Pharmacological Research 69 (2013) 1–10

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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Invited Review

The human milk microbiota: Origin and potential roles in health and disease
Leónides Fernández a , Susana Langa a,b , Virginia Martín a , Antonio Maldonado a,c ,
Esther Jiménez a , Rocío Martín d , Juan M. Rodríguez a,∗
a
Department of Nutrition, Food Science and Food Technology, Complutense University of Madrid, 28040 Madrid, Spain
b
Departament of Food Technology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Carretera de la Coruña Km 7.5, 28040 Madrid, Spain
c
Departament of Food Biotechnology, Instituto de la Grasa-CSIC, 41012 Sevilla, Spain
d
Danone Research-Centre for Specialised Nutrition, Wageningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Human milk has been traditionally considered sterile; however, recent studies have shown that it repre-
Received 22 May 2012 sents a continuous supply of commensal, mutualistic and/or potentially probiotic bacteria to the infant
Received in revised form 23 August 2012 gut. Culture-dependent and -independent techniques have revealed the dominance of staphylococci,
Accepted 1 September 2012
streptococci, lactic acid bacteria and bifidobacteria in this biological fluid, and their role on the coloniza-
tion of the infant gut. These bacteria could protect the infant against infections and contribute to the
Keywords:
maturation of the immune system, among other functions. Different studies suggest that some bacteria
Human milk
present in the maternal gut could reach the mammary gland during late pregnancy and lactation through
Breastfeeding
Bacteria
a mechanism involving gut monocytes. Thus, modulation of maternal gut microbiota during pregnancy
Mammary microbiota and lactation could have a direct effect on infant health. On the other hand, mammary dysbiosis may lead
Dendritic cells to mastitis, a condition that represents the first medical cause for undesired weaning. Selected strains
Mastitis isolated from breast milk can be good candidates for use as probiotics. In this review, their potential uses
Probiotics for the treatment of mastitis and to inhibit mother-to-infant transfer of HIV are discussed.
HIV © 2012 Elsevier Ltd. All rights reserved.

Contents

1. Bacterial diversity of human milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Functions of human milk bacteria in the infant gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Origin of the bacteria isolated from breast milk: is there a bacterial entero-mammary pathway? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Human milk bacteria as biotherapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Specific targets for human milk probiotic bacteria: lactational mastitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Specific targets for human milk probiotic bacteria: anti-HIV activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1. Bacterial diversity of human milk present in colostrum and milk, including immunocompetent
cells, immunoglobulins, fatty acids, polyamines, oligosaccharides,
Human milk is a complex species-specific biological fluid lysozyme, lactoferrin and other glycoproteins, and antimicrobial
adapted to satisfy the nutritional requirements of the rapidly grow- peptides [2], which inactivate pathogens individually, additively,
ing infant; additionally, it educates the infant immune system and and synergistically [3].
confers a certain degree of protection against pathogens [1]. These More recently, several studies have revealed that colostrum and
effects reflect the synergistic action of many bioactive molecules, breast milk are continuous sources of commensal, mutualistic and
potentially probiotic bacteria to the infant gut [4–11] (Table 1). This
is a relevant finding since intramammary human milk was tradi-
tionally considered to be sterile. In fact, human milk constitutes
Abbreviations: DCs, dendritic cells; HIV, human immunodeficiency virus; LAB,
lactic acid bacteria. one of the main sources of bacteria to the breastfed infant gut since
∗ Corresponding author. Tel.: +34 91 3943837; fax: +34 91 3943743. a baby consuming approximately 800 mL/day of milk would ingest
E-mail address: jmrodrig@vet.ucm.es (J.M. Rodríguez). between 1 × 105 and 1 × 107 bacteria daily [5]. This may explain

1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phrs.2012.09.001
2 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10
Table 1
Main bacterial genera or species isolated from human milk or which DNA sequencies
have been retrieved from this biological fluid.
Method Main species/generaa
Bacterial isolation L. acidophilus, L. fermentum, S. epidermidis,
Str. mitis, Str. salivarius
L. plantarum, S. epidermidis, Streptococcus
spp.
E. faecium, L. fermentum, L. gasseri
E. faecalis, L. crispatus, L. rhamnosus, Lc.
lactis, Leuc. mesenteroides, R. mucilaginosa,
S. aureus, S. capitis, S. epidermidis, S.
hominis, Str. mitis, Str. oris, Str. parasanguis,
Str. salivarius
L. salivarius
Corynebacterium spp., Enterococcus spp.,
Lactobacillus spp., Peptostreptococcus spp.,
Staphylococcus spp., Streptococcus spp.
