Exploring the Human Milk Microbiome: Origin, Composition, and Role

Traditionally, human milk (HML) was thought to be sterile. However, the presence of bacteria in the LH was never completely excluded. The first studies carried out between

The late 19th and early 20th centuries1,3 focused on the potentially harmful nature of the bacteriological content of LH, without considering it as it is now, a precious resource. Even so, in the late 1960s, the presence of bacteria in LH was considered a consequence of low levels of personal and environmental hygiene.4

Subsequently, in 2003, interest in the microbiology of LH resurfaced with a new perspective. Based on the detection of putatively endogenous lactic acid bacteria from the LH of eight healthy mothers, it was suggested that LH could be considered a symbiotic food, harboring safe bacteria with a potential role in the prevention of neonatal infectious diseases.5

Over time, the development of culture-independent techniques (e.g., quantitative polymerase chain reaction and next-generation sequencing - SPG), in addition to the already well-established culture-dependent techniques, has allowed the characterization of the composition, diversity and variability of the LH microflora in greater detail, although with some limitations.6

Today, LH is considered “Mother Nature’s prototypical probiotic food.”7 Growing research on this topic has led to a deeper understanding of the matter, discovering that LH is a living universe populated by bacteria, viruses, fungi and yeasts that cooperate for the present and future health of the child. This complex host-associated microbial community constitutes the LH microbiome (MLH).

The aim of this review is to provide an overview of what is currently known about the origin, composition, determinants and role of MLH, eventually suggesting possible future directions for researchers who want to advance the exploration of this field.

MLH seeding is a complex and dynamic process, still not fully understood to date. Multiple, not mutually exclusive, sources of MLH have been suggested (Table 1). It is still under debate whether the mammary gland harbors a resident microbiome (i.e., mucosal interface model) or is simply a bystander subject to a constant influx of microbes from exogenous sources (i.e., constant influx model).

This latter model is supported by the current lack of evidence for bacterial adhesion to the mammary epithelium outside of a mastitis setting and for bacterial reproduction within the mammary tissue. Instead, the mucosal interface model is supported by evidence of a pre-lactation mammary microbiome.8 However, the fact that the microbiome of the non-lactating mammary gland differs from that of the MLH does not allow us to exclude the constant influx model. 9

Although historically knowledge of MLH was limited to bacterial species15, recent evidence highlighted that MLH contains a wide variety of microorganisms , including viruses, fungi and yeasts, and new genera (Table 2).

> Bacterioma

The implementation of new SPG techniques, such as metataxonomics (16SrRNA gene sequencing) and metagenomics (shotgun sequencing), has allowed the detection of several new bacterial species, including many anaerobes, totaling more than 1300 different species .12,16,17,22–27

However, when trying to determine what constitutes the LH bacteriome, interindividual variability and the geographic location of the study as well as the methods used for collection, storage, and analysis must be considered. Thus, the definition, and the very existence, of a “core” HL bacteriome remains a topic of debate.28

Through genomic analysis, different studies have detected a wide variety of microorganisms related to soil and water, such as Bradyrhizobium , Pseudomonas and Stenotrophomonas.8,12,16,22,26,29 However, these results must be interpreted critically, since Such microorganisms could also be contained in reagents, solutions, and molecular biology kits, and their relative quantities could be amplified by DNA techniques, thus contributing to erroneous interpretations.22,30–32 Additionally, it is critical to differentiate between live and dead microorganisms. Therefore, appropriate techniques must be selected to limit potential biases.33

> Virome

The majority (95%)18 of the LH virome is made of bacteriophages, with eukaryotic viruses and other viral particles making up a minor proportion.

The HL virome has distinctive characteristics that differentiate it from other viromes (e.g., viromes from adult feces, urine, saliva, and cerebrospinal fluid).34,35 In contrast, a significant number of shared viruses have been identified among the LH and the feces of infants from mother-baby pairs, supporting their vertical inheritance through lactation.34,36

Interestingly, it has been observed34 that the virome of infant feces is more similar to LH than to adult feces.

> Mycobiome and other ‑omas

Fungi are an important component of the human microbiome.37

However, its presence in the LH is a relatively recent discovery.20 Although taking into account geographical variability, the existence of a central mycobiome has been hypothesized, suggesting that its transmission through the LH is a characteristic preserved.

