Microbial ecology and host-microbiota interactions during early life stages

Abstract

The role of human microbiota has been redefined during recent years and its physiological role is now much more important than earlier understood. Intestinal microbial colonization is essential for the maturation of immune system and for the developmental regulation of the intestinal physiology. Alterations in this process of colonization have been shown to predispose and increase the risk to disease later in life. The first contact of neonates with microbes is provided by the maternal microbiota. Moreover, mode of delivery, type of infant feeding and other perinatal factors can influence the establishment of the infant microbiota. Taken into consideration all the available information it could be concluded that the exposure to the adequate microbes early in gestation and neonatal period seems to have a relevant role in health. Maternal microbial environment affects maternal and fetal immune physiology and, of relevance, this interaction with microbes at the fetal-maternal interface could be modulated by specific microbes administered to the pregnant mother. Indeed, probiotic interventions aiming to reduce the risk of immune-mediated diseases may appear effective during early life.

Human Microbiome

The human microbiome (HM) is a complex system of many microbial communities inhabiting a diversity of environmental niches throughout the human body. The human gastrointestinal tract (GIT) is inhabited by a complex and dynamic population of around 500–1000 different microbial species. HM exhibits large variation among individuals in relation to internal and external factors such as genetic factors, age, diet and health, and remains in a complex equilibrium. Although the exact composition of the microbiota is not known, advances in genomic technologies have recently begun to unravel our microbial partners. It is estimated that each individual houses at least 160 such species from a consortium of 1000 to 1150 prevalent bacterial species¹ whose collective genome (“microbiome”) contains at least 100 times as many genes as the human genome. It is known that about 75% of the gut microbiome is covered by already known and dominant phyla (Actinobacteria, Firmicutes, and Bacteroidetes), while a fraction of about 25% is still unknown.

The microbiota has begun to receive growing attention in recent years since its remarkable relationship with health and disease has been identified. It plays an important role on human health by impacting nutrient utilization, by enhancement of gut barrier function and by stimulation of immune development. An adequate balance of the microbiota plays a critical role in maintaining the health status of the host. Intestinal colonization, a substantial antigen challenge for the newborn, is essential for the maturation of the gut-associated lymphoid tissue and for the developmental regulation of the intestinal physiology.² Moreover, on molecular levels, members of the intestinal microbiota have been shown to possess a significant capacity to modulate the expression of a large number of host genes involved not only in mucosal barrier function but also in a variety of other intestinal functions, including nutrient absorption, metabolism, angiogenesis and intestinal maturation.³

Origin and Development of Microbiota

The development of the human microbiome is a complex process that has been traditionally assumed to start at birth when the infant is exposed to the mother’s microbiota and continues to develop and change during life. Furthermore, microbiome is known to play an essential role for the maturation of the immune system and the establishment of the gut barrier. Moreover, alteration in this process of colonization has been suggested to increase the risk of disease later in life.⁴

At birth, the neonate is going to be exposed to an avalanche of new microbes and the infant gut begins to be gradually colonised by a rapidly diversifying microbiota. The first microbes to colonise are facultative anaerobes such as enterobacteria, coliforms, lactobacilli and streptococci followed on the 2nd-3rd day by anaerobes such as bifidobacteria, bacteroides, clostridia and eubacteria as the main microbes in 1–2-week-old infant faeces.⁴ The most common bifidobacteria present in infant faeces are B. longum, B. infantis and B. breve.^5,6 Molecular methods also demonstrate the presence of other types of bacteria such as Ruminococcus and Akkermansia-like bacteria already early in infant gut.^5,7 Thereafter, colonization is further modulated not only by environmental bacteria but also by early breast milk, colostrum, which contains live microbes, especially bifidobacteria and lactic acid bacteria, and a spectrum of bioactive substances. The changes in microbiota composition during weaning are more drastic in breast-fed than in formula-fed infants.⁸ After weaning the gut microbiota continues to develop toward the adult-like pattern. Throughout the first year of life, the GIT microbiota changes continuously and increases in diversity until reaching an adult-like composition. Microbiome development in the GIT has been far more studied than in other body habitats. This process will be affected by feeding habits, health status, and sanitary conditions. The early GIT microbiota is often dominated by one or a few genera, namely Escherichia, Clostridium, Bacteroides or Bifidobacterium. After the first 6 mo of life when solid food is introduced to the infant, the gut microbiota becomes more diverse. The succession of Bacteroides, Clostridium and anaerobic bacteria increases rapidly while the proportion of bifidobacteria becomes more stable. However, the specific taxa present before and after this point may vary.^9,10 Functional characterization of the infant microbiome lags behind studies of phylogenetic composition, but recent metagenomic analyses have initiated this task for gut microbiota development. In spite of taxonomical disparities, the infant gut rapidly acquires a functional gene repertoire dominated by carbohydrate metabolism genes and, as mentioned, broadly analogous to that of the adult. Specialized functional repertoires may exist at different stages, as the earliest microbiota can be enriched in genes facilitating lactate utilization whereas solid foods may promote enrichment in genes enabling utilization of a larger variety of carbohydrates, vitamin biosynthesis and xenobiotic degradation. However, genes involved in plant polysaccharide metabolism are also detected in the microbiota before the introduction of solid foods.^9,10

Factors Influencing Microbiota Development

Microbiota development of the infant is rapid and depends on the first inoculum, the mother’s microbiota, mode of delivery, type of feeding and the environment, including weaning food practices and the use of antimicrobials.¹¹

Maternal microbiota, diet and lifestyle

The maternal gut is the most important microbial source in the early colonisation of the infant’s gut. However, microbial colonization of the human body may begin earlier as bacteria have been found in umbilical cord, placenta, amniotic fluid and also in meconium.^12-14 Microbiota differences have been reported during pregnancy and also, were associated with maternal health.¹⁵ Then, pregnant women may create a circle of unfavorable environment generated by inappropriate gut microbiota which would be transferred to the infant during pregnancy and birth.⁷ In addition, a clear association on bifidobacteria colonization between mother and infant has been found. Maternal colonization by B. bifidum had the most consistent effects on the infant's bifidobacterial microbiota.¹⁶ Another study^17,18 suggested that early dietary and gut microbiological environments have a more complex effect on the metabolic programming of a child. Moreover, maternal nutritional habits during pregnancy appear to influence the type of microbes present in the meconium at the moment of birth. Infants whose mothers consumed an organic or biodynamic diet seemed to have slightly lower numbers of E. coli than did infants whose mothers consumed a regular diet.^19,20

