Theory 1. The milk microiome is populated by enteromammary transportation (= the most evidence-based hypothesis)

The dominant source of bacteria for the infant gut is now believed to be the maternal gastrointestinal tract. This hypothesis proposes that selected bacteria from the maternal gut colonise milk through an endogenous route. It's known that enteromammary traffic of immune cells occurs during late pregnancy and throughout lactation, as the breast secretes milk and becomes a highly active part of the body’s mucosal immune system. Maternal mononuclear cells transport bacteria and also fragments of gut-derived bacteria from the maternal gut to the breast.

This hypothesis is corroborated by the finding that faeces from mothers and their infants share a common bacterial signature with the same mother’s milk.1,2,3

Theory 2. The microbiome is seeded from the breast stroma (= also likely to have a role)

Viable bacteria have been found in mammary tissue of women who have never breastfed, suggesting the mammary gland itself may be a source of bacteria for milk.4

• The human milk ecosystem is exposed to the internal environment of the breast stroma through lactocyte tight junctions which are permeable at birth and only close over the next few days as the colostrum changes to transitional milk.

• A lactocyte tight junction leak in response to the mechanical effects of rising intra-alveolar pressure may facilitate bacterial translocation; alveolar rupture ensures bacterial presence in the stroma.5, 6

There is a discussion about breast stroma microbiome elsewhere in this section.

Theory 3. The microbiome is populated by retrograde spread from infant oral cavity and the nipple-areolar-complex (= less likely to be relevant)

It's been hypothesised that bacteria from the maternal skin, such as Staphylococcus and Corynebacterium, are ‘seeded’ into the mother’s milk during suckling, and then colonise the infant gut. This theory is often wrongly reported as a fact. But in light of the research, the hypothesis that the human milk microbiome is predominantly seeded by retrograde movement of planktonic oral bacteria remains unconvincing, for the following reasons.

1. The milk microbiome is exposed to the external environment through duct orifices in the nipple. Coagulase-negative Staphylococcus, Candida, and Streptococcus of mitrus and salivarious groups inhabit healthy nipple-areolar-complex skin, the infant’s mouth, and also human milk. However, these same organisms have also been isolated in antenatal colostrum, prior to contact with the newborn.7

2. The neonatal oral microbiome is highly dynamic, and altered by formula feeding. Although one study suggested that diversity increased in human milk microbiota after the first breastfeed,8 with an increased presence of oral microbes in the human milk microbiome attributed to retrograde seeding, other studies have not corroborated this finding.7

3. During milk ejection, ultrasound analysis shows that milk in the breast not subject to mechanical or suckling milk removal flows firstly towards the nipple, but then backwards into emptier ducts.9 This finding of backward flow according to pressure gradients in a non-suckled breast does not corroborate the hypothesis that bacteria spread by retrograde flow from the infant’s mouth into human milk.

4. Infant saliva contains multiple soluble factors which protect the body from potential pathogens, including antibacterial salivary lysozyme and pattern-recognition molecules which regulate inflammation (see below).

Two studies show that infant saliva and breast milk synergistically regulate the infant oral microbiome

A 2015 study had also found that mixing baby saliva with breastmilk generated hydrogen peroxide, sufficient to inhibit the growth of opportunistic pathogens like Staphylococcus aureus and Salmonella spp..10 Neonatal saliva has significantly higher concentrations of hypoxanthine and xanthine compared to adult saliva. The authors concluded that the interaction between breastmilk and infant saliva represents a unique biochemical synergism that boosts early innate immunity by regulating the oral microbiota.10

A 2018 study demonstrated that a range of micro-organisms’ growth (including of Candida albicans) was inhibited immediately, and for up to 24 hours afterwards, when saliva was mixed with breast milk, regardless of whether the organisms considered to be commensal or pathogenic.11 Breastmilk contains an abundance of the enzyme xanthine oxidase, which acts upon the high levels of xanthin and hypoxanthine in neonatal saliva to release hydrogen peroxide. Hydrogen peroxide is a key oxidative radical and antibacterial, which damages bacterial cell wall integrity.

This known synergistic antibacterial activity of infant saliva mixed with breast milk also makes the retrograde seeding hypothesis makes the retrograde seeding hypothesis of the milk microbiome (and of breast inflammation) even less convincing. Potentially pathogenic bacteria are well regulated in the infant's mouth during suckling.

References

1. Fernández L, Pannaraj PS, Rautava S, et al. The microbiota of the human mammary ecosystem. Front Cell Infect Microbiol 2020; 10: 586667

2. Sakwinska O and Bosco N. Host microbe interactions in the lactating mammary gland. Front Microbiol 2019; 10: 1863.

3. Boix-Amoros A, Collado MC, Van’t Land B, et al. Reviewing the evidence on breast milk composition and immunological outcomes. Nutr Rev 2019; 77(8): 541–556.

4. Urbaniak C, Angelini M, Gloor GB, et al. Human milk microbiota profiles in relation to birthing method, gestation and infant gender. Microbiome 2016; 4: 1.

5. Fetherstone C. Mastitis in lactating women: physiology or pathology? ​ Breastfeed Rev 2001; 9: 5–12.

6. Boix-Amoros A, Collado MC, Van’t Land B, et al. ​ Reviewing the evidence on breast milk composition and immunological outcomes. ​ Nutr Rev 2019; 77(8): 541–556.

7. Ruiz L, Bacigalupe R, Garcia-Carral C. Microbiota of human precolostrum and its potential role as a source of bacteria to the infant mouth. Scientific Reports. 2019;9(8435):https://doi.org/10.1038/s41598-41019-42514-41591.

8. Biagi E, Quercia S and Aceti A. The bacterial ecosystem of mother’s milk and infant’s mouth and gut. Front Microbiol 2017; 8: 1214.

9. Ramsay DT, Kent JC, Owens RA, et al. Ultrasound imaging of milk ejection in the breast of lactating women. Pediatrics 2004; 113(2): 361–367.

10. Al-Shehri SS, Knox CL, Liley HG. Breastmilk-saliva interactions boost innate immunity by regulating the oral microbiome in early infancy. Plos One. 2015;10(9):e0135047. doi:0135010.0131371/journal.pone.0135047.

11. Sweeney EL, Al-Shehri SS, Cowley DM, Liley HG, Bansal N, Charles BG, et al. The effect of breastmilk and saliva combinations on the in vitro growth of oral pathogenic and commensal microorganisms. Scientific Reports. 2018;8:15112.