Other microorganisms, until recently ignored, contribute to MLH. In particular, current research has been focusing on archaea. The presence of archaeal DNA has been demonstrated in 8/10 HL samples analyzed, none belonging to women with mastitis, suggesting a protective function.17 On the contrary, other authors did not identify archaeal DNA in the HL samples analyzed. .38

The complex ecosystem of LH appears to take shape over time due to many factors: maternal, neonatal, environmental, and related to LH itself. The extremely dynamic nature of MLH composition may explain the often contradictory data reported in the literature. Furthermore, it should be noted that many factors involved in determining MLH are closely intertwined.

> Maternal determinants

Some authors 26,39–41 demonstrated that, compared with women who underwent cesarean section, LH samples from women with vaginal delivery showed greater bacterial diversity and richness, with higher levels of Bifidobacterium and Lactobacillus spp . However, other studies did not confirm such results.42,43 A potential influence of the mode of acquisition of the LH virome and mycobiome has also been hypothesized.44,45

A decrease in the abundance of Lactobacillus , Bifidobacterium , Staphylococcus and Eubacterium spp was reported . in HL samples from mothers who received perinatal antibiotics.8,46,47 Maternal chemotherapy during lactation has also been associated with a reduction in HL bacterial diversity.48

Maternal diet affects MLH composition (supposedly more during pregnancy than during lactation49–51).

Dietary regimens of foods rich in fiber and fat49 as well as the intake of vitamins (vitamin C and B complex vitamins)51 have been shown to alter the composition of MLH. Furthermore, both prepregnancy body mass index (BMI) and gestational weight gain are reflected in differential abundances of bacterial strains (mainly Streptococcus , Staphylococcus , and Bifidobacterium ) in the HL.40,52–54

Compared to healthy women, mothers with celiac disease have lower levels of Bacteroides spp . and Bifidobacterium spp . in their milk.55 Likewise, mastitis determines changes in the bacterial load and microbial diversity of the MLH, which disappear once the clinical symptoms disappear.56-58

Maternal postnatal psychosocial stress (defined as symptoms of anxiety, stress or depression during the postpartum period) has been related to a lower LH bacterial diversity at 3 months after delivery, with a progressive decrease in the relative abundance of staphylococci and a parallel increase of some minority genera ( Lactobacillus , Acinetobacter and Flavobacterium ) in mothers with low psychosocial stress.59

> Neonatal determinants

Lower counts of Enterococcus spp. and higher counts of Bifidobacterium spp . have been detected in LH samples from mothers who gave birth at term compared to preterm mothers.39 On the contrary, other authors42 did not detect any difference in microbial profiles based on the duration of gestation, postulating a mechanism to test of failures that allows the mother to be "ready" to transmit her bacterial fingerprint regardless of the gestational age at birth, as part of an evolutionary pressure directed towards the benefit of the baby. Variations in the composition of the HL virome and mycobiome have recently been demonstrated based on gestational age and birth weight.44,45

The effect of the gender of the newborn on the composition of MLH60 has been hypothesized based on the detection of more streptococci and less Staphylococcus in the HL of mothers of lactating boys compared to mothers of girls. However, such differences have not been confirmed by other studies.42,61

> Environmental determinants

Analysis of LH samples collected from selected populations in Europe, Africa, and Asia suggested that the composition of MLH is related to the geographic location of the study.62 Additionally, high variability in LH metabolites has been documented across study sites, and an association between variations in the LH metabolome and specific characteristics of MLH.63 However, a new analysis of LH samples from Ethiopia, Gambia, Ghana, Kenya, United States, Peru, Spain , and Sweden, demonstrated that, although LH bacterial communities varied geographically, they consistently contained the major genera Staphylococcus and Streptococcus.64 These results have been confirmed by a recent systematic review,65 which included twelve studies using independent culture methods. to identify bacteria at the genus level in the LH of healthy women.

