DOI: 10.5061/dryad.rfj6q57j9
title: Bacterial composition of the human milk microbiome across lactation

description: Research has illustrated the presence of a diverse range of microbiota in
 human milk. The composition of the milk microbiome varies across
 different stages of lactation, emphasizing the need to consider
 the lactation stage when studying its composition. Additionally,
 the transfer of both milk and skin microbiota during
 breastfeeding is crucial for understanding their collective impact
 on infant health and development. Further exploration of the
 complete breastfeeding microbiome is necessary to unravel the
 role these organisms play in infant development. We aim to
 longitudinally assess the bacterial breastfeeding microbiome
 across stages of lactation. This includes all the bacteria that
 infants are exposed to during breastfeeding, such as bacteria
 found within human milk and any bacteria found on the breast and
 nipple. Forty-six human milk samples were collected from 15 women
 at 1, 4, 7, and 10 months postpartum. Metagenomic analysis of the
 bacterial microbiome for these samples was performed by CosmosID
 (Rockville, MD) via deep sequencing. Staphylococcus epidermidis
 and Propionibacteriaceae species are the most abundant bacterial
 species from these samples. Samples collected at 10 months showed
 higher abundances of Proteobacteria, Streptococcaceae,
 Lactobacillales, Streptococcus, and Neisseria mucosa compared to
 other timepoints. Alpha diversity varied greatly between
 participants but did not change significantly over time. As the
 bacterial breastfeeding microbiome continues to be
 studied, bacterial contributions could be used to predict and
 reduce health risks, optimize infant outcomes, and design
 effective management strategies, such as altering the maternal
 flora, to mitigate adverse health concerns.
description: Participants Samples were collected from 15
 women, who had experienced healthy pregnancies and deliveries, across
 lactation as part of the Baby Connectome Project and the Baby Connectome
 Project—Enriched, a joint effort between the University of North Carolina
 at Chapel Hill and the University of Minnesota Twin Cities. No women
 included reported having taken any antibiotics within 3 months of
 providing samples. Nine of the women exclusively breastfed through six
 months, while the other six did supplement with infant formula they still
 received more breast milk compared to infant formula through six
 months. Milk collection and processing
 Milk was collected at the University of Minnesota when the dyad
 was on site for behavioral data collection, and was timed to coincide with
 the 2nd feed of the day whenever possible. Each participant was provided a
 quiet, private space equipped with a Medela Symphony hospital grade breast
 pump and a sterilized set of pump consumables. Mothers were asked to
 completely express their right breasts. Immediately following collection,
 the entire sample was weighed and volume recorded. The entire sample was
 then vortex mixed for 2 min before being aliquoted and frozen at −80°C.
 All samples were frozen within 30 min of the end of expression.
 DNA extraction All steps of metagenomic
 analysis (including DNA extraction, library preparation, and sequencing)
 were completed at CosmosID, Rockville, MD. A 1.5 ml aliquot of untreated
 milk was thawed and then transferred to a 2 ml microcentrifuge tube. Each
 sample was centrifuged at 13,000 g, 4°C, for 20 min. The cell pellet was
 saved at the bottom of the tube (∼10 μl) as well as the top fat layer by
 carefully removing the middle liquid supernatant. 190 μl 1× PBS was added
 to the cell pellet and the pellet was resuspended using repeated
 pipetting. Twenty μl of Proteinase K was added to the resuspended cells
 and vortexed gently. The sample was incubated at 55°C for 18 h. Sample
 solution was inputted into the PowerBead Pro tube (Qiagen) and PowerSoil
 Pro extraction (Qiagen) was performed in accordance with manufacturer
 protocols. Extracted DNA samples were quantified using Qubit 4 fluorometer
 and QubitTM dsDNA HS Assay Kit (Thermofisher Scientific).
 Library preparation and sequencing DNA
 libraries were prepared using the Nextera XT DNA Library Preparation Kit
 (Illumina) and Nextera Index Kit (Illumina) with total DNA input of 1 ng.
 Genomic DNA was<br>fragmented using a proportional amount of
 Illumina Nextera XT fragmentation enzyme. Combinatory dual indexes were
 added to each sample followed by 12 cycles of PCR to construct
 libraries. DNA libraries were purified using AMpure magnetic
 beads (Beckman Coulter) and eluted in QIAGEN EB buffer. DNA libraries were
 quantified using Qubit 4 fluorometer and QubitTM dsDNA HS Assay Kit.
 Libraries were then sequenced on an Illumina NovaSeq S4 platform 2 × 150
 bp (CosmosID). Bacterial composition
 CosmosID (Rockville, MD) kmer based algorithms identify
 microorganisms based on entire genomes represented in their curated
 microbial genomics database, Genbook, with approximately 170,000 genomes
 and gene sequences. Kmers are phylogenetically stable markers identified
 in samples that are used in mapping to the CosmosID database. CosmosID
 provided filtered sequencing data, that only included high confidence
 calls based on proprietary filtering criteria. 
description: # Bacterial composition of the human milk microbiome across lactation
 [https://doi.org/10.5061/dryad.rfj6q57j9](https://doi.org/10.5061/dryad.rfj6q57j9) Longitudinal bacterial composition of human milk across lactation in 15 healthy lactating people as analyzed in a forthcoming publication. ## Description of the data and file structure Data are organized into two files, Milk_Metagenome_Taxa_Table.csv and Sample_Age_Participant.txt.  Column definitions for Milk_Metagenome_Taxa_Table.csv:      Name: Name of bacteria specified by CosmosID      Kingdom: Taxonomic kingdom      Phylum: Taxonomic phylum      Class: Taxonomic class      Order: Taxonomic order      Family: Taxonomic family      Genus: Taxonomic genus      Species: Taxonomic species      The 50 following column headers represent individual samples      Taxonomy ID: ID assigned by CosmosID      Taxonomic Lineage: Combined taxonomic lineage Column definitions for Sample_Age_Participant.csv:      Age (months): Infant age at time of milk sampling in months      Sample: Sample ID corresponding to columns in Milk_Metagenome_Taxa_Table.csv      Participant: Participant ID