Human Milk Microbiota: Origin and Potential Uses

Microbiology of Milk and Lactation: Influence on Gut Colonization

Published online: March 13, 2020 Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031)

a b Leónides Fernández Juan M. Rodríguez a Department of Galenic Pharmacy and Food Technology, Complutense University of Madrid, Madrid, b Spain; Department of Nutrition and Food Science, Complutense University of Madrid, Madrid, Spain

Abstract At the beginning of the 21st century, some pioneer studies provided evidence of the existence of a site-specific human milk microbiota. Hygienically collected milk samples from healthy women contain a relatively low bacterial load, which consist mostly of Staphylococ- cus, Streptococcus, lactic acid bacteria, and other gram-positive bacteria (Corynebacterium, Propionibacterium, and Bifidobacterium). DNA from strict anaerobic bacteria is also detected in human milk samples. The origin of human milk bacteria still remains largely unknown. Although the infant’s oral cavity and maternal skin may provide microbes to milk, selected bacteria of the maternal digestive microbiota may access the mammary glands through oral- and enteromammary pathways involving interactions with immune cells. In addition, when milk is collected using external devices, such as breast pumps, some microorganisms may arise from unhygienic handling as well as from the water used to clean and rinse the devices, for example. The human milk microbiota has a wide spectrum of potential uses. Most of them have been focused on the infant (including the preterm ones), but some bacterial strains present in human milk have also a big potential to be used to improve the mother’s health, mainly through the prevention or treatment of infectious mastitis during lactation. © 2020 Nestlé Nutrition Institute, Switzerland/S. Karger AG, Basel Human Milk Is Not Sterile

Until recently, human milk and mammary glands were thought to be sterile under physiological conditions. Therefore, the presence of microbes in such locations was traditionally considered either as an infection or as a contamination. This negative view started to change in 2003 following the publication of 2 ar- ticles describing that human milk may be a source of lactic acid bacteria [1, 2]. Such bacteria were not only generally recognized as safe, they were also considered as beneficial and with key roles in infant gut colonization. This was a relevant finding in the context of increasing awareness of the importance ascribed to human microbiota for our health throughout life. The first article proposing the existence of a site-specific microbiome in human milk was published 1 year later [3]. Nowadays, it is generally accepted that human milk contains its own microbiota, with an increasing recognition of its role in early gut colonization in infants. In the last 15 years, the number of studies on the composition of the human milk microbiota has sharply increased [4, 5]. Cultivable bacteria in hygienically collected human milk samples are usually dominated by Staphylococcus (mainly S. epidermidis and other coagulase-negative species), Streptococcus (mainly S. mitis spp. and S. salivarius spp.), Corynebacteri- um, Propionibacterium, and other taxonomically related gram-positive bacteria. Lactic acid bacteria and bifidobacteria may also be isolated from human milk but to a lesser extent [4, 6]. Among them, species belonging to the genus Lactobacillus (e.g., L. salivarius, L. gasseri, L. fermentum, and L. reuteri) and Bifidobacterium (e.g., B. longum and B. breve) have attracted particular interest from a scientific, medical, and industrial point of view due to their potential application as probiotics. The bacterial concentration in human milk from healthy women usually 3 ranges from an undetectable level to ∼10 CFU/mL when the samples are collected hygienically by manual expression or by using single-use sterile pump devices. The concentration can be much higher (up to 106 CFU/mL) in mastitis- suffering women [7]. When the milk is collected by pumping, high concentrations of contaminating gram-negative bacteria (e.g., enterobacteria, Pseudomo- nas, and Stenotrophomonas) and yeasts may arise from the rinsing water and/or poor hygienic manipulation practices [8]. Traditional culture-dependent methods have the limitation of not being able to assess the presence of viable but noncultivable microbes and, particularly, that of the strict anaerobes, the DNA of which has also been detected in the human milk microbiome. Recent developments in culturomics have shown that if prop- er culture conditions are provided, most previously unculturable bacteria can be isolated from complex microbial ecosystems. Unfortunately, culturomic approaches have not been applied yet to milk and mammary microbiota.

