the commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics

The commensalmicroflora ofhuman milk:

new perspectivesfor food

bacteriotherapyand probiotics

Rocıo Martına, Susana Langaa,Carlota Reviriegoa,

Esther Jimeneza, MarıaL. Marına, Monica Olivaresb,Julio Bozab, Jesus Jimenezb,

Leonides Fernandeza,Jordi Xausb and Juan

M. Rodrıgueza,*aDepartamento de Nutricion y Bromatologıa III,

Universidad Complutense de Madrid,28040 Madrid, Spain

(tel: +34-91-394-3747; fax: +34-91-394-3743;e-mail: [email protected])bDepartment of Immunology,

Puleva Biotech, 18004 Granada, Spain

Human milk is an important factor in the initiation, devel-opment and/or composition of the neonatal gut microflora.Bacteria commonly isolated from this biological fluidinclude staphylococci, streptococci, micrococci, lactobacilliand enterococci, which should be considered as compo-nents of the natural microflora of human milk, rather thanas mere contaminant bacteria. These bacterial groups con-tain strains with potential to be used as bacteriotherapeuticor probiotic agents. The origin of the bacteria found inhuman milk is debated and it is suggested that, at least,some species may be endogenously delivered from thematernal gut to the mammary gland. Although suchhypothesis can be a subject of controversy, it should sti-mulate further research in this fascinating area. If thishypothesis is verified, it would imply that modulation ofthe intestinal microflora of mothers can have a direct effecton the health of infants and, therefore, would open newperspectives for bacteriotherapy and probiotics.# 2003 Elsevier Ltd. All rights reserved.

Human milk as a source of commensal bacteriaDuring some months, breast milk is the best food for

the rapidly-growing infant since it fulfills all the nutri-tional requirements. Additionally, several studies haveshown that breastfeeding protects the newborn againstinfectious diseases (Lopez-Alarcon, Villalpando, &Fajardo, 1997; Wright, Bauer, Naylor, Sutcliffe, & Clark,1998). This effect may be due to the combined action ofsome breast milk components, such as immunoglobu-lins, immunocompetent cells or different antimicrobialcompounds (Saavedra, 2002). Breast milk also containsprebiotic substances, which selectively stimulate thegrowth of bacteria that exert positive effects in the gut(Dai & Walker, 1999; Drasar & Roberts, 1990).

From a microbiological point of view, studies focusedon human milk have been restricted to the identificationof potential pathogenic bacteria in stored milk and inclinical cases of maternal or infant infection (Bingen etal., 1992; El-Mohandes, Schatz, Keiser, & Jackson,1993; Le Thomas et al., 2001; Wright & Fenny, 1998).However, it seems clear that the natural flora of humanmilk contributes to prevent infant infections and thismay be one of the reasons that explain why the anti-

0924-2244/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2003.09.010

Trends in Food Science & Technology 15 (2004) 121–127

Viewpoint

* Corresponding author.

microbial activity exhibited by fresh collected humanmilk is lost after pasteurization (Ford, Law, Marshall,& Reiter, 1977). Although the knowledge of the com-mensal and/or potential probiotic bacteria that exist inmilk of healthy women is very limited, bacteria com-monly isolated from this substrate include staphylo-cocci, streptococci, micrococci, lactobacilli andenterococci (Gavin & Ostovar, 1977; Heikkila & Saris,2003; Martın et al., 2002; West, Hewitt, & Murphy,1979) (Table 1). The fact that bacteria belonging to suchgenera can be easily isolated in fresh milk of healthywomen from distant countries suggest that their pre-sence in this substrate is a common event. Therefore,they should be considered as components of the naturalmicroflora of human milk, instead of contaminantbacteria.

Human milk bacteria and colonization of theneonatal gut

Human milk is a major factor in the initiation anddevelopment of neonatal gut microflora, since this sub-strate represents a continuous source of microorganismsto the infant gut during several weeks after birth. It isestimated that an infant consuming approximately 800ml per day will ingest about 1 � 105–1 � 107 commensalbacteria while suckling (Heikkila, & Saris, 2003; Martınet al., 2002). In fact, the bacterial composition of theinfant fecal flora reflects the bacterial composition ofbreast milk (Heikkila & Saris, 2003). The presence of afew predominant Gram-positive species in breast milk

may be a reason explaining why gut microbiota ofbreast-fed infants is composed of a narrow spectrumof species, and a more diverse microbiota develops onlyafter weaning (Favier, Vaughan, de Vos, & Akkermans,2002; Hall, Cole, Smith, Fuller, & Rolles, 1990).

