Beneficial Microbes

Wageningen Academic P u b l i s h e r s

Beneficial Microbes, March 2013; 4(1): 17-30

Human milk: a source of more life than we imagine P.V. Jeurink1,2, J. van Bergenhenegouwen1,2, E. Jiménez3, L.M.J. Knippels1,2, L. Fernández3, J. Garssen1,2, J. Knol1,4, J.M. Rodríguez3 and R. Martín1 1Danone Research, Centre for Specialised Nutrition, P.O. Box 7005, 6700 CA Wageningen, the Netherlands; 2Utrecht Institute

for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, the Netherlands; 3Dpto Nutrición, Bromatología y Tecnología de los Alimentos, UCM, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain; 4Laboratory of Microbiology, Wageningen University, P.O. Box 8033, 6700 EJ Wageningen, the Netherlands; [email protected] Received: 10 July 2012 / Accepted: 8 November 2012 © 2013 Wageningen Academic Publishers

Abstract The presence of bacteria in human milk has been acknowledged since the seventies. For a long time, microbiological analysis of human milk was only performed in case of infections and therefore the presence of non-pathogenic bacteria was yet unknown. During the last decades, the use of more sophisticated culture-dependent and -independent techniques, and the steady development of the -omic approaches are opening up the new concept of the ‘milk microbiome’, a complex ecosystem with a greater diversity than previously anticipated. In this review, possible mechanisms by which bacteria can reach the mammary gland (contamination versus active migration) are discussed. In addition, the potential roles of human milk for both infant and maternal health are summarised. A better understanding of the link between the milk microbiome and health benefit, the potential factors influencing this relationship and whether or not it can be influenced by nutrition is required to open new avenues in the field of pregnancy and lactation. Keywords: human milk, human milk microbiome, contamination, active migration, health implications

1. The human milk microbiome Human milk is considered the best nutrition for new-born infants because it contains optimal ingredients for healthy growth and development. Breastfeeding confers protection against gastrointestinal infections (Duijts et al., 2010; Ip et al., 2007, 2009; Quigley et al., 2007), respiratory infections (Chantry et al., 2006; Duijts et al., 2010; Ip et al., 2007, 2009; Nishimura et al., 2009), allergic diseases (Greer et al., 2008; Ip et al., 2007) and it is also associated with a reduced long term risk of diseases such as inflammatory bowel disease (IBD), obesity or diabetes as reviewed by the American Academy of Pediatrics (AAP, 2012). The protective role of human milk seems to be the consequence of a synergistic action of the wide range of health-promoting components such as carbohydrates, nucleotides, fatty acids, immunoglobulins, cytokines, immune cells, lysozyme, lactoferrin and, other immuno

modulatory factors (Boehm and Moro, 2008; Penttila, 2010; Van ‘t Land, 2010; Walker, 2010). Recently, the presence of immunomodulatory factors in human milk like exosomes and microRNAs have been found (Kosaka et al., 2010; Zhou et al., 2012). However, to date, not much is known about their function or mechanism of action regarding their role in the development of the infant’s immune system. Most recently, extensive research has been conducted to understand the beneficial role of human milk oligosaccharides (HMOs) in infant health (reviewed by Bode, 2012; Jeurink et al., 2012; Rijnierse et al., 2011; Scholtens et al., 2012). In addition, several studies have now demonstrated that human milk contains bacteria (Beasley and Saris, 2004; Collado et al., 2009; Gueimonde et al., 2007; Heikkila and Saris, 2003; Hunt et al., 2011; Martín et al., 2003, 2009; Perez et al., 2007). It has been shown that human milk from healthy women contains approximately 103-104 cfu/ml, representing a continuous

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2012.004017

P.V. Jeurink et al.

