The Human Milk Microbiota: Origin and Potential Roles in Health and Disease

Home , Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus salivarius, Streptococcus lactarius, Weissella

Pharmacological Research 69 (2013) 1–10

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Pharmacological Research

jo urnal homepage: www.elsevier.com/locate/yphrs

Invited Review

The human milk microbiota: Origin and potential roles in health and disease

a a,b a a,c

Leónides Fernández , Susana Langa , Virginia Martín , Antonio Maldonado ,

a d a,∗

Esther Jiménez , Rocío Martín , Juan M. Rodríguez

Department of Nutrition, Food Science and Food Technology, Complutense University of Madrid, 28040 Madrid, Spain

Departament of Food Technology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Carretera de la Coru˜na Km 7.5, 28040 Madrid, Spain

Departament of Food Biotechnology, Instituto de la Grasa-CSIC, 41012 Sevilla, Spain

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

and lactation could have a direct effect on infant health. On the other hand, mammary dysbiosis may lead

Mammary microbiota

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

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,

one of the main sources of bacteria to the breastfed infant gut since lactic acid bacteria.

∗

a baby consuming approximately 800 mL/day of milk would ingest

Corresponding author. Tel.: +34 91 3943837; fax: +34 91 3943743.

5 7

× ×

E-mail address: [email protected] (J.M. Rodríguez). between 1 10 and 1 10 bacteria daily [5]. This may explain

2 L. Fernández et al. / Pharmacological Research 69 (2013) 1–10

Table 1

showed the predominance of staphylococci, streptococci, lactic acid

Main bacterial genera or species isolated from human milk or which DNA sequencies

bacteria (LAB), propionibacteria and closely related Gram-positive

have been retrieved from this biological ﬂuid.

bacteria [4,5,18,19] (Table 1), including new bacterial species, such

Method Main species/genera References

as Streptococcus lactarius [20]. Human milk has also been shown

Bacterial isolation L. acidophilus, L. fermentum, S. epidermidis, [18] to be a source of live biﬁdobacteria to the infant gut [11]. The fact

Str. mitis, Str. salivarius that bacteria belonging to such genera can be isolated from fresh

L. plantarum, S. epidermidis, Streptococcus [19]

breast milk of healthy women from distant countries suggests that

spp.

their presence in this substrate is a common event. Therefore, they

E. faecium, L. fermentum, L. gasseri [4]

should be considered as components of the natural microbiota of

E. faecalis, L. crispatus, L. rhamnosus, Lc. [5,6]

lactis, Leuc. mesenteroides, R. mucilaginosa, human mammary gland, instead of mere contaminant bacteria. The

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

mammary microbiota is somehow peculiar since it has a transient

hominis, Str. mitis, Str. oris, Str. parasanguis,

nature: its development starts during the last third of pregnancy,

Str. salivarius

reaches the highest complexity at the end of such period, remains

L. salivarius [10]

Corynebacterium spp., Enterococcus spp., [63] quite constant throughout lactation, declines sharply at weaning,

Lactobacillus spp., Peptostreptococcus spp., and rapidly disappears when there is no milk in the mammary gland

Staphylococcus spp., Streptococcus spp.

and the apoptosis process responsible for mammary involution

L. reuteri [120]

begins (Fig. 1).

S. epidermidis [7,8]

Several studies have shown that there is a mother-to-infant

B. adolescentis, B. biﬁdum, B. breve [11]

B. breve, B. longum, K. rhizophila, L. casei, L. [20,24] transfer of bacterial strains belonging, at least, to the genera Lacto-

fermentum, L. gasseri, L. gastricus, L.

bacillus, Staphylococcus, Enterococcus, and Biﬁdobacterium through

plantarum, L. reuteri, L. salivarius, L.

breastfeeding [4,7,11,21–24]. Independently of the original source

vaginalis, P. pentosaceus, R. mucilaginosa, S.

of such bacteria, human milk is the source of hundreds of bacterial

aureus, S. epidermidis, S. hominis, Str.

lactarius, Str. mitis, Str. parasanguis, Str. phylotypes to the infant gastrointestinal tract. It has been suggested

salivarius that exposure of the breast-fed infant to such a wealth of bacte-

E. durans, E. faecalis, E. faecium, E. hirae, E. [22]

rial phylotypes may exert beneﬁcial effects against diarrheal and

mundtii, L. animalis, L. brevis, L. fermentum,

respiratory diseases, and may reduce the risk of developing other

L. gasseri, L. helveticus, L. oris, L. plantarum,

diseases, such as diabetes or obesity [25,26].

