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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. Jimnez3, L.M.J. Knippels1,2, L. Fernndez3, J. Garssen1,2, J. Knol1,4,
J.M. Rodrguez3 and R. Martn1
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 Nutricin,
Bromatologa y Tecnologa 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; rocio.martin@danone.com
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 infants
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;
Martn 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

(Martn 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; Martn 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; Martn 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; Martn 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

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).

6, 17-19

spp.

Sphingobium
A. muciniphila

spp.

References2

spp.

spp.

Sphingopyxis

Verrucromicrobia Akkermansia

Class

P. aureoginosa -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.

Beneficial Microbes 4(1)

Human milk: a source of more life than we imagine

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; Martn 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 mothers skin or infants 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

Differentiation of the mammary gland

Massive migration of immune cells

Potential routes

Mammary gland
epithelium

lux
ef

Retrogr
ad

Breast skin
microbiota
Infants oral microbiota

Milk microbiota

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 mothers skin microbiota and infants
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.

Beneficial Microbes 4(1)

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 infants 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, Martn 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 (Martn 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 infants 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 infants 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 infants 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; Martn
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 (Martn
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 (Martn 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 Peyers 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).

Beneficial Microbes 4(1)

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 (Fernndez 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 (Martn 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)

Human milk: a source of more life than we imagine

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-
and IL-1, 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; Martn 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 2108 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)

Human milk: a source of more life than we imagine

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 1-2-fucosylated HMOs,
non-secretor women lack a functional FTU2 enzyme
resulting in milk that does not contain 1-2-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

P.V. Jeurink et al.

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 mothers 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 microbiomes influence on the health of both mother
and infant, thereby demanding attention in future research.

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