source of potential commensal bacteria for the infant gut (Martín et al., 2003; Perez et al., 2007). During the last decades, microbiological studies that focused on human milk were restricted to the identification of potential pathogenic bacteria in stored milk or milk retrieved from cases concerning maternal or infant infections (Bingen et al., 1992; El-Mohandes et al., 1993; Le Thomas et al., 2001; Wright and Feeney, 1998). The first descriptions of the bacterial diversity of human milk from healthy women in 2003 were based on in vitro culturing methods (Heikkila and Saris, 2003; Martín et al., 2003). Since then, several groups have studied the bacterial community of human milk using both culture-dependent and cultureindependent techniques (Table 1). Although it is questioned whether it is possible to aseptically collect human milk, culture-dependent methods have confirmed the presence of bacteria in assumedly aseptically collected milk. Cultured genera include Staphylococcus, Streptococcus, Lactococcus, Leuconostoc, Weissella, Enterococcus, Propionibacterium, Lactobacillus and Bifidobacterium. The most commonly isolated bacterial species from human milk include Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus salivarius, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus rhamnosus, Bifidobacterium breve and Bifidobacterium bifidum. Recently, a new bacterial species, Streptococcus lactarius, has been isolated from human milk (Martin et al., 2011). While more than 200 different bacterial species have been isolated from human milk up to now, the number of cultivable bacterial species that can be found within one individual is much lower, ranging from 2 to 18 different species (Martin, 2011). Culture-independent techniques, based on the amplification of the gene coding for bacterial 16S ribosomal RNA (rRNA), have allowed a more comprehensive assessment of the bacterial diversity in human milk (Gueimonde et al., 2007; Martín et al., 2007a,b, 2009). These studies have confirmed the presence of the bacterial groups identified with culture-dependent techniques, but also revealed the presence of other bacterial groups, including some Gram-negative bacteria (Hunt et al., 2011; Martín et al., 2007b). The application of -omics approaches (genomics, metagenomics, transcriptomics, proteomics, metabolomics) to the study of the human mammary gland microbiota is already in progress and there is no doubt that the results will provide a better understanding of the composition of the milk microbiome (Table 1). Recently, microbial identification techniques based on 454 pyrosequencing of the 16S rRNA gene have been used to analyse the bacterial community in human milk in more depth, both in terms of diversity and stability (Hunt et al., 2011). In the study of Hunt and co-workers, three samples each from sixteen healthy women were analysed and confirmed that Streptococcus and Staphylococcus are 18

the major genera in human milk representing, together with Serratia, more than 5% of the retrieved 16S rRNA gene sequences. Eight other genera represented ≥1% of the communities observed across the samples. It suggests that human milk contains a greater bacterial diversity than previously assumed and it indicates that a ‘core’ microbiome, as described for other bacterial communities in the human body, may also exist in human milk (Ravel et al., 2011; Turnbaugh et al., 2009). Hunt and co-workers have shown that nine genera (Streptococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacteria, Ralstonia, Propionibacterium, Sphingomonas and Bradyrhizobiaceae) were present in all three samples of all 16 women (Hunt et al., 2011). These nine ‘core’ Operational Taxonomic Units (OTUs) represented about half of the microbial community observed, although their relative abundance varied greatly between subjects. The remaining half of the community was not shared by the women participating in the study. These findings are in contrast with those of the gut, where only a low set of OTUs is shared among individuals (Turnbaugh et al., 2009) or the vaginal microbiome, which comprises several core groups (Ravel et al., 2011). Interestingly, the microbial community in human milk was stable over time within an individual, which is in line with another study that showed that the microbial communities of various sites of the human microbiome in a particular individual are highly personalised and often stable over time (Costello et al., 2009). Recently, the metagenome of five human milk samples was analysed by 454 pyrosequencing using a shotgun strategy (Jimenez et al., 2012). Two of the samples were obtained from healthy women (WH1 and WH2), one from an obese women with a body mass index above thirty (WO1) and two from women with lactational mastitis. Of these samples from mothers with lactational mastitis, one was suffering from acute mastitis caused by S. aureus (WM1), whereas the other was diagnosed with sub-acute mastitis caused by S. epidermidis (WM2). In all five samples, the most predominant phyla were Proteobacteria (58.6%), Firmicutes (12.4%), Bacteroidetes (6.7%) and Actinobacteria (1.8%). Alphaproteobacteria predominated in three of the samples (39-67%), whereas in WO1 and WM1 the Clostridia and Bacilli predominated, respectively. The most prevalent genera found in all samples except WM1 were Pseudomonas, Sphingomonas, Novosphingobium, Sphingopyxis and Sphingobium. At a lower taxonomic level, the most prevalent species in the milk samples analysed was Pseudomonas aeruginosa except in WM1, where S. aureus was the predominant species (75% of the sequences). In contrast to other studies, Hunt and co-workers neither found Lactobacillus nor Bifidobacterium as common members of the human milk microbiota (Hunt et al., 2011). These differences may be attributable to socio-economic, cultural, genetic, differences in antibiotic used or dietary Beneficial Microbes 4(1)

Beneficial Microbes 4(1)

S. mitis, S. salivarius, S. oris, S. parasanguis, S. australis, S. gallolyticus, S. vestibularis, S. lactarius

L. gasseri, L. fermentum, L. crispatus, L. rhamnosus, L. salivarius, L. reuteri, L. plantarum, L. gastricus, L. vaginalis, L. casei, L. animalis, L. brevis, L. helveticus, L. oris

