Residents or Tourists: Is the Lactating Mammary Gland Colonized by Residential Microbiota?

The existence of the human milk microbiome has been widely recognized for almost two decades, with many studies examining its composition and relationship to maternal and infant health. However, the richness and viability of the human milk microbiota is surprisingly low. Given that the lactating mammary gland houses a warm and nutrient-rich environment and is in contact with the external environment, it may be expected that the lactating mammary gland would contain a high biomass microbiome. This discrepancy raises the question of whether the bacteria in milk come from true microbial colonization in the mammary gland ("residents") or are merely the result of constant influx from other bacterial sources ("tourists"). By drawing together data from animal, in vitro, and human studies, this review will examine the question of whether the lactating mammary gland is colonized by a residential microbiome.

Maternal dietary intervention during lactation impacts the maternal faecal and human milk microbiota.

AIMS: To determine the effect of a two-week reduced fat and sugar and increased fibre maternal dietary intervention on the maternal faecal and human milk (HM) microbiomes. METHODS AND RESULTS: Faecal swabs and HM samples were collected from mothers (n = 11) immediately pre-intervention, immediately post-intervention, and 4 and 8 weeks post-intervention, and were analysed using full-length 16S rRNA gene sequencing. Maternal macronutrient intake was assessed at baseline and during the intervention. Maternal fat and sugar intake during the intervention were significantly lower than pre-intervention (P = <0.001, 0.005, respectively). Significant changes in the bacterial composition of maternal faeces were detected after the dietary intervention, with decreases in the relative abundance of Bacteroides caccae (P = <0.001) and increases in the relative abundance of Faecalibacillus intestinalis (P = 0.006). In HM, the diet resulted in a significant increase in Cutibacterium acnes (P = 0.001) and a decrease in Haemophilus parainfluenzae (P = <0.001). The effect of the diet continued after the intervention, with faecal swabs and HM samples taken 4 and 8 weeks after the diet showing significant differences compared to baseline. CONCLUSION: This pilot study demonstrates that short-term changes in maternal diet during lactation can alter the bacterial composition of the maternal faeces and HM.

Milk microbiome transplantation: recolonizing donor milk with mother's own milk microbiota.

Donor human milk (DHM) provides myriad nutritional and immunological benefits for preterm and low birthweight infants. However, pasteurization leaves DHM devoid of potentially beneficial milk microbiota. In the present study, we performed milk microbiome transplantation from freshly collected mother's own milk (MOM) into pasteurized DHM. Small volumes of MOM (5%, 10%, or 30% v/v) were inoculated into pasteurized DHM and incubated at 37 °C for up to 8 h. Further, we compared microbiome recolonization in UV-C-treated and Holder-pasteurized DHM, as UV-C treatment has been shown to conserve important biochemical components of DHM that are lost during Holder pasteurization. Bacterial culture and viability-coupled metataxonomic sequencing were employed to assess the effectiveness of milk microbiome transplantation. Growth of transplanted MOM bacteria occurred rapidly in recolonized DHM samples; however, a greater level of growth was observed in Holder-pasteurized DHM compared to UV-C-treated DHM, potentially due to the conserved antimicrobial properties in UV-C-treated DHM. Viability-coupled metataxonomic analysis demonstrated similarity between recolonized DHM samples and fresh MOM samples, suggesting that the milk microbiome can be successfully transplanted into pasteurized DHM. These results highlight the potential of MOM microbiota transplantation to restore the microbial composition of UV-C-treated and Holder-pasteurized DHM and enhance the nutritional and immunological benefits of DHM for preterm and vulnerable infants. KEY POINTS: • Mother's own milk microbiome can be successfully transplanted into donor human milk. • Recolonization is equally successful in UV-C-treated and Holder-pasteurized milk. • Recolonization time should be restricted due to rapid bacterial growth.

Effects of Different Thawing and Warming Processes on Human Milk Composition.

The composition of human milk is influenced by storage and processing practices. The effects of thawing and warming practices on human milk composition remain poorly studied despite their prevalence in home, research, and donor milk bank settings. This review comprehensively examines the impact of different thawing and warming methods on nutritional and bioactive human milk components. While some components such as carbohydrates and minerals remain stable under most typical thawing and warming conditions, others, such as fat, immune proteins, bacterial and human cells, and peptide amine hormones, are sensitive to warming. This review has identified that the data on the effects of milk thawing and warming is limited and often contradictory. Given that numerous important components of milk are diminished during cold storage, it is important that thawing and warming practices do not lead to further loss of or alterations to beneficial milk components. Further work in this field will facilitate greater standardization of thawing methods among researchers and underpin recommendations for thawing and warming of expressed milk for parents.

