Human milk: A source of more life than we imagine

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.

Full text

Beneficial Microbes, March 2013; 4(1): 17-30
WageningenAcademic
Publishers
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2012.0040 17
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 10
3
-10
4
cfu/ml, representing a continuous
Human milk: a source of more life than we imagine
P.V. Jeurink
1,2
, J. van Bergenhenegouwen
1,2
, E. Jiménez
3
, L.M.J. Knippels
1,2
, L. Fernández
3
, J. Garssen
1,2
, J. Knol
1,4
,
J.M. Rodríguez
3
and R. Martín
1
1
Danone Research, Centre for Specialised Nutrition, P.O. Box 7005, 6700 CA Wageningen, the Netherlands;
2
Utrecht Institute
for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, the Netherlands;
3
Dpto Nutrición,
Bromatología y Tecnología de los Alimentos, UCM, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain;
4
Laboratory 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

P.V. Jeurink et al.
18 Beneficial Microbes 4(1)
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 culture-
independent 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
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

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 19
Table 1. Bacteria detected in human milk by culture-dependent and -independent techniques.
Phylum Genera Culture-based methods 16S rRNA gene-based methods Metagenomics
Isolation qPCR Fingerprinting (DGGE), cloning 454 sequencing Shot-gun sequencing
Species Species Species Species Class
Actinobacteria Bifidobacterium B. breve, B. bifidum,
B. longum, B. adolescentis;
B. pseudocatenulatum
B. breve, B. bifidum, B. longum,
B. adolescentis, B. dentium,
B. animalis, B. catenulatum
B. longum
Actinobacteria
Parascovia P. denticolens
Corynebacterium spp.
Propionibacterium P. acnes spp.
Rothia R. mucilaginosa
Kocuria K. rhizophila
Bacteroidetes Bacteroides Bacteroidetes
Firmicutes Staphylococcus S. epidermidis, S.aureus, S. capitis,
S. hominis
S. aureus S. epidermidis, S. hominis spp. S. aureus Bacilli
Streptococcus S. mitis, S. salivarius, S. oris,
S. parasanguis, S. australis,
S. gallolyticus, S. vestibularis,
S. lactarius
spp. S. mitis, S. salivarius,
S. parasanguis
spp.
Lactobacillus 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
spp. L. gasseri, L. fermentum,
L. rhamnosus
Lactococcus L. lactis L. lactis
Enterorococcus E. faecium, E. faecalis, E. durans,
E. hirae, E. mundtii
spp. E. faecium, E. faecalis
Leuconostoc Leuc. mesenteroides Leuc. citreum; Leuc. fallax
Pediococcus P. pentosaceous
Weissella W. confusa, W. cibaria
Clostridia
1
spp. Clostridia

P.V. Jeurink et al.
20 Beneficial Microbes 4(1)
Table 1. Continued.
Phylum Genera Culture-based methods 16S rRNA gene-based methods Metagenomics
Isolation qPCR Fingerprinting (DGGE), cloning 454 sequencing Shot-gun sequencing
Species Species Species Species Class
Proteobacteria Bradyrhizobiaceae spp.
Pseudomonas spp. P. aureoginosa
α-Proteobacteria
Serratia spp.
Ralstonia spp.
Sphingomonas spp. spp.
Novosphingobium spp.
Sphingopyxis spp.
Sphingobium spp.
Verrucromicrobia
Akkermansia A. muciniphila
References
2
1-16 6, 17-19 20-22 23 24
1
Clostridium cluster XIVa-XIVb and cluster IV.
2
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).

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 21
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.
2. Contamination versus active migration
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
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.
Physiological changes during pregnancy Potential routes
Differentiation of the mammary gland
Intestinal
epithelium
Maternal gut
microbiota
Luminal
Bacteria transported (internalised or bound to the cell?)
M Cell
Massive migration of immune cells
Increased permeability?
Hormonal changes?
A
C
L
y
m
p
h
-
b
l
o
o
d
c
i
r
c
u
l
a
t
i
o
n
Infant’s oral microbiota
Breast skin
microbiota
Milk microbiota
Mammary gland
epithelium
B
R
e
t
r
o
g
r
a
d
e
f
l
u
x
Dendritic
cell

P.V. Jeurink et al.
22 Beneficial Microbes 4(1)
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 co-
workers 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 culture-
dependent 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
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).