L. reuteri
S. epidermidis
B. adolescentis, B. bifidum, B. breve
B. breve, B. longum, K. rhizophila, L. casei, L.
fermentum, L. gasseri, L. gastricus, L.
plantarum, L. reuteri, L. salivarius, L.
vaginalis, P. pentosaceus, R. mucilaginosa, S.
aureus, S. epidermidis, S. hominis, Str.
lactarius, Str. mitis, Str. parasanguis, Str.
salivarius
E. durans, E. faecalis, E. faecium, E. hirae, E.
mundtii, L. animalis, L. brevis, L. fermentum,
L. gasseri, L. helveticus, L. oris, L. plantarum,
P. pentosaceous, Str. australis, Str.
gallolyticus, Str. vestibularis
B. longum
DNA detection E. faecalis, E. faecium, L. fermentum, L.
gasseri, L. rhamnosus, Lc. lactis, Leuc.
citreum, Leuc. fallax, Prop. acnes, S.
epidermidis, S. hominis, Str. mitis, Str.
parasanguis, Str. salivarius, W. cibaria, W.
confusa
B. longum, Clostridium spp., Lactobacillus
spp., Staphylococcus spp., Streptococcus spp.
B. adolescentis B. animalis, B. bifidum, B.
breve, B. catenolatum, B. longum
Bifidobacterium spp., Clostridium spp.,
Enterococcus spp., Lactobacillus spp.,
Staphylococcus spp., Streptococcus spp.
B. adolescentis, B. bifidum, B. breve, B.
longum
Bradyrhizobiaceae, Corynebacterium spp.,
Propionibacterium spp., Pseudomonas spp.,
Ralstonia spp., Serratia spp., Sphingomonas
spp., Staphylococcus spp., Streptococcus spp.
a
Abbreviations: B., Bifidobacterium; E., Enterococcus; K., Kocuria; L., Lactobacil-
lus; Lc., Lactococcus; Leuc., Leuconostoc; P., Pediococcus; Prop., Propionibacterium; R.,
Rothia; S., Staphylococcus; Str., Streptococcus; W., Weissella.
why the bacterial composition of the gut microbiota of breast-
fed infants is closely related to that found in the breast milk of
their respective mothers and, also, why the development of a more
diverse microbiota coincides with the weaning period [12]. It must
be highlighted that human milk olisaccharides (HMOs) also play a
key role in driving the diversity of the infant gut microbiota [13].
HMOs are the third-largest solid component of milk and, while their
structural complexity renders them non-digestible to the host, they
are liable to hydrolytic enzymes of the infant colonic microbiota
[14]. It has been previously demonstrated that selected bifidobac-
terial and lactobacilli phylotypes use specific HMOs secreted early
in the lactation cycle [15]; more recently, the genome sequences
of two bifidobacterial strains have revealed several adaptations for
milk utilization within the infant microbiome [16,17].
The first descriptions of the bacterial diversity of breast milk
in healthy women were based on the use of culture media and
showed the predominance of staphylococci, streptococci, lactic acid
bacteria (LAB), propionibacteria and closely related Gram-positive
bacteria [4,5,18,19] (Table 1), including new bacterial species, such
References as Streptococcus lactarius [20]. Human milk has also been shown
[18] to be a source of live bifidobacteria to the infant gut [11]. The fact
that bacteria belonging to such genera can be isolated from fresh
[19] breast milk of healthy women from distant countries suggests that
their presence in this substrate is a common event. Therefore, they
[4]
[5,6] should be considered as components of the natural microbiota of
human mammary gland, instead of mere contaminant bacteria. The
mammary microbiota is somehow peculiar since it has a transient
nature: its development starts during the last third of pregnancy,
[10] reaches the highest complexity at the end of such period, remains
[63] quite constant throughout lactation, declines sharply at weaning,
and rapidly disappears when there is no milk in the mammary gland
and the apoptosis process responsible for mammary involution
[120]
begins (Fig. 1).
[7,8]
[11] Several studies have shown that there is a mother-to-infant
[20,24] transfer of bacterial strains belonging, at least, to the genera Lacto-
bacillus, Staphylococcus, Enterococcus, and Bifidobacterium through
breastfeeding [4,7,11,21–24]. Independently of the original source
of such bacteria, human milk is the source of hundreds of bacterial
phylotypes to the infant gastrointestinal tract. It has been suggested
that exposure of the breast-fed infant to such a wealth of bacte-
[22] rial phylotypes may exert beneficial effects against diarrheal and
respiratory diseases, and may reduce the risk of developing other
diseases, such as diabetes or obesity [25,26].
The application of culture-independent molecular techniques,
[23] and particularly those based on 16S rRNA genes, allowed a
[27,28] complementary biodiversity assessment of the human milk micro-
biome. The use of such techniques confirmed the dominance of
staphylococci and streptococci and the presence of LAB, propi-
onibacteria and bifidobacteria, and revealed the existence of DNA
belonging to other bacterial groups, such as some Gram-negative
[65] bacteria [11,27–29]. In addition, the application of the “-omics”
approach (genomics, metagenomics, transcriptomics, proteomics,
[29] and metabolomics) to the study of the human mammary micro-
[121]
biota is already in progress and there is no doubt that the results
provided by such techniques will open new perspectives to under-
stand the initiation and development of the infant gut microbiota
[11] [30].