P. pentosaceous, Str. australis, Str.

gallolyticus, Str. vestibularis The application of culture-independent molecular techniques,

B. longum [23]

and particularly those based on 16S rRNA genes, allowed a

complementary biodiversity assessment of the human milk micro-

DNA detection E. faecalis, E. faecium, L. fermentum, L. [27,28]

gasseri, L. rhamnosus, Lc. lactis, Leuc. biome. The use of such techniques conﬁrmed the dominance of

citreum, Leuc. fallax, Prop. acnes, S.

staphylococci and streptococci and the presence of LAB, propi-

epidermidis, S. hominis, Str. mitis, Str.

onibacteria and biﬁdobacteria, and revealed the existence of DNA

parasanguis, Str. salivarius, W. cibaria, W.

confusa belonging to other bacterial groups, such as some Gram-negative

B. longum, Clostridium spp., Lactobacillus [65] bacteria [11,27–29]. In addition, the application of the “-omics”

spp., Staphylococcus spp., Streptococcus spp. approach (genomics, metagenomics, transcriptomics, proteomics,

B. adolescentis B. animalis, B. biﬁdum, B. [29]

and metabolomics) to the study of the human mammary micro-

breve, B. catenolatum, B. longum

biota is already in progress and there is no doubt that the results

Biﬁdobacterium spp., Clostridium spp., [121]

provided by such techniques will open new perspectives to under-

Enterococcus spp., Lactobacillus spp.,

Staphylococcus spp., Streptococcus spp. stand the initiation and development of the infant gut microbiota

B. adolescentis, B. biﬁdum, B. breve, B. [11] [30].

longum

Recently, the ﬁrst microbiome study focused on human milk

Bradyrhizobiaceae, Corynebacterium spp., [25]

was published [25]. The authors used microbial identiﬁcation

Propionibacterium spp., Pseudomonas spp.,

Ralstonia spp., Serratia spp., Sphingomonas techniques based on pyrosequencing of the V1–V2 region of the

spp., Staphylococcus spp., Streptococcus spp. bacterial 16S rRNA gene to characterize the bacterial communities

a present in milk samples collected from 16 women self-described as

Abbreviations: B., Biﬁdobacterium; E., Enterococcus; K., Kocuria; L., Lactobacil-

lus; Lc., Lactococcus; Leuc., Leuconostoc; P., Pediococcus; Prop., Propionibacterium; R., healthy at three time-points over 4 weeks. Results indicated that

Rothia; S., Staphylococcus; Str., Streptococcus; W., Weissella.

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,

why the bacterial composition of the gut microbiota of breast- eight other genera represented ≥1% of the communities observed

fed infants is closely related to that found in the breast milk of across samples. Among the hundreds of operational taxonomic

their respective mothers and, also, why the development of a more units (OTUs) detected in the milk of every woman, only 9 (Strep-

diverse microbiota coincides with the weaning period [12]. It must tococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacteria,

be highlighted that human milk olisaccharides (HMOs) also play a Ralstonia, Propionibacterium, Sphingomonas, and Bradyrhizobiaceae)

key role in driving the diversity of the infant gut microbiota [13]. were present in every sample from every woman. These 9 “core”

HMOs are the third-largest solid component of milk and, while their OTUs represented approximately half of the microbial commu-

structural complexity renders them non-digestible to the host, they nity observed, although their relative abundance varied greatly

are liable to hydrolytic enzymes of the infant colonic microbiota between subjects. The remaining half of the community was not

[14]. It has been previously demonstrated that selected biﬁdobac- conserved across women. These ﬁndings are in contrast with those

terial and lactobacilli phylotypes use speciﬁc HMOs secreted early of the gut microbiome, where only a low set of OTUs is shared

in the lactation cycle [15]; more recently, the genome sequences among individuals [31], or the vaginal microbiome which com-

of two biﬁdobacterial strains have revealed several adaptations for prises several different core groups [32]. On the other hand, milk

milk utilization within the infant microbiome [16,17]. bacterial community was generally stable over time within an indi-

