Study sample characteristics
Information regarding basic anthropometrics and reproductive history of all subjects at enrolment is listed in Table 1. On average, women were 30 ± 6 years old, weighed 54 ± 8 kg prior to pregnancy, and had 1.3 ± 0.4 children. All infants were exclusively breastfed at 1 month. Most samples were collected from both mother and infant at each time point; however, we were unable to obtain all samples owing to their mishandling by study personnel. Ultimately, 19 paired samples were obtained, including maternal faeces, infant faeces, and breast milk from each of the colostrum, transition, and mature milk stages, and sIgA-coated microbiota of maternal faeces, mature milk, and infant faeces of mature milk stage, in addition to the absence of one maternal faeces, one colostrum, three mature milk, and one infant faecal sIgA-coated microbiota samples (Supplementary Table 1).
All samples were sequenced by bacterial 16S rRNA amplicon sequencing, resulting in a total of 184 high-quality metagenomes with average counts per sample of 21399. From these samples, three paired samples of maternal faeces, mature milk, and sIgA-coated microbiota samples of mature milk, infant, and maternal faeces were shotgun sequenced, yielding a total of 15 high-quality metagenomes, with an average of 5.34 (±0.13) bases per sample after quality control.
Alpha and beta diversity
As shown in Fig. 1, the bacterial diversity index (Shannon index) of maternal faeces was significantly higher than that of breast milk and infant faeces, except for transitional milk (P <0.05). There was also a significant difference (P = 0.02) between infant faeces at the colostrum stage compared with that for the mature milk stage. However, no difference was found among milk from different lactation periods, and among sIgA-coated microbiota of different samples. For bacterial richness estimation (Chao1), there were significantly higher levels in colostrum (P = 0.0.2) and transitional milk (P <0.001), and lower level in infant faeces of colostrum stage (P = 0.02) compared with maternal faeces, respectively. There were no differences between breast milk and infant faeces samples at different lactation periods and samples of sIgA-coated microbiota.
Beta diversity analysis is presented using principal coordinate analysis (PCoA) of the Brary–Curtis distance matrices (Fig. 2). For the colostrum stage sample, PC1, PC2, and PC3 explained 17.09%, 13.97%, and 10.38% of the between-sample variation, respectively, and all different sample types showed high differences (Anosim R ranged from 0.456–0.893, P = 0.001) (Fig. 2A). At the transitional milk stage, the difference between infant faeces and breast milk decreased (R = 0.339, P = 0.001) (Fig. 2B). However, at the mature stage, there was a high similarity between the breast milk and sIgA-coated microbiota in the breast (R = 0.133) (Fig. 2C). For all sIgA-coated microbiota samples, infant faeces showed high similarity with mother faeces (R = 0.188) and mature milk (R = 0.17). To a lesser extent, due to PC3 contribution (10.32%), maternal faeces showed relatively high similarity to breast milk (R = 0.395) (Fig. 2D).
Potential contribution of the maternal gut and breast milk to infant gut bacterial communities
Using Feast software for microbial source tracking, we estimated likely contributions to infant faecal bacterial communities (sink) using rarefied taxon read counts of operational taxonomic units (OTUs) from milk (source), maternal faecal (as a proxy of the maternal colonic bacteria; source), and sIgA-coated maternal faeces microbiota (source), at three lactation periods. For sIgA-coated microbiota, we also studied contributions of the maternal gut (source) and breast milk (source) to the infant gut (sink).
The contribution of maternal faeces to infant faeces microbiota increased from the colostrum to mature milk stage. At the colostrum stage, source proportions were 20%–44% in 22% of mother/infant pairs (Fig. 3A) and changed to a 18%–67% contribution in 50% of mother/infant dyads in the transitional milk stage (Fig. 3B). The contribution increased to 25%–78% at the mature milk stage (Fig. 3C). Breast milk showed a relatively stable contribution during the three stages, ranging from 12 to 86% in 22% of mother/infant dyads. It is evident that sIgA-coated microbiota of breast milk and maternal gut are the major contributors of sIgA-coated microbiota in infant gut, 14~93% of source proportion in approximately 94 mother/infant dyads (Fig. 3D).
Co-occurrence of specific genera between different sIgA-coated samples.
The unweighted pair‐group method with arithmetic means (UPGMA) analysis of sIgA-coated microbiota resulted in three typical clusters (Fig. 4A). The majority of samples of maternal faeces/breast milk, maternal faeces/infant faeces, or breast/infant faeces pairs had similar microbial patterns belonging to cluster G1, accounting for 68% of sample pairs. Clusters G2 and G3, G1 and G2 are specific samples of maternal and infant faeces, respectively. The abundance distribution of the 30 dominant genera among the three types of samples was displayed in a species abundance heatmap (Fig. 4B). The heatmap revealed several genera that exhibited co-occurrence between samples, including Bifidobacterium, Enterococcus, Streptococcus, Lactobacillus, Klebsiella, Escherichia-Shigella and an unclassified genus of Enterobacteriaceae. In addition, Staphylococcus is the dominant bacteria in breast milk and infant faeces; however, it occurs at a very low level in maternal faeces.