Recently, the first microbiome study focused on human milk
[25]
was published [25]. The authors used microbial identification
techniques based on pyrosequencing of the V1–V2 region of the
bacterial 16S rRNA gene to characterize the bacterial communities
present in milk samples collected from 16 women self-described as
healthy at three time-points over 4 weeks. Results indicated that
milk bacterial communities were generally complex and, although
a few genera (Streptococcus, Staphylococcus, and Serratia) repre-
sented greater than 5% of the relative community abundance,
eight other genera represented ≥1% of the communities observed
across samples. Among the hundreds of operational taxonomic
units (OTUs) detected in the milk of every woman, only 9 (Strep-
tococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacteria,
Ralstonia, Propionibacterium, Sphingomonas, and Bradyrhizobiaceae)
were present in every sample from every woman. These 9 “core”
OTUs represented approximately half of the microbial commu-
nity observed, although their relative abundance varied greatly
between subjects. The remaining half of the community was not
conserved across women. These findings are in contrast with those
of the gut microbiome, where only a low set of OTUs is shared
among individuals [31], or the vaginal microbiome which com-
prises several different core groups [32]. On the other hand, milk
bacterial community was generally stable over time within an indi-
vidual. Similarly to the mammary microbiota, previous studies of
various sites of the human microbiome have also revealed that the

103
Concentration of bacteria (CFU/ml)
Not detectable
Fig. 1. Schematic representation of the acquisition and development of the human mammary microbiota.
bacterial communities associated with a particular individual over
time are often stable and highly personalized [33].
The results of a second study on the breast milk microbiome
involving 18 mothers indicated that milk bacteria are not contami-
nants and suggested that this site-specific microbiome is influenced
by several factors that significantly skew its composition [34]. More
recently, our research group analyzed the metagenome of human
milk samples obtained from healthy, mastitis-suffering or obese
women, and observed that the bacterial community of breast milk
may differ depending on the individual and on the health status of
the lactating women [unpublished results].
2. Functions of human milk bacteria in the infant gut
In the last years, some studies have shown that human milk
bacteria may play several roles in the infant gut. First of all,
they can contribute to the reduction of the incidence and sever-
ity of infections in the breastfed infant by different mechanisms,
such as competitive exclusion [35], production of antimicrobial
compounds [6,9,10,35], or improvement of the intestinal barrier
function by increasing mucine production and reducing intestinal
permeability [35]. Recently, the administration of a human milk
Lactobacillus strain to infants during 6 months led to 46%, 27%, and
30% reductions in the incidence rates of gastrointestinal infections,
upper respiratory tract infections, and total number of infections,
respectively [36]. Commensal coagulase-negative staphylococci
and viridans streptococci provided by breast milk can be partic-
ularly useful to reduce the acquisition of undesired pathogens by
infants exposed to hospital environments. In fact, some Staphy-
lococcus epidermidis strains that inhibit in vivo colonization by
Staphylococcus aureus have been postulated as a future strategy to
eradicate such pathogen from the mucosal surfaces [37,38]. Sim-
ilarly, it has been shown that viridans streptococci inhibit oral
colonization by methicillin-resistant S. aureus in high-risk new-
borns exposed to hospital environments [39].
Breast milk bacteria may also participate in the correct mat-
uration of the infant immune system since some strains are able
to modulate both natural and acquired immune responses in mice
and humans [40–42]. Their function seems to have a certain degree
of flexibility depending on the conditions found in the gut envi-
ronment. As an example, Lactobacillus salivarius CECT 5713 and
Lactobacillus fermentum CECT 5716 enhanced macrophage produc-
tion of Th1 cytokines, such as IL-2 and IL-12 and the inflammatory
mediator TNF-!, in the absence of an inflammatory stimulus.
L. Fernández et al. / Pharmacological Research 69 (2013) 1–10 3
• Hormonal signalling
• Increasing recruitment of • Sharpe decrease in milk
DCs and macrophages production
• Increasing pressure of • Decreasing
the fetus on the mesenteric concentrations of lactose
vessels in the mammary
• Increased bacterial environment
translocation •Apoptosis
• Increasing filling of the (physiological involution
mammary ducts with of the mammary gland)
colostrum
Last third of pregnancy Peripartum Breastfeeding Weaning
(~3 months) (a fewdays) (a few days-a few years) (some days-some weeks)
Time
However, both strains led to a reduction of Th1 cytokines when cells
were incubated in the presence of lipopolysaccharide [40]. A recent
study confirmed that L. fermentum CECT 5716 and L. salivarius CECT
5713 have a broad array of effects on the immune system [43]. They
behaved as potent activators of NK cells and moderate activators
of CD4+ and CD8+ T cells and regulatory T cells. Thus, they had
an impact on both innate and acquired immunity. They strongly
induced a wide range of pro- and anti-inflammatory cytokines
and chemokines. The authors compared these strains with others
belonging to the same species but isolated from sources different
to breast milk and found some milk strain-specific effects, such as
a higher induction of IL-10 and IL-1 production.