76 Fernández/Rodríguez

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) The application of the first available culture-independent molecular techniques, including different PCR approaches, provided a complementary assessment of the milk microbiome [4]. Soon they were replaced by high-throughput next-generation sequencing approaches, from metataxonomics (16S rRNA am- plicon analysis) to metagenomics (total DNA sequencing). Sequencing of bacterial 16S rRNA genes does neither provide evidence for viability nor for function- ality of detected bacteria, but it is a first step in understanding the complexity of the milk microbial ecosystem and its role in infant gut colonization [5, 9, 10]. Metagenome studies involving shotgun sequencing of milk microbial DNA have been much scarcer [11, 12] and have provided data on the potential presence and roles of other components of the milk microbiome, including viruses, archaea, fungi, and protozoa. The microbiome of human milk seems to be a dynamic and complex ecosystem which is not randomly assembled but forms well-organized bacterial con- sortia and networks, which may be different among different populations [13]. At the genus level, culture-independent studies have confirmed the presence of DNA from bacterial genera previously identified with culture-dependent techniques, such as Staphylococcus, Streptococcus, Corynebacterium, Propionibacte- rium, Lactococcus, Leuconostoc, Bifidobacterium, and Lactobacillus in human milk. In addition, DNA from strict anaerobes, which are typically associated with the gut environment (e.g., Bacteroides, Eubacterium, Faecalibacterium, Roseburia, Ruminococcus, and Veillonella), has also been detected [5, 12]. Such bacteria are often noncultivable, and, in fact, they have not been isolated from human milk yet. Sequences from a third group of bacteria, typically associated with soil and water (Acinetobacter, Methylobacterium, Pseudomonas, Sphingobium, Sphin- gomonas, Stenotrophomonas, or Xanthomonas), are also frequently detected in human milk. However, they may be the result of technical artifacts derived from the presence of DNA from such genera in molecular-biological reagents. In fact, working with samples containing a low microbial biomass, such as human milk under physiological conditions, is a big challenge. Future studies will require suitable controls to determine which data are actually genuine in a given biological sample.

Where Does Human Milk Microbiota Come from?

After birth, the bacterial colonization process represents the first massive contact with microbes; the close link between early gut microbiota composition and the risk of disease later in life underlines the important role of the microbiota-

Origin and Uses of the Human Milk Microbiota 77

Other sources Water, pumps, shields, partner, Oral other breastfed brother/sister, microbiota clothes, environment…

Translocation?

Breast skin, areola, nipple and Montgomery gland microbiota Milk Infant gut

Translocation?

Infant sources Mouth Nasopharynx Skin Intestinal microbiota

Maternal vaginal, Other sources: oral, intestinal and family, medical staff, skin microbiota water, foods, environment…

Fig. 1. Potential sources of the human milk microbiota. host interactions in early life. Colostrum and milk bacteria are, obviously, among the first colonizers of the infant gut and, therefore, may play a key role in driving the development of its microbiota. Several studies have reported a mother-to- infant transfer of human milk microorganisms (at the species and/or the strain level) using both culture-dependent and -independent techniques [4, 6, 14–16]. Although it has become evident that human milk is a source of infant gut bacteria, the origin of the bacteria present in human milk still remains largely unknown and is the subject of scientific controversy. Traditionally, any bacterial cell present in human milk was considered the result of contamination aris- ing from the infant’s oral cavity or the mother’s skin. However, the detection of live bacterial cells and/or DNA from anaerobic species that are generally related to the gut environment and that do not survive in aerobic locations fueled a scientific debate on the origin of milk-associated bacteria (Fig. 1). Some bacteria from the infant’s oral cavity may contaminate milk during suckling due to milk flowing back into the mammary ducts; however, the presence of oral-related bacteria in milk may precede the first feeding since colos-

