Supplement-induced microbiota-wide changes

We first investigated the effect of the intervention on the intestinal microbiota of vaginally born infants with no antibiotic treatments. The abundance of the gut-dwelling species present in the supplement, Bifidobacterium breve and Lactobacillus rhamnosus, were significantly increased in the supplemented group, up to over tenfold in the breast-fed but less in the formula-fed infants (Fig. 1a–b). The dominant species was B. breve in 84% of the breastfed infants and in 35% of the formula-fed infants in the supplemented group. In the control group, none of the formula-fed but 12% of the breastfed infants had B. breve as the dominant species, suggesting that B. breve is a natural colonizer of breastfed infants. To validate these findings with a PCR-independent approach, we also sequenced the whole metagenome of a subset of the breastfed infants’ faecal samples. This metagenomic analysis corroborated the phylogenetic approach, and the data captured also Propionibacterium freundenreichii present in the supplement (Fig. 1c–e), which was not identified in the 16S rRNA gene data.

Fig. 1 Effect of supplement treatment and feeding mode on the microbiota composition in the vaginally born, non-antibiotic-treated infants. a–b Relative abundance of the species in the probiotic mixture in 16S rRNA gene amplicon sequences derived from faecal samples. c–e Relative abundance of the probiotic species in whole metagenome sequences of breastfed infants. The number of infants per group is noted on the bottom of each panel (a–e). f Principal coordinates analysis (Bray-Curtis dissimilarities) on the species-level 16S rRNA gene data. g Composition of the Bifidobacterium population by treatment group and feeding type Full size image

The supplement affected the overall microbiota composition (Fig. 1f). Among the vaginally born infants with no antibiotic treatments, the main driver of inter-individual differences in the species-level intestinal microbiota composition was the treatment group, explaining 19% of the variation (p = 0.001 in permutational multivariate ANOVA, Fig. 1f). This result should be interpreted with caution due to the compositional nature of the data (with relative abundances summing up to 1). When inspecting the bifidobacterial community, we observed weaker responses to the supplement in the formula-fed compared to the breastfed infants (Fig. 1g).

Further evidence of a mediating effect of feeding type came from analysis of specific bacterial taxa (Fig. 2): most of the supplement-induced changes observed in the breast-fed infants were not present in the formula-fed infants. In the breast-fed group, the abundance of lactobacilli was 100% (twofold) increased and that of bifidobacteria 29% increased in response to the supplement (p < 0.0001, generalized least squares model, GLS, Additional file 1: Table S1). Most other taxa were reduced in abundance (Fig. 2a, Additional file 1: Table S1): Clostridia by 66% (p < 0.0001, GLS) and Gammaproteobacteria by 58% (p < 0.0001, generalized linear model, GLM, with negative binomial distribution). In contrast, in the formula-fed infants, the total abundance of bifidobacteria was slightly but significantly decreased in the supplemented group (by 7%, p < 0.0001, GLM, Additional file 1: Table S2), despite the specific increase in B. breve. In addition, several Firmicutes and Proteobacteria taxa were increased in the formula-fed supplemented group compared to the formula-fed control group (Fig. 2): Anaerostipes by fourfold (p = 0.05, GLM), Veillonella by sevenfold (p < 0.0001, GLM) and Klebsiella by sixfold (p = 0.05, GLM).

Fig. 2 Effect of supplement treatment in the vaginally born, non-antibiotic-treated breastfed infants and formula-fed infants. The fold changes represent the difference in the relative abundance of the taxon between the supplement-treated group and the control group. The asterisks indicate the significance of the difference (based on GLM or GLS, see Additional file 1: Tables S1 and S2): *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

Supplementation prevented caesarean birth-associated loss of bifidobacteria and normalized microbiota functions

After establishing a strong overall effect of the treatment, we tested if the supplement ameliorated some of the caesarean-induced changes in microbiota composition. In the control group, the microbiota of the caesarean-delivered infants was clearly different from the vaginally born infants (Fig. 3a), most notably due to a lower abundance of the most abundant genera, Bifidobacterium (75% decline, p = 0.01, GLS, Additional file 1: Table S3) and Bacteroides (96% decline, p < 0.0001, GLM). Overall, 6% (p = 0.001) of the inter-individual variation in microbiota composition was statistically attributable to birth mode in the control group according to permutational multivariate ANOVA. Remarkably, in the supplemented group, birth mode did not have a significant impact on microbiota composition (1% p = 0.08). The relative increases in Enterococcaceae, Clostridiaceae and Veillonellaceae that were observed in the section-born infants of the control group were not present in the supplemented group (Fig. 3b, Additional file 1: Table S3). Furthermore, the decline in Bifidobacteriaceae was prevented, and the declines in Coriobacteriaceae, Porphyromonadaceae and Bacteroidaceae were reduced in magnitude by the supplement (Fig. 3b, Additional file 1: Table S3).