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 23
Therefore, once inside DCs, gut bacteria could spread
to other locations due to the circulation of immune cells
within the mucosal-associated lymphoid system. Antigen-
stimulated 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 (Donnet-
Hughes 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
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

P.V. Jeurink et al.
24 Beneficial Microbes 4(1)
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 follicle-
stimulating 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
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 2×10
8
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

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 25
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.
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

P.V. Jeurink et al.
26 Beneficial Microbes 4(1)
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.
References
American Academy of Paediatrics (AAP), 2012. Breastfeeding and
the use of human milk. Pediatrics 129: e827-841.
Aas, J.A., Paster, B.J., Stokes, L.N., Olsen, I. and Dewhirst, F.E., 2005.
Defining the normal bacterial flora of the oral cavity. Journal of
Clinical Microbiology 43: 5721-5732.
Albesharat, R., Ehrmann, M.A., Korakli, M., Yazaji, S. and Vogel, R.F.,
2011. Phenotypic and genotypic analyses of lactic acid bacteria in
local fermented food, breast milk and faeces of mothers and their
babies. Systematic and Applied Microbiology 34: 148-155.
Albrecht, S., Schols, H.A., Van den Heuvel, E.G., Voragen, A.G. and
Gruppen, H., 2011. Occurrence of oligosaccharides in feces of breast-
fed babies in their first six months of life and the corresponding
breast milk. Carbohydrate Research 346: 2540-2550.
Alp, G. and Aslim, B., 2010. Relationship between the resistance to
bile salts and low pH with exopolysaccharide (EPS) production of
Bifidobacterium spp. isolated from infants feces and breast milk.
Anaerobe 16: 101-105.
Arroyo, R., Martin, V., Maldonado, A., Jimenez, E., Fernández, L.
and Rodriguez, J.M., 2010. Treatment of infectious mastitis during
lactation: antibiotics versus oral administration of lactobacilli
isolated from breast milk. Clinical Infectious Diseases 50: 1551-1558.
Barbosa-Cesnik, C., Schwartz, K. and Foxman, B., 2003. Lactation
mastitis. The Journal of the American Medical Association 289:
1609-1612.
Beasley, S.S. and Saris, P.E., 2004. Nisin-producing Lactococcus lactis
strains isolated from human milk. Applied and Environmental
Microbiology 70: 5051-5053.
Beischer, N., Mackay, E.V. and Colditz, P.B., 1997. Obstetrics and the
newborn. Saunders, London, UK.
Bertotto, A., Castellucci, G., Scalise, F., Tognellini, R. and Vaccaro,
R., 1991. ‘Memory’ T cells in human breast milk. Acta Paediatrica
Scandinavica 80: 98-99.
Bingen, E., Denamur, E., Lambert-Zechovsky, N., Aujard, Y., Brahimi,
N., Geslin, P. and Elion, J., 1992. Analysis of DNA restriction
fragment length polymorphism extends the evidence for breast
milk transmission in Streptococcus agalactiae late-onset neonatal
infection. Journal of Infectious Diseases 165: 569-573.
Blander, J.M. and Medzhitov, R., 2004. Regulation of phagosome
maturation by signals from Toll-like receptors. Science 304: 1014-
1018.
Bode, L., 2012. Human milk oligosaccharides: every baby needs a
sugar mama. Glycobiology, in press. DOI: http://dx.doi.org/10.1093/
glycob/cws074.
Boehm, G. and Moro, G., 2008. Structural and functional aspects
of prebiotics used in infant nutrition. Journal of Nutrition 138:
1818S-1828S.
Butts, C.L., Shukair, S.A., Duncan, K.M., Bowers, E., Horn, C.,
Belyavskaya, E., Tonelli, L. and Sternberg, E.M., 2007. Progesterone
inhibits mature rat dendritic cells in a receptor-mediated fashion.
International Immunology 19: 287-296.
Capone, K.A., Dowd, S.E., Stamatas, G.N. and Nikolovski, J., 2011.
Diversity of the human skin microbiome early in life. Journal of
Investigative Dermatology 131: 2026-2032.
Carbone, F., Procaccini, C., De Rosa, V., Alviggi, C., De Placido, G.,
Kramer, D., Longobardi, S. and Matarese, G., 2010. Divergent
immunomodulatory effects of recombinant and urinary-derived
FSH, LH, and hCG on human CD4+ T cells. Journal of Reproductive
Immunology 85: 172-179.
Cephas, K.D., Kim, J., Mathai, R.A., Barry, K.A., Dowd, S.E., Meline,
B.S. and Swanson, K.S., 2011. Comparative analysis of salivary
bacterial microbiome diversity in edentulous infants and their
mothers or primary care givers using pyrosequencing. PloS ONE
6: e23503.
Chantry, C.J., Howard, C.R. and Auinger, P., 2006. Full breastfeeding
duration and associated decrease in respiratory tract infection in
US children. Pediatrics 117: 425-432.
Charoenphandhu, N., Nakkrasae, L.I., Kraidith, K., Teerapornpuntakit,
J., Thongchote, K., Thongon, N. and Krishnamra, N., 2009. Two-
step stimulation of intestinal Ca(2+) absorption during lactation by
long-term prolactin exposure and suckling-induced prolactin surge.
American Journal of Physiology - Endocrinology and Metabolism
297: E609-E619.
Collado, M.C., Delgado, S., Maldonado, A. and Rodriguez, J.M., 2009.
Assessment of the bacterial diversity of breast milk of healthy women
by quantitative real-time PCR. Letters in Applied Microbiology
48: 523-528.
Collado, M.C., Laitinen, K., Salminen, S. and Isolauri, E., 2012.
Maternal weight and excessive weight gain during pregnancy modify
the immunomodulatory potential of breast milk. Pediatric Research
72: 77-85.
Contreras, G.A. and Rodriguez, J.M., 2011. Mastitis: comparative
etiology and epidemiology. Journal of Mammary Gland Biology
and Neoplasia 16: 339-356.
Costello, E.K., Lauber, C.L., Hamady, M., Fierer, N., Gordon, J.I. and
Knight, R., 2009. Bacterial community variation in human body
habitats across space and time. Science 326: 1694-1697.
Delgado, S., Arroyo, R., Jimenez, E., Marin, M.L., Del Campo, R.,
Fernández, L. and Rodriguez, J.M., 2009. Staphylococcus epidermidis
strains isolated from breast milk of women suffering infectious
mastitis: potential virulence traits and resistance to antibiotics.
BMC Microbiology 9: 82.
Delves, P.J., Martin, S.J., Burton, D.R., Roitt, I.M., 2011. Roitt’s essential
immunology. Wiley Blackwell, Hoboken, NY, USA.

