The milk microbiome: composition, origins and perturbations

How do human microbiomes interact with the immune system?

Half of the cells in the human body are microbial, with each niche’s microbiome as unique to individuals as fingerprints. Microbiomes contribute many more genes than the human genome does to our human body. Microbiomes shape phenotypic traits of the host, including nutritional and metabolic traits.

Within the human immune system, microbiomes use chemical and metabolic signals to regulate the abundance and activities of immune cells, including lymphocytes. As lymphocytes respond to encounters with antigens, they regulate the constantly changing genetic sequences produced in immunoglobulins.

The complex cross talk between a microbiome and the body’s adaptive immune system determines whether the body

• Recognizes a specific molecular pattern as non-self, or as a sign of endogenous cell and tissue damage, and

• How vigorously the immune system responds.1

What is the composition of the human milk microbiome?

The human milk microbiome comprises organised networks of bacteria, viruses, fungi, archaea, and protozoa.

• The median bacterial load of healthy human milk is 10 6 cells/ml, and about 200 different species of bacteria have been identified, including a high load of bacteria which have been previously labelled as pathogenic (Staphylococcus aureus, Esherishia coli, Group B streptococci). It’s generally agreed that the core bacterial genera of the milk microbiome, universal across lactating women, are Staphylococcus, Streptococcus and Propionibacterium. Much smaller and more variable populations of Lactobacillus and Bifidobacterium may occur, but not in all breastfeeding women.2-6

• The viral fraction of human milk, the virome, is dominated by bacteriophages, which comprise 95% of human milk viruses. Bacteriophages modulate bacterial ecology by killing certain species.

• The fungal fraction of human milk, the mycobiome, interacts with and stabilises the microbial domain in protective association networks, which together strengthen host health and immunity and resist overgrowth of any particular bacterial species (previously referred to as pathogen colonisation).

  • Candida albicans is the most common fungal commensal in the human body, and Candida spp including C. albicans occur commonly in human milk, having a beneficial, probiotic effect, interacting with and containing bacteria.2, 7-12

Methods of human milk microbiome sampling are not yet standardised

Neither culture nor PCR is yet able to determine the true composition of a human milk microbiome inside a lactating woman’s breast. It is not clear if findings from milk analysed in vitro can be extrapolated back to apply to milk which is still in the alveoli and ducts.

• Culture-based methods select out bacteria from expressed breast milk on specific growth media, identifying species and numbers of colony forming units.

• Molecular polymerase chain reaction (PCR) methods of analysing expressed breast milk identify DNA, which may be from viable or non-viable bacteria, non-cultivable bacteria, and bacterial fragments. Non-viable bacteria are thought to be a significant component of the mammary gland immune system, acting as antigens which interact with host immune cells, much like inactivated vaccines.

Human milk microbiomes are highly variable between mothers and in the one mother over time

The milk microbiome participates in the activation of myriad immune feedback loops within multiple complex systems. Micro-organisms interact together, with the milk metabolome, with milk oligosaccharides, with milk leukocytes, and many other factors to maintain physiological integrity and health of the lactating breast.4

The bacterial communities in human milk are highly dynamic. Antimicrobial-induced disturbance of milk microbiota is quickly reversed.

Human milk microbiome composition differs between colostrum, transitional and mature milk.2 Researchers agree that the composition of the human milk microbiome is also impacted by

• Genetic predisposition

• Ethnicity

• Geographical location

• Circadian rhythm

• Age

• Body mass index

• Maternal nutrient intake, including of fatty acids, carbohydrates or proteins

• Some studies have found differences in the milk microbiome depending on the infant’s mode of delivery, others haven’t.

What is the origin of the human milk microbiome?

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. Enteromammary traffic of immune cells occurs during late pregnancy and lactation, as the lactating breast becomes part of the body’s mucosal immune system. Maternal mononuclear cells transport bacteria and also fragments of gut-derived bacteria to the breast during pregnancy and lactation.