In relation to allergic conditions, the human milk strain Lacto-
bacillus gasseri CECT 5714 reduced the incidence and severity of the
allergic response in an animal model of cow’s milk protein allergy
[44]. It is also interesting to note that the presence of viridans strep-
tococci, one of the dominant bacterial groups in human milk, seems
to be a feature of the healthy infant gut in contrast to that of the
atopy-suffering infants [45].
Finally, human milk bacteria have a remarkable potential to play
metabolic roles in the infant. The glycobiome of some lactobacilli
and bifidobacteria, including that of species that have been isolated
from human milk, may help to create a specific “healthy” micro-
biota in the infant gut [14,46]. These microorganisms might also
contribute to infant digestion through the breakdown of sugars and
proteins; this possibility is particularly attractive having in account
that transit of food through the gastrointestinal tract is shorter in
infants than in adults and, also, that the pH of the infant’s stomach
is higher than that of the adult. In this context, human milk lacto-
bacilli strains are metabolically active in the infant gut and increase
the production of functional metabolites such as butyrate, which is
the main energy source for colonocytes and a relevant compound
in the modulation of intestinal function [47,48]. As a result, they
improve the intestinal habit, with an increase in fecal moisture,
and in stool frequency and volume [47,48].
3. Origin of the bacteria isolated from breast milk: is there
a bacterial entero-mammary pathway?
The origin of the bacteria present in breast milk has become a
controversial issue in the last years. Traditionally, it was believed
that milk harbored bacteria that were just the result of contamina-
tion with bacteria from the mother’s skin or the infant’s oral cavity
[19].
4 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10
Fig. 2. Potential sources of the bacteria present in human colostrum and milk. DC: dendritic cell.
Infrared photography [49] has shown that a certain degree of
retrograde flow back into the mammary ducts can occur during
suckling. Obviously, such back flow may provide an ideal route for
the exchange of bacteria from the infant’s mouth into the mam-
mary gland (Fig. 2). But it is also obvious that breast milk can also
be a source of bacteria to the infant’s mouth. Ecological niches in the
human microbiome are not thought to be isolated environments,
but rather a network of interrelated communities experiencing
constant exchange [33]. Therefore, it is very likely that milk or
mammary bacterial communities are no the exception, and that
they are constantly influenced by exposure to other microbial pop-
ulations associated with the mother and her infant. Little is known
about the infant human salivary microbiome but investigations on
adults have revealed that Streptococcus species are the dominant
phylotype in this fluid [50–52] while such dominance is even higher
in edentulous infants [53]. Streptococcus species are among the
most abundant phylotype in colostrum and milk samples [7,8,25],
supporting the hypothesis that the maternal milk bacteria may play
an important role in establishing salivary bacterial communities
and/or vice versa.
Some bacterial phylotypes usually present on adult skin, such
as Staphylococcus, Corynebacteria, and Propionibacteria [54,55], are
also common in human milk. This presents the possibility that
interactions with the maternal skin microbiota may also contribute
to shape the composition of milk microbiota (Fig. 2). However, a
comparison of the bacterial communities detected in milk to those
of the sebaceous skin found on the breast indicates that although
the two communities share some phylotypes, major differences
also exist [25]. Such relevant differences between the two commu-
nities indicate that bacterial communities in milk are not simply
a result of skin contamination. Sampling of breast milk for micro-
biological analysis must take into account that skin contamination
is almost unavoidable and that, logically, doubts on the original
location (internal mammary gland or skin) of the isolated bacteria
may arise. However, independent studies have shown that lacto-
bacilli or bifidobacteria could not be isolated from breast skin swabs
obtained from women that provided milk samples from which such
bacteria were isolated [11,29]. Previously, it has been reported that
lactobacilli and enterococcal isolates present in human milk are
genotypically different from those isolated in the skin, within a
same bacterial species and a same host [4]. In addition, bifidobac-
teria belongs to a strictly anaerobic genus and, therefore, skin is, at
least, a very unlikely source [29]. These and other findings suggest
that at least some of the bacteria present in the maternal gut could
reach the mammary gland through an endogenous route [56].
Although the pathway and mechanisms that some bacteria
could exploit to cross the intestinal epithelium and reach the mam-
mary gland and other locations has not been elucidated yet, some
works have offered a plausible scientific basis. It has been demon-
strated that dendritic cells (DCs) can penetrate the gut epithelium
to take up non-pathogenic bacteria directly from the gut lumen.