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 27
Donnet-Hughes, A., Perez, P.F., Dore, J., Leclerc, M., Levenez, F.,
Benyacoub, J., Serrant, P., Segura-Roggero, I. and Schiffrin, E.J.,
2010. Potential role of the intestinal microbiota of the mother in
neonatal immune education. Proceedings of the Nutrition Society
69: 407-415.
Duijts, L., Jaddoe, V.W., Hofman, A. and Moll, H.A., 2010. Prolonged
and exclusive breastfeeding reduces the risk of infectious diseases
in infancy. Pediatrics 126: e18-25.
El-Mohandes, A.E., Schatz, V., Keiser, J.F. and Jackson, B.J., 1993.
Bacterial contaminants of collected and frozen human milk used
in an intensive care nursery. American Journal of Infection Control
21: 226-230.
Fanaro, S., Chierici, R., Guerrini, P. and Vigi, V., 2003. Intestinal
microflora in early infancy: composition and development. Acta
Paediatrica 92 Suppl. s442: 48-55.
Fanning, S., Hall, L. and Van Sinderen, D., 2012. Bifidobacterium breve
UCC2003 surface exopolysaccharide production is a beneficial trait
mediating commensal-host interaction through immune modulation
and pathogen protection. Gut Microbes 3: 420-425.
Fernández, L., Marín, M.L., Langa, S., Martín, R., Reviriego, C.,
Fernández, A., Olivares, M., Xaus, J. and Rodríguez, J.M., 2004.
A novel genetic label for detection of specific probiotic lactic acid
bacteria. Food Science and Technology 10: 101-108.
Foxman, B., D’Arcy, H., Gillespie, B., Bobo, J.K. and Schwartz, K., 2002.
Lactation mastitis: occurrence and medical management among
946 breastfeeding women in the United States. American Journal
of Epidemiology 155: 103-114.
Frank, J.A., Reich, C.I., Sharma, S., Weisbaum, J.S., Wilson, B.A. and
Olsen, G.J., 2008. Critical evaluation of two primers commonly
used for amplification of bacterial 16S rRNA genes. Applied and
Environmental Microbiology 74: 2461-2470.
Gao, Z., Tseng, C.H., Pei, Z. and Blaser, M.J., 2007. Molecular analysis
of human forearm superficial skin bacterial biota. Proceedings of
the National Academy of Sciences of the USA 104: 2927-2932.
Gavin, A. and Ostovar, K., 1977. Microbiological characterization of
human milk. Journal of Food Protection 40: 614-616.
Gillman, M.W., Rifas-Shiman, S.L., Camargo Jr., C.A., Berkey, C.S.,
Frazier, A.L., Rockett, H.R., Field, A.E. and Colditz, G.A., 2001. Risk
of overweight among adolescents who were breastfed as infants.
The Journal of the American Medical Association 285: 2461-2467.
Gouon-Evans, V., Rothenberg, M.E. and Pollard, J.W., 2000. Postnatal
mammary gland development requires macrophages and
eosinophils. Development 127: 2269-2282.
Greer, F.R., Sicherer, S.H. and Burks, A.W., 2008. Effects of early
nutritional interventions on the development of atopic disease
in infants and children: the role of maternal dietary restriction,
breastfeeding, timing of introduction of complementary foods,
and hydrolyzed formulas. Pediatrics 121: 183-191.
Grice, E.A., Kong, H.H., Conlan, S., Deming, C.B., Davis, J., Young,
A.C., Bouffard, G.G., Blakesley, R.W., Murray, P.R., Green, E.D.,
Turner, M.L. and Segre, J.A., 2009. Topographical and temporal
diversity of the human skin microbiome. Science 324: 1190-1192.
Gueimonde, M., Laitinen, K., Salminen, S. and Isolauri, E., 2007.
Breast milk: a source of bifidobacteria for infant gut development
