Future Directions of Milk Microbiome Research

Concluding thesis chapter

Scientific considerations

Milk microbiota is a hot research topic in the field of developmental origins of health and disease. Building on earlier studies, our recent findings shed new light on the composition and determinants of the milk microbiota, and raise new research questions about the origins, function and analysis of milk bacteria.

Unanswered questions about milk microbiota

  • Do specific bacteria get selected from the maternal gut and/or oral microbiota for translocation to the mammary gland? How does this occur?

    • Is the translocation specifically directed to the mammary gland
    • Do bacteria transferred to the mammary gland remain viable?
    • Are these bacteria released or do they remain within immune cells upon reaching the mammary gland?
    • Do these bacteria permanently colonise the mammary gland and remain viable regardless of lactation?
    • Is this process specifically stimulated or upregulated during pregnancy and postpartum? If so, how is this process regulated?
  • Do external bacteria enter the mammary gland through the nipple? How does this occur?

    • Are infant oral bacteria transferred into the mammary gland during suckling?
    • Are bacteria from breast pumps transferred into the mammary gland during pumping?
    • Does the mechanical force of pumping affect the intramammary milk microbiota?
  • How do breast pump cleaning practices and milk storage conditions affect the microbiota of expressed milk?

  • How do the qualities and mechanics of different breast pumps and accessories affect the microbiota of expressed milk?

  • What best practices should be applied to milk microbiota analysis to promote accurate and reproducible results (e.g. collection, storage, processing, DNA extraction, and sequencing quality controls)?

  • How does milk microbiota composition vary throughout lactation?

  • How do other microorganisms (e.g. fungi and viruses) contribute to milk microbial communities?

  • How do other milk components and characteristics influence the milk microbiota?

  • Do milk microbes colonize the infant gut?

  • What is the relative importance of the prebiotic (e.g. milk oligosaccharides) vs. probiotic (i.e. live microorganisms) components of human milk?

So far, milk microbiota studies have mainly been observational cross-sectional studies based on 16S rRNA amplicon sequencing, a method that is susceptible to reagent contamination when analyzing low biomass samples such as milk, and cannot distinguish between live or dead bacteria. Better understanding of the dynamics and function of milk microbiota requires a comprehensive multi-pronged strategy that 1) assesses viability, activity, and function of milk bacteria; 2) examines the temporality of the microbial load and composition throughout lactation; 3) studies other members of the microbial community including viruses, which can conceivably be vertically transferred more readily than the bacteria, and fungi, which could have important health implications; 4) evaluates the interaction between milk microbiota with the maternal and infant immune systems; and 5) experimentally establishes the functional significance of milk microbiota. In addition, the origins of milk bacteria should be rigorously assessed using appropriate experimental study designs and suitable animal models that distinguish between intramammary milk and the expressed milk ingested by the offspring.

Technical considerations

Best practices in microbiome studies specifically for low biomass samples should include: 1) negative controls for culture, DNA extraction, and sequencing library preparation, 2) sequencing sufficient numbers of negative controls, 3) biological replicates in different runs, and 4) limit of detection controls using serial dilution.

I propose that culture-informed milk microbiota studies could enhance the success of identification and exclusion of potential reagent contaminants and thereby provide a more accurate representation of the microbiota profile.

The current biobanking gold standard for microbiome research aims to preserve the original bacterial profile for downstream DNA and/or RNA-based sequencing. Frozen stool samples have been successfully used for culturomics. However, I could not isolate bacteria from long-term frozen milk samples which could potentially be due to variability in the bacterial load of milk samples and the overall low biomass of milk microbiota. Although, the results of this thesis should be verified in larger studies and milk samples of mothers of healthy term infant, it is recommended that milk samples be stored using cryopreservatives such as glycerol to maintain the viability of bacteria for future culture-dependent downstream investigation.

There is a large variation in the absolute concentration of the total bacterial load in milk. Differences in microbiome composition based on relative vs. absolute quantification have been previously shown. The more accurate absolute quantification was specifically important in identifying bacteria associated with health outcomes highlighting the importance of absolute quantification in the study of the milk microbiota composition specifically in relation to infant health outcomes.

Bacteria exhibit a considerable amount of heterogeneity in metabolic activity. The activity of bacteria could be assessed based on various cellular processes including proliferation. By experimentally separating bacteria into multiple activity groups using appropriate targeted labels prior to sequencing, it is possible to profile the microbiota based on the activity levels. This approach has been successfully applied to the study of gut microbiota.

Practical and translational considerations

Study of human milk microbiota has two immediate and potentially highly important applications. It must be acknowledged that, for a variety of reasons, pumping may be the only way for some mothers to provide their own breast milk to their infant, and this is still beneficial compared to formula feeding. We have shown that pumping has the most consistent impact on the milk microbiota composition potentially introducing environmental opportunistic bacteria into the milk. While the significance of this observation for the health of the infant is not clear, it could facilitate renewed discussions about the choice of the pump (e.g. tubing material resistant to biofilm formation) and cleaning practices among the stakeholders.

The current standard in processing donor milk in human milk banks involves culture-based bacteriological assessment for the common pathogens (including group B Streptococcus, Staphylococcus aureus, coagulase-negative Staphylococcus, α-Streptococcus, Enterococcus, and Bacillus sp.) followed by pasteurization (62.5 °C for 30 min) which eliminates most milk bacteria except the spore-forming Bacillus species. Subsequently, donor milk will be excluded if any of the following criteria are met: 1) isolation of any pathogen, 2) non-pathogenic bacterial load > 104 colony-forming units/mL before pasteurization, or 3) any positive culture after pasteurization. Donor milks are critically important for the premature infants admitted to NICU who are especially vulnerable to microbiome-mediated conditions, which can be fatal, including necrotizing enterocolitis. Breastmilk provides optimal nutrition for premature infants, and human donor milk is the best alternative when mothers cannot provide their own milk. While donor milk processing aims to minimise the risk of pathogen transmission to the highly vulnerable infant, it also eliminates the non-pathogenic components of the milk microbiota. While the role of milk microbiota in infant health is not elucidated, it is conceivable that milk microbiota influences the development of the infant respiratory tract and gut microbiota compositions. It is therefore an open question if reconstitution of the donor milk with the infant’s own mother milk microbiota would impact the health outcomes in premature infants.

Shirin Moossavi
Shirin Moossavi
Postdoctoral Fellow

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