78 Fernández/Rodríguez

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) trum collected within 24 h after birth has been found to contain typical oral bacteria like Veillonella, Leptotrichia, and Prevotella [10]. In addition, contamination from the breastfed baby’s oral cavity does not explain why precolostrum secreted by some women before delivery already contains some of the micro organisms that characterizes human milk [3]. A recent study has revealed the presence of typical oral bacteria (such as streptococci) in precolostrum collected during the first pregnancy and before contact with the newborn, a fact that in- dicates that they are not a contamination from the infant mouth [17]. The fact that some of these strains were shared by the mother’s milk and the infant mouth suggests that at least some oral bacteria reach the infant’s mouth through breastfeeding. Microbes inhabiting maternal skin (particularly the external surfaces closer to the ejected milk: nipples, mammary areolas, and Montgomery glands) may be transferred during ejection. Some bacteria commonly isolated in human milk, such as Propionibacterium acnes, Corynebacterium spp. or, particularly, S. epidermidis, are also common in some regions of the human skin. However, although staphylococci, corynebacteria, and propionibacteria have been traditionally associated with the skin, they are widespread in most, if not all, human mucosal surfaces; in fact, the populations of such bacterial groups reach their highest concentrations in the mucosal layers of the digestive and genitourinary tracts. A recent study comparing the bacterial composition of human milk, areolar skin, and infant stool samples of healthy mother-infant pairs found that the bacterial communities were distinct in these 3 ecosystems, differing in both composition and diversity [16]. Despite sharing some phylotypes, the comparison between the bacterial communities detected in milk and those found on breast skin or in the infant’s mouth reveals major differences between them. Both the skin and the infant’s mouth are highly improbable sources of strict anaerobic bacteria typically associated with the adult gut microbiome. Sharing of Bifidobacterium, Bacteroides, Para- bacteroides, and members of the clostridial class (Blautia, Clostridium, and Col- linsella) between maternal feces, human milk, and neonatal feces has already been reported [5, 18]. The assessment of bifidobacterial communities in several mother-infant pairs through bifidobacterial culturing and profiling analyses using an internal transcribed spacer revealed a large number of bifidobacterial strains that were commonly identified in maternal and infant fecal samples, as well as in the corresponding human milk sample [15]. All these studies reinforce the hypothesis that at least some bacteria, including obligate anaerobes, may be vertically transferred from mother to neonate via breastfeeding. Selected bacteria of the maternal digestive microbiota may access the mammary glands through oral and enteromammary pathways [3, 19, 20]. It has pre-

Origin and Uses of the Human Milk Microbiota 79

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) viously been observed that certain bacteria from the maternal digestive tract may spread to extradigestive locations in healthy hosts. In addition, some studies have offered a scientific plausible basis for such physiological translocation. The mechanism would involve mononuclear cells (dendritic cells and macrophages), which would be able to take up nonpathogenic bacteria from the gut lumen and, subsequently, carry them to other locations, including the lactating mammary gland [reviewed in 19, 20]. In vitro and in vivo data reinforcing this hypothesis have been obtained by different groups. It must be highlighted that there is an intense efflux of intestinal immune cells to the mammary gland during late pregnancy and lactation and that, in fact, the existence of an enteromammary circu- lation of IgA-producing cells is long known. An increased bacterial translocation from the gut to the mesenteric lymph nodes and the mammary gland in pregnant and lactating mice was observed by Perez et al. [18]. Bacteria could be observed histologically in the subepithelial dome and interfollicular regions of Peyer’s patches, and in the lamina propria of the small bowel, and associated with cells in the glandular tissue of the mammary gland. The Peyer’s patches of pregnant and lactating mice were macro- scopically larger than those of control animals and had a more prominent subepithelial dome and more dilated draining lymphatic vessels containing mononuclear cells. Similarly, oral administration of the strains Lactobacillus rhamnosus GG and L. gasseri K7 to mice during pregnancy and lactation led to changes in the mesenteric lymph nodes and mammary gland microbiota [21]. Live lactic acid bacteria were detected in blood, mesenteric lymph nodes, and mammary gland, while the Lactobacillus genus was detected exclusively in the mammary gland of the mice that ingested the strains. More recently, de Andrés et al. [22] also tried to elucidate if some lactic acid bacteria were able to translocate and colonize the mammary gland and milk in a murine model. For this purpose, L. lactis MG1614 and L. salivarius PS2 were transformed with a plas- mid containing the lux genes; subsequently, the transformed strains were orally administered to pregnant mice. The murine model allowed the visualization, isolation, and PCR detection of the transformed bacteria in different body locations, including mammary tissue and milk, reinforcing the hypothesis that physiological translocation of maternal bacteria during pregnancy and lactation may contribute to the composition of the mammary and milk microbiota. While some may argue that their presence in milk might be the result of superficial fecal contamination in mice, such a route can hardly explain their isolation and detection from mammary biopsies. In relation to human species, a study cited above [18] showed that human milk contains viable bacteria, including Streptococcus, Lactobacillus, and Bifido- bacterium, while acridine orange staining of milk and blood cytopreparations