The ﬁrst descriptions of the bacterial diversity of breast milk vidual. Similarly to the mammary microbiota, previous studies of

in healthy women were based on the use of culture media and various sites of the human microbiome have also revealed that the

L. Fernández et al. / Pharmacological Research 69 (2013) 1–10 3

• Hormonal signalling • Increasing recruitment of • Sharpe decrease in milk

103 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 Concentration of bacteria (CFU/ml)

Not detectable

Last third of pregnancy Peripartum Breastfeeding Weaning (~3 months) (a fewdays) (a few days-a few years) (some days-some weeks)

Time

Fig. 1. Schematic representation of the acquisition and development of the human mammary microbiota.

bacterial communities associated with a particular individual over However, both strains led to a reduction of Th1 cytokines when cells

time are often stable and highly personalized [33]. were incubated in the presence of lipopolysaccharide [40]. A recent

The results of a second study on the breast milk microbiome study conﬁrmed that L. fermentum CECT 5716 and L. salivarius CECT

involving 18 mothers indicated that milk bacteria are not contami- 5713 have a broad array of effects on the immune system [43]. They

nants and suggested that this site-speciﬁc microbiome is inﬂuenced behaved as potent activators of NK cells and moderate activators

by several factors that signiﬁcantly skew its composition [34]. More of CD4+ and CD8+ T cells and regulatory T cells. Thus, they had

recently, our research group analyzed the metagenome of human an impact on both innate and acquired immunity. They strongly

milk samples obtained from healthy, mastitis-suffering or obese induced a wide range of pro- and anti-inﬂammatory cytokines

women, and observed that the bacterial community of breast milk and chemokines. The authors compared these strains with others

may differ depending on the individual and on the health status of belonging to the same species but isolated from sources different

the lactating women [unpublished results]. 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-

2. Functions of human milk bacteria in the infant gut

bacillus gasseri CECT 5714 reduced the incidence and severity of the

allergic response in an animal model of cow’s milk protein allergy

In the last years, some studies have shown that human milk

[44]. It is also interesting to note that the presence of viridans strep-

bacteria may play several roles in the infant gut. First of all,

tococci, one of the dominant bacterial groups in human milk, seems

they can contribute to the reduction of the incidence and sever-

to be a feature of the healthy infant gut in contrast to that of the

ity of infections in the breastfed infant by different mechanisms,

atopy-suffering infants [45].

such as competitive exclusion [35], production of antimicrobial

Finally, human milk bacteria have a remarkable potential to play

compounds [6,9,10,35], or improvement of the intestinal barrier

metabolic roles in the infant. The glycobiome of some lactobacilli

function by increasing mucine production and reducing intestinal

and biﬁdobacteria, including that of species that have been isolated

permeability [35]. Recently, the administration of a human milk

from human milk, may help to create a speciﬁc “healthy” micro-

Lactobacillus strain to infants during 6 months led to 46%, 27%, and

biota in the infant gut [14,46]. These microorganisms might also

30% reductions in the incidence rates of gastrointestinal infections,

contribute to infant digestion through the breakdown of sugars and

upper respiratory tract infections, and total number of infections,

proteins; this possibility is particularly attractive having in account

respectively [36]. Commensal coagulase-negative staphylococci

that transit of food through the gastrointestinal tract is shorter in

and viridans streptococci provided by breast milk can be partic-

infants than in adults and, also, that the pH of the infant’s stomach

ularly useful to reduce the acquisition of undesired pathogens by

is higher than that of the adult. In this context, human milk lacto-

infants exposed to hospital environments. In fact, some Staphy-

bacilli strains are metabolically active in the infant gut and increase

lococcus epidermidis strains that inhibit in vivo colonization by

the production of functional metabolites such as butyrate, which is

Staphylococcus aureus have been postulated as a future strategy to

the main energy source for colonocytes and a relevant compound

eradicate such pathogen from the mucosal surfaces [37,38]. Sim-

in the modulation of intestinal function [47,48]. As a result, they

ilarly, it has been shown that viridans streptococci inhibit oral

improve the intestinal habit, with an increase in fecal moisture,

colonization by methicillin-resistant S. aureus in high-risk new-

and in stool frequency and volume [47,48].