and maturation? Neonatology 92: 64-66.
Heikkila, M.P. and Saris, P.E., 2003. Inhibition of Staphylococcus aureus
by the commensal bacteria of human milk. Journal of Applied
Microbiology 95: 471-478.
Huang, S., Yang, F., Zeng, X., Chen, J., Li, R., Wen, T., Li, C., Wei,
W., Liu, J., Chen, L., Davis, C. and Xu, J., 2011. Preliminary
characterization of the oral microbiota of Chinese adults with and
without gingivitis. BMC Oral Health 11: 33.
Hunt, K.M., Foster, J.A., Forney, L.J., Schutte, U.M., Beck, D.L., Abdo,
Z., Fox, L.K., Williams, J.E., McGuire, M.K. and McGuire, M.A.,
2011. Characterization of the diversity and temporal stability of
bacterial communities in human milk. PloS ONE 6: e21313.
Inglis, G.D., Thomas, M.C., Thomas, D.K., Kalmokoff, M.L., Brooks,
S.P. and Selinger, L.B., 2012. Molecular methods to measure
intestinal bacteria: a review. Journal of AOAC International 95: 5-23.
Ip, S., Chung, M., Raman, G., Chew, P., Magula, N., DeVine, D.,
Trikalinos, T. and Lau, J., 2007. Breastfeeding and maternal and
infant health outcomes in developed countries. Evidence Report/
Technology Assessesment 153: 1-186.
Ip, S., Chung, M., Raman, G., Trikalinos, T.A. and Lau, J., 2009. A
summary of the Agency for Healthcare Research and Quality’s
evidence report on breastfeeding in developed countries.
Breastfeeding Medicine 4 Suppl. 1: S17-S30.
Jeurink, P.V., Rijnierse, A., Martín, R., Garssen, J. and Knippels, L.M.,
2012. Difficulties in describing allergic disease modulation by pre-,
pro- and synbiotics. Current Pharmaceutical Design 18: 2369-2374.
Jimenez, E., Delgado, S., Fernández, L., Garcia, N., Albujar, M.,
Gomez, A. and Rodriguez, J.M., 2008a. Assessment of the bacterial
diversity of human colostrum and screening of staphylococcal and
enterococcal populations for potential virulence factors. Research
in Microbiology 159: 595-601.
Jiménez, E., Delgado, S., Maldonado, A., Arroyo, R., Albujar, M.,
Garcia, N., Jariod, M., Fernández, L., Gomez, A. and Rodriguez,
J.M., 2008b. Staphylococcus epidermidis: a differential trait of the
fecal microbiota of breast-fed infants. BMC Microbiology 8: 143.
Jiménez, E., Espinosa, I., Arroyo, R., Fernández, L. and Rodríguez, J.M.,
2012. Metagenomic analysis of human milk. In: Book of abstracts,
3
rd
TNO Beneficial Microbes Conference, 26-28 March 2012,
Noordwijkerhout, the Netherlands, p. 76.
Jiménez, E., Fernández, L., Maldonado, A., Martín, R., Olivares,
M., Xaus, J. and Rodríguez, J.M., 2008c. Oral administration of
Lactobacillus strains isolated from breast milk as an alternative for
the treatment of infectious mastitis during lactation. Applied and
Environmental Microbiology 74: 4650-4655.
Klein, B.Y., Tamir, H. and Welch, M.G., 2011. PI3K/Akt responses
to oxytocin stimulation in Caco2BB gut cells. Journal of Cellular
Biochemistry 112: 3216-3226.
Kosaka, N., Izumi, H., Sekine, K. and Ochiya, T., 2010. microRNA as
a new immune-regulatory agent in breast milk. Silence 1: 7.
Lamberti, L.M., Fischer Walker, C.L., Noiman, A., Victora, C. and
Black, R.E., 2011. Breastfeeding and the risk for diarrhea morbidity
and mortality. BMC Public Health 11 Suppl. 3: S15.
Lane, J.A., Mehra, R.K., Carrington, S.D. and Hickey, R.M., 2011.
Development of biosensor-based assays to identify anti-infective
oligosaccharides. Analytical Biochemistry 410: 200-205.