The composition of the gut microflora is deeply influ-enced by the diet of the infant. Introduction of solidfood and withdrawal of breast milk coincide with majormicrobiota changes (Favier et al., 2002). The major dif-ferences between the microbiological composition ofhuman milk and infant formula are probably the mainfactor responsible for the differences repeatedlyobserved between the gut microflora of breast- and for-mula-fed infants (Benno, Sawada, & Mitsuoka, 1984;Favier et al., 2002; Harmsen et al., 2000; Mackie, Sghir,& Gaskins, 1999; Yoshioka, Iseki, & Fugita, 1983).

Human milk bacteria as biotherapeutic agentsBreast milk is the unique food ingested by many neo-

nates, a population that is very sensitive to infectiousdiseases (Morris & Potter, 1997), specially when theyhave to stay in neonatal units for a prolonged time. Inrecent years, the problems associated to the spread ofclinical antibiotic resistances among bacteria has led toa new interest in bacteriotherapy, a practice that makesuse of commensal or beneficial bacteria to prevent ortreat colonization of the host by pathogens (Huovinen,2001; Reid, Howard & Gan, 2001; Strauss, 2000). Thisapproach is based on the competitive exclusion princi-ple, where the harmless commensal or probiotic bacteriasuccessfully compete with potential pathogens for thesame site (Mackie et al., 1999). Therefore, if bacteriawith the ability to provide infant health benefits, such asprotection against pathogenic bacteria, were isolatedfrom human milk, they would be immediately regardedas particularly attractive organisms since they wouldfulfill some of the main criteria generally recommendedfor human probiotics, such as human origin, a historyof safe prolonged intake by infants, and adaptation tomucosal and dairy substrates (Klaenhammer & Kullen,1999). In this context, Heikkila and Saris (2003) haverecently shown that commensal bacteria isolated fromhuman milk have potential use as bacteriotherapeuticagents in preventing neonatal and maternal breastinfections caused by Staphylococcus aureus.

Among the bacteria isolated from human milk, spe-cies like Lactobacillus gasseri, Lactobacillus rhamnosusor Enterococcus faecium are considered among thepotential probiotic bacteria (Holzapfel, Haberer, Snell,Schillinger, & Huis in’t Veld, 1998) and some strains areincluded in a variety of commercial probiotic products.Therefore, milk of healthy woman may be a source ofpotentially probiotic or biotherapeutic LAB with a rolein protecting mothers and/or infants against infectiousdiseases (Gavin & Ostovar, 1977). In addition to LAB,other bacteria found in milk, such as streptococci, can

Table 1. Bacterial groups and species commonly isolated fromfresh human milk of healthy womena

Bacterial group
Main species
Staphylococcus sp.
S. epidermidis S. hominis S. capitis S. aureus
Streptococcus sp.
S. salivarius S. mitis S. parasanguis S. peroris
Lactobacillus sp.
L. gasseri L. rhamnosus L. acidophilus L. plantarum L. fermentum
Enterococcus sp.
E. faecium E. faecalis
a Summarized from studies of Eidelman and Szilagyi (1979), El-Mohandes et al. (1993), Gavin and Ostovar (1977), Heikkila andSaris (2003), Martæn et al. (2002), West et al. (1979), Wright andFenny (1998), and Xaus et al. (2003).

122 Rocıo Martın et al. / Trends in Food Science & Technology 15 (2004) 121–127

be useful to reduce the acquisition of undesired patho-gens by high-risk newborn infants exposed to hospitalenvironments (Sprunt & Leidy, 1988; Tannock, 1997).For example, Uehara et al. (2001) have shown thatviridans streptococci inhibit oral colonization by methi-cillin-resistant S. aureus in infants.

Exclusive breastfeeding during the first months of lifehas been linked to lower asthma (Gdalevich, Mimouni,& Mimouni, 2001) and atopic dermatitis (Gdalevich,Mimouni, David, & Mimouni, 2001) rates during child-hood and, as a result, this practice has been stronglyrecommended to mothers with a family of atopy, as apossible means to prevent atopic eczema. Human milkLAB may play a role in this protective effect since it hasbeen described that probiotic lactobacilli can be effec-tive to prevent atopy and atopic diseases through avariety of mechanisms (Kalliomaki et al., 2001). Gutbacteria are considered the earliest and most importantstimulus for development of gut-associated lymphoidtissue and they can promote anti-allergenic processes(Kalliomaki et al., 2001). It is interesting to notethat the presence of viridans streptococci (organismshighly prevalent in milk) seems to be a feature of thehealthy infant gut in contrast with the atopic infant gut(Kirjavainen et al., 2001).

