Human Milk Oligosaccharide-Stimulated Bifidobacterium Species Contribute to Prevent Later Respiratory Tract Infections

Abstract

(1) Background: Human milk oligosaccharides (HMOs) may support immune protection, partly via their action on the early-life gut microbiota. Exploratory findings of a randomized placebo-controlled trial associated 2'fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) formula feeding with reduced risk for reported bronchitis and lower respiratory tract illnesses (LRTI), as well as changes in gut microbiota composition. We sought to identify putative gut microbial mechanisms linked with these clinical observations. (2) Methods: We used stool microbiota composition, metabolites including organic acids and gut health markers in several machine-learning-based classification tools related prospectively to experiencing reported bronchitis or LRTI, as compared to no reported respiratory illness. We performed preclinical epithelial barrier function modelling to add mechanistic insight to these clinical observations. (3) Results: Among the main features discriminant for infants who did not experience any reported bronchitis (n = 80/106) or LRTI (n = 70/103) were the 2-HMO formula containing 2'FL and LNnT, higher acetate, fucosylated glycans and Bifidobacterium, as well as lower succinate, butyrate, propionate and 5-aminovalerate, along with Carnobacteriaceae members and Escherichia. Acetate correlated with several Bifidobacterium species. By univariate analysis, infants experiencing no bronchitis or LRTI, compared with those who did, showed higher acetate (p < 0.007) and B. longum subsp. infantis (p ≤ 0.03). In vitro experiments demonstrate that 2'FL, LNnT and lacto-N-tetraose (LNT) stimulated B. longum subsp. infantis (ATCC15697) metabolic activity. Metabolites in spent culture media, primarily due to acetate, supported epithelial barrier protection. (4) Conclusions: An early-life gut ecology characterized by Bifidobacterium-species-driven metabolic changes partly explains the observed clinical outcomes of reduced risk for bronchitis and LRTI in infants fed a formula with HMOs. (Trial registry number NCT01715246.).

Keywords: Bifidobacterium; acetate; bronchitis; fucosylated glycans; machine learning based classification models; respiratory infections.

Figure 1

Subject flow chart showing the number of per protocol formula-fed infants with stool sample at three months of age, by controls and cases who experienced at least one reported bronchitis or lower respiratory tract infection (LRTI) between three and 12 months of age.

Figure 2

Representative sPLS-DA plots and loadings of the five independent rounds of sPLS-DA modeling with down-sampling to separate controls and cases for bronchitis (n = 52) (a,b) and LRTI (n = 66) (c,d). Results from all five sPLS-DA modeling rounds for both bronchitis and LRTI are provided in Table S1.

Figure 3

Comparison of the identified main features from the sPLS-DA model by feeding group: (a,b) relative abundance of metabolites; (c) relative abundance of microbial taxa; (d) concentration of calprotectin. (n = 106; * indicate p < 0.05, ** p < 0.01 by non-parametric t-test).

Figure 4

Association between relative acetate and Bifidobacterium species in infant stool samples. (a) Spearman correlation; (b) linear regression analysis of the sum of Bifidobacterium species and relative amounts of acetate.

Figure 5

Comparison of relative amounts of short-chain fatty acids and Bifidobacterium species in the stool of infants at three months of age between infants who did not (controls) or did (cases) experience any bronchitis and LRTI thereafter from 3 to 12 months of age: (a,b) relative abundance of the short-chain fatty acids for bronchitis or LRTI cases and controls; (c,d) relative abundance of the Bifidobacterium species for bronchitis or LRTI cases and controls (* indicate p < 0.05, ** p < 0.01 by non-parametric t-test).

Figure 6

In vitro experiments with B. longum subsp. infantis conditioned with glucose (Glc), lactose (Lac), 2′fucosyllactose (2′FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and 6′sialyllactose (6′SL): (a) bacterial growth monitoring using optical density at 600 nm (OD600); (b) quantification of acetate in spent culture media from conditioned B. longum subsp. infantis; (c) measurement of conditioned spent culture medium pH; (d) measurement of NFkB activation by TNFα in the presence of control and conditioned (cond) spent culture media; (e) measurement of Salmonella invasion in cultured epithelial cells in the presence of control and conditioned spent culture media; (f) measurement of Salmonella invasion with different amounts of acetate added to fresh or spent culture media in comparison to control and spent culture media. Symbols stand for independent experiments each with 3 replicates (* p < 0.05, ** p < 0.01 by Kruskal–Wallis or non-parametric t-test).