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 3. Origin of the bacteria isolated from breast milk: is there

to modulate both natural and acquired immune responses in mice a bacterial entero-mammary pathway?

and humans [40–42]. Their function seems to have a certain degree

of ﬂexibility depending on the conditions found in the gut envi- The origin of the bacteria present in breast milk has become a

ronment. As an example, Lactobacillus salivarius CECT 5713 and controversial issue in the last years. Traditionally, it was believed

Lactobacillus fermentum CECT 5716 enhanced macrophage produc- that milk harbored bacteria that were just the result of contamina-

tion of Th1 cytokines, such as IL-2 and IL-12 and the inﬂammatory tion with bacteria from the mother’s skin or the infant’s oral cavity

mediator TNF-␣, in the absence of an inﬂammatory stimulus. [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 least, a very unlikely source [29]. These and other ﬁndings suggest

retrograde ﬂow back into the mammary ducts can occur during that at least some of the bacteria present in the maternal gut could

suckling. Obviously, such back ﬂow may provide an ideal route for reach the mammary gland through an endogenous route [56].

the exchange of bacteria from the infant’s mouth into the mam- Although the pathway and mechanisms that some bacteria

mary gland (Fig. 2). But it is also obvious that breast milk can also could exploit to cross the intestinal epithelium and reach the mam-

be a source of bacteria to the infant’s mouth. Ecological niches in the mary gland and other locations has not been elucidated yet, some

human microbiome are not thought to be isolated environments, works have offered a plausible scientiﬁc basis. It has been demon-

but rather a network of interrelated communities experiencing strated that dendritic cells (DCs) can penetrate the gut epithelium

constant exchange [33]. Therefore, it is very likely that milk or to take up non-pathogenic bacteria directly from the gut lumen.

mammary bacterial communities are no the exception, and that DCs are able to open the tight junctions between intestinal epithe-

they are constantly inﬂuenced by exposure to other microbial pop- lial cells, send dendrites outside the epithelium and directly sample

ulations associated with the mother and her infant. Little is known bacteria, while preserving the integrity of the epithelial barrier

about the infant human salivary microbiome but investigations on through the expression of tight-junction proteins [57]. Using such

adults have revealed that Streptococcus species are the dominant mechanism, a Salmonella typhimurium strain that was deﬁcient in

phylotype in this ﬂuid [50–52] while such dominance is even higher invasion genes encoded by Salmonella pathogenicity island 1 (SPI1)

in edentulous infants [53]. Streptococcus species are among the was still able to reach the spleen after oral administration to mice

most abundant phylotype in colostrum and milk samples [7,8,25], [57]. This mechanism may not be exclusive to DCs, as CD18+ cells,

supporting the hypothesis that the maternal milk bacteria may play including macrophages, have been shown to be essential for extra-

an important role in establishing salivary bacterial communities intestinal dissemination of non-invasive Salmonella [58].

and/or vice versa. It has also been shown that intestinal DCs can retain small num-

Some bacterial phylotypes usually present on adult skin, such bers of live commensal bacteria for several days in the mesenteric

as Staphylococcus, Corynebacteria, and Propionibacteria [54,55], are lymph nodes [59]. Once inside DCs and/or macrophages, gut bac-

also common in human milk. This presents the possibility that teria could spread to other locations since there is a circulation

interactions with the maternal skin microbiota may also contribute of lymphocytes within the mucosal associated lymphoid system.

