Microbiome Profiling of Pretreated Human Breast Milk Using Shotgun Metagenomic Sequencing

This study explored the metagenomic sequencing methodology for analyzing the breast milk microbiome and elucidated its composition. Twenty-two breast milk samples were collected from 11 healthy lactating women. By optimizing microbial cell wall disruption parameters and developing a nucleic acid extraction method, microbial DNA/RNA libraries were constructed and subjected to metagenomic next-generation sequencing (mNGS), microbial standards spiked into breast milk at serial dilutions served to validate the method's reliability. The sequencing data underwent rigorous quality control and classification using the Kraken2 software and a self-generated database. The breast milk microbiome was found to comprise 21 phyla, 234 genera, and 487 species, with <i>Firmicutes</i> and <i>Proteobacteria</i> being the dominant phyla. At the genus level, <i>Staphylococcus</i> and <i>Streptococcus</i> were the most abundant, while at the species level, <i>Staphylococcus aureus</i>, <i>Streptococcus bradystis</i>, and <i>Staphylococcus epidermidis</i> were the most prevalent. The microbial profiles of the left and right breast milk samples were consistent at the phylum, genus, and species levels. Besides common bacteria, diverse viral, eukaryotic, and archaeal sequences were also detected. Functional profiling revealed that the "lactose and galactose degradation I" pathway accumulated the highest read count, whereas the L-valine biosynthesis pathway was detected most frequently. This study provides a comprehensive understanding of the healthy breast milk microbiome, highlighting the presence of specific flora colonization and the distinct yet correlated microbial environments in bilateral breast milk, laying the groundwork for future research into the interactions between breast milk microbiota and maternal and infant health outcomes.