P.V. Jeurink et al.
28 Beneficial Microbes 4(1)
Langa, S., 2006. Interactions between lactic acid bacteria, intestinal
epithelial cells and immune cells. Development of in vitro models.
PhD Thesis, Complutense University of Madrid, Madrid, Spain.
Le Thomas, I., Mariani-Kurkdjian, P., Collignon, A., Gravet, A.,
Clermont, O., Brahimi, N., Gaudelus, J., Aujard, Y., Navarro, J.,
Beaufils, F. and Bingen, E., 2001. Breast milk transmission of a
Panton-Valentine leukocidin-producing Staphylococcus aureus
strain causing infantile pneumonia. Journal of Clinical Microbiology
39: 728-729.
Ling, Z., Kong, J., Liu, F., Zhu, H., Chen, X., Wang, Y., Li, L., Nelson,
K.E., Xia, Y. and Xiang, C., 2010. Molecular analysis of the diversity
of vaginal microbiota associated with bacterial vaginosis. BMC
Genomics 11: 488.
Lopez-Alarcon, M., Villalpando, S. and Fajardo, A., 1997. Breast-
feeding lowers the frequency and duration of acute respiratory
infection and diarrhea in infants under six months of age. Journal
of Nutrition 127: 436-443.
Ma, B., Forney, L.J. and Ravel, J., 2012. Vaginal microbiome: rethinking
health and disease. Annual Review of Microbiology 66: 371-389.
Macpherson, A.J. and Uhr, T., 2004. Induction of protective IgA by
intestinal dendritic cells carrying commensal bacteria. Science
303: 1662-1665.
Makino, H., Kushiro, A., Ishikawa, E., Muylaert, D., Kubota, H.,
Sakai, T., Oishi, K., Martín, R., Ben Amor, K., Oozeer, R., Knol, J.
and Tanaka, R., 2011. Transmission of intestinal Bifidobacterium
longum subsp. longum strains from mother to infant, determined
by multilocus sequencing typing and amplified fragment length
polymorphism. Applied and Environmental Microbiology 77:
6788-6793.
Maldonado, J., Canabate, F., Sempere, L., Vela, F., Sanchez, A.R.,
Narbona, E., Lopez-Huertas, E., Geerlings, A., Valero, A.D., Olivares,
M. and Lara-Villoslada, F., 2012. Human milk probiotic Lactobacillus
fermentum CECT5716 reduces the incidence of gastrointestinal and
upper respiratory tract infections in infants. Journal of Pediatric
Gastroenterology and Nutrition 54: 55-61.
Martín, R., Heilig, G.H., Zoetendal, E.G., Smidt, H. and Rodriguez,
J.M., 2007a. Diversity of the Lactobacillus group in breast milk and
vagina of healthy women and potential role in the colonization of
the infant gut. Journal of Applied Microbiology 103: 2638-2644.
Martín, R., Heilig, H.G., Zoetendal, E.G., Jimenez, E., Fernandez, L.,
Smidt, H. and Rodriguez, J.M., 2007b. Cultivation-independent
assessment of the bacterial diversity of breast milk among healthy
women. Research in Microbiology 158: 31-37.
Martín, R., Jimenez, E., Heilig, H., Fernandez, L., Marin, M.L.,
Zoetendal, E.G. and Rodriguez, J.M., 2009. Isolation of bifidobacteria
from breast milk and assessment of the bifidobacterial population by
PCR-denaturing gradient gel electrophoresis and quantitative real-
time PCR. Applied and Environmental Microbiology 75: 965-969.
Martín, R., Jimenez, E., Olivares, M., Marin, M.L., Fernandez, L.,
Xaus, J. and Rodriguez, J.M., 2006. Lactobacillus salivarius CECT
5713, a potential probiotic strain isolated from infant feces and
breast milk of a mother-child pair. International Journal of Food