Mode of delivery

Differences in microbiota composition depending on delivery mode will affect the early stage of neonatal microbial colonisation.²¹ Of note, infants born by caesarean section have a different microbiota composition as compared with vaginally delivered newborn infants.²² This, unequivocally will have an impact on their health and indicates a mother–child transmission of microbiota during vaginal delivery. Indeed, an increased risk for atopic diseases such as allergic rhinitis, asthma and celiac disease among other was reported in children born by caesarean section.^21,23,24

Vaginally delivered infants come in contact with the maternal vaginal and faecal microbiota which results in the gut colonisation by microbes from mother’s birth canal. In infants born by caesarean section (C-section) the establishment of gut microbiota is delayed. These infants are more often colonised with clostridia and bacteroides and less with bifidobacteria and lactobacilli.^19,20,25,26 16S rRNA pyrosequencing has revealed that vaginally delivered infants acquire bacterial communities resembling their own mother’s vaginal microbiota, dominated by Lactobacillus, Prevotella or Sneathia, whereas C-section infants acquire bacterial communities similar to those found on the skin, dominated by Staphylococcus, Corynebacterium and Propionibacterium.²²

Infant’s diet

Transfer of microbiota continues during breastfeeding which plays a key role during the infants first months of life. Hence, exclusively breastfed infants exhibit significant differences in gut microbiota as compared with formula fed.^5,6,27 Breast milk has been shown to be an excellent and continuous source of bacteria including staphylococci, streptococci, lactic acid bacteria and bifidobacteria.^28-30 In addition, components such as oligosaccharides support the growth of beneficial bacteria, which may critically contribute to the establishment of a healthy microbiota in breast-fed infants. Among breast-fed infants, Bifidobacterium-dominated microbiotae which can reach up to 60–90% of the total faecal microbiota are more frequent than among infants fed with formula, but other compositions are not uncommon.^5,6,17 Other microbes present are Staphylococcus, Streptococcus, Corynebacterium, Lactobacillus, Micrococcus and Propionibacterium originating from nipple, milk ducts and the surrounding skin. Transmission of specific intestinal Bifidobacterium strains from mothers to infants have been recently reported^31,32 suggesting that each mother-infant pair might have unique family-specific strains and breastfeeding could contribute to the microbial transfer from the mother to the infant and, therefore, impact on the infant gut colonization.³³ Formula fed microbiota is more complex and similar to that of adults, and is characterized by the predominance of facultative anaerobes such as Bacteroides and Clostridium followed by Staphylococcus, Streptococcus and Enterobacteriaceae, and delayed colonisation by bifidobacteria.^25,34, The target of infant formula research has been to develop product composition that closely resembles breast milk. There are, however, very little data on changes in the composition of the intestinal microbiota during early weaning and how the influence of pre-weaning feeding methods and perinatal events impact on the microbiota after the introduction of complementary foods.²⁶

Lifestyle and geographical location

Single children tend to have lower counts of bifidobacteria in the gut at one month of age,¹⁹ non-E. coli enterobacteria and clostridia, and a lower ratio of anaerobic to facultative bacteria by one year of age compared with children with siblings.³⁵ It has been also described, that infants living on a farm had a low diversity and a pronounced dominance of some Clostridium and Eubacterium species while other children had a more diverse microbiota.³⁶

Based on numerous reports, it seems that the geographical differences in microbiota composition result from the different dietary habits in the region or the country. A recent study³⁷ showed a “geographical gradient“ in the infant microbiota across Europe where northern infants showed higher levels of Bifidobacteria, Atopobium, C. perfringens, C. difficile while Southern infants presented higher proportion of Bacteroides, Eubacteria and Lactobacillus. Higher counts of lactobacilli, eubacteria and enterococci have been described in Estonian infants while Swedish infants showed high numbers of clostridia and bacteroides.³⁸ Other studies also reported higher levels of bifidobacteria in Italian adult subjects as compared to Swedish, French and German adults.³⁹ It has also been reported that Bifidobacteria genus, Bacteroides-Prevotella and Cl. histolyticum levels were higher in Malawi infants than Finnish infants at age of 6 months.⁴⁰ A similar study demonstrated that children living in a rural village in Burkina Faso are characterised by an enrichment of Bacteroides while Italian children of Enterobacteriaceae.⁴¹

Antibiotics, probiotics and prebiotics

Antibiotics are very commonly used to treat specific infections but often without considering their impact on gut microbiota. Antimicrobials are not exclusively selective for pathogens and indiscriminately affect all members of the commensal gut microbial ecosystem, especially decreasing the levels of anaerobic bacteria, Bacteroides, E. coli and beneficial bifidobacteria and increasing the levels of potentially harmful clostridia and Klebsiella.^19,42 Oral use of antibiotics (mainly amoxicillin) and antimycotics (miconazole) by the infant during the first month of life resulted in decreased numbers of bifidobacteria and B. fragilis-group species.^19,20 The efficacy of probiotics reducing antibiotic-induced diarrhea has been extensively studied and their ability to efficaciously modulate the gut microbiota and restore the disturbed microbial ecosystem after antibiotic therapy proven.⁴³ Meta-analyses conducted in this area⁴⁴ concluded that available data provide sufficient evidence for the role of probiotics in the prevention of antibiotic-associated diarrhea (2874 participants; RR 0.52; 95% CI 0.38 to 0.72; I(2) = 56%).