In particular, it has been speculated that at least some of the geographic variability in MLH composition may be related to differences in the environment and in the procedure for collecting, storing, and analyzing MLH.66 As for collection methods, It has been observed61 that LH from mothers who use breast pumps has a higher microbial load and lower abundance of culturable staphylococci compared to manually collected LH samples. In contrast, other authors found no differences in ɑ diversity between samples collected by manual extraction or by pumping with a single-use sterile device.67

Analysis of MLH from women living in the same region but with different lifestyles (traditional vs. Western) revealed that HL samples from "rural women" had greater diversity and greater abundance of subdominant bacterial lineages than those from "rural women." urban women.”68

A study conducted in the Central African Republic within a small-scale society suggested that seasonality may influence the relative abundance of specific taxa in the MLH, although it may be difficult to determine whether variation in composition depends on differences in seasonal environmental exposure and/or seasonal variation in diet.69 The same study69 explored the relationship between the size of the mother-child social relationship network and the composition and diversity of the MLH, showing how the LH of mothers with larger networks large and babies with more caregivers, had greater microbial uniformity (but not microbial richness) than the LH of mothers whose babies had fewer caregivers.

Cabrera-Rubio et al. [26] were the first to describe the changes that MLH undergoes over time, from colostrum to transitional and mature milk. These authors reported progressively greater abundance of typical oral inhabitants (e.g., Veillonella , Leptotrichia , and Prevotella spp .) in the transitional and mature LH, and higher Bifidobacterium counts in later stages of lactation. Other authors39 subsequently reported a greater influence of the lactation stage on the counts of Bifidobacterium and Enterococcus spp ., which showed a progressive increase in their concentration from colostrum to mature LH, as did Lactobacillus and Staphylococcus spp .

Different patterns have been described over time. Analyzing LH samples collected at 3 time points over a 4-week interval, a set of 9 “basic operational taxonomic units” was identified.16 However, in some samples, the LH bacterial communities were quite consistent over time. time, while, in others, the relative abundance of bacterial genera changed over time.16

Some authors60 observed relative stability of MLH over time, with only small changes in some minority genera, while others43 observed no effect of lactation stage on MLH composition. Regarding the virome, it was recently documented44 that, although bacteriophages are predominant in transitional and mature LH samples, transitional LH has a higher abundance of Podoviridae and Myoviridae , while in mature LH Podoviridae decreases, and Siphoviridae become in the most abundant family.

For the mycobiome, a recent study45 analyzed LH samples from different stages of lactation and found that, in transitional LH samples, Saccharomyces cerevisiae and Aspergillus glaucus were the most abundant species, while Penicillium rubens and Aspergillus glaucus predominated in samples of Mature LH.

It has been speculated that other components of LH, such as LH oligosaccharides (OLH, prebiotics), milk fatty acids, hormones, immune cells and antibodies, could modulate the composition of MLH.70,71 In particular, OLH may promote the growth of Staphylococcus spp . in the lactating mammary gland.72

When breast milk is not available or insufficient, donation of LH (DLH) is the second best alternative.73-75 However, pasteurization , necessary to guarantee microbiological safety standards, inevitably inactivates several of the nutritional properties and biological functions of the LH,76 including the MLH. In fact, pasteurization eliminates most bacteria in milk (except spore-forming Bacillus species).77–79

However, the viability of the MLH is no longer considered essential. In fact, it has been hypothesized that the probiotic effect of beneficial microbes in LH depends on the ability of host cells to recognize specific bacterial components or products, thereby activating the immune system. These “non-viable (most often heat-inactivated)” microbial cells (intact or disrupted) or crude cell extracts (i.e. nucleic acids, cell wall components) are known as para-probiotics or ghost probiotics [80]. .

MLH seeds the infant gastrointestinal tract with pioneer bacteria, thus contributing to the establishment of both the infant oral and intestinal microbiota.81,82 However, not all of the bacteria present in HL are found in the infant gut, but rather, only a select few appear to colonize the newborn.42 However, it has been hypothesized that transient exposure could be as effective as persistent colonization.83,84 Additionally, bacteria in LH may upregulate protective factors such as antibodies, immune cells, lactoferrin and beta-defensins that would then be transmitted to the newborn through breastfeeding.42 The LH virome, especially bacteriophages, probably also contributes to the intestinal ecology of the baby.18