to shape the composition of milk microbiota (Fig. 2). However, a Antigen-stimulated cells move from the intestinal mucosa to colo-

comparison of the bacterial communities detected in milk to those nize distant mucosal surfaces, such as those of the respiratory and

of the sebaceous skin found on the breast indicates that although genitourinary tracts, salivary and lachrymal glands, and, most sig-

the two communities share some phylotypes, major differences niﬁcantly, that of the lactating mammary gland [60]. In addition, it is

also exist [25]. Such relevant differences between the two commu- known that, during the lactation period, colonization of the mam-

nities indicate that bacterial communities in milk are not simply mary gland by cells of the immune system is a selective process

a result of skin contamination. Sampling of breast milk for micro- regulated by the lactogenic hormones [61]. This process is respon-

biological analysis must take into account that skin contamination sible for the abundance of such cells in human milk. A hypothetical

is almost unavoidable and that, logically, doubts on the original model to explain how some maternal bacteria could be transferred

location (internal mammary gland or skin) of the isolated bacteria to the neonatal gut is shown in Fig. 2.

may arise. However, independent studies have shown that lacto- The suggestion that the origin of the live bacteria found in breast

bacilli or biﬁdobacteria could not be isolated from breast skin swabs milk could be the maternal gut and that the bacteria would arrive

obtained from women that provided milk samples from which such to the mammary gland through an endogenous route, involving

bacteria were isolated [11,29]. Previously, it has been reported that maternal DCs and macrophages, has been conﬁrmed recently by

lactobacilli and enterococcal isolates present in human milk are independent research groups. Initially, our group showed that the

genotypically different from those isolated in the skin, within a exposure of mouse immature DCs to two bacterial strains isolated

same bacterial species and a same host [4]. In addition, biﬁdobac- from human milk led to a high stimulation of two DC activation

teria belongs to a strictly anaerobic genus and, therefore, skin is, at surface markers: the class II major histocompatibility complex and

L. Fernández et al. / Pharmacological Research 69 (2013) 1–10 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

Fig. 3. Speciﬁc interactions between cells of a L. gasseri strain isolated from breast

transiently, other body locations in healthy hosts, where they could

milk (A) and DC cells (B), as assessed by transmission electron microscopy [62].

contribute to extraintestinal probiotic effects. Additionally, human

The interactions were studied using transwell bicompartmental assays in which

bacterial cells and immature DC cells were initially separated by a monolayer of clinical studies have also indicated that oral intake of speciﬁc bac-

Caco-2 cells. terial strains at deﬁned doses play a key role in the prevention

or improvement of extraintestinal conditions, and such probiotic

the B7.2 protein [62,63]. It seems clear that bacterial induction of effects would be hardly explainable if they were exclusively con-

DC maturation is an active process since it has been shown that ﬁned to the gut [74]. Globally, these studies indicate that certain

dead bacteria or inert particles, such as latex beads, cannot activate intestinal bacteria may have a rather underrated ability to spread

DCs even if they are rapidly phagocytosed [64]. On the other hand, L. from the gut to extra-intestinal locations in healthy hosts.

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). 4. Human milk bacteria as biotherapeutic agents

Later, other study showed that bacterial translocation from the

gut to mesenteric lymph nodes and mammary gland occurred dur- In recent years, the problems associated to the spread of clin-

ing late pregnancy and lactation in mice [65]. In addition, this ical antibiotic resistances among pathogenic bacteria and to the

work revealed that human breast milk cells contain a number of rise of allergic or inﬂammatory diseases in developed countries

viable bacteria and a range of bacterial DNA signatures, which are have led to a new interest in probiotics, which are deﬁned as live

also found in maternal peripheral blood mononuclear cells. Those microorganisms that confer a health beneﬁt on the host when

peripheral blood mononuclear cells showed greater biodiversity administered in adequate amounts [75]. Obviously, probiotic bac-

than did those obtained from control women. Taken together, teria that are originally isolated from human milk are particularly

their results suggest that intestinally derived bacteria and bac- attractive organisms since they would fulﬁll some of the main crite-

terial components are transported to the lactating breast within ria generally recommended for human probiotics, such as human

mononuclear cells. The authors speculated that this programs the origin, a history of safe prolonged intake by a particularly sensi-

neonatal immune system to recognize speciﬁc bacterial molecular tive population (neonates, infants), and adaptation to mucosal and

patterns and to respond appropriately to pathogenic and com- dairy substrates [76].

mensal organisms [65]. More recently, two successive studies that Among the bacteria isolated from human milk, species like L.