Other factors

Gestational age has a profound role in determining the gut microbiota.^19,45 In newborn infants, especially those prematurely born, who are hospitalised and therefore separated from their mothers for long periods immediately after birth, the environment becomes an important source of colonising bacteria.^25,42

Microbiota and Human Health

In recent years, the increase in microbiota-related research has provided important advances toward establishing the identity of specific microbes and microbial groups or microbial molecules contributing to various aspects of host physiology and health. Studies on human microbiota should include microbial ecology and analysis of the complex metabolism of the microbial community, as well as various host–microbial interactions occurring at the interface between microbes and host intestinal epithelia. Such studies should lead to an understanding of the impact of the microbiota on human health and disease. Concurrently, host factors involved in various aspects of development and maturation targeted by the microbiota have been identified.

A balance among microbial groups present in the human gut is crucial for maintaining health. When this balance is disturbed, the host–microbe relationship can progress toward a disease state. The importance of the microbiome in relation to human disease has been stressed by a recent National Institutes of Health (NIH) initiative, the “Human Microbiome Project,” which aims to assess how changes in the microbiome correlate with human disease using genomic and metagenomic sequencing.⁴⁶

Altered intestinal colonization by commensal microorganisms, increased occurrence of potential pathogens, as well as high interindividual variability and reduced microbial diversity have been reported in preterm infants⁴⁵ increasing the risk to develop later disease. Several gastrointestinal pathologies such as IBD, IBS, NEC, obesity, various forms of colitis and even autism has been linked to disturbances in human-associated microbiota or alterations of the intimate cross-talk between these microbes and human cells.⁴⁷ Numerous studies have linked early gut microbiota to the development of atopic diseases, but no specific microbes have yet been identified with consistently harmful or protective roles regarding atopy.^48,49 Several studies indicate a link between bifidobacteria deficiency and increased incidence of atopy.^50,51 However, large prospective case-control studies have failed to confirm this association²⁰ and conflicting results have been obtained regarding the protective role of different Bifidobacterium species.⁵¹ The development of metabolic complications associated with obesity during childhood tracks into adulthood is related to risk factors such as diet, lifestyle, decreased physical activity and rapid infancy weight gain. However, some reports have suggested that the gut microbiota is an important factor affecting the regulation of host energy homeostasis and adiposity, as differences in microbial composition can explain an increased capacity of the obesity-associated microbiome to harvest energy from the diet.^52,53 A subsequent metagenomic study⁵⁴ with 154 individuals showed that obesity was associated with a markedly reduced bacterial diversity, a relative depletion of Bacteroidetes, and a higher proportion of Actinobacteria compared with lean subjects. However, lower ratios of Firmicutes to Bacteroidetes in overweight human adults compared with lean controls have recently been reported.⁵⁵ In addition, diets based on a high intake of protein and/or low intake of carbohydrate or low fat consumption may alter microbial composition and activity in the large intestine and thus impact gut health.⁵⁶ Other studies have examined gut microbiota composition in human obesity and type-2 diabetes and the impact of weight reduction on microbiota,^57,58 some of which contest the link between the proportion of Bacteroidetes and Firmicutes and human obesity.⁵⁹ In addition, it has been shown that infants with high numbers of Bifidobacterium and low numbers of Staphylococcus in early life may be protected from weight gain during later life.⁶⁰ Hence, infants born to women with normal weight gain during pregnancy showed higher levels of bifidobacteria than those from women with excessive weight gain, suggesting a potential role for Bifidobacterium on infant microbiota and weight development.^8,15 A recent study reported that high numbers of bifidobacteria may positively correlate with normalization of inflammatory status and improved glucose tolerance and glucose-induced insulin secretion.⁵⁶ Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion, action, or both. A link between gut microbiota composition and activity and the management of glycemia associated with overweight and diabetes has been reported.⁶¹ In type-1 diabetes, a combination of multiple factors leading to the development of autoimmunity appears to include an aberrant intestinal microbiota, a leaky intestinal mucosal barrier and an altered intestinal immune responsiveness.⁶² Type 2 diabetes is a metabolic disease and the primary cause is obesity-linked insulin resistance. Recent research⁵⁸ reported that type 2-diabetes is associated with compositional changes in the intestinal microbiota. The relative abundance of Firmicutes was significantly lower, while the proportion of Bacteroidetes and Proteobacteria was higher in diabetic persons compared with non-diabetic counterparts. Accordingly, the ratios of Bacteroidetes to Firmicutes positively correlated with reduced glucose tolerance. These results, assuming that diabetes and impaired glucose tolerance are linked to obesity, are in agreement with the recent evidence obtained for overweight persons by Schwiertz and colleagues.⁵⁵

Early Microbial Exposure

As we outlined above, the gastrointestinal tract of the fetus has been generally considered microbiologically sterile but there are indications of microbial exposure already prior to delivery.^12-14,63 Microbial colonization of the human body may begin earlier through transport of maternal bacteria via the bloodstream to the placenta from where they could reach the umbilical cord or the amniotic fluid, which is constantly being swallowed by the fetus.^12,13,64 Different bacterial species in the amniotic fluid of preterm pregnancies were detected and further demonstrated that women whose amniotic fluid was PCR-positive for bacteria exhibited elevated levels of IL-6, histological chorioamnionitis and funisitis, which were strongly associated with the development of neonatal sepsis.⁶⁵ In other study, Leptotrichia spp and other related bacterial species were detected using culture-independent molecular approaches in the amniotic fluid of women in preterm labor with a strong dose-dependent relationship between bacterial abundance in the amniotic fluid and gestational age at delivery.⁶⁴ Furthermore, bacteria and/or their DNA have been also isolated from the meconium of healthy neonates.^13,14,66,67 Of note, bacterial colonization of placenta in non-pathologic situations has been recently reported even in subjects delivered by elective caesarean section with a very low risk of contamination.⁶⁶ Interestingly, translocation of bacteria from the oral cavity had been previously described.⁶⁵ All these studies provide evidence for direct in utero microbial exposure and the possibility that introduction of the developing fetus to microbial products may play a role in shaping postnatal immune development.⁶⁸ As contact with microbes takes place already in utero the impact of maternal intestinal microbiota may be more important than earlier understood.