L. lactis

E. faecium, E. faecalis, E. durans, E. hirae, E. mundtii

Leuc. mesenteroides

P. pentosaceous

Streptococcus

Lactobacillus

Lactococcus

Enterorococcus

Leuconostoc

Pediococcus

Clostridia1

Weissella

S. epidermidis, S.aureus, S. capitis, S. hominis

Staphylococcus

Firmicutes

K. rhizophila

Bacteroides

Kocuria

Bacteroidetes

R. mucilaginosa

L. gasseri, L. fermentum, L. rhamnosus

spp.

spp.

spp.

S. mitis, S. salivarius, S. parasanguis

spp.

W. confusa, W. cibaria

Leuc. citreum; Leuc. fallax

E. faecium, E. faecalis

L. lactis

S. epidermidis, S. hominis

S. aureus

spp.

spp.

spp.

Rothia

spp. P. acnes

B. longum

454 sequencing

Propionibacterium

P. denticolens

Parascovia

B. breve, B. bifidum, B. longum, B. adolescentis, B. dentium, B. animalis, B. catenulatum

Species

Fingerprinting (DGGE), cloning

Corynebacterium

B. breve, B. bifidum, B. longum, B. adolescentis; B. pseudocatenulatum

Species

Species

Bifidobacterium

qPCR

Isolation

Actinobacteria

16S rRNA gene-based methods

Culture-based methods

Genera

Phylum

Table 1. Bacteria detected in human milk by culture-dependent and -independent techniques.

S. aureus

Species

Clostridia

Bacilli

Bacteroidetes

Actinobacteria

Class

Shot-gun sequencing

Metagenomics

Human milk: a source of more life than we imagine

19

20 spp. spp. spp.

Serratia

Ralstonia

Sphingomonas

20-22

23

24

2

Clostridium cluster XIVa-XIVb and cluster IV. References numbers are: 1. Gavin and Ostovar (1977); 2. West et al. (1979); 3. Martin et al. (2003); 4. Heikkila and Saris (2003); 5. Martin et al. (2006); 6. Martin et al. (2009); 7. Sinkiewicz and Nordstrom (2005); 8. Martin et al. (2011); 9. Martin et al. (2012); 10. Makino et al. (2011); 11. Albesharat et al. (2011); 12. Jimenez et al. (2008a); 13. Jimenez et al. (2008b); 14. Delgado et al. (2009); 15. Solis et al. (2010); 16. Alp and Aslim (2010); 17. Gueimonde et al. (2007); 18. Collado et al. (2009); 19. Collado et al. (2012); 20. Martin et al. (2007a); 21. Martin et al. (2007b); 22. Perez et al. (2007); 23. Hunt et al. (2011); 24. Jimenez et al. (2012).

1

6, 17-19

spp.

Sphingobium A. muciniphila

spp.

References2

spp.

spp.

Sphingopyxis

Verrucromicrobia Akkermansia

Class

P. aureoginosa α-Proteobacteria

Species

Shot-gun sequencing

Metagenomics

Novosphingobium

1-16

spp.

Species

Pseudomonas

454 sequencing

spp.

Species

Species

Fingerprinting (DGGE), cloning

Bradyrhizobiaceae

qPCR

Isolation

Proteobacteria

16S rRNA gene-based methods

Culture-based methods

Genera

Phylum

Table 1. Continued.

P.V. Jeurink et al.



2. Contamination versus active migration

differences, since studies were performed in Europe and the USA. Furthermore, it could also be due to the well documented technical limitations of molecular techniques to study bacterial communities (Inglis et al., 2012; Zoetendal et al., 2004). Specific issues such as biased DNA isolation and amplification with specific primers that are not optimal for certain bacterial groups have been described (Frank et al., 2008; Sim et al., 2012). The use of new techniques such as pyrosequencing or metagenomics have recently revealed the presence of ‘rare’ bacterial species in human milk (Hunt et al., 2011). The fact that specific species were never isolated before may be due to a fastidious growth requirement or a scattered presence. New techniques such as single cell cultivation and sequencing, allowing bacterial cell isolation and characterisation, are rapidly evolving and will allow us to expand our knowledge on the milk microbiota composition and functionality.

Several studies have shown the transmission of bacterial strains from mother-to-infant through breastfeeding (Albesharat et al., 2011; Jimenez et al., 2008c; Makino et al., 2011; Martin, 2012; Martín et al., 2006; Matsumiya et al., 2002). However, the exact mechanisms by which bacteria reach the mammary gland have been the subject of much debate over the years (Figure 1).

