Human milk (HM) contains an incredible array of microorganisms. These likely contribute to the seeding of the infant gastrointestinal microbiome, thereby influencing infant immune and metabolic development and later-life health. Given the importance of the HM microbiota in this context, there has been an increase in research efforts to characterize this in different populations and in relation to different maternal and infant characteristics. However, despite a decade of intensive research, there remain several unanswered questions in this field. In this review, the “5 W+H” approach (who, what, when, where, why, and how) is used to comprehensively describe the composition, function, and origin of the HM microbiome. Here, existing evidence will be drawn together and critically appraised to highlight avenues for further research, both basic and applied. Perhaps the most interesting of these is the potential to modulate the HM microbiome using pre/probiotics or dietary interventions. Another exciting possibility is the personalization of donor milk for women with insufficient supply. By gaining a deeper understanding of the HM microbiome, opportunities to intervene to optimize infant and lifelong health may be identified.

The antibacterial activity of honeys derived from the endemic flora of the southwest corner of Western Australia, including the trees Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla), remains largely unexplored. Investigation of these honeys showed minimum inhibitory concentrations (MICs) of 6.7–28.0% (w/v) against Gram positive and negative bacteria. Honey solutions showed enhanced antibacterial activity after hydrogen peroxide was allowed to accumulate prior to testing, with a mean MIC after accumulation of 14.3% compared to 17.4% before accumulation. Antibacterial activity was reduced after treatment with catalase enzyme, with a mean MIC of 29.4% with catalase compared to 15.2% without catalase. Tests investigating the role of the Gram negative outer membrane in honey susceptibility revealed increases in activity after destabilisation of the outer membrane. Honeys reduced both the formation of biofilm and the production of bacterial pigments, which are both regulated by quorum sensing. However, these reductions were closely correlated with global growth inhibition. Honey applied to existing biofilms resulted in decreased metabolic activity and minor decreases in viability. These results enhance our understanding of the mechanisms of antibacterial action of Jarrah and Marri honeys, and provide further support for the use of honey in the treatment of infected wounds.

Human milk provides all of the elements necessary for infant growth and development. Previous studies have reported associations between breastfeeding and a reduced risk of developing obesity and late-onset metabolic disorders; however, the underlying mechanisms are poorly understood. Recently, intakes of human milk components have been associated with infant body composition, which is likely partially implicated in the reduced risk of developing childhood obesity among breastfed infants. In this systematic review, we searched electronic bibliographic databases for studies that explored relationships between the 24 h intakes of human milk macronutrients and bioactive components and infant body composition and/or growth parameters. Of 13 eligible studies, 10 assessed relationships of infant body composition and growth outcomes with human milk macronutrients, while 8 studies assessed relationships with human milk bioactive components. Significant time-dependent relationships with infant anthropometrics and body composition were found for intakes and no relationships for concentrations of several human milk components, such as lactose, total protein, and human milk oligosaccharides, suggesting that measuring concentrations of human milk components without quantifying the intake by the infant may provide a limited understanding. Future studies investigating the effect of human milk components on infant growth and body composition outcomes should consider measuring the actual intake of components and employ standardised methods for measuring milk intake.

It is known that human milk (HM)¹ antimicrobial protein composition varies during lactation. However, the impact of maternal diet on these antimicrobial proteins, particularly lactoferrin and lysozyme remains unknown. In addition, it is unclear whether daily, circadian, and between breast variations exist for lactoferrin and lysozyme concentrations. We investigated the impact of a low sugar, low fat, high fibre dietary intervention on HM lysozyme and lactoferrin concentrations. HM was sampled across a 3-week period; daily, at different times of day, and from both breasts to measure the level of intraindividual variation. The intervention significantly reduced maternal sugar, total fat, and saturated fat intake. HM lactoferrin concentration declined significantly over the course of the intervention however the effect size was relatively small. In addition, lactoferrin and lysozyme concentrations were variable over time, and differed significantly within and across the day but not between breasts.

Human milk is composed of complex microbial and non-microbial components that shape the infant gut microbiome. Although several maternal and infant factors have been associated with human milk microbiota, no study has investigated this in an Australian population. Therefore, we aimed to investigate associations between human milk bacterial composition of Australian women and maternal factors (body mass index (BMI), mode of delivery, breast pump use, allergy, parity) and infant factors (sex, mode of feeding, pacifier use, and introduction of solids). Full-length 16S rRNA gene sequencing was used to characterise milk bacterial DNA profiles. Milk from mothers with a normal BMI had a higher relative abundance ofStreptococcus australisthan that of underweight mothers, while milk from overweight mothers had a higher relative abundance ofStreptococcus salivariuscompared with underweight and obese mothers. Mothers who delivered vaginally had a higher relative abundance ofStreptococcus mitisin their milk compared to those who delivered via emergency caesarean section. Milk of mothers who used a breast pump had a higher relative abundance ofStaphylococcus epidermidisandStreptococcus parasanguinis. Milk of mothers whose infants used a pacifier had a higher relative abundance ofS.australisandStreptococcus gwangjuense. Maternal BMI, mode of delivery, breast pump use, and infant pacifier use are associated with the bacterial composition of human milk in an Australian cohort. The data from this pilot study suggests that both mother and infant can contribute to the human milk microbiome.

The human milk (HM) microbiome plays an important role in shaping the infant gut microbiota, with potential implications for immune development and both short- and long-term health. Among the maternal and infant factors influencing HM microbial composition, maternal diet represents a modifiable determinant. However, evidence regarding the impact of diet on the HM microbiota remains limited, and the methodological quality of available studies is variable. This review synthesises findings from 15 observational and interventional studies, critically evaluating dietary assessment approaches, milk collection protocols, microbiome analysis methods, and control of confounding factors. Current evidence suggests that maternal intake of macronutrients, micronutrients, and bioactive compounds may influence HM bacterial composition and functional potential, though results are inconsistent. Key limitations across studies include small sample sizes, short intervention periods, lack of appropriate control groups, variable aseptic sampling methods, inadequate contamination controls, and insufficient adjustment for confounders. To advance the field, we recommend larger, multicentre randomised controlled trials with longer intervention durations, incorporation of dietary biomarkers, standardised HM collection and processing protocols, and advanced multi-omics approaches. Strengthening methodological rigour is essential to generate robust evidence that can guide dietary interventions aimed at optimising the HM microbiota and improving infant health outcomes.