It’s hypothesised that selected bacteria from the maternal gut colonise milk through this endogenous route. This is supported by the finding that faeces from mothers and their infants share a common bacterial signature with the same mother’s milk.2-4

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

Viable bacteria are found in mammary tissue of women who have never breastfed, suggesting the mammary gland itself may be a source of bacteria for milk.13, 14

• 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.6, 15

Theory 3. The microbiome is populated by retrograde spread from infant oral cavity and the nipple-areolar-complex (= unlikely 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. 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.2

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, with an increased presence of oral microbes in the human milk microbiome attributed to retrograde seeding, other studies have not corroborated this finding.16

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.17 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.

A 2018 study demonstrated that a range of micro-organisms’ growth was inhibited by saliva mixed with breast milk, regardless of whether the organisms considered to be commensal or pathogenic.18 Microbial growth including of Candida albicans is inhibited for up to 24 hours when breastmilk and saliva are mixed. 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 antibacterial activity of infant saliva also makes the retrograde seeding hypothesis less than convincing.

Is the concept of dysbiosis useful?

The pathogenicity of most bacterial species depends, firstly, on the state of the host, and secondly, the strain of the bacteria. The terms commensal and pathogenic are unhelpful in discussions of milk microbiome and breast inflammation, because pathogenicity in the context of human milk is on a spectrum and context specific. A potentially pathogenic microbe which exists quite normally within the human milk microbiome is only pathogenic when regulatory feedback loops have been overwhelmed, resulting in prolonged or severe illness and the need for antibiotics.

Increasingly, human microbiome researchers are concerned about the term dysbiosis, which is now widespread in use, since the concept of dysbiosis has been based on an outdated assumption of a normative, categoriseable eubiotic state. Yet human microbiomes are not yet able to be taxonomically categorized due to their astonishing complexity and are highly variable between individuals and over time. It's increasingly recognised that a focus on cataloguing microbiomes will yield little extra clinical advantage.

Researchers also point out that microbial diversity is not always associated with improved health, as is currently assumed.4, 5

The NDC Clinical Guidelines for Breast Inflammation draws upon the work of microbiome researchers who argue that 'dysbiosis' is most accurately used to refer a host-microbiome interaction which leads to suboptimal health outcomes (rather than to lists and comparisons between the myriad kinds of micro-organisms comprising a microbiome). This theoretical re-framing acknowledges that critical host factors control or modulate microbiome populations, and that the human microbiome is really a DNA fusion or 'chimera' made up of microbiome DNA and human cell DNA, blended together.[16-19]

Selected references

Please note that the referencing in this module is still under development. Comprehensive citations are found in the two research publications which the breast inflammation module is built (Douglas 2022 mechanobiological mode; Douglas 2022 classification, prevention, management; Douglas 2023)

Douglas P. Re-thinking benign inflammation of the lactating breast: a mechanobiological model. Women's Health. 2022;18:17455065221075907.

Douglas PS. Re-thinking benign inflammation of the lactating breast: classification, prevention, and management. Women's Health. 2022;18:17455057221091349.

Douglas PS. Does the Academy of Breastfeeding Medicine Clinical Protocol #36 'The Mastitis Spectrum' promote overtreatment and risk worsened outcomes for breastfeeding families? Commentary. International Breastfeeding Journal. 2023;18:Article no. 51 https://doi.org/10.1186/s13006-13023-00588-13008.