From hype to hope: Considerations in conducting robust microbiome science.

Microbiome science has been one of the most exciting and rapidly evolving research fields in the past two decades. Breakthroughs in technologies including DNA sequencing have meant that the trillions of microbes (particularly bacteria) inhabiting human biological niches (particularly the gut) can be profiled and analysed in exquisite detail. This microbiome profiling has profound impacts across many fields of research, especially biomedical science, with implications for how we understand and ultimately treat a wide range of human disorders. However, like many great scientific frontiers in human history, the pioneering nature of microbiome research comes with a multitude of challenges and potential pitfalls. These include the reproducibility and robustness of microbiome science, especially in its applications to human health outcomes. In this article, we address the enormous promise of microbiome science and its many challenges, proposing constructive solutions to enhance the reproducibility and robustness of research in this nascent field. The optimisation of microbiome science spans research design, implementation and analysis, and we discuss specific aspects such as the importance of ecological principals and functionality, challenges with microbiome-modulating therapies and the consideration of confounding, alternative options for microbiome sequencing, and the potential of machine learning and computational science to advance the field. The power of microbiome science promises to revolutionise our understanding of many diseases and provide new approaches to prevention, early diagnosis, and treatment.

Impact of breastfeeding and other early-life factors on the development of the oral microbiome.

The oral cavity is home to the second most diverse microbiome in the human body. This community contributes to both oral and systemic health. Acquisition and development of the oral microbiome is a dynamic process that occurs over early life; however, data regarding longitudinal assembly of the infant oral microbiome is scarce. While numerous factors have been associated with the composition of the infant oral microbiome, early feeding practices (breastfeeding and the introduction of solids) appear to be the strongest determinants of the infant oral microbiome. In the present review, we draw together data on the maternal, infant, and environmental factors linked to the composition of the infant oral microbiome, with a focus on early nutrition. Given evidence that breastfeeding powerfully shapes the infant oral microbiome, the review explores potential mechanisms through which human milk components, including microbes, metabolites, oligosaccharides, and antimicrobial proteins, may interact with and shape the infant oral microbiome. Infancy is a unique period for the oral microbiome. By enhancing our understanding of oral microbiome assembly in early life, we may better support both oral and systemic health throughout the lifespan.

Characterisation of Mid-Gestation Amniotic Fluid Cytokine and Bacterial DNA Profiles in Relation to Pregnancy Outcome in a Small Australian Cohort.

A well-established association exists between intrauterine bacteria and preterm birth. This study aimed to explore this further through documenting bacterial and cytokine profiles in Australian mid-gestation amniotic fluid samples from preterm and term births. Samples were collected during amniocenteses. DNA was extracted and the full-length 16S rRNA gene was amplified and sequenced. Levels of the cytokines IL-1ß, IL-6, IL-10, TNF-α and MCP-1 were determined using the Milliplex MAGPIX system. Bacterial DNA profiles were low in diversity and richness, with no significant differences observed between term and preterm samples. No differences in the relative abundance of individual OTUs between samples were identified. IL-1ß and TNF-α levels were significantly higher in samples containing reads mapping to Sphingomonas sp.; however, this result should be interpreted with caution as similar reads were also identified in extraction controls. IL-6 levels were significantly increased in samples with reads that mapped to Pelomonas sp., whilst TNF-α levels were elevated in fluid samples from pregnancies that subsequently delivered preterm. Bacterial DNA unlikely to have originated from extraction controls was identified in 20/31 (64.5%) mid-gestation amniotic fluid samples. Bacterial DNA profiles, however, were not predictive of preterm birth, and although cytokine levels were elevated in the presence of certain genera, the biological relevance of this remains unknown.

Effect of Holder pasteurization and UV-C irradiation on bacteriophage titres in human milk.