Observational and interventional studies suggest that exposure to microbes during pregnancy may influence the metabolic and immunologic profiles of the pregnant uterus and, hence, the risk of disease developing in the offspring later in life.^69,70 It has been reported microbiota differences during pregnancy associated with serum biochemical variables of relevance to the nutritional and health status during pregnancy (eg, cholesterol, folic acid, ferritin, and reduced transferrin) and therefore with possible consequences on fetal health programming.^15,57 Moreover, in case of pregnant obese women, a vicious circle of unfavorable metabolic development may be generated if abnormal gut microbiota related to overweight, or excessive weight gain during pregnancy, is transferred to the infant.^7,15 However, there is no direct evidence on the roles and mechanisms of action of each bacterial group in those instances. Under these circumstances, the possible role played by the composition of the gut microbiota in women’s health during pregnancy and its possible influence on the maternal-fetal interactions in utero have also been recently investigated.^71-73 Of note is the fact that these studies led to the conclusion that maternal microbial exposure during pregnancy, and perinatal and postnatal periods may have a significant impact on infants’ health.

Altogether, the complex interactions between microbes and host combined with recent clinical observations and epidemiologic trends may point to the convergence of two hypotheses: the “hygiene hypothesis” and the “fetal programming hypothesis.” The hygiene hypothesis supports that the rapid increase of the incidence of allergy could be related to lower microbe exposure at early life and subsequent lower number of infections during infancy.⁷⁴ Until recently it has been assumed that the human fetus develops in a sterile environment and is therefore born immunologically inexperienced and immature. However, it is established that maternal exposures during pregnancy may induce long-lasting or even permanent changes in fetal physiology and thereby have an impact on disease risk in later life (Fetal programming hypothesis).⁷⁵ We are beginning to understand that exposure to microbes before conception, during gestation, and in the neonatal period have profound effects on health,⁷⁶ since maternal microbial environment during pregnancy may remarkably influence the newborn immune development and, consequently, impact infant’s health early and later on in life. Recent evidence indicates the importance of prenatal and postnatal developmental processes in terms of maturation of balanced immune responses. It is well established that early microbial colonization events provide the neonate with vital stimuli that guide the immune maturation. Disturbances in this process may result in the development of immune disorders such as atopic disease, allergy and asthma, which are regarded as a failure in the balanced microbial composition and immune response.^48,49,68 Experimental murine models demonstrate that maternal treatment with lipopolysaccharide or the commensal Acinetobacter lwoffii during gestation attenuates allergic sensitization and airway inflammation in the offspring.⁷⁷ Also, epidemiological studies indicate that maternal farm environment exposure during pregnancy protects against allergic sensitization, asthma and disease, and have an upregulation of receptors of the innate immune system whereas exposures during infancy alone are under discussion.^78,79 Furthermore, maternal infection with other organisms, such as parasites, viruses and exposure to viral antigens during gestation have also been recently shown to modulate the immune response in the offspring. These data, along with observations from probiotic intervention studies,^80,81 show that maternal microbial exposure during gestation may lead to long-term health consequences for the offspring by influencing fetal immune development. However, continuous enhanced post-natal high microbial exposure may be required for optimal health protection⁷⁸ and breastfeeding practice support the microbiota transfer as well as immune factors supporting equilibrated balance (Fig. 1). In this respect, the microbiota is quantitatively the most important environmental source of immune stimulation and may provide a primary signal for the maturation of a balanced post-natal innate and adaptive immune system^51,82.

Figure 1. Factors influencing early microbiota colonization and its impact on infant health. Neonatal bacterial colonization is determined by environmental factors (e.g., maternal environment and diet) combined with host influences (genetics and gut characteristics). Maternal microbial environment affects maternal and fetal immune physiology and, of relevance, this interaction with microbes at the fetal-maternal interface could be modulated by specific microbes administered to the pregnant mother and/or neonates. The genetically-determined gut characteristics (structure, function, immunity), and the time and mode of birth (preterm, term, cesarean, vaginal), are crucial factors as well as maternal microbiota and nutritional status together diet (parenteral nutrition, formula, breast milk, and also, use of probiotics, prebiotics and/or antibiotics).

Due to the wide complexity of the microbiota, more detailed, basic microbial ecology studies, in clinically and immunologically well-characterized mother-infant pairs are needed, which are now made possible by advances in DNA sequencing technologies. Recent findings showed that adult gut microbiota can be classified according to 3 distinct microbial profiles or “enterotypes” that respond differently to diet and drug intake.⁸³ These enterotypes are likely established during early life explaining why neonatal gut microbiota composition has a lasting effect on health and immunity⁸⁴; however, the origin and factors affecting their development remain unclear. Moreover, how the maternal microbiota impacts on offspring’s microbiota development coupled to the effects on immune maturation during early life forms a key issue that deserves further investigation.

Breast Milk: Postnatal Link Between Mothers and their Offspring

Mother transfers the environmental information to her fetus through the placenta or to her infant through lactation (Fig. 1). Breast milk is recognized as the single most important postnatal element in metabolic and immunological programming of the child’s health.⁸⁵ Breastfeeding emerged as a nutritional strategy following the divergence of mammals millions of years ago that was favored by natural selection.⁸⁶ Despite the fact that milk production is very costly for mammalian females, it brings great benefits to the offspring. Today, breast milk is considered the gold standard for infant nutrition.⁸⁷ Compilations of long-term studies have shown that breast-fed infants have lower risk of diabetes,⁸⁸ hypercholesterolemia,⁸⁹ cardiovascular disease⁹⁰ and obesity⁹¹ in adulthood than formula-fed infants, although the causality is difficult to ascertain. It is likely that the beneficial impact achieved for the infant with breast-feeding is a combination of a balanced supply of nutrients, bioactive proteins and indigestible oligosaccharides, as well as bifidogenic bacteria in breast milk.⁸⁷