The traditional hypothesis: ‘a contamination’ Traditionally, it is believed that the presence of bacteria in human milk is a result of a mere contamination with bacteria from the mother’s skin or infant’s oral cavity. It is assumed that infants acquire bacteria from the maternal gut and vaginal microbiota during birth and transfer these

Physiological changes during pregnancy

A

Differentiation of the mammary gland Massive migration of immune cells

Potential routes

B

Mammary gland epithelium

lux ef

Retrogr ad

Breast skin microbiota Infant’s oral microbiota

Milk microbiota

C

Luminal

Maternal gut microbiota

Intestinal epithelium

Increased permeability? Hormonal changes?

Dendritic cell

M Cell

Lymph-blood circ ulation

Bacteria transported (internalised or bound to the cell?)

Figure 1. Potential mechanisms of the human milk microbiome establishment. Physiological changes during and after pregnancy facilitate the migration of bacteria to the mammary gland. (A) Hormonal changes occurring in this period may have an influence on gut permeability, which could facilitate bacterial uptake. (B) Through the retrograde flux, the mother’s skin microbiota and infant’s oral microbiota may contribute to the establishment of the human milk microbiome. (C) Bacteria from the maternal intestinal tract may be taken up by different immune cells. The massive migration of immune cells to the mammary glands could provide another possible route to alter the human milk microbiome.


21

P.V. Jeurink et al.

bacteria from the mouth to the breast skin and from there to the mammary gland during breastfeeding. The exchange of bacteria from the infant’s mouth to the mammary gland might be facilitated by a certain degree of retrograde flow into the mammary ducts during suckling, as demonstrated by Ramsay et al. (2004) (Figure 1B). It is very likely that milk or mammary bacterial communities are constantly influenced by exposure to other microbial communities associated with the mother and her infant. Human milk microbiota, as any other ecological niche in the human microbiome, is not thought to be an isolated environment, but rather a network of interrelated communities (Costello et al., 2009). As mentioned above, birth is considered to be a natural ‘transplant’ of bacteria from the maternal gut and vagina microbiota to the infant. Indeed, Makino and co-workers have recently shown that strains originating from the maternal gut are transferred to the infant gut (Makino et al., 2011). However, the role of the vaginal microbiota as a source of bacteria to the infant remains unclear. A molecular epidemiological study on the transmission of vaginal Lactobacillus species from the mother to the new-born infant showed that less than one-fourth of the infants acquired maternal vaginal lactobacilli at birth, and that one month later, these vaginal lactobacilli had been outcompeted by lactobacilli associated with human milk (Matsumiya et al., 2002). In addition, Martín and coworkers showed that the profiles of Lactobacillus sequences retrieved from infant faeces were similar to those retrieved from human milk of the respective mothers, whereas the lactobacilli in the faeces did not resemble the maternal lactobacilli community of the vagina (Martín et al., 2007a). In conclusion, these studies suggest that although some vaginal lactobacilli are transferred to the infant at birth, they do not seem to successfully colonise the neonatal gut. Besides the maternal gut and vaginal microbiota, it has also been suggested that the infant’s mouth and the maternal skin serve as a source of bacteria that are detectable in human milk (Figure 1B). It has been shown, both by culturedependent and -independent techniques, that Streptococcus, a dominant phylotype in the salivary microbiome (Aas et al., 2005; Cephas et al., 2011; Nasidze et al., 2009) is also frequently found in colostrum and human milk (Hunt et al., 2011; Jimenez et al., 2008a,b). At first glance, it would support the theory that the infant’s mouth supplies bacteria to the mammary gland, but it might also indicate that bacteria in human milk may play a role in the establishment of the infant’s salivary microbiome. Bacterial phylotypes commonly found in human milk are also thought to originate from the skin. Indeed, Staphylococcus, Propionibacterium and Corynebacteria, which are dominant in adult skin, are found in human milk (Capone et al., 2011; Gao et al., 2007; Grice et al., 2009; Hunt et al., 2011; Jimenez et al., 2008a,b). However, when the bacterial communities found in human 22

milk are compared to those of the sebaceous skin of the type found on the breast, major differences arise especially in terms of relative abundance of shared phylotypes (Hunt et al., 2011). Moreover, it has been shown that Lactobacillus, enterococcal and bifidobacterial isolates from human milk were genotypicallly different from those isolated from skin or were not even detectable (Gueimonde et al., 2007; Martín et al., 2003, 2009). In addition, several arguments support the idea that the presence of bacteria in human milk is not a result of a mere contamination and exclude the infant as the only vehicle. Firstly, bifidobacteria are very strict anaerobes which makes it unlikely that they are transported from the infant mouth to the breast skin despite oxygen-stress (Xiao et al., 2011). However, this possibility cannot be excluded since it may be strain dependent. Secondly, bacteria can be isolated from colostrum before the infant is even born and last, but not least, live bacteria orally administered to lactating women in a capsule can be retrieved from human milk (Arroyo et al., 2010; Jimenez et al., 2008c).