Fig. 3 Effects of supplement and birth mode on the microbiota. Overall average composition of the microbiota at class level in the different groups, based on the 16S rRNA amplicon data (a). Significant family-level group differences compared to the vaginally born control group (b). Effect of supplement treatment and birth mode on the metaproteome (c) and metagenome (d) in principal coordinates analysis (Bray-Curtis dissimilarities) Full size image

The caesarean section-associated reduction of Bifidobacterium and Bacteroides spp., the two most important groups of bacteria in terms of carbohydrate degradation in infants, was reflected in the predicted carbohydrate degradation potential of the microbiota. Based on the taxonomic composition (abundance of taxa in the samples) and the CAZy database [25] (carbohydrate-active enzymes present in the taxa), the carbohydrate degradation potential in the samples was predicted. The predicted summarized total abundances of carbohydrate-active enzymes (CAZys) involved in the degradation of different types of carbohydrates, including human milk oligosaccharides, were significantly reduced among the caesarean-born infants (Additional file 2: Figure S1). The predicted abundances of these enzyme groups were increased in the supplemented infants, regardless of birth mode.

We conducted a metaproteome analysis in a subset of the cohort, including 11 vaginally born control infants, 12 caesarean-born control infants, 13 vaginally born supplemented infants and 12 caesarean-born supplemented infants. All were fully breastfed and had received no antibiotic treatments. The bacterial metaproteomes differed between birth modes in the control group, but were similar between birth modes in the supplemented group (Fig. 3c). Treatment with the supplement thus seemed to eliminate the effect of birth mode on the metaproteome-derived functions (Additional file 1: Table S4). This is illustrated by the finding that the supplemented groups (both caesarean-born and vaginal-delivered infants) compared to the vaginally born control group showed a high level of induction (up to 50-fold; Additional file 1: Table S4) of beta-galactosidase and beta-galactosyl N-acetyl hexosaminephosphorylase (LNBP), common bifidobacterial enzymes involved in the degradation of lactose and human milk oligosaccharides (HMOs), respectively [26]. In contrast, the bacteria in the caesarean-born infants expressed comparatively higher levels of aspartate aminotransferase and aspartate ammonia lyase, enzymes involved in potentially undesired protein degradation. The metaproteome data were also used to predict the taxonomic origin of the proteins, and the obtained results appeared similar to those from the 16S rRNA gene data (Additional file 2: Figure S2).

While the sample size in our metagenomic analysis was small, based on visual inspection of the PCoA plot, the genomic content of the microbiota appeared to cluster by birth mode and treatment: the metagenomes separated according to birth modes in the control group, but clustered together in the supplemented group (Fig. 3d). As the birth modes did not clearly differ in the supplemented group, we grouped together the faecal metagenomes of the supplemented infants for statistical analysis. Compared to the metagenomes in the vaginally born control group, a strong increase was observed for the lactose/galactose and rhamnose degradation genes (approximately 20-fold and 4-fold, respectively) while several amino acid and vitamin B (notably folic acid) synthesis pathways were significantly reduced (almost 30-fold) in the caesarean-born control group (Additional file 1: Table S5). These pathways were increased or unchanged in the supplemented group (Additional file 1: Table S5).

Supplement prevented antibiotic-associated microbiota distortion

Next, we assessed the effects of antibiotics on the microbiota and whether the supplement prevented these effects. In the control group, infants who had been treated with one or more antibiotic courses showed a clearly different microbiota composition compared to those that had received no antibiotics (Fig. 4a, Additional file 1: Table S6). In the control group, antibiotic use explained 4% (p = 0.001) of the microbiota composition, while in the treatment group antibiotic use did not have significant overall impact (< 1%, p = 0.56). In the control group, antibiotic use was associated with a decline in bifidobacteria by 17% (p = 0.015) and increases in Enterococcus and the Gram-negative classes Gammaproteobacteria and Bacteroidia by two-, two- and sixfold (p = 0.017, p = 0.04 and p < 0.0001), respectively. The supplement prevented or corrected the antibiotic-associated increases in Bacteroidaceae, Enterococcaceae and Enterobacteriaceae and the decline in Bifidobacteriaceae (Fig. 4b).