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ScienceDirect Probiotics tailored to the infant: a window of opportunity Christophe Chassard, Tomas de Wouters and Christophe Lacroix Initial neonatal gut colonization is a crucial stage for developing a healthy physiology, beneficially influenced by breast-feeding. Breast milk has been shown not only to provide nutrients and bioactive immunological compounds, but also commensal bacteria, including gut-associated anaerobic bacteria such as Bifidobacterium species. Infant formulas are increasingly supplemented with probiotic bacteria despite uncertainties regarding their efficacy, and lack of mechanistic understanding. Breast milk may be a valuable source of such bacteria which, upon validation of their mechanism of action, might open a window of opportunity for developing probioticsupplemented infant formula with proven efficacy. Addresses Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zu¨rich, Schmelzbergstrasse 7, Zu¨rich CH-8092, Switzerland Corresponding author: Lacroix, Christophe ([email protected])
Current Opinion in Biotechnology 2014, 26:141–147 This review comes from a themed issue on Food biotechnology Edited by Mattheos AG Koffas and Jan Marienhagen
Available online XXX 0958-1669/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.12.012
Introduction The human gastrointestinal tract harbors one of the densest bacterial ecosystems found in nature, conferring its host with increased digestive capacity, beneficial metabolites and vitamins, and protection against pathogens. In fact the collective microbial activity in the colon resembles that of an additional human organ, which is why it has been referred to as ‘the forgotten organ’ or ‘virtual organ within an organ’ [1,2]. The mutualistic interactions between the enteric microbiota and the host are essential for health [3,4]. Imbalances in host–microbe communication can lead to aberrant responses towards the microbiota, resulting in immune-related disorders, of which the most studied are inflammatory bowel diseases (IBD). Establishment of the neonatal gut microbiota starts from birth. It has been suggested that the first contacts with pioneer bacteria could be deterministic for subsequent gut maturation, metabolic and immunologic programming, and consequently for short-term and long-term health. www.sciencedirect.com
The infant microbiome is partly inherited from the mother and the environment during the first 2 years of life. Recent research has indicated that breast milk also contains a broad diversity of microbes present in the maternal gut and are transmitted to the baby [5,6,7]. A recent hypothesis suggests a novel way of mother– neonate communication, in which maternal gut bacteria reach breast milk via intestinal translocation and blood carriage, describing an internal entero-mammary pathway to influence neonatal gut colonization and maturation of the immune system. This novel pathway of bacterial transfer would support the addition of carefully selected bacteria from mother’s breast milk to formula and opens new windows of opportunity for designing probiotics tailored to the infant. Probiotics are defined as live organisms, which when administered in sufficient amounts, can have a beneficial effect on the host’s health [8]. This review will discuss the current knowledge of the infant gut colonization considering both phylogenetic and functional diversity in the establishment of a balanced trophic chain. The potential of ‘traditional probiotics’ in shaping the infant gut microbiota and influencing intestinal functions and host health over the long term will be addressed. Opportunities to develop efficient probiotic strategies based on increased understanding of the roles and functions of the gut microbiota and on mechanisms of probiotic activity that support development and health of the infant will also be discussed.