The presence of lactobacilli in breast milk could alsoexplain, at least partially, the existence of prebioticoligosaccharides in this fluid, since synthesis of suchcompounds by fructosyl- and glucosyltransferases fromhuman lactobacilli has been demonstrated recently(Kralj et al., 2002; van Hijum, van Geel-Schutten,Rahaoui, van der Maarel, & Dijkhuizen, 2002).

Origin of LAB isolated from breast milkCommensal staphylococci and streptococci seem to be

among the predominant bacterial species in breast milk(Eidelman & Szilagyi, 1979; Heikkila & Saris, 2003;West et al., 1979). It has been suggested that coagulase-negative staphylococci, with S. epidermidis as the pre-dominant species, may have originated from the mater-nal skin during breastfeeding (West et al., 1979). Incontrast, viridans streptococci, such as Streptococcussalivarius, are rarely found as skin residents but they arecommon in the infant mouth. Therefore, it is generallythought that streptococci are transferred from the infantmouth to the breast and from there to the milk (Heik-kila & Saris, 2003; West et al., 1979). Most authorsthink that the oral cavity, along with the rest of thedigestive tract, is initially colonized during the birthprocess by the vaginal and fecal microflora of themother (Mackie et al., 1999). However, our group hasfound that the milk that is expressed by the mammaryglands during the weeks previous to labor (and, there-fore, not submitted to any kind of infant contact) con-tains bacterial species similar to those isolated fromfresh milk obtained after labor. In addition, similar

results have been obtained in milk of women whichneonates were born by cesarean delivery (unpublisheddata). Such observations open new challenges on theorigin of, at least, some of the bacterial species found inhuman milk.

The origin of some of LAB species commonly foundin this biological substrate seems to be particularlypuzzling. Human lactobacilli and enterococci areusually mucosal-related bacteria and can be easily iso-lated from both the gastrointestinal and the urogenitaltracts. Heikkila and Saris (2003) obtained a few Lacto-bacillus crispatus isolates from milk and, speculated thatthey could have a vaginal origin, since this species is thepredominant vaginal lactobacillus in healthy women(Song et al., 1999). The infant would have resulted con-taminated during delivery and then would have trans-mitted it to the breast skin during nursing. The sameauthors were able to isolate L. rhamnosus isolates with aRAPD profile identical to the commercial strain GGand also a nisin-producing Lactococcus lactis from milkof Finnish women. L. rhamnosus GG is a frequentlyused probiotic strain in milk products in Finland, whilenisin-producing L. lactis strains are often isolated fromfermented foods. Therefore, it is very improbable thatsuch bacteria were present in the milk samples as aconsequence of mere external contamination.

These findings suggest that at least some of the LABpresent in the maternal gut can reach the mammarygland through a endogenous route, and that intestinalLAB may have a rather underrated ability to spreadfrom gut to extra-intestinal locations in healthy hosts.Cases reports of side effects of lactobacilli are veryscarce but a few cases of Lactobacillus endocarditis,sepsis or liver abscess have been reported, usually inpatients with a severe underlying illness and/oran immunocompromised status (Alvarez-Olmos &Oberhelman, 2001; Rautio et al., 1999). Interestingly,the liver abscess case was due to L. rhamnosus GG in apatient who daily ate big quantities of a GG-containingdairy product (Rautio et al., 1999). Although suchconditions can not be considered representative ofwhat may happen inside a healthy host, they illustratethe fact that lactobacilli can have mechanisms to spreadfrom the gut to other organs and that they are justoverlooked in healthy hosts because, under suchcircumstances, they do not lead to a pathogenic condi-tion. In fact, the very few number of case reports due tolactobacilli, which affected to severely ill or immuno-compromised hosts, means that lactobacilli (and otherLAB) intake can be considered as extraordinarily safe,since billion of doses of dairy probiotic products areingested each year (Reid, 2002) and a lot of pseudo-probiotic products are available in the market despitetheir health claims and safety have not been scientifi-cally evaluated (Hamilton-Meyer, Shah, & Winkler,1999).

