# Viable bacterial communities in freshly pumped human milk and their changes during cold storage conditions

## TL;DR

This study explores how bacterial communities in human milk change during refrigeration and freezing, finding that freezing affects bacterial viability more than refrigeration.

## Contribution

The study introduces the use of PMA to specifically analyze viable bacteria in human milk under different storage conditions.

## Key findings

- Streptococcus, Staphylococcus, Diaphorobacter, Cutibacterium, and Corynebacterium are the most common viable bacteria in fresh human milk.
- Freezing significantly affects the viability and relative abundances of some bacterial genera, unlike refrigeration.
- PMA-treated samples showed lower diversity and significant differences compared to untreated samples.

## Abstract

Human milk harbors diverse bacterial communities that contribute to infant health. Although pumping and storing milk is a common practice, the viable bacterial composition of pumped milk and the impact of storage practice on these bacteria remains under-explored. This metagenomic observational study aimed to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions.

In 2023, twelve lactating mothers from the CELSPAC: TNG cohort (Czech Republic) provided freshly pumped milk samples. These samples were stored under various conditions (refrigeration for 24 h, 48 h, or freezing for six weeks) and treated with propidium monoazide (PMA) to selectively identify viable cells. The DNA extracted from individual samples was subsequently analyzed using 16S rRNA amplicon sequencing on the Illumina platform.

The genera Streptococcus, Staphylococcus, Diaphorobacter, Cutibacterium, and Corynebacterium were the most common viable bacteria in fresh human milk. The median sequencing depth and Shannon index of fresh human milk samples treated with PMA (+ PMA) were significantly lower than in untreated (-PMA) samples (p < 0.05 for all), which was true also for each time point. Also, significant changes in these parameters were observed between fresh human milk samples and their paired frozen samples (p < 0.05), while no differences were found between fresh human milk samples and those refrigerated for up to 48 h (p > 0.05). Of specific genera, only + PMA frozen human milk samples showed a significant decrease in the central log-ratio transformed relative abundances of the genera Diaphorobacter and Cutibacterium (p < 0.05) in comparison to + PMA fresh human milk samples.

The study demonstrated that the bacterial profiles significantly differed between human milk samples treated with PMA, which represent only viable bacteria, and those untreated. While storage at 4 °C for up to 48 h did not significantly alter the overall diversity and composition of viable bacteria in human milk, freezing notably affected both the viability and relative abundances of some bacterial genera.

The online version contains supplementary material available at 10.1186/s13006-025-00738-0.