The enteric microbiota, a complex and adaptive organ The human gastrointestinal tract (GIT) is colonized by a highly diverse, commensal microbiota, comprising mainly bacteria, which with a total number of 1014 cells form one of the densest known ecosystems and outnumber the cells and gene repertoire of their host by an estimated factor of 10 and 150, respectively [9]. Using advanced molecular tools, it has been estimated that each individual harbors at least 100–200 different bacterial species and that the collective human gut microbiota encompasses over 1800 genera, 16 000 phylotypes at the species-level and 35 000 phylotypes at the strain-level [10,11]. More than 99% of the gut microbiota are obligate anaerobes, most belonging to the Clostridia class within the Firmicutes phylum, as well as to the Bacteroidetes phylum. The gut microbiota is involved in the regulation of multiple host physiological pathways, connecting the gastrointestinal tract, liver, immune system, adipose tissue, muscle, and brain [12,13]. Current Opinion in Biotechnology 2014, 26:141–147
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Establishment of the gastrointestinal microbiota in the infant It is generally accepted that the fetal GIT is sterile and that the establishment of the gut microbiota is immediately initiated at birth and characterized by a dynamic succession of bacterial populations until a homeostatic adult-like microbiota is established by the age of 2–3. The diversity of the early microbiota is relatively low, and inter-individual variations in diversity and functionality are higher as compared to adults [14]. The neonatal gut microbiota establishment is non-random, as opposed to chaotic, and follows a smooth temporal gradient with gradually increasing diversity [15,16]. Early culture-dependent research performed in the second half of the 20th century led to the still widely accepted classical colonization dogma, according to which facultative anaerobic bacteria, mainly Staphylococcus, Enterococcus, Streptococcus spp. and members of the Enterobacteriaceae, act as pioneer bacteria to reach high densities within the first days of life. By depletion of oxygen these facultative anaerobes create a reduced environment, which enables the successive establishment of obligate anaerobic populations, such as Bifidobacterium, and members of the Bacteroidetes and Clostridia [17]. Culture-independent studies confirmed these findings. As such butyrate-producing members of the Clostridia (e.g. Roseburia and Faecalibacterium spp.) are essential for adult colonic health but are not usually detected in neonates [6,18]. Using both anaerobic culture-dependent (i.e. culture, isolation and 16S rRNA gene sequencing) and culture-independent methods (i.e. qPCR and pyrosequencing), Jost et al. [5] recently demonstrated that obligate anaerobes, such as members of the Bacteroidetes, may reach adult-like population densities already within the first week of life and thus earlier than previously assumed. These populations have never been extensively studied in neonates. The fact that other strict anaerobes are able to colonize early suggests that their establishment is rather directed by metabolic cross-feeding between members of the microbiota than by oxygen pressure, as generally assumed.
Factors influencing the infant microbiota The composition and activity of the gut microbiota codevelop with the host starting from birth, and are subjected to a complex interplay that depends on the hosts’ genetics, nutrition, and life-style. Natural delivery and feeding mode (i.e. vaginal delivery at term and exclusive breast-feeding) have been associated with beneficial health effects related to gut colonization and development, such as protection against infection, reduced infant morbidity and mortality, and low incidence of immunological disorders [19,20,21]. Natural delivery and breast feeding provide early contact with the maternal microbiota and are thought to be crucial for gut maturation, metabolic and immunologic programming, host–microbe homeostasis establishment [22]. The major potential Current Opinion in Biotechnology 2014, 26:141–147
sources of pioneer bacteria and factors that may influence neonatal gut microbiota composition include transmission to the fetal gut in utero [23], delivery mode, environmental factors such as geographical location [24], familial environment, sanitary conditions, mode of feeding, and medical treatment of the mother or the infant [19]. Human breast milk is a complex nutrient shaped by millions of years of evolutionary adaptation. It provides tailored nutritional, protective, immunological and developmental functions to suit all the needs of the developing neonate in an age-adapted manner while sustaining a healthy microbiota establishment [25]. This makes mother’s milk the undisputable gold standard for early nutrition that no formula milk can compete with [26]. Breast milk itself has been identified as a continuous source of live commensal maternal bacterial, including strict anaerobes, able to colonize the neonatal gut, and thus to influence early host-microbe interactions and neonatal development [5,27,28,29]. The way of entry of bacteria into the mammary gland and breast milk remains unclear to date. Bacteria may enter the mammary gland by contamination from outside, and/or from within, via a bacterial entero-mammary pathway [7]. In a recent study, using a combination of culture and culture-independent methods, Jost et al. [5] showed that gut-associated obligate anaerobic genera, such as Bifidobacterium, Bacteroides, Parabacteroides, and members of the Clostridia (Blautia, Clostridium, Collinsella and Veillonella) were shared between maternal feces, breast milk and neonatal feces. Furthermore, the same strain of Bifidobacterium breve could be isolated in all three ecosystems within one mother–neonate pair. Several butyrate-producing members of the Clostridia (Coprococcus, Faecalibacterium, Roseburia and Subdoligranulum) were shared between maternal feces and breast milk but remained undetected in neonate feces. Despite this considerable diversity of breast milk bacteria [6], their impact on neonatal gut microbiota establishment, gut maturation, immunity development and consequences on later health status, remain largely unknown.
