Downloaded from British Journal of Nutrition (2014), 111, 1602–1610 doi:10.1017/S0007114513004200 q The Authors 2013 https://www.cambridge.org/core Selective proliferation of intestinal Barnesiella under fucosyllactose supplementation in mice Gisela A. Weiss1,2, Christophe Chassard3 and Thierry Hennet1* . IP address: 1Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland 170.106.33.14 2Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, Zurich, Switzerland 3 Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Switzerland (Submitted 29 May 2013 – Final revision received 14 November 2013 – Accepted 3 December 2013 – First published online 10 January 2014) , on 26 Sep 2021 at 14:57:53 Abstract The oligosaccharides 2-fucosyllactose and 3-fucosyllactose are major constituents of human breast milk but are not found in mouse milk. Milk oligosaccharides have a prebiotic action, thus affecting the colonisation of the infant intestine by microbiota. To determine the specific effect of fucosyllactose exposure on intestinal microbiota in mice, in the present study, we orally supplemented newborn mice with pure 2-fucosyllactose and 3-fucosyllactose. Exposure to 2-fucosyllactose and 3-fucosyllactose increased the levels of bacteria of the Porphyro- , subject to the Cambridge Core terms of use, available at monadaceae family in the intestinal gut, more precisely members of the genus Barnesiella as analysed by 16S pyrosequencing. The ability of Barnesiella to utilise fucosyllactose as energy source was confirmed in bacterial cultures. Whereas B. intestinihominis and B. viscericola did not grow on fucose alone, they proliferated in the presence of 2-fucosyllactose and 3-fucosyllactose following the secretion of linkage- specific fucosidase enzymes that liberated lactose. The change in the composition of intestinal microbiota mediated by fucosyllactose supplementation affected the susceptibility of mice to dextran sulphate sodium-induced colitis, as indicated by increased resistance of mice subjected to 2-fucosyllactose supplementation for 6 weeks. The present study underlines the ability of specific milk oligosaccharides to change the composition of intestinal microbiota and thereby to shape an intestinal milieu resilient to inflammatory diseases. Key words: Fucosyllactose: Oligosaccharides: Breast milk: Microbiota Oligosaccharides are major constituents of human breast milk. Disialyllacto-N-tetraose was shown to lower the incidence of British Journal ofMilk Nutrition oligosaccharides are built up by multiple glycosyl- necrotising enterocolitis in rats(8). Finally, 3-sialyllactose was transferases expressed in the secretory mammary gland by shown to influence the intestinal abundance of clostridial cluster https://www.cambridge.org/core/terms extending the lactose core, thereby forming about 200 various IV bacteria in mice and hence their susceptibility to dextran structures(1). Milk oligosaccharides do not deliver energy to sulphate sodium (DSS)-induced colitis(9). the newborn as the human gut lacks the glycosidase machinery Fucosyllactose is a major constituent of human milk oligosac- required to break down these complex carbohydrates. Rather, charides(10), whereas only trace amounts are found in mouse milk oligosaccharides have a prebiotic action providing a milk(11). Fucosyllactose occurs as Fuc(a1–2)Gal(b1–4)Glc, also nutrient milieu for the microbial colonisation of the intestine(2), called 2-fucosyllactose (2-FL), and Fuc(a1–3)(Gal(b1–4))Glc, thereby influencing the development of the mucosal immune called 3-fucosyllactose (3-FL). 2-FL is similar to the blood group system of breast-fed infants(3). Milk oligosaccharides shape H-antigen and 3-FL to the Lewis-X antigen found on glyco- the intestinal flora by providing fermentable carbohydrates to proteins and glycolipids(12). 2-FL accounts for 20 % of total (13) . selected bacterial groups and by acting as soluble receptors human milk oligosaccharides and 3-FL for less than 1 % .The https://doi.org/10.1017/S0007114513004200 preventing the adhesion of other bacteria(4 – 6). The remarkable abundance of 2-FL in human milk suggests an evolutionary diversity of milk oligosaccharides hampers the attribution of pressure pertaining to the development of beneficial effects on specific functions to individual structures. To date, only a few infant physiology. Along the way, ex vivo and in vivo studies studies have unravelled the biological impact of single milk oligo- have shown that 2-FL inhibits the mucosal attachment and saccharides. For example, lacto-N-fucopentaose III was shown to intestinal colonisation of Campylobacter jejuni, hence lowering induce the production of IL-10 and PGE2 in spleen cells(7). the incidence