The revolutionary hypothesis: ‘active migration’ The findings mentioned in the previous paragraphs are suggestive and support the hypothesis that at least some of the bacteria present in the maternal gut could reach the mammary gland through an endogenous route (Martín et al., 2004). However, the exact mechanisms by which bacteria could cross the intestinal epithelium, evade the immune system and reach the mammary gland are not clear yet. It is possible that intestinal tissue-resident innate cells, like dendritic cells (DCs) or macrophages, play an important role in this migration process, as they may act as carriers of bacteria from the maternal gut to the mammary gland (Martín et al., 2004; Perez et al., 2007). It has been demonstrated that DCs are able to open the tight junctions between intestinal epithelial cells and penetrate the gut epithelium with their dendrites, enabling DCs to sample commensal bacteria directly from the gut lumen without damaging the integrity of the epithelial barrier (Rescigno et al., 2001). This mechanism has been demonstrated for a Salmonella typhimurium strain that, although it was deficient of invasion genes, was able to reach the spleen alive after oral administration to mice (Rescigno et al., 2001). Macrophages have also been shown to be essential for extra-intestinal dissemination of non-invasive bacteria (Vazquez-Torres et al., 1999). Moreover, the specialised M cell layer of Peyer’s patches and lymphoid follicles have been shown to sample commensal bacteria, after which resident DCs take up the bacteria and transport them to the mesenteric lymph nodes where they stayed alive 10 to 60 hours after intra-gastric administration (Macpherson and Uhr, 2004).


Therefore, once inside DCs, gut bacteria could spread to other locations due to the circulation of immune cells within the mucosal-associated lymphoid system. Antigenstimulated cells migrate from the intestinal mucosa to colonise distant mucosal surfaces, such as those of the respiratory and genitourinary tracts, salivary and lachrymal glands and, most significantly, that of the lactating mammary gland (Delves et al., 2011). Over the years, evidence supporting this theory is growing. For example, labelled bacteria, administered to pregnant mice during the last 2 weeks of pregnancy, were found in the stomach of the offspring just after lactation and not before (Fernández et al., 2004). Indeed, it has been shown in mice that bacteria migrate from the gut to the mesenteric lymph nodes (MLN) and mammary gland during late pregnancy and lactation. Perez and co-workers have shown that 70% of the MLN of pregnant mice contained bacteria compared to 10% of MLN of conventional non-pregnant mice. Within 24 hours after birth, only 10% of MLN contained bacteria whereas 80% of mammary gland was colonised (Perez et al., 2007). This implies that bacteria that are located in the MLN before delivery start migrating to the mammary gland, possibly under the influence of labour-induced hormones. Furthermore, in a human mother-infant pair, the bacterial DNA signature found in human milk, maternal faecal samples, infant faeces and maternal peripheral blood mononuclear cells was the same, suggesting that bacteria translocate via the blood circulation. Additionally, the peripheral blood mononuclear cells from lactating women showed greater diversity in bacterial gene sequences than those obtained from non-lactating women (DonnetHughes et al., 2010; Perez et al., 2007). Taken together, these results suggest that intestine-derived bacteria and bacterial components are transported to the lactating breast within mononuclear cells (Figure 1C). Moreover, the presence of bacteria in blood of healthy humans has been shown, which supports the hypothesis that mononuclear cells transport bacteria through the circulation (McLaughlin et al., 2002; Nikkari et al., 2001; Vankerckhoven et al., 2004). Despite these interesting findings, several questions arise and need to be answered before the ‘migration hypothesis’ could be generally accepted. It remains unclear how a bacterium interacts with the immune cell and is actually transported to the mammary gland. While it was initially suggested that the bacterial migration would occur inside the DCs (Martín et al., 2004), the results of Langa and Perez and co-workers suggest that bacteria may be transported being attached to the surface of cells instead of being internalised (Langa, 2006; Perez et al., 2007). Moreover, the mechanisms by which bacteria avoid being phagocytosed and killed by the hosts’ innate immunity is yet unknown. However, innate cell education by the pregnancy hormone, progesterone, might play a role. Progesterone has been shown to suppress Toll-like receptor-triggered immune signalling, thereby interfering with the regulation of Beneficial Microbes 4(1)


