Colonization by B. infantis EVC001 modulates enteric inflammation in exclusively breastfed infants

Abstract

Background: Infant gut dysbiosis, often associated with low abundance of bifidobacteria, is linked to impaired immune development and inflammation-a risk factor for increased incidence of several childhood diseases. We investigated the impact of B. infantis EVC001 colonization on enteric inflammation in a subset of exclusively breastfed term infants from a larger clinical study.

Methods: Stool samples (n = 120) were collected from infants randomly selected to receive either 1.8 × 1010 CFU B. infantis EVC001 daily for 21 days (EVC001) or breast milk alone (controls), starting at day 7 postnatal. The fecal microbiome was analyzed using 16S ribosomal RNA, proinflammatory cytokines using multiplexed immunoassay, and fecal calprotectin using ELISA at three time points: days 6 (Baseline), 40, and 60 postnatal.

Results: Fecal calprotectin concentration negatively correlated with Bifidobacterium abundance (P < 0.0001; ρ = -0.72), and proinflammatory cytokines correlated with Clostridiaceae and Enterobacteriaceae, yet negatively correlated with Bifidobacteriaceae abundance. Proinflammatory cytokines were significantly lower in EVC001-fed infants on days 40 and 60 postnatally compared to baseline and compared to control infants.

Conclusion: Our findings indicate that gut dysbiosis (absence of B. infantis) is associated with increased intestinal inflammation. Early addition of EVC001 to diet represents a novel strategy to prevent enteric inflammation during a critical developmental phase.

Conflict of interest statement

B.M.H., S.C., G.C., H.K.B., and S.A.F. are employees of Evolve Biosystems, a company focused on restoring the infant microbiome. J.T.S. previously worked as a consultant for Evolve Biosystems. The other authors declare no competing interests.

Figures

Fig. 1

Microscopic analysis of the infant gut microbiome. Gram stain light microscopy (a, b) and scanning electron microscopy (c, d) micrographs of diluted fecal samples on day 40 postnatal from the control infants (a, c) and infants fed EVC001 (b, d). Scale bars: 50 µm (a, b) and 5 µm (c, d)

Fig. 2

Fecal calprotectin levels are dependent on the abundance of Bifidobacteriaceae. Forty fecal samples from day 40 postnatal were evaluated for the concentration of fecal calprotectin and Bifidobacteriaceae abundance (****P < 0.0001; rs = −0.72; a) and subdivided based on Bifidobacteriaceae abundance < or >25% (b). The data set is representative of at least three different experiments completed in duplicate and a non-parametric Wilcoxon rank-sum test was used to determine significance with the corresponding P values adjusted and considered statistically significant if *P < 0.05. **P < 0.01

Fig. 3

Fecal cytokine signature of infants who received B. infantis EVC001. Radar plot representations of median cytokine concentrations [pg/mg] detected in fecal samples from the controls (n = 20) and infants fed Bifidobacterium infantis EVC001 (n = 20; EVC001) on a day 6 (Baseline), b day 40 postnatal, and c day 60 postnatal. Median values were adjusted to log scale, then normalized within each cytokine group from 0 to 1. Statistical analysis was completed using Wilcoxon rank-sum test. P values were adjusted using Bonferonni–Holm method and considered statistically significant if *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Principal coordinate analysis (PCoA) of global cytokine profiles according to group status. PCoA based on Bray–Curtis dissimilarity of global cytokine profiles between EVC001-fed infants and controls at a day 6 (Baseline), b day 40, and c day 60 postnatal

Fig. 5

Correlations between specific gut taxa and intestinal inflammatory cytokine responses. Heatmap shows correlation between bacterial families and specific cytokines computed via Spearman correlation. P values are corrected using Benjamini–Hochberg procedure (false discovery rate) to estimate significant correlations between microbial taxonomic composition and specific cytokine concentration detected in the feces of exclusively breastfed infants at three time points (day 6 (Baseline), day 40, and day 60 postnatal). Each cytokine was tested in duplicate at three different time points. P values were adjusted and considered to be statistically significant if P < 0.05 (empty circle); P < 0.01 (semi-solid circle); P < 0.001 (solid circle)