DCs are able to open the tight junctions between intestinal epithe-
lial cells, send dendrites outside the epithelium and directly sample
bacteria, while preserving the integrity of the epithelial barrier
through the expression of tight-junction proteins [57]. Using such
mechanism, a Salmonella typhimurium strain that was deficient in
invasion genes encoded by Salmonella pathogenicity island 1 (SPI1)
was still able to reach the spleen after oral administration to mice
[57]. This mechanism may not be exclusive to DCs, as CD18+ cells,
including macrophages, have been shown to be essential for extra-
intestinal dissemination of non-invasive Salmonella [58].
It has also been shown that intestinal DCs can retain small num-
bers of live commensal bacteria for several days in the mesenteric
lymph nodes [59]. Once inside DCs and/or macrophages, gut bac-
teria could spread to other locations since there is a circulation
of lymphocytes within the mucosal associated lymphoid system.
Antigen-stimulated cells move from the intestinal mucosa to colo-
nize distant mucosal surfaces, such as those of the respiratory and
genitourinary tracts, salivary and lachrymal glands, and, most sig-
nificantly, that of the lactating mammary gland [60]. In addition, it is
known that, during the lactation period, colonization of the mam-
mary gland by cells of the immune system is a selective process
regulated by the lactogenic hormones [61]. This process is respon-
sible for the abundance of such cells in human milk. A hypothetical
model to explain how some maternal bacteria could be transferred
to the neonatal gut is shown in Fig. 2.
The suggestion that the origin of the live bacteria found in breast
milk could be the maternal gut and that the bacteria would arrive
to the mammary gland through an endogenous route, involving
maternal DCs and macrophages, has been confirmed recently by
independent research groups. Initially, our group showed that the
exposure of mouse immature DCs to two bacterial strains isolated
from human milk led to a high stimulation of two DC activation
surface markers: the class II major histocompatibility complex and
 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10
Fig. 3. Specific interactions between cells of a L. gasseri strain isolated from breast
milk (A) and DC cells (B), as assessed by transmission electron microscopy [62].
The interactions were studied using transwell bicompartmental assays in which
bacterial cells and immature DC cells were initially separated by a monolayer of
Caco-2 cells.
the B7.2 protein [62,63]. It seems clear that bacterial induction of
DC maturation is an active process since it has been shown that
dead bacteria or inert particles, such as latex beads, cannot activate
DCs even if they are rapidly phagocytosed [64]. On the other hand, L.
gasseri CECT 5715, other strain isolated from breast milk, showed a
high level of binding to DCs and ability to translocate across a Caco-2
cell monolayer through a DC-mediated mechanism [62] (Fig. 3).
Later, other study showed that bacterial translocation from the
gut to mesenteric lymph nodes and mammary gland occurred dur-
ing late pregnancy and lactation in mice [65]. In addition, this
work revealed that human breast milk cells contain a number of
viable bacteria and a range of bacterial DNA signatures, which are
also found in maternal peripheral blood mononuclear cells. Those
peripheral blood mononuclear cells showed greater biodiversity
than did those obtained from control women. Taken together,
their results suggest that intestinally derived bacteria and bac-
terial components are transported to the lactating breast within
mononuclear cells. The authors speculated that this programs the
neonatal immune system to recognize specific bacterial molecular
patterns and to respond appropriately to pathogenic and com-
mensal organisms [65]. More recently, two successive studies that
focused on the oral administration of three lactobacilli strains iso-
lated from human milk (L. salivarius CECT 5713, L. gasseri CECT 5714,
and L. fermentum CECT 5716) provided new evidences that show the
existence of a bacterial entero-mammary pathway during lactation
[66,67].
The mammary gland prepares for lactation through a series of
developmental steps that occur during adolescence and pregnancy.
The principal feature of mammary growth in pregnancy is a great
increase in ducts and alveoli under a multi-hormonal influence. At
the end of this period, the lobules of the alveolar system are maxi-
mally developed and small amounts of colostrum may be released
for several weeks prior to delivery. Additionally, the nipple and
areola markedly enlarge and the sebaceous glands within become
more prominent [68]. The increased lymph and blood supply to
the mammary gland and the oxytocin release that causes contrac-
tion of the mioepithelial cells that invest the mammary alveoli may
also facilitate the presence of endogenous bacteria in breast milk.
These changes provide good conditions for biofilm formation on
the mammary areola and/or in the mammary duct system, leading
to the formation of a specific and transitory mammary microbiota.
5
At the same time, the whole body (including immune, cardiovascu-
lar, respiratory, digestive and genitourinary systems) experiences
diverse physiological adaptations during pregnancy and lactation,
and most of them are compatible with an increase in the bacterial
translocation rate in the gut.