Human milk is the optimal nutrition source for infants and contains a complex mix of bioactive compounds and microorganisms. When unavailable, pasteurized donor milk may be provided, particularly to preterm infants. Holder pasteurization (HP) is typically implemented in human milk banks to prevent pathogen transmission. Given the impact of heat on milk bioactives, ultraviolet-C irradiation (UV-C) is an alternative being explored and has demonstrated effective bactericidal activity. In addition to bacteria, milk contains viruses, including primarily bacteriophages (phages) and which likely influence the developing bacterial microbiome of infants. However, the effect of pasteurization on human milk phages is unknown. This study assessed the effect of HP and UV-C on titres of exogenous bacteriophages inoculated into human milk. Ten donor human milk samples were tested in parallel with water controls. Milk samples or water controls were inoculated to a final concentration of 1 × 104 PFU/mL (±1 log) each of a thermotolerant Escherichia coli phage (T4) and a thermosensitive Staphylococcus aureus phage (BYJ20) and subjected to HP and UV-C treatments. UV-C inactivated both phages within milk and water controls, however, HP was ineffective against the thermotolerant T4 phages. Initial data suggest that UV-C treatment may eliminate phage with potential to affect preterm infant gut colonization. Further studies should extend this to other phages.

Human Milk Lipids and Small Metabolites: Maternal and Microbial Origins.

Although there has been limited application in the field to date, human milk omics research continues to gain traction. Human milk lipidomics and metabolomics research is particularly important, given the significance of milk lipids and metabolites for infant health. For researchers conducting compositional milk analyses, it is important to consider the origins of these compounds. The current review aims to provide a summary of the existing evidence on the sources of human milk lipids and small metabolites. Here, we describe five major sources of milk lipids and metabolites: de novo synthesis from mammary cells, production by the milk microbiota, dietary consumption, release from non-mammary tissue, and production by the gut microbiota. We synthesize the literature to provide evidence and understanding of these pathways in the context of mammary gland biology. We recommend future research focus areas to elucidate milk lipid and small metabolite synthesis and transport pathways. Better understanding of the origins of human milk lipids and metabolites is important to improve translation of milk omics research, particularly regarding the modulation of these important milk components to improve infant health outcomes.

Characterisation of human milk bacterial DNA profiles in a small cohort of Australian women in relation to infant and maternal factors.

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 of Streptococcus australis than that of underweight mothers, while milk from overweight mothers had a higher relative abundance of Streptococcus salivarius compared with underweight and obese mothers. Mothers who delivered vaginally had a higher relative abundance of Streptococcus mitis in their milk compared to those who delivered via emergency caesarean section. Milk of mothers who used a breast pump had a higher relative abundance of Staphylococcus epidermidis and Streptococcus parasanguinis. Milk of mothers whose infants used a pacifier had a higher relative abundance of S. australis and Streptococcus 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.

Methodological approaches for studying the human milk microbiome.

Human milk contains a low-biomass, low-diversity microbiome, consisting largely of bacteria. This community is of great research interest in the context of infant health and maternal and mammary health. However, this sample type presents many unique methodological challenges. In particular, there are numerous technical considerations relating to sample collection and storage, DNA extraction and sequencing, viability, and contamination. Failure to properly address these challenges may lead to distortion of bacterial DNA profiles generated from human milk samples, ultimately leading to spurious conclusions. Here, these technical challenges are discussed, and various methodological approaches used to address them are analyzed. Data were collected from studies in which a breadth of methodological approaches were used, and recommendations for robust and reproducible analysis of the human milk microbiome are proposed. Such methods will ensure high-quality data are produced in this field, ultimately supporting better research outcomes for mothers and infants.

Human Milk Microbiome and Microbiome-Related Products: Potential Modulators of Infant Growth.

Infant growth trajectory may influence later-life obesity. Human milk provides a wide range of nutritional and bioactive components that are vital for infant growth. Compared to formula-fed infants, breastfed infants are less likely to develop later-onset obesity, highlighting the potential role of bioactive components present in human milk. Components of particular interest are the human milk microbiota, human milk oligosaccharides (HMOs), short-chain fatty acids (SCFAs), and antimicrobial proteins, each of which influence the infant gut microbiome, which in turn has been associated with infant body composition. SCFAs and antimicrobial proteins from human milk may also systemically influence infant metabolism. Although inconsistent, multiple studies have reported associations between HMOs and infant growth, while studies on other bioactive components in relation to infant growth are sparse. Moreover, these microbiome-related components may interact with each other within the mammary gland. Here, we review the evidence around the impact of human milk microbes, HMOs, SCFAs, and antimicrobial proteins on infant growth. Breastfeeding is a unique window of opportunity to promote optimal infant growth, with aberrant growth trajectories potentially creating short- and long-term public health burdens. Therefore, it is important to understand how bioactive components of human milk influence infant growth.

Effect of a reduced fat and sugar maternal dietary intervention during lactation on the infant gut microbiome.