80 Fernández/Rodríguez

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) identified bacterial cells in association with maternal mononuclear cells. These results strongly suggest the involvement of mononuclear cells in the transport of intestinal bacteria to the mammary gland in late pregnancy. Other studies have reported that oral administration of L. reuteri, L. gasseri, L. fermentum, and L. salivarius strains isolated from human milk to pregnant or lactating women led to their presence in milk [reviewed in 23]. Gut bacterial translocation has usually been associated with pathogenic conditions, but a low rate of bacterial translocation (involving Bacteroides, lactobacilli, bifidobacteria, or enterococci) occurs in healthy hosts and may be associated with physiological immunomodulation of the infant [18]. Some bacterial strains seem to specifically mediate their own translocation without collateral translocation of other bacteria from the host digestive tract [24]. Many transient anatomical and physiological changes that occur during pregnancy and lactation may favor an increased bacterial translocation during such periods [reviewed in 19, 20]. Further studies are required to elucidate the mechanisms by which some bacterial strains may translocate physiologically in certain hosts or life stages. The existence of such bacterial oral- and enteromammary pathways would provide new opportunities for manipulating altered maternal-fetal microbiota, reducing the risk of preterm birth or infant diseases.

Potential Uses of the Human Milk Microbiota: The Mother’s Side

Up to the present, most of the research on potential applications of the human milk microbiota has been focused on infant health, including that of the preterm neonate. However, human milk bacteria may also be relevant for breast health. The lactating mammary gland ecosystem is hospitable to many microorganisms, including bacterial groups that have the potential to cause mastitis. Upon dis- turbance of this balanced state, infection can occur, and, in fact, some studies have reported that mastitis is a process characterized by a mammary bacterial dysbiosis, including a lower microbial diversity, increased abundance of opportunistic pathogens, and depletion of commensal obligate anaerobes (Fig. 2) [7, 25]. The etiopathogenesis of the different types of lactational mastitis (acute mastitis, subacute mastitis, and granulomatous lobular mastitis), as well as the factors that may predispose to or protect from mastitis, has recently been reviewed [23]. Since the resistance to antibiotics and the ability to drive the formation of biofilms are common properties among mastitis-causing bacteria, many cases are refractory to antibiotic therapy. In this context, the development of new

Origin and Uses of the Human Milk Microbiota 81

Host factors Infant factors Microbial factors Environmental factors Medical factors Other factors

Predisposing factors Protecting factors

Milk microbiota Low diversity High divesity High concentration Dysbiosis Low concentration

Healthy mammary gland Mastitis Correct treatment

Predisposing factors

Fig. 2. The human milk microbiota: from physiology to dysbiosis and mastitis. strategies for mastitis management based on human milk probiotics, as an alternative or complement to antibiotic therapy, is particularly appealing. To date, oral administration of a few Lactobacillus strains isolated from human milk has proven to be an excellent approach for the treatment and prevention of lactational mastitis [7]. The results of the first placebo-controlled clinical trial aimed to test the potential of the combination of L. salivarius CECT 5713 and L. gasseri CECT 5714 for the treatment of staphylococcal mastitis revealed that they were an efficient alternative for the treatment of lactational mastitis [23], leading to the disap- pearance of clinical symptoms and to significant reductions in milk staphylococcal concentrations. Subsequently, the efficacy of L. fermentum CECT 5716 or L. salivarius CECT 5713 for the same target was evaluated and compared to antibiotic therapy in a clinical trial involving 352 women with infectious mastitis [26]. The probiotic treatment led to significant reductions (1.7–2.1 log10 CFU/mL) in the staphylococcal and/or streptococcal counts in milk and to a rapid improvement in the condition. On the basis of the bacterial counts, pain