It has to be highlighted that the presence of live LAB (similar to
those found in human milk) in the bloodstream of healthy human
hosts is not an uncommon event [69–72]. It is interesting to note
that 125 of the 485 lactobacilli strains deposited in the PROSAFE
collection were originally isolated from human blood, and it is
also illustrative that these 125 strains belong to 16 different Lacto-
bacillus species [69]. A study investigating the influence of the oral
microbiota composition on pregnancy outcome in 300 pregnant
women revealed that some bacteria, such as Actinomyces naselundii,
were linked to lower birth weight and earlier delivery while oth-
ers, including Lactobacillus casei, were associated with a slightly
higher birth weight and normal delivery [73]. These authors con-
cluded that oral bacteria can enter the uterine environment through
the bloodstream. Probably, in addition to the digestive and geni-
tourinary tracts, LAB and other bacteria can inhabit, permanent- or
transiently, other body locations in healthy hosts, where they could
contribute to extraintestinal probiotic effects. Additionally, human
clinical studies have also indicated that oral intake of specific bac-
terial strains at defined doses play a key role in the prevention
or improvement of extraintestinal conditions, and such probiotic
effects would be hardly explainable if they were exclusively con-
fined to the gut [74]. Globally, these studies indicate that certain
intestinal bacteria may have a rather underrated ability to spread
from the gut to extra-intestinal locations in healthy hosts.
4. Human milk bacteria as biotherapeutic agents
In recent years, the problems associated to the spread of clin-
ical antibiotic resistances among pathogenic bacteria and to the
rise of allergic or inflammatory diseases in developed countries
have led to a new interest in probiotics, which are defined as live
microorganisms that confer a health benefit on the host when
administered in adequate amounts [75]. Obviously, probiotic bac-
teria that are originally isolated from human milk are particularly
attractive organisms since they would fulfill some of the main crite-
ria generally recommended for human probiotics, such as human
origin, a history of safe prolonged intake by a particularly sensi-
tive population (neonates, infants), and adaptation to mucosal and
dairy substrates [76].
Among the bacteria isolated from human milk, species like L.
gasseri, L. salivarius, Lactobacillus reuteri, L. fermentum or Bifidobac-
terium breve are considered among those with probiotic potential
and enjoy the Qualified Presumption of Safety (QPS) status con-
ceded by the European Food Safety Authority (EFSA). In contrast
to other bacteria, these seem to be uniquely adapted to reside
in the human digestive tract and to interact with us in symbio-
sis from the time we are born. Additionally, some of the strains
isolated from this biological fluid have been shown to play anti-
infectious, anti-inflammatory, immunomodulatory and metabolic
roles, both in vitro and in vivo, including human studies (see Sec-
tion 2). The recent genome sequencing of some of these strains
[77–80] is providing some new clues to understand the relation-
ship between phenotypic properties and their subjacent molecular
basis [63]; in the future, such approach may contribute to a more
rational selection, design and/or application of probiotic bacteria.
Exclusive breastfeeding during the first month of life has been
linked to lower asthma [81] and atopic dermatitis [82] rates during
childhood and, as a result, this practice has been strongly recom-
mended to mothers with a family of atopy, as a possible means
to prevent atopic eczema. Human milk LAB may play a role in
6 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10
this protective effect since it has been described that probiotic
lactobacilli can be effective to prevent atopy and atopic diseases
through a variety of mechanisms [83]. Gut bacteria are considered
the earliest and most important stimulus for development of gut-
associated lymphoid tissue and they can promote antiallergenic
processes [83]. Therefore, milk of healthy women can be consid-
ered as a source of potentially probiotic or biotherapeutic bacteria
with a role in protecting mothers and/or infants against a variety
of allergic, inflammatory or infectious diseases.
4.1. Specific targets for human milk probiotic bacteria:
lactational mastitis
Mastitis is a common disease during lactation since it has an
incidence of up to 33% of the lactating mothers [84,85]. This inflam-
mation of one or more lobules of the mammary gland usually has an
infectious origin [86], involving staphylococci, streptococci and/or
corynebacteria [85]. 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 [87–90]. Multiresistance to
antibiotics, formation of biofilms and mechanisms for evasion of the
host immune response are common among clinical isolates of these
two staphylococcal species [91–95]. This explains why this condi-
tion uses to be elusive to antibiotic therapy, and why it constitutes
one of the main reasons to cease breastfeeding [85]. In this con-
text, the development of new strategies for mastitis management
based on probiotics, as an alternative or complement to antibiotic
therapy, is particularly appealing.
For this purpose, our research group selected some lactobacilli
strains on the basis of specific properties required for success in
mastitis treatment after oral administration: a high survival rate
during transit through the gastrointestinal tract, specific interac-
tions with DCs, ability to colonize the mammary gland and, once
there, mechanisms for competitive exclusion of mastitis-causing
staphylococci and streptococci. Initially, a pilot trial highlighted
the potential of L. salivarius CECT 5713 and L. gasseri CECT 5714,
two strains isolated from breast milk, for the treatment of staphy-
lococcal mastitis [66]. After 30 days, probiotics led to a significant
reduction (∼2 log10 CFU/mL) in the mean staphylococcal counts of
the milk cultures, in contrast, the values achieved in the placebo
group remained unchanged at the end of the study. This fact was
correlated with the clinical evolution since, while no clinical signs
of mastitis were observed in women assigned to the probiotic group
at day 14, they persisted throughout the study in the placebo group.