Human milk contains large quantities of secretory immunoglobulins mainly IgA. These antibodies can bind to pathogens and prevent their attachment to an infant's cell. In addition to these immunologic components, breast milk contains several nonspecific factors, such as lysozyme, lactoferrin and oligosaccharides, which are able to block attachment of pathogens or virus to the infant's mucosa, preventing infections and supporting the growth of beneficial bacteria. Lysozyme inhibits the growth of many bacterial species by disrupting the bacterial cell wall. Lactoferrin, known as a multifunctional protein in human breast milk, also limits bacterial growth by removing essential iron and by stimulating cytokine production, and enhancing mucosal immunity, natural killer (NK) cell activity and macrophage cyto-toxicity. Nucleotides in human breast milk have been shown to enhance the immune function in infants. Breast milk is a continuous source of microbes and their growth factors and components are regulating host-microbe interactions.^28,92-94 A delicate balance of such components is transferred from mother to infant via breastfeeding. Breast milk composition shows marked individual variation,^34,95 and factors that determine human milk composition remain poorly understood, although maternal lifestyle, nutritional and immunological status, dietary habits and lactation time influence breast milk composition and quality.^96,97

The origin and route of transfer of these bacteria have not yet been unveiled. The translocation of commensal bacteria has been demonstrated to occur in different parts of the body including the intestinal epithelium and skin.^98-101 It has been suggested that bacteria could be transferred from the human gut to the mammary gland through an endogenous route (the so-called entero-mammary pathway) via the mucosa-associated lymphoid system.¹⁰² Another theory is endocytosis, which could be supported by our observed differences between elective and non-elective C-section, if the stress of labor and delivery affected gut permeability. This is further supported by reports sustaining that the enteric nerve system affects the sampling of bacteria in the gut and their transfer to Peyer’s patches.¹⁰³ Another source of colonizing bacteria could be the skin and oral cavity of newborns exposed to the mother’s vaginal and intestinal microbiota during childbirth.²² In placenta, the penetration of bacteria has been observed in intrauterine infections, preterm birth and spontaneous abortion due to particular pathogens⁶⁶ although bacterial colonization of placenta in non-pathologic situations was reported recently (see above).

Altogether this information leads to the speculation that it could be of relevance for the infant’s health to monitor bacterial population in pregnant women at different sites and to appropriately modify it when requested to adequately control microbiota transferred to the infant in the early vital inoculums.

Early Health Programming with Probiotics

There is a growing interest in beneficial microbes with specific functions in the human microbiota, which could be used in foods or supplements to improve human health and prevent and treat diseases. The latest evidence demonstrates that maternal lifetime nutrition influences the placental morphology, with a potential impact on the later health of their offspring.¹⁰⁴ Pregnancy and early infancy are, to the current understanding, the most interesting critical stages and targets for dietary and food-based interventions aiming to reduce disease risk (Fig. 2). Hence, it is generally agreed that the faecal microbiota of the healthy, full-term, vaginally delivered, breast-fed infant constitutes the gold standard and therefore the target for any intervention in this regard.

Figure 2. Perinatal and postnatal probiotic interventions and their potential benefits for infant development and health. Early modulation driving healthy microbiota would be the aim of probiotic therapy and opens new prevention and treatment possibilities of diseases in which alteration of the microbiota plays relevant roles. Several studies have shown that administration of probiotics to pregnant women, nursing mothers, or newborns can influence the establishment and composition of infant gut microbiota and also, the development and maturation of immune system impacting early and later in life. NEC, necrotizing enterocolitis.

A probiotic has been defined as a “live microorganism which when administered in adequate amounts confers a health benefit on the host.”¹⁰⁵ The most common probiotic bacteria are species belonging to Lactobacillus, Bifidobacterium, Enterococcus and also some Propionibacterium strains. During the last few decades, a large number of studies have been conducted to analyze the beneficial effects of probiotics, using different formula and focusing to the prevention or treatment of diseases. Therefore, modulation of an unbalanced indigenous microbiota would be the aim of probiotic therapy and opens new prevention and treatment possibilities of diseases in which alteration of the microbiota plays relevant roles. Studies have shown that administration of probiotics to pregnant women, nursing mothers, or newborns can influence the establishment and composition of infant gut microbiota^16,71,72 impacting early and later in life (Fig. 2).

Prenatal interventions

Use of probiotics to promote infant microbiota development

Many clinical trials have been performed, in the perinatal period and during the first months of life aiming to test the influence of probiotic administration on the fecal microbial composition. It could be proved that prenatal maternal administration of L. rhamnosus LGG affected the transfer and initial establishment of bifidobacteria in neonates¹⁰⁶ and was associated with modulation of the infant intestinal Bifidobacterium pattern toward that of a healthy breastfed infant.¹⁰⁷ In particular, infants whose mothers received LGG during pregnancy showed higher prevalence of B. breve and a lower prevalence of B. adolescentis than placebo group.¹⁰⁶ Another study showed that LGG temporarily colonized the infant’s gut after administration of the probiotic to the pregnant mother before delivery; this colonization was stable for up to 6 mo.¹⁰⁸ Although, these results suggest that bacteria are transferred from mother to newborn maternal LGG consumption increased the bifidobacterial diversity in infants at 3 weeks and reduced the similarity of Bifidobacterium species pattern between mother and infant.¹⁰⁶ Nevertheless, there are also studies where prenatal consumption of LGG failed to modulate the diversity of early infant gut microbiota despite promoting a beneficial bifidobacteria profile.¹⁰⁹ Bifidobacteria also have a potential as prenatal probiotics as maternal colonization by B. bifidum had the most consistent effects on the infant's bifidobacterial microbiota.¹⁶

Use of probiotics in prevention of allergy diseases

The prevalence of allergic diseases, atopic eczema, allergic rhinitis and asthma, continues growing in the developed world and constitutes a common health problem among children.¹¹⁰ While the precise etiology is unclear, certain conditions or elements have a crucial role in the development of allergic diseases, such as environmental factors, aberrant gut microbiota composition and activity, impairment of intestinal mucosal barrier and shift of the Th1/Th2 –type cytokines balance toward a Th2 response.¹¹¹