focused on the oral administration of three lactobacilli strains iso- gasseri, L. salivarius, Lactobacillus reuteri, L. fermentum or Biﬁdobac-

lated from human milk (L. salivarius CECT 5713, L. gasseri CECT 5714, terium breve are considered among those with probiotic potential

and L. fermentum CECT 5716) provided new evidences that show the and enjoy the Qualiﬁed Presumption of Safety (QPS) status con-

existence of a bacterial entero-mammary pathway during lactation ceded by the European Food Safety Authority (EFSA). In contrast

[66,67]. to other bacteria, these seem to be uniquely adapted to reside

The mammary gland prepares for lactation through a series of in the human digestive tract and to interact with us in symbio-

developmental steps that occur during adolescence and pregnancy. sis from the time we are born. Additionally, some of the strains

The principal feature of mammary growth in pregnancy is a great isolated from this biological ﬂuid have been shown to play anti-

increase in ducts and alveoli under a multi-hormonal inﬂuence. At infectious, anti-inﬂammatory, immunomodulatory and metabolic

the end of this period, the lobules of the alveolar system are maxi- roles, both in vitro and in vivo, including human studies (see Sec-

mally developed and small amounts of colostrum may be released tion 2). The recent genome sequencing of some of these strains

for several weeks prior to delivery. Additionally, the nipple and [77–80] is providing some new clues to understand the relation-

areola markedly enlarge and the sebaceous glands within become ship between phenotypic properties and their subjacent molecular

more prominent [68]. The increased lymph and blood supply to basis [63]; in the future, such approach may contribute to a more

the mammary gland and the oxytocin release that causes contrac- rational selection, design and/or application of probiotic bacteria.

tion of the mioepithelial cells that invest the mammary alveoli may Exclusive breastfeeding during the ﬁrst month of life has been

also facilitate the presence of endogenous bacteria in breast milk. linked to lower asthma [81] and atopic dermatitis [82] rates during

These changes provide good conditions for bioﬁlm formation on childhood and, as a result, this practice has been strongly recom-

the mammary areola and/or in the mammary duct system, leading mended to mothers with a family of atopy, as a possible means

to the formation of a speciﬁc and transitory mammary microbiota. 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 respectively. In contrast, the effectiveness of antibiotics prescribed

lactobacilli can be effective to prevent atopy and atopic diseases to group C women differed signiﬁcantly, both in the reduction of

through a variety of mechanisms [83]. Gut bacteria are considered bacterial counts and in the improvement of the pain score.

the earliest and most important stimulus for development of gut-

associated lymphoid tissue and they can promote antiallergenic 4.2. Speciﬁc targets for human milk probiotic bacteria: anti-HIV

processes [83]. Therefore, milk of healthy women can be consid- activity

ered as a source of potentially probiotic or biotherapeutic bacteria

with a role in protecting mothers and/or infants against a variety Although breastfeeding is a source of new pediatric HIV-1 infec-

of allergic, inﬂammatory or infectious diseases. tions worldwide [96,97], most breastfed infants of HIV-positive

women remain uninfected despite repeated exposure of their oral

4.1. Speciﬁc targets for human milk probiotic bacteria: and gastrointestinal mucosal surfaces to the virus. Therefore, breast

lactational mastitis milk has been recognized both as a vector of transmission and as

vehicle of protection against HIV-1 [96].

Mastitis is a common disease during lactation since it has an Most breast milk-transmitted HIV infections take place in devel-

incidence of up to 33% of the lactating mothers [84,85]. This inﬂam- oping countries where breastfeeding usually remains the best

mation of one or more lobules of the mammary gland usually has an election for providing optimal infant nutrition and protection

infectious origin [86], involving staphylococci, streptococci and/or against morbidity and mortality associated with diarrheal and

corynebacteria [85]. Traditionally, S. aureus has been considered as lower respiratory infections [98,99]. In such countries, the choice

the main etiological agent of acute mastitis although S. epidermidis of the most suitable infant feeding option for an HIV-infected

is emerging as the leading cause of subacute and chronic mastitis mother should depend on her individual circumstances, including

both in human and veterinary medicine [87–90]. Multiresistance to her health status and the local situation, and should also consider

antibiotics, formation of bioﬁlms and mechanisms for evasion of the the health services available and the counseling and support she is

host immune response are common among clinical isolates of these likely to receive [100].