More recently, the efficacy of L. fermentum CECT 5716 or L.
salivarius CECT 5713, two lactobacilli strains isolated from breast
milk, to treat lactational mastitis when administered orally was
evaluated and compared to antibiotic therapy [67]. A total of
352 women with infectious mastitis were randomly divided
in three groups. Those in groups A (n = 124) and B (n = 127)
ingested daily 9 log10 CFU of L. fermentum CECT 5716 or L. sali-
varius CECT 5713, respectively, for 3 weeks while those in group
C (n = 101) were submitted to antibiotic therapy prescribed in
their respective Primary Care Centres. On day 0, the mean bac-
terial counts in milk samples of the three groups were similar
(4.35–4.47 log10 CFU/mL) and lactobacilli could not be detected.
On day 21, the mean bacterial counts in the probiotic groups (2.61
and 2.33 log10 CFU/mL) were lower than that of the control group
(3.28 log10 CFU/mL). The probiotic treatment led to a significant
reduction (1.7–2.1 log10 CFU/mL) in the milk bacterial count and
to a rapid improvement of the condition. The final bacterial count
was approximately 2.5 log10 CFU/mL, an acceptable bacterial load
in milk of healthy women [5,64]. After the probiotic treatment, L.
salivarius CECT 5713 and L. fermentum CECT 5716 could be isolated
from the milk samples of women of the probiotic groups A and B,
respectively. In contrast, the effectiveness of antibiotics prescribed
to group C women differed significantly, both in the reduction of
bacterial counts and in the improvement of the pain score.
4.2. Specific targets for human milk probiotic bacteria: anti-HIV
activity
Although breastfeeding is a source of new pediatric HIV-1 infec-
tions worldwide [96,97], most breastfed infants of HIV-positive
women remain uninfected despite repeated exposure of their oral
and gastrointestinal mucosal surfaces to the virus. Therefore, breast
milk has been recognized both as a vector of transmission and as
vehicle of protection against HIV-1 [96].
Most breast milk-transmitted HIV infections take place in devel-
oping countries where breastfeeding usually remains the best
election for providing optimal infant nutrition and protection
against morbidity and mortality associated with diarrheal and
lower respiratory infections [98,99]. In such countries, the choice
of the most suitable infant feeding option for an HIV-infected
mother should depend on her individual circumstances, including
her health status and the local situation, and should also consider
the health services available and the counseling and support she is
likely to receive [100].
As a general rule, exclusive breastfeeding has been recom-
mended for HIV-infected women during the first 6 months of
life unless replacement feeding is acceptable, feasible, affordable,
sustainable and safe [97,100]. Such feeding option may offer HIV-
1-infected women in developing countries an affordable, culturally
acceptable, and effective means of reducing mother-to-child HIV-
1 transmission while maintaining the benefits of breastfeeding
[101]. In fact, recent studies confirm that exclusive breastfeeding
can be successfully supported in HIV-infected women [102,103]. In
contrast, there seems to be an association between mixed breast-
feeding and increased HIV transmission risk [104].
Several factors may modulate the risk of HIV-1 transmission
via breastfeeding but the results of different studies focused on
the potential role of specific milk components are not conclusive
[105–107]. Recently, it was shown that breast milk contains innate
factors that blocked infection of CD4+ cells with cell-free HIV, but
did not affect infection with cell-associated virus [107]. On the other
hand, humoral mucosal immunity to HIV does not appear to be
a protective factor against viral transmission through breast milk
[105,108]. Therefore, factors associated to protection against ver-
tical transmission of HIV-1 through breastfeeding remain poorly
understood [96].
Interestingly, the potential of some human milk strains to inhibit
the infectivity of HIV with tropism to different co-receptors has
been described recently [109]. A total of 38 bacterial strains iso-
lated from breast milk were investigated for their ability to inhibit
HIV-1 infection in vitro. The heat-killed bacteria and cell-free super-
natants obtained from some strains inhibited HIV-1 infection; the
highest levels corresponded to killed bacteria from Lactobacillus
curvatus VM25 (55.5%), L. fermentum VM31 (52.5%), Pediococcus
pentosaceous VM95 (49.0%), and P. pentosaceous VM21 (45.5%).
HIV-inhibition was observed with supernatants from eight strains,
although the most effective were L. salivarius VM5 (42%) and L.
gasseri VM22 (40%) supernatants. Four strains (L. salivarius VM5, L.
gasseri VM22, Lactococcus lactis VM17, and Streptococcus salivarius
VM18) were selected for further evaluation and demonstrated dis-
tinct inhibition patterns against R5-, X4- and R5/X4-tropic HIV-1.