In human allergy intervention studies, perinatal maternal microbial supplementation with probiotics may have important preventive effects.^73,81 Specific probiotics administered to allergic infants are effective in the prevention of atopic eczema, as well as modulating the immune system.^112,113 The frequency of atopic eczema in the probiotic group was half that of the placebo group (15/64 [23%] vs 31/68 [46%]; relative risk 0.51 [95% CI 0.32–0.84]). LGG was administered to pregnant women for four weeks prior to delivery, then to newborns at high risk of allergy for six months, as result a significant reduction in early atopic disease was achieved.¹¹³ In prospective clinical studies ingestion of probiotic strains LGG and B. lactis Bb12 alleviated allergy-associated symptomatology in infants allergic to cow’s milk and atopic dermatitis.¹¹⁴ The effect of probiotics on the prevention of atopic dermatitis has been demonstrated in double-blinded randomized controlled studies where LGG was given to pregnant and lactating mothers, who had a strong family history of eczema, allergic rhinitis or asthma and to their infants for the first 6 mo after birth. The frequency of developing atopic dermatitis in the offspring was significantly reduced by 2, 4, and 7 y.^{50,80,112,113} Furthermore, several well-designed studies have provided evidence that specific strains of probiotics can be somewhat effective for the treatment of established atopic dermatitis.¹¹⁵ Pregnant women carrying high-risk children were given a mixture of 4 probiotic strains (Lactobacillus rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve Bb99, Propionibacterium freudenreichii ssp. shermanii JS) before delivery and later their infants received the same probiotics along with galacto-oligosaccharides for 6 mo after birth.¹¹⁶ Of note, prospective clinical studies showed that supplementation of probiotic bacteria failed to reduce the risk of atopic dermatitis and even resulted in increased sensitization to allergens in one of them.^72,117,118 However, a meta-analysis report which included seven randomized, placebo-controlled trials comparing the development of atopic eczema in children whose mothers took probiotics vs placebo during pregnancy showed a significant reduction in the incidence of atopic eczema(reduction 5.7%; p = 0.022) in children aged 2 to 7 y whose mothers received probiotics during pregnancy.¹¹⁹ However, the beneficial effect was only significant for lactobacilli (reduction 10·6%; p = 0·045), but not for a probiotic combinationss (difference 3·06%, p = 0·204). Other recent reports indicate that probiotics had no effect on asthma development and related episodes,^120,121 and airway inflammation.¹¹⁶ A major reason for these discrepancies may be the use of different probiotic strains, the food or vehicle used to deliver the probiotics and doses used. Furthermore, host factors such as genetic susceptibility, microbiota composition and environmental factors, such as geographic region and diet, may influence the results of these studies.

Use of probiotics in prevention of obesity

The prevalence of obesity is rapidly increasing in Western society constituting an important health issue. Genetic and environmental factors such as diet, socioeconomic status, maternal obesity, rapid infancy weight gain and reduced physical activity have unequivocal influence on the incidence of obesity and its related diseases. In addition, some reports have suggested that gut microbiota is an important factor affecting energy disposal and storage in adipocytes.^53,54 Of note, gut microbiota composition may play a pivotal role in the microbial, metabolic and immunological programming of the infant although information in this regard is still scant.³ The association between gut microbiota composition and maternal nutritional status during pregnancy has been published by Collado et al.¹⁵ and by Santacruz et al.⁵⁷ Interestingly, both studies are supportive to the view that a gut microbiota profile favoring a higher number of bifidobacteria and a lower number of Staphylococcus aureus may provide protection against maternal overweight development. Other studies provided clinical evidence of consistently improved plasma glucose concentrations and insulin sensitivity in healthy women during pregnancy and 12 mo postpartum when an adequate dietary intake was combined with probiotics; moreover those effects were extended to neonates and infants.¹²² Interestingly, nutrition counselling and probiotic intervention had a distinct effect on gestational diabetes; thus, while probiotics diminished the risk of gestational diabetes (13% (diet/probiotics) vs. 36% (diet/placebo) and 34% (control); p = 0.003), dietary counselling reduced the risk of associated fetal overgrowth [p = 0.035 for birth weight and p = 0.028 for birth length].^17,18 In line with these observations, perinatal probiotic intervention with LGG moderated excessive weight gain especially among children who later became overweighed during the first years of life with a most pronounced impact at the age of 4 y.^17,18 In another clinical placebo-controlled study a probiotic combination (LGG + Bifidobacterium lactis Bb12) administered during the perinatal period combined with an advantageous diet consistently improved plasma glucose concentrations and insulin sensitivity in healthy women during pregnancy and 12 mo postpartum.^73,122 This intervention study also provided clinical evidence that probiotic consumption lowers the risk of central adiposity over the 6-mo postpartum period.¹²³ The risk of central adiposity defined as waist circumference 80 cm or more was lowered in women in the diet/probiotics group compared with the control/placebo group (OR 0.30, 95%CI 0.11–0.85, p = 0.023 adjusted for baseline BMI), while the diet/placebo group did not differ from the controls (OR 1.00, 95% CI 0.38–2.68, p = 0.994) at 6 mo postpartum.

Use of probiotics in prevention of preterm birth

The effectiveness of probiotics in preventing preterm labor and birth has been the focus of recent studies since maternal infection may increase 30–50% the risk of preterm delivery.¹²⁴ It has been suggested that specific probiotics could displace and inhibit pathogens responsible for triggering the inflammatory cascade that leads to preterm labor and delivery. Relevant clinical trials have suggested that oral administration of lactobacilli, could increase the number of vaginal Lactobacillus, restore the vaginal microbiota to normal, and treat women suffering bacterial vaginosis.¹²⁵ Nevertheless, some trials found that intra-vaginal administration had no significant effect, and although most of the available studies and results concerning the effectiveness of the administration of lactobacilli for the treatment of bacterial vaginosis are mostly positive, further studies to conclude definitively that probiotics are useful are needed.¹²⁵

Randomized controlled trials assessing the prevention of preterm birth by administration of probiotics in pregnant women and women planning pregnancy were recently reviewed.¹²⁴ One of the studies enrolled women after 34 weeks’ gestation received fermented milk as a probiotic. In another study women with bacterial vaginosis in early pregnancy received commercially available yogurt vaginally. The results showed an 81% reduction in the risk of genital infection after the probiotics were administered (OR 0.19; 95% CI 0.08–0.48). However, there are insufficient data available to assess the actual effect on preterm birth and its complications^126,127