phagosome maturation, which is necessary for bacterial killing (Blander and Medzhitov, 2004; Sun et al., 2012). Furthermore, progesterone treatment of DCs suppressed production of the pro-inflammatory cytokines TNF-α and IL-1β, but did neither affect the production of the anti-inflammatory cytokine IL-10 nor the DC capacity for phagocytosis (Butts et al., 2007). The migration process is suggested to be selective. It was initially thought that certain strains could be recognised by the immune cells and transported to the mammary gland while others do not. More likely is the option that all bacteria are recognised by immune cells, but certain strains are equipped to remain silent and/or evade killing by the innate immune cells. In addition, the relative proximity and abundance of bacteria at the mucosal surface and its capacity to adhere to the mucus are affecting the likelihood that bacteria get ‘sampled’ either via M cells or via direct sampling by DCs (Figure 1C). Furthermore, the capacity of the bacteria to survive in or stay attached to immune cells is also influencing the possibility to be transported to the mammary gland. The production of exopolysaccharides (EPS) might be an example of such bacterial capacities to enhance survival. Recently, it has been shown that B. breve strain UCC2003 produces an EPS that is suggested to confer the ability to remain immunologically silent by evading the adaptive B-cell host response (Fanning et al., 2012). In addition, the EPS positive strains were responsible for reduced colonisation levels of a gut pathogen, which might explain why B. breve is one of the most commonly bifidobacteria species found in human milk (Alp and Aslim, 2010; Martín et al., 2009; Solis et al., 2010; Turroni et al., 2011). However, as also some pathogens produce EPS, it remains to be elucidated which exact mechanism(s) is/are involved in the decision to (be) kill(ed) or stay alive. Another question that needs further research is related to the ‘window of opportunity’ in which the migration of bacteria can occur. The results from Perez and co-workers showed how the bacteria migrate from the MLN to the mammary gland one day after delivery (Donnet-Hughes et al., 2010). However, it remains unknown when the migration process starts and ends and what are the factors that limit that period. During pregnancy, the mammary gland prepares for lactation through a series of developmental steps. The principal feature of mammary growth in pregnancy is a great increase in ducts and alveoli under a multi-hormonal influence (Figure 1A). At the end of this period, the lobules of the alveolar system are maximally developed and small amounts of colostrum may be released for several weeks prior to delivery. Additionally, the nipple and areola enlarge markedly and the sebaceous glands become more prominent (Beischer et al., 1997). Hormones produced during pregnancy and lactation play a crucial role in this 23

P.V. Jeurink et al.

process. The increased lymph and blood supply to the mammary gland and the oxytocin release, which causes contraction of the mioepithelial cells, may also facilitate the presence of endogenous bacteria in human milk (Figure 1A). Furthermore, oxytocin is produced throughout the entire human gastrointestinal tract (Ohlsson et al., 2006) and can directly modulate gut cell functions, as shown both in vitro with the increased permeability of human gut cell line Caco2BB cells (Klein et al., 2011) and in vivo colonic motility in rats (Matsunaga et al., 2009). Moreover, it is known that progesterone plays a role inhibiting the immune response and helps the dilatation of the milk ducts (Yoshinaga, 2008). Gonadotrophins, like folliclestimulating hormone (FSH), luteinising hormone (LH), and human chorionic gonadotropin (hCG), can also modulate immune responses. For example, recombinant (r)LH alone promotes proliferation of CD4+ T cells from normal healthy women, whereas the combination of rLH and rFSH inhibit proliferation. Addition of rhCG even further potentiated the inhibitory effect, suggesting that simultaneous secretion of these hormones, as seen in the follicular phase, can positively influence the CD4+ T cell tolerance towards embryo implantation (Carbone et al., 2010). Furthermore, lactogenic hormones are responsible for the regulation of the massive migration of immune cells towards the mammary gland (Bertotto et al., 1991). Prolactin has been described as initiator of increased transcellular transport via the alteration of one of the claudin-proteins that are regarded as the most important components of the tight junctions (Charoenphandhu et al., 2009). Moreover, culturing mammary gland epithelial cells in the presence of pregnancy hormones induced the production of inflammatory cytokines, promoting innate cell recruitment and adhesion (Santos et al., 2009). Inflammatory processes are required for tissue remodelling and angiogenesis and essential for normal mammary gland development (Gouon-Evans et al., 2000). In conclusion, hormonal and physiological changes during late pregnancy and lactation may provide the right conditions for immune cells to transport bacteria to the MLN. Future research should reveal which factors are determining the timing at which the bacteria are allowed to be transported to and subsequently colonise the mammary gland.

