P2.I166: Gut Microbiota Establishment in Infants Fed Formulas with an Age-Adapted Synbiotic Blend of Six Human Milk Oligosaccharides, B. infantis LMG11588, and B. lactis CNCM I-3446: A Staged Analysis of a Double-Blind, Randomized Trial

Background: Human milk oligosaccharides (HMOs) drive gut microbiome development in early life via microbes able to metabolize specific HMOs. B. longum subsp. infantis (B. infantis) can do so and is increasingly recognized to contribute to early postnatal immune development. Globally, a progressive loss of HMO-metabolizing bifidobacteria is observed. In this ongoing trial we assessed gut microbiome modulating effects in infants fed formulas supplemented with 6 HMOs and two probiotics from birth to age 15 months (mo). Here, we report staged endpoint analysis results for all infants up to age 6mo.

Methods: In a multicenter European trial (Clinical trials.gov: NCT04962594), formula-fed infants aged ≤14 days were randomized to control (n=117, partially hydrolyzed 100% whey-based formula), or test formula (n=119, same formula supplemented with 1.77g/L HMOs [2’FL, DFL, 3-FL, LNT, 3’SL, 6’SL], B. lactis [CNCM I-3446; 1x10^6 CFU/g], and B. infantis [LMG11588; 5x10^5 CFU/g]). Non-randomized breastfed infants (n=82) were enrolled as reference. Complementary diet was allowed from 4mo. Fecal samples collected at baseline, 3 and 6mo were analyzed for microbiota via shotgun metagenomics. Microbiome differences between control and test formula groups were assessed analyzing differential abundance using a compositionality-bias-corrected mixed linear model adjusting for baseline age and study site, as well as differential prevalence using a binomial generalized linear regression model.

Results: At baseline, diversity measures were similar between test and control groups, while at 6mo both intra- and interindividual diversity measures showed significant differences with test formula-fed infants being closer to breastfed (Figure 1). At 3 and 6mo, test formula-fed infants had a significantly higher abundance of all (Figure 2) and infant-type Bifidobacterium species (B. longum subsp. longum, B. infantis, B. breve, B. bifidum, and B. scardovii) compared to control formula-fed infants and closer to breastfed. Ruminococcus gnavus, a mucolytic species associated with inflammatory conditions, was significantly higher in control formula-fed infants at both 3 and 6mo compared to test formula and breastfed infants. B. infantis and B. lactis were significantly higher in test formula-fed infants compared to controls at 3 and 6mo. and strain tracking of B. infantis revealed high prevalence of the provided LMG11588 strain in the test formula group and no replacement of autochthonous strains. Pathogen analysis via virulence factors revealed primarily the presence of toxigenic Clostridiodes difficile with a high prevalence and abundance at age 3 and 6mo in the control formula group and significantly lower prevalence in test formula-fed and breastfed infants.

Conclusion: An infant formula with a specific synbiotic blend of 6 HMOs and B. infantis plus B. lactis supported the expansion of beneficial HMO metabolizing bifidobacteria to 6mo, especially infant-type species in all infants. The lower presence of mucolytic R. gnavus and toxigenic C. difficile in test formula-fed infants indicates improved gut ecology via reduction of risk factors.