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Page 6 of 20 Horwell et al. Microbiome Res Rep 2025;4:1 https://dx.doi.org/10.20517/mrr.2024.32

unknowns. Moreover, several of these variables, such as maternal co-morbidity, also independently increase
the likelihood of having a CS, potentially compounding the effect.

Breastfeeding (the developmental phase)
During this period, even when accounting for the mode of delivery, the most significant factor associated
[14]
with the structure of the microbiome is the receipt of breast milk, either exclusively or partially . Contrary
[83]
to previously held dogma, human breast milk is not sterile and has been found to be rich in the genera
Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Weisella, Staphylococcus,
6
and Streptococcus [84-87] . One paper has recorded a median bacterial load of 10 cells/mL of breast milk,
suggesting that exclusively breastfed (EBF) infants consume approximately 8 billion bacteria per day . The
[88]
origin of the lactational mammary duct microbiome is complex and likely multifaceted. DNA sequencing of
the milk has demonstrated species otherwise typically found in the oral cavity and maternal skin. It is
reasonable to assume colonisation occurs during suckling of the infant as well as from direct contact with

adjacent skin microbiomes. This, however, does not explain the presence of obligate anaerobes found in
milk that are prototypical of an adult’s colonic microbiome - Enterococcus, Bifidobacterium, Bacteroides,
Clostridium, and Faecalibacterium [89,90] . Recent evidence has suggested an entero-mammary pathway
whereby bacterial cells from the colonic lumen are phagocytosed by dendritic and CD18 cells, taken into
+
the systemic lymphatic and cardiovascular circulation and then deposited in the mammary gland . This
[91]
mechanism is analogous to the established entero-mammary circulation, where B cells migrate to the
mammary epithelium and release secretory (S) IgA into the milk supply . If this mode of bacterial
[92]
translocation is confirmed with mechanistic studies, it would provide a clear route for the initial
colonisation of strict anaerobic species that are not typically found on surface environments, and the
absence of this mode of transmission could have potentially profound consequences on the development of
the colonic immune system. This may go some way to explain how certain bacterial species that are
exquisitely vulnerable to atmospheric conditions can reach the human GIT. For example, Akkermansia
mucinophilia, a species that is inversely related to IBD and obesity, is notoriously difficult to culture and
[93]
survives for very short periods under atmospheric conditions ; thus, it has no known extra-colonic
environmental niche to provide a stable supply for colonisation. A recent study has detected this species in
the colostrum and milk supply from mothers , perhaps being a key moment for this, and similar species,
[94]
to establish themselves for permanent colonisation of the newborn’s GIT. It has previously been
[95]
demonstrated that maternal diet affects the milk microbiome , and this may be a branch of interesting
future research to develop specialised maternal diets to modulate the developing neonatal microbiome with
the aim of improving long-term health outcomes.

Infants fed exclusively formula milk (FM) do not receive these bacterial species. Accordingly, they have
remarkably different microbiomes to EBF infants. One meta-analysis of 684 infants from five countries
[96]
showed an increased abundance of Bacteroides, Eubacterium, Firmicutes, and Veillonella in formula-fed
babies. EBF infants had a lower alpha diversity due to the increased abundance of the genus
Bifidobacterium. This increased Bifidobacterium abundance is associated with a growth in microbial
metabolic pathways favouring lipid and vitamin metabolism. Several studies have taken these findings,
along with the increased Bacteroides [associated with increased body mass index (BMI)], to suggest that FM
may cause obesity, diabetes, and other adverse health outcomes in adulthood as a result of the altered
nutrient metabolism in the colon [97,98] . Furthermore, the differences in microbiomes between infants who are
EBF and FM appear to be subtly more pronounced if the mode of delivery was CS. The evidence suggests
that the relative absence of anaerobes (typically found in the microbiota of CS-delivered children) is
exacerbated for infants who are then subsequently on FM , particularly in Proteobacteria, suggesting an
[99]
increased importance of EBF after CS. Another interesting finding is the microbiomic difference between
infants who are EBF and those who receive human breast milk from a bottle . For instance, while
[100]