1. Rees T, Bosch T, Douglas AE. How the microbiome challenges our concept of self. Plos Biology. 2018(February 9 ):https://doi.org/10.137/journal.pbio.2005358.
2. Fernandez L, Pannaraj PS, Rautava S, Rodriguez JM. The microbiota of the human mammary ecosystem. Frontiers in cellular and infection microbiology. 2020;10:Article 5866667.
3. Sakwinska O, Bosco N. Host microbe interactions in the lactating mammary gland. Frontiers in Microbiology. 2019;10:doi:10.3389/fmicb.2019.01863.
4. Boix-Amoros A, Collado MC, Land VtB, Calvert A, Le Doare K, Garssen J. Reviewing the evidence on breast milk composition and immunological outcomes. Nutrition Reviews. 2019;77(8):541-56.
5. Ruiz L, Garcia-Carral C, Rodriguez JM. Unfolding the human milk microbiome landscape in the omics era. Frontiers in Microbiology. 2019;10(1378):doi:10.3389/fmicb.2019.01378.
6. Oikonomou G, Addis MF, Chassard C. Milk microbiota: what are we exactly talking about? Frontiers in Microbiology. 2020;11(60):doi:10.3389/fmicb.2020.00060.
7. Douglas PS. Overdiagnosis and overtreatment of nipple and breast candidiasis: a review of the relationship between the diagnosis of mammary candidiasis and Candida albicans in breastfeeding women. Women's Health. 2021;17:DOI: 10.1177/17455065211031480.
8. Dinleyici M, Perez-Brocal V, Arslanoglu S, Aydemir O, Ozumut SS, Tekin N. Human milk mycobiota composition: relationship with gestational age, delivery mode, and birth weight. Beneficial Microbes. 2020;11(2):doi:10.3910/BM2019.0158.
9. Moossavi S, Fehr K, Derakhshani H, Sbihi H, Robertson B, Bode L. Human milk fungi: environmental determinants and inter-kingdom associations with milk bacteria in the CHILD Cohort Study. BMC Microbiology. 2020;20:146.
10. Boix-Amoros A, Puente-Sanchez F, Du Toit E, Linderborg K. Mycobiome profiles in breast milk from healthy women depend on mode of delivery, geographic location, and interaction with bacteria. Applied and Environmental Microbiology. 2019;85(9):e02994-18.
11. Heisel T, Nyaribo L, Sadowsky MJ, Gale CA. Breastmilk and NICU surfaces are potential sources of fungi for infant mycobiomes. Fungal and Genetic Biology. 2019;128:29-35.
12. Lai GC, Tan TG, Pavelka N. The mammalian mycobiome: a complex system in a dynamic relationship with the host. WIRES Systems Biology and Medicine. 2018;11:e1438.
13. Ingman WV, Glynn DJ, Hutchinson MR. Inflammatory mediators in mastitis and lactation insufficiency. Journal of Mammary Gland Biology and Neoplasia. 2014;19:161-7.
14. Urbaniak C, Angelini M, Gloor GB, Reid G. Human milk microbiota profiles in relation to birthing method, gestation and infant gender. Microbiome. 2016;4(1):doi:10.1186/s40168-015-0145-y.
15. Fetherstone C. Mastitis in lactating women: physiology or pathology? Breastfeeding Review. 2001;9:5-12.
16. Winter S, Baumler A. Gut dysbiosis: ecological causes and causative effects on human disease. PNAS. 2023;120(50):e2316579120.
17. Tiffany CR, Baumler AJ. Dysbiosis: from fiction to function. American Journal of Physiology - Gastrointestinal and Liver Physiology. 2019;317:G602-G608.
18. Hooks KB, O'Malley MA. Dysbiosis and its discontents. mBio. 2017;8(5):e01492-17.
19. Brussow H. Problems with the concept of gut microbiota dysbiosis. Microbial Biotechnology. 2020;13(2):423-34.

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Excess

16. Biagi E, Quercia S, Aceti A. The bacterial ecosystem of mother's milk and infant's mouth and gut. Frontiers in Microbiology. 2017:doi: 10.3389/fmicb.2017.01214.

17. 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.

18. Wilson E, Woodd SL, Benova L. Incidence of and risk factors for lactational mastitis: a systematic review. Journal of Human Lactation. 2020;36(4):673-86.

19. Kvist L, Larsson BW, Hall-Lord ML, Steen A, Schalen C. The role of bacteria in lactational mastitis and some considerations of the use of antibiotic treatment. International Breastfeeding Journal. 2008;3(6):doi:10.1186/746-4358-3-6.

20. Delgado S, Arroyo R, Martin R, Rodriguez JM. PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis. BMC Infectious Diseases. 2008;8(Article 51):https://doi.org/10.1186/471-2334-8-51.

21. Cullinane M, Amir LH, Donath SM, Garland SM, Tabrizi SN, Payne MS, et al. Determinants of mastitis in women in the CASTLE study: a cohort study. BMC Family Practice. 2015;16:181.

22. Douglas PS. Re-thinking benign inflammation of the lactating breast: classification, prevention, and management. Women's Health. 2021:Under review.