Box 1.

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 colonize 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? What is the functional significance of milk microbiota?