Lactate-utilizing community is associated with gut microbiota dysbiosis in colicky infants

Abstract

The aetiology of colic, a functional gastrointestinal disorder in infants, is not yet resolved. Different mechanisms have been suggested involving the gut microbiota and intermediate metabolites such as lactate. Lactate can be metabolized by lactate-utilizing bacteria (LUB) to form different end-products. Using a functional approach, we hypothesized that H₂ production and accumulation by LUB is associated with the development of colic. The LUB communities in the feces of forty infants, including eight colicky infants, were characterized using a combination of culture- and molecular-based methods, and metabolite concentrations were measured by HPLC. Interactions among LUB strains isolated from feces were investigated with pure and mixed cultures using anaerobic techniques. We emphasized high prevalence of crying, flatulence, colic and positive correlations thereof in the first 3 months of life. Crying infants showed significantly higher ratio of LUB non-sulfate-reducing bacteria (LUB non-SRB) (H₂-producer), to LUB SRB (H₂-utilizer) at 3 months. Colicky infants had significantly higher number of H₂-producing Eubacterium hallii at 2 weeks compared to non-colicky infants. We revealed the function of Desulfovibrio piger and Eubacterium limosum to reduce H₂ accumulation in co-cultures with H₂-producing Veillonella ratti. Our data suggest that the balance between H₂-producing and H₂-utilizing LUB might contribute to colic symptoms.

Figure 1

Prevalence and correlations of infant gastrointestinal symptoms in the first 6 months of life. Prevalence of flatulence (a), stomach cramps (b), and crying hours (c) of infants from 2 weeks to 6 months (n = 40); >1h/d: infants who cried more than 1 h/d; <1h/d: infants who cried less than 1 h/d. (d) Spearman pairwise correlation map of infant gastrointestinal symptoms during the first 3 months of life (n = 150). The color gradient denotes Spearman R value. Colic criteria 1: paroxysms of irritability, fussing, or crying that start and stop without obvious cause; Colic criteria 2: episodes lasting 3 or more hours per day and occurring at least 3 days per week for at least 1 week; Colic: infants diagnosed by Rome III criteria (see Methods).

Figure 2

(a–c) Total counts of LUB SRB, LUB non-SRB, and LUB non-SRB/LUB SRB ratio in feces of infant crying <1 h/d and infant crying >1 h/d at 2 weeks, 1 month, 3, and 6 months. (d) E. hallii levels between colicky (n = 8) and non-colicky (n = 32) infants at 2 weeks, 1 month, 3, and 6 months. Colic: infants diagnosed by Rome III criteria (see Methods). Central horizontal line shows the median, upper and lower box border show the 90th and 10th centile respectively, upper and lower whisker show the 95th and 5th centile respectively. Open circle designates outliers; asterisk designates extreme values using a step of 1.5 x IQR (interquartile range). *P < 0.05; Ŧ P = 0.056; LUB, lactate-utilizing bacteria; SRB, sulfate-reducing bacteria.

Figure 3

Consumption of lactose, glucose, and lactate and production of formate, acetate, propionate, and butyrate of LUB after 48 h incubation in YCFA medium supplied with lactose, DL-lactate, L-lactate, and glucose. Positive and negative values indicate production and consumption, respectively.

Figure 4

(a) Production of formate, acetate, propionate, and butyrate by single and co-culture of V. ratti, E. limosum, and D. piger (DSM 749) grown in triplicate in YCFA medium containing 60 mM DL-lactate. (b) OD₆₀₀ and lactate consumption by single and co-culture of V. ratti and D. piger. V. ratti and E. limosum strains were isolated from infant feces.

Figure 5

Production of H₂ by V. ratti (V), D. piger (D), E. limosum (E) in single and co-cultures. Values are means ± SD (n = 2).

Figure 6

Schematic overview of lactate, H₂, and H₂S metabolism in infant gut.