Rocıo Martın et al. / Trends in Food Science & Technology 15 (2004) 121–127 123

Probably, in addition to the digestive and genito-urinary tracts, LAB can inhabit, permanently or tran-siently, a variety of other body locations in healthyhosts, where they could contribute to extra-intestinalprobiotic effects. Recently, clinical evidence that pro-biotic lactobacilli can be endogenously delivered to thehuman vagina following oral intake has been provided(Reid, Bruce et al., 2001). Additionally, human clinicalstudies have also indicated that oral intake of specificLAB strains at defined doses play a key role in the pre-vention or improvement of extraintestinal conditions, andsuch probiotic effects would be hardly explainable if LABwere exclusively confined in the gut (Aso et al., 1995).

Although the exact pathway that LAB could exploitto cross the intestinal epithelium and reach the mam-mary gland and other locations remains unclear, recentworks offer a reasonable explanation (Qutaishat et al.,2003; Rescigno et al., 2001). Penetration of the gutmucosa by pathogens expressing invasion genes hasbeen believed to occur mainly through M cells, specia-lized epithelial cells that are located in Peyer’s patches.Recently, it has been demonstrated that dendritic cellscan penetrate the gut epithelium to take up bacteriadirectly from the gut lumen (Rescigno et al., 2001).Dendritic cells are able to open the tight junctionsbetween intestinal epithelial cells, send dendrites outsidethe epithelium and directly sample bacteria, while pre-serving the integrity of the epithelial barrier through theexpression of tight-junction proteins (Rescigno et al.,2001). Using such mechanism, a Salmonella typhimur-ium strain that was deficient in invasion genes encodedby Salmonella pathogenicity island 1 (SPI1) was stillable to reach the spleen after oral administration tomice (Rescigno et al., 2001). Once inside dendritic cells,other lymphocytes types and/or macrophages, bacteriacould spread to other locations since there is a circula-tion of lymphocytes within the mucosal associated lym-phoid system (Roitt, 1994). Antigen-stimulated cellsmove from the intestinal mucosa to colonize distantmucosal surfaces, such as those of the respiratory andgenitourinary tracts, salivary and lachrymal glands,and, most significantly, that of the lactating mammarygland (Roitt, 1994). In addition, it is known that, duringthe lactation period, colonization of the mammarygland by cells of the immune system is a selective pro-cess regulated by the lactogenic hormones (Bertotto etal., 1991). This process is responsible for the abundanceof such cells in human milk. Recently, transmission ofS. typhimurium DT104 to infants through maternalbreast milk has been reported (Qutaishat et al., 2003).The authors showed that the pathogen was initiallylocated in the maternal gut and reached the mammarygland following an endogenous route similar to thatdescribed above. Figure 1 shows an hypothetical modelthat may explain how some maternal LAB strains couldbe transferred to the neonatal gut.

The mammary gland prepares for lactation through aseries of developmental steps that occur during adoles-cence and pregnancy. The principal feature of mam-mary growth in pregnancy is a great increase in ductsand alveoli under the influence of many hormones. Latein pregnancy, the lobules of the alveolar system aremaximally developed and small amounts of colostrummay be released for several weeks prior to delivery.Additionally, the nipple and areola markedly enlargesand the sebaceous glands within become more promi-nent (Beischer, Mackay, & Colditz, 1997). The increasedlymph and blood supply of the mammary gland and theoxytocin release that causes contraction of the mioe-pithelial cells that invest the mammary alveoli may alsofacilitate the presence of endogenous bacteria in breastmilk (Beischer et al., 1997). These changes provide goodconditions for biofilm formation on the mammaryareola and/or in the mammary duct system.

Can maternal LAB cross the placenta barrier andreach the fetal gut?

Since the old studies of Tissier (1900) concerning theacquisition of the infant gut microflora, the idea thatfetuses are sterile in utero and that bacterial coloniza-tion of the newborn intestinal tract starts during thetransit through the labour channel, due to cross-con-tamination with vaginal and faecal bacteria of thematernal microflora, has been widely accepted (Isolauri,Sutas, Kankaanpaa, Arvilommi, & Salminen, 2001;Mackie et al., 1999; Tannock, 1995). However, ourgroup has found that LAB and other bacteria can beisolated, although at low counts, from placenta, amnio-tic fluid, umbilical chord vessels and meconium of heal-thy pre-breastfed newborns, including those deliveredby cesarean section. Interestingly, although some of theisolates shared identical RAPD and RFLP profiles withisolates of the same species obtained from maternalmilk and faeces, we were unable to establish relation-ships with vaginal, perineal or skin isolates (Xaus et al.,2003). Experiments with pregnant mice orally adminis-tered with a labeled E. faecium strain showed a low leveltransfer of the strain to the fetal gut and a high leveltransfer to the mammary glands (Xaus et al., 2003).Globally, these results strongly suggest that a few com-mensal species may cross the placenta and, therefore,that gut colonization may begin before birth.

