Background: Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants’ feces (n = 5, each) and mothers’ feces (n = 3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. Results: The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants’ and mothers’ feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P < 0.05). The human milk metagenome also contained a similar occurrence of immune-modulatory DNA motifs to that of infants’ and mothers’ fecal metagenomes. Conclusions: Our results further expand the complexity of the human milk metagenome and enforce the benefits of human milk ingestion on the microbial colonization of the infant gut and immunity. Discovery of immune-modulatory motifs in the metagenome of human milk indicates more exhaustive analyses of the functionality of the human milk metagenome are warranted.

Communication between mother and offspring in mammals starts at implantation via the maternal–placental–fetal axis, and continues postpartum via milk targeted to the intestinal mucosa. MicroRNAs (miRNAs), short, noncoding single-stranded RNAs, of about 22 nucleotides in length, are actively involved in many developmental and physiological processes. Here we highlight the role of miRNA in the dynamic signaling that guides infant development, starting from implantation of conceptus and persisting through the prenatal and postnatal periods. miRNAs in body fluids, particularly in amniotic fluid, umbilical cord blood, and breast milk may offer new opportunities to investigate physiological and/or pathological molecular mechanisms that portend to open novel research avenues for the identification of noninvasive biomarkers.