two staphylococcal species [91–95]. This explains why this condi- As a general rule, exclusive breastfeeding has been recom-

tion uses to be elusive to antibiotic therapy, and why it constitutes mended for HIV-infected women during the ﬁrst 6 months of

one of the main reasons to cease breastfeeding [85]. In this con- life unless replacement feeding is acceptable, feasible, affordable,

text, the development of new strategies for mastitis management sustainable and safe [97,100]. Such feeding option may offer HIV-

based on probiotics, as an alternative or complement to antibiotic 1-infected women in developing countries an affordable, culturally

therapy, is particularly appealing. acceptable, and effective means of reducing mother-to-child HIV-

For this purpose, our research group selected some lactobacilli 1 transmission while maintaining the beneﬁts of breastfeeding

strains on the basis of speciﬁc properties required for success in [101]. In fact, recent studies conﬁrm that exclusive breastfeeding

mastitis treatment after oral administration: a high survival rate can be successfully supported in HIV-infected women [102,103]. In

during transit through the gastrointestinal tract, speciﬁc interac- contrast, there seems to be an association between mixed breast-

tions with DCs, ability to colonize the mammary gland and, once feeding and increased HIV transmission risk [104].

there, mechanisms for competitive exclusion of mastitis-causing Several factors may modulate the risk of HIV-1 transmission

staphylococci and streptococci. Initially, a pilot trial highlighted via breastfeeding but the results of different studies focused on

the potential of L. salivarius CECT 5713 and L. gasseri CECT 5714, the potential role of speciﬁc milk components are not conclusive

two strains isolated from breast milk, for the treatment of staphy- [105–107]. Recently, it was shown that breast milk contains innate

lococcal mastitis [66]. After 30 days, probiotics led to a signiﬁcant factors that blocked infection of CD4+ cells with cell-free HIV, but

∼

reduction ( 2 log10 CFU/mL) in the mean staphylococcal counts of did not affect infection with cell-associated virus [107]. On the other

the milk cultures, in contrast, the values achieved in the placebo hand, humoral mucosal immunity to HIV does not appear to be

group remained unchanged at the end of the study. This fact was a protective factor against viral transmission through breast milk

correlated with the clinical evolution since, while no clinical signs [105,108]. Therefore, factors associated to protection against ver-

of mastitis were observed in women assigned to the probiotic group tical transmission of HIV-1 through breastfeeding remain poorly

at day 14, they persisted throughout the study in the placebo group. understood [96].

More recently, the efﬁcacy of L. fermentum CECT 5716 or L. Interestingly, the potential of some human milk strains to inhibit

salivarius CECT 5713, two lactobacilli strains isolated from breast the infectivity of HIV with tropism to different co-receptors has

milk, to treat lactational mastitis when administered orally was been described recently [109]. A total of 38 bacterial strains iso-

evaluated and compared to antibiotic therapy [67]. A total of lated from breast milk were investigated for their ability to inhibit

352 women with infectious mastitis were randomly divided HIV-1 infection in vitro. The heat-killed bacteria and cell-free super-

in three groups. Those in groups A (n = 124) and B (n = 127) natants obtained from some strains inhibited HIV-1 infection; the

ingested daily 9 log10 CFU of L. fermentum CECT 5716 or L. sali- highest levels corresponded to killed bacteria from Lactobacillus

varius CECT 5713, respectively, for 3 weeks while those in group curvatus VM25 (55.5%), L. fermentum VM31 (52.5%), Pediococcus

C (n = 101) were submitted to antibiotic therapy prescribed in pentosaceous VM95 (49.0%), and P. pentosaceous VM21 (45.5%).

their respective Primary Care Centres. On day 0, the mean bac- HIV-inhibition was observed with supernatants from eight strains,

terial counts in milk samples of the three groups were similar although the most effective were L. salivarius VM5 (42%) and L.

(4.35–4.47 log10 CFU/mL) and lactobacilli could not be detected. gasseri VM22 (40%) supernatants. Four strains (L. salivarius VM5, L.