Microbiology 112: 35-43.
Martín, R., Langa, S., Reviriego, C., Jiménez, E., Marín, M.L., Xaus, J.,
Fernández, L. and Rodríguez, J.M., 2003. Human milk is a source
of lactic acid bacteria for the infant gut. The Journal of Pediatrics
143: 754-758.
Martín, R., Langa, S., Reviriego, C., Jiménez, E., Marín, M.L., Olivares,
M., Boza, J., Jiménez, J., Fernández, L., Xaus, J. and Rodríguez, J.M.,
2004. The commensal microflora of human milk: new perspectives
for food bacteriotherapy and probiotics. Trends in Food Science
and Technology 15: 121-127.
Martin, V., 2011. Interactions between the microbiota of human milk
and human immonodeficiency virus type 1 (HIV-1). PhD Thesis,
Complutense University of Madrid, Madrid, Spain.
Martin, V., Maldonado-Barragan, A., Moles, L., Rodriguez-Banos,
M., Campo, R.D., Fernandez, L., Rodriguez, J.M. and Jimenez, E.,
2012. Sharing of bacterial strains between breast milk and infant
feces. Journal of Human Lactation 28: 36-44.
Martin, V., Maldonado, A., Fernandez, L., Rodriguez, J.M. and Connor,
R.I., 2010. Inhibition of human immunodeficiency virus type 1 by
lactic acid bacteria from human breastmilk. Breastfeeding Medicine
5: 153-158.
Martin, V., Manes-Lazaro, R., Rodriguez, J.M. and Maldonado-
Barragan, A., 2011. Streptococcus lactarius sp. nov., isolated from
breast milk of healthy women. International Journal of Systematic
and Evolutionary Microbiology 61: 1048-1052.
Matsumiya, Y., Kato, N., Watanabe, K. and Kato, H., 2002. Molecular
epidemiological study of vertical transmission of vaginal
Lactobacillus species from mothers to newborn infants in Japanese,
by arbitrarily primed polymerase chain reaction. Journal of Infection
and Chemotherapy 8: 43-49.
Matsunaga, M., Konagaya, T., Nogimori, T., Yoneda, M., Kasugai, K.,
Ohira, H. and Kaneko, H., 2009. Inhibitory effect of oxytocin on
accelerated colonic motility induced by water-avoidance stress in
rats. Neurogastroenterology and Motility 21: 856-e859.
McLaughlin, R.W., Vali, H., Lau, P.C., Palfree, R.G., De Ciccio, A., Sirois,
M., Ahmad, D., Villemur, R., Desrosiers, M. and Chan, E.C., 2002.
Are there naturally occurring pleomorphic bacteria in the blood of
healthy humans? Journal of Clinical Microbiology 40: 4771-4775.
Nasidze, I., Li, J., Quinque, D., Tang, K. and Stoneking, M., 2009. Global
diversity in the human salivary microbiome. Genome Research
19: 636-643.
Nikkari, S., McLaughlin, I.J., Bi, W., Dodge, D.E. and Relman, D.A.,
2001. Does blood of healthy subjects contain bacterial ribosomal
DNA? Journal of Clinical Microbiology 39: 1956-1959.
Nishimura, T., Suzue, J. and Kaji, H., 2009. Breastfeeding reduces
the severity of respiratory syncytial virus infection among young
infants: a multi-center prospective study. Pediatrics International
51: 812-816.
Ohlsson, B., Truedsson, M., Djerf, P. and Sundler, F., 2006. Oxytocin is
expressed throughout the human gastrointestinal tract. Regulatory
Peptides 135: 7-11.
Owen, C.G., Martin, R.M., Whincup, P.H., Smith, G.D. and Cook, D.G.,
2006. Does breastfeeding influence risk of type 2 diabetes in later
life? A quantitative analysis of published evidence. The American
Journal of Clinical Nutrition 84: 1043-1054.

