3. Discussion
CMPA, inflammation, infections, EHF, intestinal bile acid
MUCOSAL INFLAMMATION, INFLAMMATION, INFECTIONS, PATHOPHYSIOLOGY
The pathophysiology of CMPA is complex and multifactorial [CMPA symptoms may manifest at different ages in infancy; therefore, management with an EHF may commence before or after the introduction of complementary solid foods. In the present analysis of the fecal microbiome, we stratified the cohort into infants enrolled before 3 months of age (EE cohort) and those enrolled after 3 months of age (LE cohort). This stratification aimed to reduce the confounding effects of age and complementary diet. The EE cohort was exclusively formula-fed to about 4 months of age, i.e., from enrollment to the 1-month follow-up visit. We hypothesized that the HMO effects on the gut microbiome and metabolome would be easier to identify before solid foods and dietary fiber were introduced.Global fecal microbial changes were investigated by computing phylogenetic alpha diversity. Using a classifying-based analytical approach, we defined five clusters of samples, i.e., fecal community types (FCT), characterized by similar taxonomic groups at the genus level. Overall, we observed a more diverse and complex gut ecosystem as a function of age which is in accordance with the gut microbiome maturation trajectory described by others [Our FCT model showed that the intake of HMO slowed the developmental progression toward a mature, adult-type microbiome composition, illustrated by a reduced microbial diversity and an enrichment of early-stage FCTs in the HMO group compared to the control group.In the EE cohort, these ecological differences were particularly visible after 3 months of HMO intake (V3). A survival analysis showed that, at the same age, fewer infants in the HMO group transitioned to a later FCT stage compared to control infants. The HMO group exhibited enrichment in bifidobacteria and depletion in proteobacteria (e.g., In the LE cohort, in which infants were already exposed to solid food before enrolment, HMO-mediated changes in the gut microbiome were less pronounced. Significant differences in alpha diversity and FCT distribution between feeding groups could only be demonstrated at 12 months of age, suggesting that a longer intervention may be required to induce an effect in this population. In the LE cohort, bifidobacteria abundance tended to increase in the HMO group already after 1 month, although this trend did not pass the significance threshold at the species or genus level and disappeared at later visits. Several aspects of dietary intake may explain these observations. The frequency of formula feeding is generally reduced with age; therefore, the overall formula intake and HMO exposure are lower in infants in the LE cohort. Intake of dietary fibers from the complementary diet is likely to confound the HMO effect due to an increased capacity of the microbiome to ferment fiber. As a result, the effects of HMO may be less clear in the LE cohort compared to infants who received the HMO-supplemented EHF before 3 months of age as part of exclusive formula feeding.Exclusively breastfeeding for 4 to 6 months is known to decrease fecal microbial diversity, delay the maturation of the gut microbiome, and promote the enrichment of HMO-utilizing-bifidobacteria [In our cohort of infants with CMPA, HMO feeding was able to reconstitute microbiome patterns closer to those seen in breastfed infants, especially when HMOs were introduced during exclusive feeding with the HMO-supplemented formula. These findings are consistent with other studies in healthy-term infants. Berger et al. reported that formula-fed infants receiving 2′-FL and LNnT exhibited higher levels of In the EE cohort, HMO intake was negatively associated with fecal metabolites derived from the bacterial oxidative catabolism (Ehrlich pathway) of branched-chain amino acids (BCAAs) and aromatic amino acids (i.e., isobutyric acid, isovaleric acid, phenylacetic acid, 3,4-hydroxyphenylacetic acid, and 4-cresol sulfate). These reactions are characterized by the oxidation of the amino acid as an electron donor into a volatile carboxylic acid. Oxidative metabolisms provide energy in the form of adenosine triphosphate (ATP) through substrate-level phosphorylation and are often coupled with reductive metabolic pathways to maintain the redox balance [Feeding an HMO-supplemented EHF reduced the abundance of several amino acid fermentation catabolites, particularly in the EE cohort. Importantly, some metabolites, such as phenols, ammonia, and hydrogen sulfide, can be toxic to colonocytes and may contribute to mucosal inflammation [We found evidence of HMO-mediated metabolic remodeling of bile acid profiles, which was more marked in the EE cohort. Bile acids play a central role in nutrient digestion, fat absorption, and cholesterol metabolism. Primary bile acids are synthesized from cholesterol, conjugated with glycine or taurine in the liver, and secreted into the bile before entering the enterohepatic circulation. A large proportion (80–90%) of the intestinal bile acid pool is reabsorbed in the ileum [Not all prebiotics and types of fiber have the same effect on bile acid conjugation and related outcomes. In contrast to HMO, feeding of inulin has recently been shown to increase microbial BSH-derived unconjugated bile acids and promote type 2 inflammation [The present analysis of the microbiome and metabolome has several limitations. Due to a relatively small number of stool samples from study subjects, the statistical power of the analysis was limited. As subjects were enrolled at different ages, the high variability in age at each time point limited the ability to identify HMO effects due to confounding by age and complementary diet. This issue was partially mitigated by stratifying the population in EE and LE cohorts. However, this stratification further limited the statistical power. Nevertheless, we identified a robust HMO-mediated microbial signature that was consistent with existing literature and in accordance with the changes in the fecal metabolic profile, particularly in the EE cohort. The metabolomic analysis was carried out using a targeted approach that allowed us to accurately quantify metabolites from a variety of pathways. However, the assessment of amino acid metabolism was limited as some metabolites were not included in the initial selection of target metabolites.In conclusion, the microbiome analysis in the present study of infants with CMPA demonstrated that the supplementation of a whey-based EHF with 2′-FL and LNnT enriched the microbiome with HMO-utilizing bifidobacteria and slowed the progression of the microbiome composition towards an adult-type pattern. HMO supplementation partially reversed the dysbiosis common to infants with CMPA and shifted the microbiome composition closer to a pattern typical of breastfed infants. Specific HMO-mediated changes in fecal amino acid degradation and bile acid conjugation were identified in this cohort, with the greatest effect seen in infants who commenced the HMO-supplemented EHF before 3 months of age. The clinical significance of these observations in the context of a reduced incidence of infections and potentially other health benefits requires further investigation.