We defined core milk microbiota genera present in at least 90% of individuals with a minimum mean relative abundance of 0.01 % , as shown in Fig. 3C. Colostrum is rich in core bacteria and 11 genera. In the transitional stage, there were seven co-occurrence core genera with colostrum including Streptococcus, Bifidobacterium, Escherichia-Shigella, the three typically core genera of maternal faeces, and Staphylococcus, Klebsiella, Acinetobacter, and Lactobacillus. However, only Staphylococcus, Klebsiella, and Acinetobacter remained in the mature stage. Streptococcus, Bifidobacterium, Staphylococcus and Escherichia-Shigella because core genus of infant gut of corresponding lactation period. At the mature milk stage, infant gut and sIgA-coated microbiota in the mature milk stage, the intestinal flora of infants, and the sIgA-coated microbiota share similar five core bacteria. Among them, Streptococcus, Bifidobacterium, and Klebsiella are also core genera of sIgA-coated breast milk microbiota. The most prominent feature was that the mother's gut contained 11 specific core bacteria, suggesting that the infant's gut microbiota is much simpler than that of the adult. Meanwhile, only Escherichia-Shigella and Enterococcus were in sIgA-binding bacteria of maternal faeces.
Different genera and families between sample types
As lactation progressed, Streptococcus abundance in breast milk gradually decreased, while Enterococcus and Bifidobacterium gradually increased. Staphylococcus maintains a considerable superior abundance. Enterococcus and Escherichia-Shigella increased in mature milk at the cost of reduced Lactobacillus (Fig. S1). Edge analysis identified several families significantly decreased in abundance in mature milk compared with those in colostrum, including Ruminococcaceae, Corynebacteriaceae, Lachnospiraceae, Peptostreptococcaceae, and Microboccaceae (Fig. 4).
In infant faeces, Klebsiella and Escherichia_ Shigella abundance increased and decreased gradually, respectively, during the lactation stages. The abundance of adult-specific families, Lachnospiraceae and Bacteroidaceae, significantly increased in mature milk compared with that in colostrum (Fig. 4). In sIgA-coated microbiota, Lactobacillus and Bifidobacterium became gradually enriched from maternal faeces to breast milk to infant (Fig. S1). Lactobacillaceae in breast milk and infant faeces significantly increased compared with maternal faeces (Fig. 5).
Identification of co-occurrence species by shotgun sequencing
According to the principal component analysis of shotgun sequenced results in Fig, 6A, there was a significant difference between breast milk, maternal faeces, and sIgA-coated microbiota. Three different sIgA-coated microbiota showed no separation. In contrast with 16S amplicon sequencing, two additional co-occurrence genera were identified by shotgun sequencing, Clostridium and Gardneralla (Fig, 6B). Among the classified species, B. longum was the dominant co-occurrence, followed by Bifidobacterium breve. Co-occurrences of Lactobacillus in sIgA-coated bacteria are primarily L. salivarius, L. reuteri, Lactobacillus gasseri, Lactobacillus jonsonii, and Lactobacillus oris. Among them, L. reuteri and L. gasseri were the dominant species in sIgA-coated bacteria in the infant gut.
Kyoto Encyclopedia of Genes and Genomes (KEGG) functional categories shared in metagenomes of sample dyads of sIgA-coated microbiota
Eight KEGG pathways (level 4) shared between shotgun sequencing samples were identified (Fig. 7). Two pathways were associated with energy metabolism, including sucrose-6-phosphatase, which is involved in starch and sucrose metabolism (K07024) and the LacI family transcriptional regulator involved in maltosaccharide utilisation (K02529). Four pathways were associated with the survival of bacteria in an ever-changing and hostile environment. Among them, the putative ABC transport system permease protein (K02004) is involved in negative regulation of biofilm formation. The putative ABC transport system ATP-binding protein (K02003) can facilitate the acquisition of essential compounds from the extracellular environment. The ATP-binding cassette, subfamily B (K06147), and ABC-2 type transport system ATP-binding protein ABC importers (K01990) evolved the use of multiple mechanisms to transport nutrients across the membrane that aid survival in an ever-changing and hostile environment. Another two enzyme that is widely used in bacteria is the putative transposase K07497 and the ABC-2 type transport system permease protein (K01992).