Human milk: a source of more life than we imagine
Beneficial Microbes 4(1) 29
Penttila, I.A., 2010. Milk-derived transforming growth factor-β and
the infant immune response. The Journal of Pediatrics 156: S21-S25.
Perez-Cano, F.J., Gonzalez-Castro, A., Castellote, C., Franch, A. and
Castell, M., 2010. Influence of breast milk polyamines on suckling
rat immune system maturation. Developmental and Comparative
Immunology 34: 210-218.
Perez, P.F., Dore, J., Leclerc, M., Levenez, F., Benyacoub, J., Serrant,
P., Segura-Roggero, I., Schiffrin, E.J. and Donnet-Hughes, A., 2007.
Bacterial imprinting of the neonatal immune system: lessons from
maternal cells? Pediatrics 119: e724-732.
Pflughoeft, K.J. and Versalovic, J., 2012. Human microbiome in health
and disease. Annual Review of Pathology 7: 99-122.
Quigley, M.A., Kelly, Y.J. and Sacker, A., 2007. Breastfeeding and
hospitalization for diarrheal and respiratory infection in the United
Kingdom Millennium Cohort Study. Pediatrics 119: e837-842.
Ramsay, D.T., Kent, J.C., Owens, R.A. and Hartmann, P.E., 2004.
Ultrasound imaging of milk ejection in the breast of lactating
women. Pediatrics 113: 361-367.
Ravel, J., Gajer, P., Abdo, Z., Schneider, G.M., Koenig, S.S., McCulle,
S.L., Karlebach, S., Gorle, R., Russell, J., Tacket, C.O., Brotman,
R.M., Davis, C.C., Ault, K., Peralta, L. and Forney, L.J., 2011. Vaginal
microbiome of reproductive-age women. Proceedings of the
National Academy of Sciences of the USA 108 Suppl. 1: 4680-4687.
Renz, H., Brandtzaeg, P. and Hornef, M., 2011. The impact of perinatal
immune development on mucosal homeostasis and chronic
inflammation. Nature Reviews Immunology 12: 9-23.
Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta,
G., Bonasio, R., Granucci, F., Kraehenbuhl, J.-P. and Ricciardi-
Castagnoli, P., 2001. Dendritic cells express tight junction proteins
and penetrate gut epithelial monolayers to sample bacteria. Nature
Immunology 2: 361-367.
Rijnierse, A., Jeurink, P.V., Van Esch, B.C., Garssen, J. and Knippels,
L.M., 2011. Food-derived oligosaccharides exhibit pharmaceutical
properties. European Journal of Pharmacology 668 Suppl. 1: S117-
123.
Santos, S.J., Aupperlee, M.D., Xie, J., Durairaj, S., Miksicek, R., Conrad,
S.E., Leipprandt, J.R., Tan, Y.S., Schwartz, R.C. and Haslam, S.Z.,
2009. Progesterone receptor A-regulated gene expression in
mammary organoid cultures. Journal of Steroid Biochemistry and
Molecular Biology 115: 161-172.
Scholtens, P., Oozeer, R., Martin, R., Ben Amor, K. and Knol, J., 2012.
The early settlers: intestinal microbiology in early life. Annual
Reviews of Food Science and Technology 3: 425-447.
Sekirov, I., Russell, S.L., Antunes, L.C. and Finlay, B.B., 2010. Gut
microbiota in health and disease. Physiological Reviews 90: 859-904.
Sim, K., Cox, M.J., Wopereis, H., Martin, R., Knol, J., Li, M.S.,
Cookson, W.O., Moffatt, M.F. and Kroll, J.S., 2012. Improved
detection of bifidobacteria with optimised 16S rRNA-gene based
pyrosequencing. PloS ONE 7: e32543.
Sinkiewicz, G. and Nordstrom, E.A., 2005. Occurrence of Lactobacillus
reuteri, lactobacilli and bifidobacteria in human breast milk.
Pediatric Research 58: 415.
Smith, K., McCoy, K.D. and Macpherson, A.J., 2007. Use of axenic
animals in studying the adaptation of mammals to their commensal
intestinal microbiota. Seminars in Immunology 19: 59-69.
Solis, G., De Los Reyes-Gavilan, C.G., Fernandez, N., Margolles, A.
and Gueimonde, M., 2010. Establishment and development of lactic
acid bacteria and bifidobacteria microbiota in breast-milk and the
infant gut. Anaerobe 16: 307-310.
Stuebe, A., 2009. The risks of not breastfeeding for mothers and infants.
