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. This metagenomic observational study aimed to characterize viable bacterial communities in freshly pumped human milk and its changes under different storage conditions. METHODS: 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. RESULTS: 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. 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 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.

• Keywords
• 16S rRNA, Human milk, Microbiome, Milk expression, Next-generation sequencing, Propidium monoazide, Pumped milk, Storage, Viable bacteria,
• MeSH
• Azides MeSH
• Bacteria * isolation & purification   genetics   classification   MeSH
• Refrigeration MeSH
• Adult MeSH
• Humans MeSH
• Milk, Human * microbiology   MeSH
• Microbiota * MeSH
• Propidium analogs & derivatives   MeSH
• RNA, Ribosomal, 16S MeSH
• Food Storage * methods   MeSH
• Freezing MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Azides MeSH
• propidium monoazide MeSH Browser
• Propidium MeSH
• RNA, Ribosomal, 16S MeSH

Intrapartum antibiotic prophylaxis (IAP) is commonly used during C-section delivery and in Group B Streptococcus-positive women before vaginal delivery. Here, we primarily aimed to investigate the effect of IAP on the neonatal oral and fecal bacteriomes in the first week of life. In this preliminary study, maternal and neonatal oral swabs and neonatal fecal (meconium and transitional stool) swabs were selected from a pool of samples from healthy mother-neonate pairs participating in the pilot phase of CELSPAC: TNG during their hospital stay. The DNA was extracted and bacteriome profiles were determined by 16S rRNA amplicon sequencing (Illumina). In the final dataset, 33 mother-neonate pairs were exposed to antibiotics during C-section or vaginal delivery (cases; +IAP) and the vaginal delivery without IAP (controls, -IAP) took place in 33 mother-neonate pairs. Differences in alpha diversity (Shannon index, p=0.01) and bacterial composition (PERMANOVA, p<0.05) between the +IAP and -IAP groups were detected only in neonatal oral samples collected ≤48 h after birth. No significant differences between meconium bacteriomes of the +IAP and -IAP groups were observed (p>0.05). However, the IAP was associated with decreased alpha diversity (number of amplicon sequence variants, p<0.001), decreased relative abundances of the genera Bacteroides and Bifidobacterium, and increased relative abundances of genera Enterococcus and Rothia (q<0.01 for all of them) in transitional stool samples. The findings of this study suggest that exposure to IAP may significantly influence the early development of the neonatal oral and gut microbiomes. IAP affected the neonatal oral bacteriome in the first two days after birth as well as the neonatal fecal bacteriome in transitional stool samples. In addition, it highlights the necessity for further investigation into the potential long-term health impacts on children.

• Keywords
• 16S rRNA, Antibiotics, Diversity, Infant, Microbiome, Mother, Next-generation sequencing,
• MeSH
• Anti-Bacterial Agents * administration & dosage   MeSH
• Antibiotic Prophylaxis * methods   MeSH
• Bacteria genetics   classification   isolation & purification   drug effects   MeSH
• Cesarean Section MeSH
• Adult MeSH
• Feces * microbiology   MeSH
• Humans MeSH
• Meconium microbiology   MeSH
• Infant, Newborn MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome drug effects   MeSH
• Pregnancy MeSH
• Mouth * microbiology   MeSH
• Delivery, Obstetric MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• RNA, Ribosomal, 16S MeSH