Probiotics, cognitive function and mental development

A recent study surprisingly revealed that gut microbes which interact with the fetus before birth can modulate brain developmental pathways.¹²⁸ This study shows that the commensal microbiota is necessary not only for the development of the immune system but also for brain development and that this regulation has explicit time constraints. It has been reported that gut microbes acquired early in life can impact brain development in mice and subsequent behavior such as decreasing physical activity and increasing anxiety.¹²⁹ Furthermore, understanding the window of vulnerability when alterations in immune function can lead to brain impairment, and the therapeutic window when normal brain function can be restored is of clear importance. One study has shown that exposure to high levels of IL-6 in utero alters brain development and leads to permanent behavioral impairments.¹³⁰ Consumption of certain probiotic strains may affect cytokine patterns and it has been shown that these molecules may affect cognitive functions¹³¹ and gut hormones and neuropeptides may affect the learning process.¹³²

Postnatal interventions

Infants are one of the populations that may benefit more from the use of probiotics due the existence of a critical “window period” during the first months of life where there is an important opportunity for immune education when the intestinal microbiota and maturation of the immune system are not yet completed.^19,48,49 Different newborn infants including preterm, formula-fed, C-section delivered, and those who required antibiotics or intensive care are prone to develop diseases including infections such as diarrhea, necrotizing enterocolitis, atopic eczema and other related allergic diseases.²⁵ The main aim of probiotic intervention in these populations should be to avoid an aberrant microbiota development which may lead to impaired gut barrier function and abnormal immune responsiveness.¹³³

Use of probiotics to promote infant microbiota development

It has been showed that the administration of L. rhamnosus GG to mothers during 3 weeks after delivery modifies the initial infant establishment of bifidobacteria than those receiving placebo.¹⁰⁶ Infants whose mothers received probiotic had a higher prevalence of B. breve and a lower prevalence of B. adolescentis than did those in the placebo group at 5 d of age. It has been recently shown that prenatal administration to the mother of Lactobacillus rhamnosus GG (LGG) in late pregnancy modulated infant’s intestinal Bifidobacterium microbiota rendering it similar to that of a healthy breastfed infant.⁷³ It has also been reported that specific probiotic combinations during early feeding via the mother or incorporated in early formula-feeding, shaped the intestinal microbiota composition in infants from Germany and Finland.¹³⁴ Results showed that maternal intake of a probiotic mixture consisting of L. rhamnosus LPR and B .longum BL999, promoted the growth of lactic acid bacteria in the infant’s gut and that the impact was still evident 4 mo after discontinuation of probiotic administration. On the other hand, the specific probiotic combinations administered to formula-fed infants had only minor effects on microbiota composition toward lowering the bacterial counts of B. bifidum compared with the placebo group.

Use of probiotics for the prevention of allergic disease

An epidemic rise in allergic disease in “westernized” countries, especially food allergy and asthma, has occurred in parallel with many lifestyle changes. Studies have linked alterations in the intestinal microbiota in infants with the later development of allergic disease.^48-50 These abnormalities particularly involved Bifidobacterium species^51,135 and microbial diversity.¹³⁶

A Cochrane Library meta-analysis of probiotic supplementation for the prevention of allergic disease in term infants found a significant reduction in eczema in infants who received probiotics compared with those that did not [typical RR 0.82, 95% CI 0.70, 0.95] and further trials are recommended to determine the reproducibility of the results.¹³⁷ A recent meta-analysis⁷¹ including 12 randomized-controlled trials (781 participants) concluded that probiotics are not a clinically effective treatment for eczema (mean difference −0.90 points on a 20-point visual analog scale; 95% CI −2.84- 1.04), although when given during early infancy for the prevention rather than the treatment of eczema the results for some probiotic strains are promising. Another recent study¹³⁸ evaluated the clinical evidence for the use of probiotics as a therapeutic modality for allergic rhinitis and asthma by reducing symptom severity and medication use, although, further good-quality studies are needed. Another study¹³⁹ showed that beneficial effects of probiotic bacteria were obtained when treatment was given within the first 3 mo of life. Parental-reported eczema during the first 3 mo of life was significantly lower in the intervention group compared with placebo, 6/50 vs 15/52 (p = 0.035). It reported a relative risk reduction of 58% of parental reported eczema at 3 mo of age being the effect sustained until the age of 2 y, although relative risk reduction decreased with age. A more recent review and meta-analysis reported that a protective effect against allergic disease was only observed if probiotic supplementation were initiated in the prenatal period and continued postnatal, but not if probiotic supplementation was commenced postnatal.⁸¹ Some studies have indicated that probiotics during weaning may be effective in the prevention of eczema.¹⁴⁰ The cumulative incidence of eczema at 13 mo was 11% (4–17%, 95% CI) and 22% (13–31%, 95% CI) in the probiotic and placebo groups, respectively (p < 0.05).Therefore, further studies are required to determine if postnatal administration of probiotics might be beneficial for prevention of allergic disease.

Use of probiotics in obesity

Of note, weight increments outweighing that of length during the first 6 mo of life were associated with increased risk of obesity at 3 y of age. Changes in weight status in infancy may influence risk of later obesity more than weight status at birth.¹⁴¹ It has been demonstrated that the infant fecal microbial composition is related to maternal weight and weight gain over pregnancy.^7,15 In this study, Bacteroidetes and Staphylococcus levels were significantly higher in infants from overweighed mothers during the first six months of life and infants of women with normal weight gain over pregnancy showed higher levels of bifidobacteria than those born to women with excessive weight gain. Thus, higher infant weight correlates with lower numbers of bifidobacteria.⁷

Interestingly, differences in early fecal microbiota composition have been shown in one study to predict overweight in children early in life. Kalliomäki and colleagues⁶⁰ have compared over time groups of children and observed that those who became overweight by 7 y of age had lower levels of bifidobacteria and higher levels of Staphylococcus aureus at 6 and 12 mo of age compared with those remaining normal-weight. Overall, these few studies support the hypothesis that bifidobacteria which comprises the predominant microbiota of healthy breast-fed infants¹⁴² might affect weight gain through mucosal host-microbe crosstalk, immune regulation, and control of inflammation.¹⁴³ Clinical evidence was also provided that probiotic consumption lowers the risk of central adiposity over the 6-mo postpartum period.¹²³