3. Can bacteria in human milk influence maternal and infants’ health? Independent from the origin of bacteria in human milk, its relevance may lay in the potential implications on the health of women and their infants. The milk microbiome may be regarded as an inoculum for the infant gut. The exposure of the breastfed infant to the bacterial richness in milk may be one factor contributing 24

to the differential faecal microbiota between breastfed and formula-fed infants (Fanaro et al., 2003). The study of Donnet-Hughes suggests that the milk microbiome plays a key role in programming the neonatal immune system (Donnet-Hughes et al., 2010). It is widely known that, to achieve neonatal mucosal tissue homeostasis, the gut needs to develop tolerance to ingested antigens and to components of the indigenous bacterial microbiota. Neonatal defects in establishing tolerance have been linked to the development of disease and chronic inflammation of the mucosa (Renz et al., 2011). In addition, studies performed in germ-free mice have taught us that early life colonisation is required for the development of a fully functional immune system and affects many physiological processes within the host (Smith et al., 2007). Therefore, bacteria present in human milk may be essential in programming the immune system to respond appropriately to (food-)antigens, pathogens and commensal bacteria (Donnet-Hughes et al., 2010). Perez-Cano and co-workers have shown that two strains isolated from human milk, L. fermentum CECT5716 and L. salivarius CECT5713, are able to activate NK cells, CD4+ T cells, CD8+ T cells and regulatory T cells. They suggest that these strains have an impact on both innate and acquired immunity and strongly induce a wide range of pro- and anti-inflammatory cytokines and chemokines. Moreover, an infant formula supplemented with 2×108 cfu/day of L. fermentum CECT 5716, a strain isolated from human milk, has been shown to reduce the incidence of gastrointestinal and upper respiratory tract infections in infants (Maldonado et al., 2012). The comparison with other strains belonging to the same species but with a different origin suggests that there is a milk strain-specific effect, such as a higher induction of IL-10 and IL-1 production (Perez-Cano et al., 2010). From this, it can be hypothesised that the ecosystem aids the concept of colonisation resistance also in the mammary gland in order to potentially protect the host against pathogens and/or viruses. Support for this hypothesis can be found in the fact that some lactic acid bacteria strains isolated from human milk are able to inhibit an in vitro infection of HIV-susceptible TZM-1b cells by the HIV-1 virus (Martin et al., 2010). In conclusion, the ingestion of such a wealth of bacterial genera may play a key role in early colonisation and contribute to the protective effects of breastfeeding against diarrhoea and respiratory disease, and reduced risk of developing obesity (Gillman et al., 2001; Lamberti et al., 2011; Lopez-Alarcon et al., 1997; Van ‘t Land, 2010; Von Kries et al., 1999). It is widely known that breastfeeding is not only the optimal nutrition for the infant, but it also confers several health benefits to the lactating women. For example, women who breastfed for at least 6 months, are less likely to develop diabetes or breast cancer later in life compared to women that do not breastfeed or less than 6 months (Owen et al., 2006; Stuebe, 2009). It can therefore be speculated that the Beneficial Microbes 4(1)

