ,Rosen MJ,Johnson AJ.DADA2: high-resolution sample inference from Illumina amplicon data.Nat Methods2016;13:581-3 PMCID:PMC4927377

[36] Constancias F.fconstancias/metabaRpipe: v0.9. 2022.

[37] Quast C,Yilmaz P.The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.Nucleic Acids Res2013;41:D590-6 PMCID:PMC3531112

[38] Vidal RM,Tennant SM.Colonization factors among enterotoxigenic Escherichia coli isolates from children with moderate-to-severe diarrhea and from matched controls in the Global Enteric Multicenter Study (GEMS).PLoS Negl Trop Dis2019;13:e0007037 PMCID:PMC6343939

[39] McMurdie PJ.phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.PLoS One2013;8:e61217 PMCID:PMC3632530

[40] Oksanen AJ,Kindt R. vegan: an R package for community ecologists. Available from: https://github.com/vegandevs/vegan. [Last accessed on 17 Dec 2024]

[41] Valero-Mora PM.ggplot2: elegant graphics for data analysis.J Stat Soft2010;35:1-3

[42] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550 PMCID:PMC4302049

[43] Dostal A,Chassard C,Lacroix C.Low iron availability in continuous in vitro colonic fermentations induces strong dysbiosis of the child gut microbial consortium and a decrease in main metabolites.FEMS Microbiol Ecol2013;83:161-75 PMCID:PMC3511601

[44] Celis AI,Huang KC.The impact of iron and heme availability on the healthy human gut microbiome in vivo and in vitro.Cell Chem Biol2023;30:110-26.e3 PMCID:PMC9913275

[45] Fanaro S,Bagna R.Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study.J Pediatr Gastroenterol Nutr2009;48:82-8

[46] Scholtens PA,Bindels JG,Tolboom JJ.Bifidogenic effects of solid weaning foods with added prebiotic oligosaccharides: a randomised controlled clinical trial.J Pediatr Gastroenterol Nutr2006;42:553-9

[47] Souza DDS,Weber TK,de Morais MB.Randomized, double-blind, placebo-controlled parallel clinical trial assessing the effect of fructooligosaccharides in infants with constipation.Nutrients2018;10:1602 PMCID:PMC6266108

[48] Logtenberg MJ,Hobé RG.Structure-specific fermentation of galacto-oligosaccharides, isomalto-oligosaccharides and isomalto/malto-polysaccharides by infant fecal microbiota and impact on dendritic cell cytokine responses.Mol Nutr Food Res2021;65:e2001077 PMCID:PMC8459273

[49] Le Blay G, Chassard C, Baltzer S, Lacroix C. Set up of a new in vitro model to study dietary fructans fermentation in formula-fed babies.Br J Nutr2010;103:403-11

[50] Yao D,Dong Y.In vitro fermentation of fructooligosaccharide and galactooligosaccharide and their effects on gut microbiota and SCFAs in infants.J Funct Foods2022;99:105329

[51] Martinez FA,Converti A,de Souza Oliveira RP.Bacteriocin production by Bifidobacterium spp. a review.Biotechnol Adv2013;31:482-8

[52] Vazquez-Gutierrez P,Werder J,Lacroix C.High iron-sequestrating bifidobacteria inhibit enteropathogen growth and adhesion to intestinal epithelial cells in vitro.Front Microbiol2016;7:1480 PMCID:PMC5031772

[53] McDonald JAK,Pechlivanis A.Inhibiting growth of clostridioides difficile by restoring valerate, produced by the intestinal microbiota.Gastroenterology2018;155:1495-507.e15 PMCID:PMC6347096

[54] Hryckowian AJ,Smits SA.Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model.Nat Microbiol2018;3:662-9 PMCID:PMC6126909

[55] Kondepudi KK,Nilsson I,Ljungh A.Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile.Anaerobe2012;18:489-97

[56] Embleton N,Cummings S,Ewer A.Lactoferrin impact on gut microbiota in preterm infants with late-onset sepsis or necrotising enterocolitis: the MAGPIE mechanisms of action study.Effic Mech Eval2021;8:1-88

[57] Sherman MP,Arcinue R.Randomized control trial of human recombinant lactoferrin: a substudy reveals effects on the fecal microbiome of very low birth weight infants.J Pediatr2016;173:S37-42

[58] Chichlowski M,Harris CL.Effect of bovine milk fat globule membrane and lactoferrin in infant formula on gut microbiome and metabolome at 4 months of age.Curr Dev Nutr2021;5:nzab027 PMCID:PMC8105244

[59] Young G,Cummings S.Mechanisms affecting the gut of preterm infants in enteral feeding trials: a nested cohort within a randomised controlled trial of lactoferrin.Arch Dis Child Fetal Neonatal Ed2023;108:272-9 PMCID:PMC10176413

[60] Haiwen Z,Bingxi Z.Oral administration of bovine lactoferrin-derived lactoferricin (Lfcin) b could attenuate enterohemorrhagic Escherichia coli O157:H7 induced intestinal disease through improving intestinal barrier function and microbiota.J Agric Food Chem2019;67:3932-45

[61] Teraguchi S,Ozawa K.Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk.Appl Environ Microbiol1995;61:501-6 PMCID:PMC167309

[62] Rastogi N,Alam H.Preparation and antimicrobial action of three tryptic digested functional molecules of bovine lactoferrin.PLoS One2014;9:e90011 PMCID:PMC3940724

[63] Lönnerdal B,Jiang R.Biological activities of commercial bovine lactoferrin sources.Biochem Cell Biol2021;99:35-46

[64] Chilton CH,Śpiewak K.Potential of lactoferrin to prevent antibiotic-induced Clostridium difficile infection.J Antimicrob Chemother2016;71:975-85 PMCID:PMC4790624

[65] Kortman GAM,Swinkels DW.Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface.PLoS One2012;7:e29968 PMCID:PMC3260200

[66] Bhakat D,Mukhopadhyay AK.Iron influences the expression of colonization factor CS6 of enterotoxigenic Escherichia coli.Microbiology2021;167:001089

[67] Hinrichsen F,Westermann M.Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis.Cell Metab2021;33:2355-66.e8

[68] Salvi PS.Butyrate and the intestinal epithelium: modulation of proliferation and inflammation in homeostasis and disease.Cells2021;10:1775 PMCID:PMC8304699