Use of probiotics in diarrhea-types

Diarrhea is an obvious target for probiotics and their role in prevention and treatment of infant’s diarrhea is the best studied probiotic effect. In fact, probiotics are considered among the alternatives for management of acute gastroenteritis in children. However, evidence of efficacy is limited to a few strains.¹⁴⁴ In addition, specific probiotic strains have been shown to exert a protective effect against acute diarrhea, rotavirus diarrhea, antibiotic-associated diarrhea (AAD).^44,145-147 The beneficial effects maybe a combination of different mechanisms as balancing the commensal gut microbiota and modulating of immune response as well as physical exclusion of pathogens by probiotics

Based on recent meta-analysis, most of the tested probiotics, such as Lactobacillus rhamnosus GG (LGG), B. lactis and S. thermophilus and S. boulardii can significantly reduce the incidence of antibiotic-associated diarrhea (AAD) in children.¹⁴⁸ The combined risk ratio (RR) of developing AAD was significantly lower with probiotic compared with placebo (RR 0.35, 95% CI 0.19–0.67). In several RCTs it has been shown that probiotics mixed with milk or infant formula or given as an oral supplement are effective in the prevention of acute rotavirus diarrhea reducing and shortening the episodes in children¹⁴⁴

Probiotics and prevention of sepsis and NEC

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in low birth weight infants. Delayed enteral feeding, frequent use of antibiotic therapy and altered acquisition of normal digestive microbiota are the most likely factors contributing to the development of NEC in preterm infants. Current evidence indicates that probiotic supplementation significantly reduces all-cause mortality and definite necrotising enterocolitis without significant adverse effects in preterm neonates.^149,150 Further, certain probiotics can reduce both the severity and incidence of NEC in preterm neonates while no effect has been observed with other probiotic strains underlining the high strain-specificity of probiotics. Also, several RCTs have reported that enteral probiotic supplementation significantly reduced the incidence of NEC and mortality among premature infants.¹⁵¹ A meta-analysis study concluded that probiotics may be effective in reducing the risk of NEC in low-birth weight infants but more research is needed to clarify their safety, appropriate dose and type of probiotic to be used.^150,152 It has also been hypothesized that very preterm infants, who have less microbial diversity in their GIT, may benefit from colonization with probiotics. Cultivation and molecular-based studies have demonstrated quantitative and qualitative changes in ffecalecal samples of infants suffering NEC; however, no causative microorganism has been identified. Depending on the strain(s) administered, these probiotics could potentially induce a similar microbial community to that of the term infant or adult gastrointestinal microbiome.¹⁵³ These studies support the hypothesis that augmentation of the intestinal microbiome of premature infants with exogenous probiotics (healthy organisms) may favorably alter the microbiota resulting in an improvement in clinical outcomes. However, there is a lack of data regarding the optimal strain(s), dose, time to start and duration of probiotic therapy, as well as the taxonomy and quality of currently available probiotics formulations and products. Further large clinical trials are required to resolve these issues and to support the development of probiotic products.

Probiotics and autism spectrum disorders

On this area, autism is a biologically based disorder of brain development. The reported prevalence of autism spectrum disorders (ASDs) has increased by 5- to 10-fold over the past 20 y. Autism spectrum symptoms may be associated with the high prevalence of neurocognitive impairment in preterm children, particularly extremely preterm children, reflecting a different etiology and associated risk factors such as genetics and environmental factors. However, research is focused in early diagnostics and detection of environmental factors.

Further, as recently reviewed,^46,154 alterations in the microbiome have been associated with autism. It has been shown that a significantly higher number of Clostridium spp is present in the fecal samples of autistic children when compared with healthy individuals. Children with autism have often been reported to have gastrointestinal problems that are more frequent and more severe than in children from the general population.¹⁵⁵ Then, the potential use of probiotics would be beneficial for these infants. Whether the use of probiotics by children with autism can lead to improvements in behaviors needs to be established in well-controlled trials with sufficient group sizes. Important for these trials is the choice of the bacterial strains, as effects of probiotic bacteria can be highly strain specific.¹⁵⁶

Probiotics and infant colic and crying

Infantile colic is a common problem within the first 3 mo of life and causes considerable distress for parents and pediatricians. Despite 40 y of research, its pathogenesis is incompletely understood and treatment remains an open issue.¹⁵⁷ The etiology is complex and includes dietary protein hypersensitivity. The administration of probiotic L. reuteri ATCC 55730 has been shown to be effective in the treatment of colic in children.^158,159

Conclusions

The first contact between fetus and microorganisms occurs in utero; however, the influence of maternal microbiota composition upon the offspring has not been consistently evaluated. What is known is that infant’s gut microbes’ colonization and establishment as well as its composition play a pivotal role in the metabolic and immunological development of the child. Moreover, infant’s microbiota will unequivocally influence its metabolic capacities and prompt future development of asthma and atopic disease among others. The first microbes are influenced also by other factors as the mode of delivery, environment during birth, prematurity, hygiene measures, maternal perinatal factors and the type of infant feeding. Taken into consideration all the available information it could be concluded that microbial exposition before birth and exposure to microbes during pregnancy and lactation may influence the metabolic and immunologic profiles of the pregnant uterus and the risk of disease development by the offspring later in life (Fig. 1). Therefore, exposure to the adequate microbes early in gestation and neonatal period seems to be highly relevant to the future well-being.

It has been demonstrated that an intimate interrelationship between diet, microbiome and immune system influences human health. Of note, those disorders associated with an aberrant microbiota or inappropriate immune responses are potential targets for probiotic intervention. Clinical efficacy of specific probiotic strains has been demonstrated in some diseases such as diarrhea, irritable bowel syndrome and food allergies. In addition, new evidences support the use of probiotics in the prevention and treatment of a number of diseases including atopic diseases, immune disorders, obesity, and diabetes. We are still far from specific clinical indications for the different conditions susceptible for being prevented or ameliorated with probiotics (Fig. 2). We need further studies adequately powered and comprising specific and uniform types of probiotics before we can reach protocols applicable in the clinical practice.