milk microbiome also plays a key role in the mammary health of lactating women. The bacterial richness of human milk might modulate the human milk composition and therefore the presence of immune parameters, metabolites or bacteria causing diseases. However, the physiological state of the bacteria and the ability of the bacteria to divide inside the mammary gland or within human milk remains to be elucidated. During the course of lactation, up to 30% of women suffer from acute, subacute or recurrent mastitis, sometimes leading to fever, redness or swelling and always to breast pain (Barbosa-Cesnik et al., 2003). Mastitis, usually caused by staphylococci, streptococci and/or corynebacteria (Contreras and Rodriguez, 2011), is one of the main reasons for early cessation of breastfeeding (Walker, 2008). Traditionally, S. aureus has been considered as the main etiological agent of acute mastitis, although S. epidermidis is emerging as the leading cause of subacute and chronic mastitis both in human and veterinary medicine (Delgado et al., 2009). Recurrent episodes are frequent among women who experience mastitis, while others report no problems throughout the course of several lactations (Foxman et al., 2002). Mechanisms of immune evasion by staphylococci and streptococci, and use of antibiotics during late pregnancy and peripartum seem to predispose to suffer this condition (Contreras and Rodriguez, 2011). Hunt et al. (2011) have shown that the composition of the milk microbiome is host-dependent. Therefore, it may be that this composition is an important factor that determines whether a woman will suffer from mastitis. It is possible that mechanisms such as competitive exclusion for nutrients and other resources or production of bacteriocins by particular members of the commensal communities in milk repress potential pathogens and the subsequent signs and symptoms of mastitis (Heikkila and Saris, 2003). Interestingly, orally administered probiotics have proven to be an effective alternative to treat mastitis versus the use of antibiotics (Arroyo et al., 2010; Jimenez et al., 2008c). The probiotic strains L. salivarius CECT5713 and L. fermentum CECT5716 were able to modulate the human milk microbiome by decreasing the total bacterial count by 2 log and replacing the mastitis-causing Staphylococcus by Lactobacillus. Moreover, the use of these probiotic strains prevented the mother from suffering of side effects often associated with antibiotic treatment such as vaginal infections and recurrent mastitis episodes. However, in case of women receiving L. fermentum CECT5716 only, mild complaints like flatulence were reported. The altered milk microbiome induced by probiotic treatment with L. salivarius and L. fermentum has been shown to facilitate breastfeeding, which in turn leads to health benefits for both the mother and her infant. Further research should focus on identifying the components of the milk microbiome associated with health benefits and identify any other factor influencing these communities. Beneficial Microbes 4(1)


Related to this, HMOs present in human milk are able to modulate the microbiota of breastfed infants (Bode, 2012). Therefore, it can be speculated that HMOs are also able to modulate the bacterial communities in the mammary gland. Interestingly, HMOs mirror blood group characteristics; four different milk groups have been identified based on secretor and Lewis blood group systems (Albrecht et al., 2011; Thurl et al., 2010). While milk of ‘secretor’ women is rich in 2’-fucosyllactose and other α1-2-fucosylated HMOs, ‘non-secretor’ women lack a functional FTU2 enzyme resulting in milk that does not contain α1-2-fucosylated HMOs. Interestingly, some strains of Staphylococcus, the major cause of mastitis, bind to 2’-fucosyllactose (Lane et al., 2011). Therefore, it is possible that susceptibility to suffer from mastitis is determined not only by the bacterial composition of the human milk, but also by the blood group and corresponding type of HMOs in the milk. The link between differential microbiota composition in healthy versus diseased states has been described for the gastrointestinal tract, vagina and oral cavity (Huang et al., 2011; Ling et al., 2010; Ma et al., 2012; Pflughoeft and Versalovic, 2012; Sekirov et al., 2010). It therefore seems logical that the milk microbiome is also influenced by the health status of the mother. Interestingly, it was recently shown that the milk metagenome and microbiome of obese or overweight women differ from healthy-weight controls (Collado et al., 2012; Jimenez et al., 2012). Besides the health status, potential factors influencing the milk microbiome could include parity, mode of delivery and maternal diet, but also the genetic background. Further research is needed in order to better understand the associations between the health status and actual microbial communities and the implications these associations may have for the both the mother and her infant.

4. Conclusions The use of more sophisticated culture-dependent and -independent techniques to study the human milk microbiome has revealed a complex ecosystem with a much greater diversity than previously anticipated. Furthermore, literature provides increasing evidence supporting the hypothesis that at least some gut bacteria can reach the mammary gland through an endogenous extra-intestinal route and that the establishment of the milk microbiome is not a result of a mere contamination. However, the exact mechanisms by which bacteria could cross the intestinal epithelium, evade the immune system and reach the mammary gland requires further research. The potential role of the milk microbiome appears to have implications not only on short- and long-term infant health but also on the mammary health. A better understanding of the link between the milk microbiome and health benefits and the potential factors influencing this association will open new avenues in the field of pregnancy and lactation. As an 25

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example, if the composition of the human milk microbiota could be modified through the diet (including the use of pre- and/or probiotics), then it could lead to a reduction in the risk of mastitis or in the duration or severity of the symptoms usually associated to this condition. Such approach would aid mothers to exclusively breastfed their infants for up to six months, as recommended by WHO. Such a breastfeeding period would be particularly beneficial for the infant, not only from a nutritional point of view but also for the development of a fully functional immune system and the interconnected physiological processes. In addition, the mother’s risk for a diversity of diseases, such as diabetes, osteoporosis or breast cancer, would be reduced. These examples emphasise the possible magnitude of the milk microbiome’s influence on the health of both mother and infant, thereby demanding attention in future research.

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