of diarrhoea(14,15). Fucosyllactose also acts as Abbreviations: 2-FL, 2-fucosyllactose; 3-FL, 3-fucosyllactose; DGGE, denaturing gradient gel electrophoresis; DSS, dextran sulphate sodium; YCFA, yeast extract, casitone, fatty acid. * Corresponding author: T. Hennet, fax þ41 44 635 6814, email [email protected] Downloaded from Enrichment of Barnesiella by fucosyllactose 1603 a protected source of lactose for bacteria expressing fucosidase in lysis buffer was increased to 958C. The DNA concentrations https://www.cambridge.org/core enzymes, such as bifidobacteria and Bacteroides, whereas of extracts were measured using NanoDrop (Witec AG). Escherichia coli and Clostridium perfringens cannot utilise 2-FL Aliquots of 100 ng of extracted DNA were subjected to PCR as nutrient(16). using the 16S rDNA universal heteroduplex analysis (HDA) The potential of specific oligosaccharides to influence primers HDA1-GC 50-CGCCCGGGGCGCGCCCCGGGCGGG- intestinal colonisation by commensal microbiota in vivo is not GCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAGT-30 known. To address the potential contribution of fucosyllactose and HDA2 50-TTACCGCGGCTGCTGGCA-30 at 568C for strand to shaping of the distribution of intestinal microbiota, we supple- annealing. Initial denaturation at 948C for 4 min was followed mented newborn mice with 2-FL and 3-FL, which are naturally by thirty cycles of 30 s at 948C, 30 s at 568C and 1 min at 728C. IP address: not found in mouse milk. The lack of fucosylated oligo- The quality of PCR products was verified by agarose gel saccharides in mouse milk may decrease the selection of electrophoresis. fucosidase-expressing bacteria. The exposure of infant mice to 170.106.33.14 soluble 2-FL and 3-FL may also impair the adhesion of bacteria Denaturing gradient gel electrophoresis and species to fucosylated glycans in the gastrointestinal mucosa or interfere identification with the bacterial regulation of host-derived fucosylated glycans , on through interaction with bacterial carbohydrate sensors, such as Amplified 16S rDNA fragments were separated by denaturing 26 Sep 2021 at 14:57:53 the FucR of Bacteroides thetaiotaomicron (17).Previousexper- gradient gel electrophoresis (DGGE) using an INGENYphorU iments have demonstrated the effect of the milk oligosaccharide (Ingeny International BV) system equipped with 6 % 3-sialyllactose on microbial composition as well as on the extent polyacrylamide gels with a denaturant in the range of (9) of chemically induced intestinal inflammation . Beyond the 30–55 %, where 100 % denaturant is equivalent to 7 M-urea effect on intestinal microbial colonisation, the question as to and 40 % formamide. Electrophoresis was carried out at 130 V whether fucosyllactose supplementation has an impact on the for 4·5 h at 608C. Polyacrylamide gels were stained with , subject to the Cambridge Core terms of use, available at severity of an inflammatory disease was addressed using the GelRede nucleic acid stain (catalogue no. 41 003; Biotium) for DSS-induced colitis model. 45 min, destained in ultrapure water and viewed under UV light. Bands of interest were excised from gels and lysed in ultrapure water. Extracted DNA was reamplified using the same Experimental methods primers and PCR conditions. To purify the bacterial DNA, PCR Fucosyllactose supplementation in mice products were reloaded on a denaturant gradient gel followed by excision and lysis of selected bands. DNA samples recovered Newborn C57BL/6 mice were fed orally with 500 mmol/l of from lysed bands of the second DGGE were reamplified by 2-FL and 3-FL (Glycom) daily from day 1 to day 20 after birth PCR before purification using the QIAquick PCR Purification starting with 5 ml, increasing by 2·5 ml every 2–3d reaching a Kit (catalogue no. 28 104; Qiagen) and sequenced (Microsynth daily amount of 25 ml on day 20. Sterile water was given as AG). Species identification was done using the Ribosomal negative control. For supplementation after weaning, 2-FL Microbiome Database Project Classifier tool(18). and 3-FL were added to drinking-water (3·3 mmol/l) from day British Journal of21 Nutrition to day 45. Fresh faeces were collected on day 21 and day 45 and immediately stored at 2808C until further analysis. Pyrosequencing and data processing https://www.cambridge.org/core/terms The V5–V6 region of 16S rRNA from faecal DNA samples was 0 Dextran sulphate sodium-induced colitis amplified using the primers 784F 5 -AGGATTAGATACCCT- GGTA-30 and 1061R 50-CRRCACGAGCTGACGAC-30 3640(19). From day 45 postpartum, 2·75 % (w/v) of DSS (catalogue no. Amplicons were pyrosequenced using a Roche 454 GS-FLX 0216011090; MP Biomedicals) was added to drinking-water for system (DNAVision). Sequences