Objective: A growing body of literature has shown that maternal diet during pregnancy is associated with infant gut bacterial composition. However, whether maternal diet during lactation affects the exclusively breastfed infant gut microbiome remains understudied. This study sets out to determine whether a two-week of a reduced fat and sugar maternal dietary intervention during lactation is associated with changes in the infant gut microbiome composition and function. Design: Stool samples were collected from four female and six male (n = 10) infants immediately before and after the intervention. Maternal baseline diet from healthy mothers aged 22-37 was assessed using 24-h dietary recall. During the 2-week dietary intervention, mothers were provided with meals and their dietary intake was calculated using FoodWorks 10 Software. Shotgun metagenomic sequencing was used to characterize the infant gut microbiome composition and function. Results: In all but one participant, maternal fat and sugar intake during the intervention were significantly lower than at baseline. The functional capacity of the infant gut microbiome was significantly altered by the intervention, with increased levels of genes associated with 28 bacterial metabolic pathways involved in biosynthesis of vitamins (p = 0.003), amino acids (p = 0.005), carbohydrates (p = 0.01), and fatty acids and lipids (p = 0.01). Although the dietary intervention did not affect the bacterial composition of the infant gut microbiome, relative difference in maternal fiber intake was positively associated with increased abundance of genes involved in biosynthesis of storage compounds (p = 0.016), such as cyanophycin. Relative difference in maternal protein intake was negatively associated with Veillonella parvula (p = 0.006), while positively associated with Klebsiella michiganensis (p = 0.047). Relative difference in maternal sugar intake was positively associated with Lactobacillus paracasei (p = 0.022). Relative difference in maternal fat intake was positively associated with genes involved in the biosynthesis of storage compounds (p = 0.015), fatty acid and lipid (p = 0.039), and metabolic regulator (p = 0.038) metabolic pathways. Conclusion: This pilot study demonstrates that a short-term maternal dietary intervention during lactation can significantly alter the functional potential, but not bacterial taxonomy, of the breastfed infant gut microbiome. While the overall diet itself was not able to change the composition of the infant gut microbiome, changes in intakes of maternal protein and sugar during lactation were correlated with changes in the relative abundances of certain bacterial species.Clinical trial registration: Australian New Zealand Clinical Trials Registry (ACTRN12619000606189).

Effect of Cold Storage on the Viable and Total Bacterial Populations in Human Milk.

Expression and cold storage of human milk is a common practice. Current guidelines for cold storage of expressed milk do not take into account the impact on the milk microbiome. Here, we investigated the impact of cold storage on viable bacterial populations in human milk. Freshly expressed milk samples (n = 10) were collected and analysed immediately, stored at 4 °C for four days, −20 °C for 2.25 months and 6 months, and −80 °C for 6 months. Samples were analysed using propidium monoazide (PMA; a cell viability dye) coupled with full-length 16S rRNA gene. An aliquot of each sample was additionally analysed without PMA to assess the impact of cold storage on the total DNA profile of human milk. Cold storage significantly altered the composition of both the viable microbiome and total bacterial DNA profile, with differences in the relative abundance of several OTUs observed across each storage condition. However, cold storage did not affect the richness nor diversity of the samples (PERMANOVA all p > 0.2). Storage of human milk under typical and recommended conditions results in alterations to the profile of viable bacteria, with potential implications for infant gut colonisation and infant health.

Exclusively Breastfed Infant Microbiota Develops over Time and Is Associated with Human Milk Oligosaccharide Intakes.

Temporal development of maternal and infant microbiomes during early life impacts short- and long-term infant health. This study aimed to characterize bacterial dynamics within maternal faecal, human milk (HM), infant oral, and infant faecal samples during the exclusive breastfeeding period and to document associations between human milk oligosaccharide (HMO) intakes and infant oral and faecal bacterial profiles. Maternal and infant samples (n = 10) were collected at 2−5, 30, 60, 90 and 120 days postpartum and the full-length 16S ribosomal RNA (rRNA) gene was sequenced. Nineteen HMOs were quantitated using high-performance liquid chromatography. Bacterial profiles were unique to each sample type and changed significantly over time, with a large degree of intra- and inter-individual variation in all sample types. Beta diversity was stable over time within infant faecal, maternal faecal and HM samples, however, the infant oral microbiota at day 2−5 significantly differed from all other time points (all p < 0.02). HMO concentrations and intakes significantly differed over time, and HMO intakes showed differential associations with taxa observed in infant oral and faecal samples. The direct clinical relevance of this, however, is unknown. Regardless, future studies should account for intakes of HMOs when modelling the impact of HM on infant growth, as it may have implications for infant microbiota development.