On day 21, the mean bacterial counts in the probiotic groups (2.61 gasseri VM22, Lactococcus lactis VM17, and Streptococcus salivarius

and 2.33 log10 CFU/mL) were lower than that of the control group VM18) were selected for further evaluation and demonstrated dis-

(3.28 log10 CFU/mL). The probiotic treatment led to a signiﬁcant tinct inhibition patterns against R5-, X4- and R5/X4-tropic HIV-1.

reduction (1.7–2.1 log10 CFU/mL) in the milk bacterial count and These results demonstrate for the ﬁrst time that LAB from human

to a rapid improvement of the condition. The ﬁnal bacterial count breast milk can signiﬁcantly inhibit HIV-1 infection in vitro, and

was approximately 2.5 log10 CFU/mL, an acceptable bacterial load suggest a possible role for these bacteria in mucosal protection

in milk of healthy women [5,64]. After the probiotic treatment, L. against HIV-1 in breastfeed infants.

salivarius CECT 5713 and L. fermentum CECT 5716 could be isolated Human milk bacteria may exert an anti-HIV action in the

from the milk samples of women of the probiotic groups A and B, infant gut through a variety of mechanisms (Fig. 4), including

L. Fernández et al. / Pharmacological Research 69 (2013) 1–10 7

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 in gut microbiota and intestinal inﬂammatory parameters [117].

[110–112]. DC–HIV interaction is critical for the infection out- These ﬁndings support the hypothesis that gastrointestinal tract

come. Although DCs are able to induce speciﬁc immune responses alterations are a key factor in HIV pathogenesis, and may explain

against a broad variety of pathogens, HIV-1 has evolved ways to why infants who are exclusively breastfed have a signiﬁcantly

exploit DCs as cellular reservoirs, therefore facilitating viral dis- lower risk of being HIV-infected than infants who are bottle-fed

semination, persistence in lymphoid tissues and evasion of antiviral or mixed-fed [118,119].

immunity [113,114]. Additionally, oral lactobacilli isolated from

healthy humans can capture HIV-1 in vitro through lectin-like

5. Conclusion

interactions between mannose residues on the viral envelope gly-

coprotein gp120 and the bacteria [115]. This physical interaction

Human milk is a source of bacteria to the infant gut, where

(the so-called “HIV-1 trap”) may prevent viral attaching to cellular

they may play a variety of antiinfectious, immunomodulatory, and

receptors on mucosal tissues.

metabolic roles. In fact, recent studies indicate that the mam-

Recently, it was observed that the genome of L. salivarius CECT

mary gland contains its own microbiota during late pregnancy and

5713 contains genes encoding four proteins (1184, 1230, 1306, and

lactation. This bacterial community may differ depending on the

1686) potentially involved in human molecular mimetism [63].

individual and the health status of the lactating women. It seems

Among them, protein 1230 was considered of particular relevance

that certain bacteria from the maternal gut can use mononuclear

because of its similarity with proteins related to DC functionality,

immune cells to colonize, ﬁrst, the mammary gland and, later, the

such as CD209 antigen (DC-SIGN1) and the C-type lectin domain

infant gut through breast-feeding. If further studies conﬁrm these

family 4 member M (DC-SIGN2). In addition, protein 1230 contains

ﬁndings, they would have practical consequences since it would

a recognition motif for high mannose N-linked oligosaccharides

imply that modulation of the maternal intestinal microbiota can

present in a variety of pathogen antigens, including HIV gp120.

have a direct effect on her infant’s health, opening new perspectives

Consequently, it might have the potential to block gp120 from bind-

for bacteriotherapy and probiotics.

ing to target cells and, therefore, to inhibit HIV infectivity. To test

such predictions, the ability of L. salivarius CECT 5713 to stimu-

Acknowledgements

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

This study was partly supported by the FUN-C-FOOD

reduction in viral infectivity of TZM-bl target cells with R5 (HIV-

(Consolider-Ingenio 2010) and AGL2010-15420 projects from

1BaL), CXCR4 (HIV-1HC4) and R5/X4 (HIV-1C7/86) of 42.3, 58.9, and

the Ministerio de Economía y Competitividad (Spain).

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 References

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