It is well-known that a few extracellular pathogenicbacteria can enter the central nervous system becausethey can interact with and cross the monolayer of tight-junction-forming cells that constitute the cerebral endo-thelium or blood–brain barrier (Nassif, Bourdoulous,Eugene, & Couraud, 2002). These pathogens includeStreptococcus pneumoniae and group B streptococci,bacteria closely related to the predominant speciesfound in human milk. The bacterial characteristicsrequired to interact with the brain barriers might have


been selected initially to allow mucosal colonization(Nassif et al., 2002). One in the bloodstream, a similarmechanism could be used by specific gut bacteria tocross the placental barrier. The placenta is derived fromthe trophoblast as a specialized development of thatportion which is in closest relation to the underlyinguterine decidua. Initially, three layers (the wall of thefetal capillary in the villus, a variable amount of villousconnective tissue and the trophoblastic epitheliumwhich cover the villus) separate the maternal and foetal

circulations, but they become progressively more inti-mate as these tissues thin out during pregnancy. Nearterm, there is an increase in the efficiency of theexchange of nutrients, waste products and gases betweenmother and fetus because of the thinning of this barrier(Mahan & Arlin, 1992). This increased exchange couldalso involve selected commensal bacteria that, sub-sequently, would initiate gut colonization as a firstadaptation of the fetal gut for life outside the mother(Fig. 1).

Fig. 1. A hypothetical model to explain how some maternal LAB strains to could be transferred to the foetal and neonatal gut. (A) Dendriticcells could penetrate the gut epithelium to take up LAB directly from the gut lumen as previously shown for non-invasive bacteria byRescigno et al. (2001). (B) Once inside antigen-stimulated cells, LAB could move from the intestinal mucosa to colonize distant mucosalsurfaces, since there is a circulation of lymphocytes within the mucosal associated lymphoid tissues, including those found in the respiratoryand genitourinary tracts, salivary and lachrymal glands, and, most significantly, that of the lactating mammary gland (Roitt, 1994). Abbre-viations: GUM, genitourinary tract mucosa; LMGM, mucosa of the lactating mammary gland; MLN, mesentric lymph node; PP, Peyer patches

and associated lymphoid tissue; RM, respiratory tract mucosa; SLGM, mucosa of the salivary and lacrimal glands.


ConclusionsHuman milk is a consistent source of commensal

bacteria to the neonatal gut and, therefore, it is notstrange that the bacterial composition of the infant fecalflora reflects the bacterial composition of breast milk. Inthis paper, we suggest that may exist an efflux of LABfrom the gut of healthy (pre)lactating women to theirfetuses/neonates; firstly, there would be a low-leveltransit of selected bacterial species across the placentalbarrier that allows their transference to the prenatal gut;secondly, but most important in quantitative terms,there is an efflux to the neonate gut through breastfeed-ing. Although such hypothesis will surely becontroversial, we hope that it can serve to stimulatefurther research in this fascinating area. Modernmolecular ecology techniques can facilitate the study ofthe complex microbial relationships among differentmucosal ecosystems. If our hypothesis was verified, itwould have practical consequences since they wouldimply that modulation of the maternal intestinal micro-flora can have a direct effect on the health of infants.This would open new perspectives for bacteriotherapy(including food delivery of selected strains) and probio-tics. As Favier et al. (2002) stated, adequate knowl-edge of the mucosal-associated microorganisms, as wellas the events that influence the timing of colonization,may provide opportunities to modulate the microbiotawhen modulation is necessary to enhance importantfunctions ranging from postnatal intestinal maturationto maintenance of mucosal barrier and nutrientabsorption.

AcknowledgementsThis study was partly supported by a grant from the

Ministerio de Ciencia y Tecnologia (Spain) and by agrant of Puleva Biotech (Granada, Spain).

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the commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics

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