Abstract
Background
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.
Aim
This study aims to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions.
Methods
We conducted a metagenomic observational study in lactating mothers (n = 12) who 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 extracted DNA from individual samples was subsequently analyzed using 16S rRNA amplicon sequencing on the Illumina platform.
Results
Fresh milk samples treated with PMA had significantly lower median sequencing depth, diversity indices, and number of amplicon sequence variants than untreated samples (p < 0.05 for all). Moreover, the relative abundance of the bacterial genus Streptococcus was significantly reduced in PMA-treated samples when compared to those without PMA treatment (p < 0.05). The significant difference in the sequencing depth, amplicon sequence variants, and diversity indices between the paired PMA-untreated and PMA-treated human milk samples (p < 0.05) was observed at each storage interval. 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 and refrigerated samples (p > 0.05). Of specific genera, only PMA-treated samples stored at -20°C for six weeks (but not refrigerated samples) showed a significant decrease in the relative abundance of the genus Streptococcus (p < 0.05) and, contrary, increase in the relative abundance of the genus Staphylococcus (p < 0.05) in comparison to fresh PMA-treated samples.
Conclusions
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 two-day storage at 4°C did not significantly alter the overall diversity and composition of viable bacteria in human milk, freezing notably affected the viability and relative abundances of some bacterial genera.