Early microbial exposure is essential to provide antigenic stimuli that promote maturation of the intestinal immune system by promoting a shift from the predominant immune milieu of intrauterine T helper (TH) 2 cells to a balanced TH1/TH2 response and triggering the differentiation of regulatory T cells. 85

Through modifications of the childhood intestinal microbiota and through the gut-brain axis, MLH can also influence the development of a more desirable behavioral phenotype in the offspring, as proposed for other LH bioactives.86 In fact, In early childhood, LH can promote the colonization of a specific microbiota that influences the regulation of offspring biobehavior. A milk-oriented infant gut microbiota may produce a less energetically costly behavioral phenotype to more optimally allocate maternal energy investment.86

An association between breastfeeding and upper respiratory microbiota composition was reported at 6 weeks, with breastfed infants showing a significantly different microbial composition than formula-fed ones.87 Interestingly, such an association appears to disappear at 6 weeks. months of age (when weaning normally begins).87,88

Finally, it has been hypothesized that MLH may also benefit the mother, protecting her against infections such as mastitis.42

Breastfeeding represents a valuable route of transmission of maternal microbes in both humans and other animals (e.g., rhesus monkeys, cows, sheep, goats).89–92 Since MLH transmission appears to be a conserved characteristic between different species , a possible evolutionary purpose can be hypothesized.

Maternal microbial transmission provides offspring with important microbes early in life, rather than leaving their acquisition to chance during later stages of development. By shaping the offspring’s own microbiome, such microbes may determine evolutionary advantages in the recipient.11,93,94 Consequently, within a broader evolutionary context, MLH transmission could be seen as at least partially capable of shaping to the microbiome of the entire species over evolutionary time, as microbes that promote host fitness will increase their chances of reaching the next generation.

Despite the progress made in recent decades, there are still many questions to answer. However, the lack of internationally recognized “best practices” in MLH analysis (e.g., LH collection, storage and processing, DNA extraction and sequencing) often limits comparison between studies. Therefore, standardized and rigorous study designs are needed to promote accuracy and reproducibility of results.

Many of the topics addressed in the present review represent interesting fields to explore. First, the sources and seeding pathways of MLH need to be further examined, possibly through experimental studies in animal models. Furthermore, interactions between mother, baby and environment should be better investigated, thus uncovering hidden mechanisms of co-regulation between different microbiomes. Furthermore, all members of the LH microbial community must be equally considered. Until now, bacteria have been the most studied microorganisms.

Progressively, attention has shifted to viruses (although with a strong bias towards DNA viruses), fungi and yeasts. The next frontier will be to explore the archaeome and deepen understanding of the potential child health implications of “minor” components of the MLH. Finally, the functional significance of MLH and its impact on the gastrointestinal tract microbiome, the immune system, and later infant health would benefit from appropriate experimental, possibly longitudinal, studies.

The practical and translational implications of MLH research should also be considered. For example, studies on the reconstitution of DLH through the inoculation of defined quantities of the infant’s own breast milk should be encouraged with the aim of restoring live MLH, as described by Cacho et al.95 Likewise, the possible role of maternal dietary supplementation with pre- or postbiotics aimed at modulating MLH, as well as the most appropriate time for such supplementation (e.g., during pregnancy and/or during lactation).

Although traditionally considered sterile, it is now clear that the LH harbors a wide variety of microorganisms, ranging from bacteria to viruses, fungi and yeasts, and minor genera. The transmission of such microorganisms to the baby may help determine its present and future health, primarily shaping the neonatal GI tract microbiome and immune system. The complexities of the LH ecosystem warrant further research to deepen knowledge about the origin, determinants, and implications for infant health.

Traditionally, human milk was thought to be sterile. However, with the passage of time and the development of new technology, the characterization of the composition, diversity and variability of the LH microflora in greater detail has been achieved. Currently, HL is considered a living universe composed of a complex microbial community of bacteria, viruses, fungi and yeasts that constitute the microbiome and cooperate for the present and future health of the child. The present review provides an overview on the origin, composition, determinants and role of the human milk microbiome, and suggests possible directions for future research in this field.

Table 1 Summary of the main hypothetical sources of the MLH

Table 2. Composition of the human milk microbiome.