These results demonstrate for the first time that LAB from human
breast milk can significantly inhibit HIV-1 infection in vitro, and
suggest a possible role for these bacteria in mucosal protection
against HIV-1 in breastfeed infants.
Human milk bacteria may exert an anti-HIV action in the
infant gut through a variety of mechanisms (Fig. 4), including
 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10
Fig. 4. Potential mechanisms by which human milk bacteria can inhibit HIV infectivity. (A) Interactions between DC, HIV, and bacteria: (A1) blocking of DC receptor through
proteins or exopolysaccharides and (A2) blocking of the gp120 glycoprotein from binding to target cells. (B) Potential effects of human milk bacteria at the mucosal sites.
interactions with and modulation of DCs immunological functions
[110–112]. DC–HIV interaction is critical for the infection out-
come. Although DCs are able to induce specific immune responses
against a broad variety of pathogens, HIV-1 has evolved ways to
exploit DCs as cellular reservoirs, therefore facilitating viral dis-
semination, persistence in lymphoid tissues and evasion of antiviral
immunity [113,114]. Additionally, oral lactobacilli isolated from
healthy humans can capture HIV-1 in vitro through lectin-like
interactions between mannose residues on the viral envelope gly-
coprotein gp120 and the bacteria [115]. This physical interaction
(the so-called “HIV-1 trap”) may prevent viral attaching to cellular
receptors on mucosal tissues.
Recently, it was observed that the genome of L. salivarius CECT
5713 contains genes encoding four proteins (1184, 1230, 1306, and
1686) potentially involved in human molecular mimetism [63].
Among them, protein 1230 was considered of particular relevance
because of its similarity with proteins related to DC functionality,
such as CD209 antigen (DC-SIGN1) and the C-type lectin domain
family 4 member M (DC-SIGN2). In addition, protein 1230 contains
a recognition motif for high mannose N-linked oligosaccharides
present in a variety of pathogen antigens, including HIV gp120.
Consequently, it might have the potential to block gp120 from bind-
ing to target cells and, therefore, to inhibit HIV infectivity. To test
such predictions, the ability of L. salivarius CECT 5713 to stimu-
late the maturation of immature DCs and to inhibit the in vitro
HIV-1 infectivity was assessed [63]. The heat-killed cells led to a
reduction in viral infectivity of TZM-bl target cells with R5 (HIV-
1BaL ), CXCR4 (HIV-1HC4 ) and R5/X4 (HIV-1C7/86 ) of 42.3, 58.9, and
49.8%, respectively (mean values). Although to a lesser extent, cell-
free conditioned supernatant from L. salivarius CECT 5713 cultures
also inhibited HIV-1BaL (23.5%), HIV-1HC4 (21.4%) and HIV-1C7/86
(17.6%) infectivity. The results of this work suggest that molecular
mimetism displayed by L. salivarius CECT 5713 may have an antag-
onist role toward HIV gp120 and, therefore, may explain, at least
partly, its activity against HIV infectivity in vitro.
Intestinal permeability decreases faster in breast-fed infants
than those given formula [116], suggesting that some components
in breast milk influence the maturation of the gut mucosal epithe-
lium. Introduction of food proteins or enteric pathogens during
formula or mixed replacement feeding may increase the likeli-
hood of HIV transmission by stimulating intestinal inflammatory
responses in the neonatal gut, leading to increased permeability
and to facilitation of virus penetration. In fact, gastrointestinal tract
impairment in HIV-positive patients is present in the early phases
of HIV disease, and this impairment is associated with alterations
7
in gut microbiota and intestinal inflammatory parameters [117].
These findings support the hypothesis that gastrointestinal tract
alterations are a key factor in HIV pathogenesis, and may explain
why infants who are exclusively breastfed have a significantly
lower risk of being HIV-infected than infants who are bottle-fed
or mixed-fed [118,119].
5. Conclusion
Human milk is a source of bacteria to the infant gut, where
they may play a variety of antiinfectious, immunomodulatory, and
metabolic roles. In fact, recent studies indicate that the mam-
mary gland contains its own microbiota during late pregnancy and
lactation. This bacterial community may differ depending on the
individual and the health status of the lactating women. It seems
that certain bacteria from the maternal gut can use mononuclear
immune cells to colonize, first, the mammary gland and, later, the
infant gut through breast-feeding. If further studies confirm these
findings, they would have practical consequences since it would
imply that modulation of the maternal intestinal microbiota can
have a direct effect on her infant’s health, opening new perspectives
for bacteriotherapy and probiotics.
Acknowledgements
This study was partly supported by the FUN-C-FOOD
(Consolider-Ingenio 2010) and AGL2010-15420 projects from
the Ministerio de Economía y Competitividad (Spain).
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