82 Fernández/Rodríguez

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) scores and clinical evolution, women in any of the two probiotic groups had a significantly better outcome than those in the antibiotic group. The potential of oral administration of L. salivarius PS2 during pregnancy to prevent mastitis in women who had suffered infectious mastitis after, at least, one previous pregnancy was the subject of a subsequent trial [27]. For this purpose, 108 pregnant women were randomly divided in two groups: (a) a probiotic group, who ingested daily 9 log10 CFU of L. salivarius PS2 from 30 weeks of pregnancy until delivery, and (b) a control group, who received a placebo. The occurrence of mastitis was evaluated during the first 3 months after delivery. At the end of the study, the percentage of women suffering mastitis in the probiotic group (25%) was significantly lower than that in the control group (56%). Therefore, oral administration of such Lactobacillus strain during late pregnancy was an efficient method to prevent mastitis in a susceptible population. A parallel trial involving lactating women with mastitis was carried out in order to identify microbiological, biochemical, and/or immunological biomarkers of the probiotic effect of L. salivarius PS2 [28]. Samples of milk, blood, and urine were collected before and after the probiotic intervention and screened for a wide spectrum of microbiological, biochemical, and immunological parameters. In the mastitis group, L. salivarius PS2 intake led to a reduction in milk bacterial counts, milk and blood leukocyte counts, and IL-8 level in milk, increased levels of IgE, IgG3, EGF, and IL-7, a modification in the milk electrolyte profile, and a reduction in some oxidative stress biomarkers. In the same cohort, the characterization of the urine metabolic profiles at the beginning of the probiotic intervention showed increased energy metabolism (lactate, citrate, formate, acetate, and malonate) and decreased branched- chain amino acid catabolism (isocaproate and isovalerate) when compared to those obtained after the intervention [29]. In addition, probiotic supplementa- tion led to a normalization of breast permeability. Changes in the levels of acetate and 2-phenylpropionate after probiotic intake suggested immunomodulation while an increased malonate level indicated an important antagonistic strategy of L. salivarius PS2 since this catabolite is a well-known repressor of the tricarboxylic acid cycle, which may alter staphylococcal and streptococcal metabolism and negatively affect their survival, virulence, and ability for bio- film formation. Transcriptomic profiling of human milk somatic cells and blood leukocytes was also applied to this cohort to explore potential targets responsive to probiotic intervention [30]. Despite the interindividual variability in the gene expression changes in both types of cells, their results showed the involvement of inflammatory and cell growth-related pathways and genes in the human milk somatic cells following the intake of L. salivarius PS2. Indi- vidual analyses of selected genes supported the upregulation of STC1 and IL19

Origin and Uses of the Human Milk Microbiota 83

Ogra PL, Walker WA, Lönnerdal B (eds): Milk, Mucosal Immunity and the Microbiome: Impact on the Neonate. Nestlé Nutr Inst Workshop Ser. Basel, Karger, 2020, vol 94, pp 75–85 (DOI:10.1159/000505031) and the downregulation of PLAUR and IFNGR1 in somatic cells of the patients as potential targets responsive to the probiotic. Other potential mechanisms by which some Lactobacillus strains may be able to control mastitis-causing agents in the breast after oral administration, including immunomodulation and local competitive exclusion and production of an- timicrobials, have been reviewed by Fernández et al. [27]. Mastitis represents the first medical cause of undesired weaning. Therefore, this condition should be considered as a major public health issue since breastfeeding is associated with a reduced risk of many diseases in infants and mothers.

Disclosure Statement and Funding Sources

The authors are co-inventors in patents WO 2004/003235 A2 and PCT/NL2013/050924, involving the application of Lactobacillus strains for the prevention or treatment of mastitis. Work of the research group dealing with the potential origin of the human milk microbiota has been funded by grant AGL2016-75476-R (Spanish Ministry of Economy and Competitiveness).

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