Reviews in Obstetrics and Gynecology 2: 222-231.
Sun, Y., Cai, J., Ma, F., Lü, P., Huang, H. and Zhou, J., 2012. miR-
155 mediates suppressive effect of progesterone on TLR3, TLR4-
triggered immune response. Immunology Letters 146: 25-30.
Thurl, S., Munzert, M., Henker, J., Boehm, G., Muller-Werner,
B., Jelinek, J. and Stahl, B., 2010. Variation of human milk
oligosaccharides in relation to milk groups and lactational periods.
British Journal of Nutrition 104: 1261-1271.
Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan,
A., Ley, R.E., Sogin, M.L., Jones, W.J., Roe, B.A., Affourtit, J.P.,
Egholm, M., Henrissat, B., Heath, A.C., Knight, R. and Gordon,
J.I., 2009. A core gut microbiome in obese and lean twins. Nature
457: 480-484.
Turroni, F., Foroni, E., Serafini, F., Viappiani, A., Montanini, B.,
Bottacini, F., Ferrarini, A., Bacchini, P.L., Rota, C., Delledonne,
M., Ottonello, S., Van Sinderen, D. and Ventura, M., 2011. Ability of
Bifidobacterium breve to grow on different types of milk: exploring
the metabolism of milk through genome analysis. Applied and
Environmental Microbiology 77: 7408-7417.
Van ‘t Land, B., Boehm, G. and Garssen, J., 2010. Breast milk:
components with immune modulating potential and their possible
role in immune mediated disease resistance. In: Watson, R.R.,
Zibadi, S. and Preedy, V.R. (eds.) Dietary components and immune
function. Springer, Berlin, Germany, pp. 25-41.
Vankerckhoven, V.V., Van Autgaerden, T., Huys, G., Vancanneyt, M.,
Swings, J. and Goossens, H., 2004. Establishment of the PROSAFE
collection of probiotic and human lactic acid bacteria. Microbial
Ecology in Health and Disease 16: 131-136.
Vazquez-Torres, A., Jones-Carson, J., Baumler, A.J., Falkow, S., Valdivia,
R., Brown, W., Le, M., Berggren, R., Parks, W.T. and Fang, F.C., 1999.
Extraintestinal dissemination of Salmonella by CD18-expressing
phagocytes. Nature 401: 804-808.
Von Kries, R., Koletzko, B., Sauerwald, T., Von Mutius, E., Barnert,
D., Grunert, V. and Von Voss, H., 1999. Breast feeding and obesity:
cross sectional study. BMJ 319: 147-150.
Walker, A., 2010. Breast milk as the gold standard for protective
nutrients. The Journal of Pediatrics 156: S3-S7.
Walker, M., 2008. Conquering common breast-feeding problems.
Journal of Perinatal and Neonatal Nursing 22: 267-274.
West, P.A., Hewitt, J.H. and Murphy, O.M., 1979. The influence of
methods of collection and storage on the bacteriology of human
milk. Journal of Applied Bacteriology 46: 269-277.
Wright, K.C. and Feeney, A.M., 1998. The bacteriological screening
of donated human milk: laboratory experience of British Paediatric
Association’s published guidelines. Journal of Infection 36: 23-27.
Xiao, M., Xu, P., Zhao, J., Wang, Z., Zuo, F., Zhang, J., Ren, F., Li, P.,
Chen, S. and Ma, H., 2011. Oxidative stress-related responses of
Bifidobacterium longum subsp. longum BBMN68 at the proteomic
level after exposure to oxygen. Microbiology 157: 1573-1588.

P.V. Jeurink et al.
30 Beneficial Microbes 4(1)
Yoshinaga, K., 2008. Review of factors essential for blastocyst
implantation for their modulating effects on the maternal immune
system. Seminars in Cell and Developmental Biology 19: 161-169.
Zhou, Q., Li, M., Wang, X., Li, Q., Wang, T., Zhu, Q., Zhou, X., Gao,
X. and Li, X., 2012. Immune-related microRNAs are abundant in
breast milk exosomes. International Journal of Biological Sciences
8: 118-123.
Zoetendal, E.G., Collier, C.T., Koike, S., Mackie, R.I. and Gaskins,
H.R., 2004. Molecular ecological analysis of the gastrointestinal
microbiota: a review. The Journal of Nutrition 134: 465-472.