Human Milk Oligosaccharides and Bacterial Profile Modulate Infant Body Composition during Exclusive Breastfeeding.

Human milk is a complex and variable ecosystem fundamental to the development of newborns. This study aimed to investigate relationships between human milk oligosaccharides (HMO) and human milk bacterial profiles and infant body composition. Human milk samples (n = 60) were collected at two months postpartum. Infant and maternal body composition was measured with bioimpedance spectroscopy. Human milk bacterial profiles were assessed using full-length 16S rRNA gene sequencing and 19 HMOs were quantitated using high-performance liquid chromatography. Relative abundance of human milk bacterial taxa were significantly associated with concentrations of several fucosylated and sialylated HMOs. Individual human milk bacteria and HMO intakes and concentrations were also significantly associated with infant anthropometry, fat-free mass, and adiposity. Furthermore, when data were stratified based on maternal secretor status, some of these relationships differed significantly among infants born to secretor vs non-secretor mothers. In conclusion, in this pilot study the human milk bacterial profile and HMO intakes and concentrations were significantly associated with infant body composition, with associations modified by secretor status. Future research designed to increase the understanding of the mechanisms by which HMO and human milk bacteria modulate infant body composition should include intakes in addition to concentrations.

Microbial metabolites: the next frontier in human milk.

Microbial metabolites mediate the impact of the microbiome on host physiology. Here, we highlight metabolites of interest for early-life programming and synthesise early evidence that maternally derived microbial metabolites are transmitted to the infant via human milk. We advocate for the use of targeted and untargeted metabolomics to compliment current milk microbiome work.

Environmental determinants of human milk composition in relation to health outcomes.

Humans are exposed to environmental factors at every stage of life including infancy. The aim of this mini-review was to present a narrative of environmental factors influencing human milk composition. Current literature shows lactation is a dynamic process and is responsive to multiple environmental challenges including geographical location, lifestyle, persistent pollutants and maternal factors (ethnicity, diet, stress, allergy and adiposity) that may influence human milk composition in a synergistic manner and should be considered in order to improve infant and maternal outcomes on a populations scale. Further interventional studies on larger international cohorts are needed to elucidate these complex relationships. Lactating women should aim for a healthy lifestyle and maintain a healthy body composition prior to and throughout the reproductive period, including during lactation.

The Viable Microbiome of Human Milk Differs from the Metataxonomic Profile.

Bacteria in human milk contribute to the establishment of the infant gut microbiome. As such, numerous studies have characterized the human milk microbiome using DNA sequencing technologies, particularly 16S rRNA gene sequencing. However, such methods are not able to differentiate between DNA from viable and non-viable bacteria. The extent to which bacterial DNA detected in human milk represents living, biologically active cells is therefore unclear. Here, we characterized both the viable bacterial content and the total bacterial DNA content (derived from viable and non-viable cells) of fresh human milk (n = 10). In order to differentiate the living from the dead, a combination of propidium monoazide (PMA) and full-length 16S rRNA gene sequencing was used. Our results demonstrate that the majority of OTUs recovered from fresh human milk samples (67.3%) reflected DNA from non-viable organisms. PMA-treated samples differed significantly in their bacterial composition compared to untreated samples (PERMANOVA p < 0.0001). Additionally, an OTU mapping to Cutibacterium acnes had a significantly higher relative abundance in PMA-treated (viable) samples. These results demonstrate that the total bacterial DNA content of human milk is not representative of the viable human milk microbiome. Our findings raise questions about the validity of conclusions drawn from previous studies in which viability testing was not used, and have broad implications for the design of future work in this field.

Human Milk Lactose, Insulin, and Glucose Relative to Infant Body Composition during Exclusive Breastfeeding.

Human milk (HM) components may influence infant growth and development. This study aimed to investigate relationships between infant body composition (BC) and HM lactose, insulin, and glucose (concentrations and calculated daily intakes (CDI)) as well as 24-h milk intake and maternal BC at 3 months postpartum. HM samples were collected at 2 months postpartum. Infant and maternal BC was assessed with bioimpedance spectroscopy. Statistical analysis used linear regression accounting for infant birth weight. 24-h milk intake and CDI of lactose were positively associated with infant anthropometry, lean body mass and adiposity. Higher maternal BC measures were associated with lower infant anthropometry, z-scores, lean body mass, and adiposity. Maternal characteristics including BC and age were associated with concentrations and CDI of HM components, and 24-h milk intake. In conclusion, 24-h intake of HM and lactose as well as maternal adiposity are related to development of infant BC.