Establishment of a healthy trophic equilibrium in the gut Major sources of carbohydrates for the infant colonic microbiota are human milk oligosaccharides (breast milk), prebiotics (formula), endogenous glycans (mucines) of host origin, undigested monosaccharides and lactose [30]. Complex carbohydrates are mainly degraded by Bifidobacterium, Lactobacillus and Bacteroides (human milk oligosaccharides and mucin degraders) into small sugars. Simple sugars are further metabolized together with lactose by numerous glycolytic microbes, including the predominant lactate producing bacteria such as Streptococcus, Staphylococcus, Enterococcus and enterobacteria (Figure 1). Large amounts of lactate are produced by these species during infant gut fermentation. Lactate can www.sciencedirect.com
Probiotics tailored to the infant Chassard, de Wouters and Lacroix 143
Figure 1
Primary Substrates
Secondary Substrates
Final Products
Lactate utilizers
Source of Dietary Carbohydrates
Lactobacillus* Propionibacteria* Veillonella *
Lactate
Lactose
Propionate
Lactose utilisers Staphylococcus Streptococcus
Lactobacillus*
Breast milk
Propionibacterium* Bifidobacterium* Staphylococcus Streptococcus ...
Propionibacterium* Bacteroides
H2 Glucose
Host metabolism
Other mono- and oligosaccharides
Clostridium
CO2
HMO HMO degraders Acetate producers
Lactobacillus* Bacteroides* Bifidobacterium*
Bacteroides
Butyrate Clostridium ?
Endogenous
Mucins
Mucin degraders Bacteroides Bifidobacterium*
Acetate
*Viable bacteria found in breast milk HMO = human milk oligosaccharides
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Trophic chain and corresponding functional microbial groups involved in intestinal carbohydrate fermentation in infants.
be subsequently converted into propionate by Veillonella and propionibacteria species (lactate-utilizing bacteria) or butyrate, while lactate accumulation may have detrimental effects on gut health. Although not indicative of potential local effects, fecal lactate concentrations can be considered as a marker of metabolic dysbiosis which has been associated with diseases or antibiotic treatments [31,32]. Butyrate concentrations found in infant feces are usually very low and correlate with the low population of Clostridium species colonizing the gut in the first weeks of life. Small amounts of H2 are also produced and potentially used by gut microbes or excreted in expired breath (Figure 1). Optimal colonization of the infant gut is thus characterized by a balance of metabolic activities of the gut microbiota, emphasizing the importance of considering the trophic contributions of microbial groups when modulating infant microbiota with probiotics.
Tailored probiotics towards infant development and long life health In infants, health and well-being are tightly linked to the development of the intestine and its digestive and immune capacities. Manipulation of the microbiota at this early stage could therefore have a high and www.sciencedirect.com
long-lasting impact. Because a dynamic succession of colonizers, influenced by host and environmental effects, takes place during colonization of the infant’s gut, the timing of administration of probiotics could be important to maximize effectiveness. Furthermore, tailoring probiotics for infants would require the identification of key colonizers for each step of this succession, and methods to promote their establishment. Most studies conducted with probiotics in pediatrics were carried out with strains of Bifidobacterium and Lactobacillus isolated from infants, and to a limited extent Propionibacterium and Streptococcus [33,34]. Even though some positive effects with probiotics supplemented infant formula were observed for the treatment and prevention of short-term diseases, such as antibiotic-associated and infectious diarrhea, there is only scarce but promising evidence on growth, host metabolism, atopic disease, allergy, and intestinal inflammation [35]. The ESPGHAN Committee on Nutrition concluded in 2011 that there is insufficient data to recommend the routine use of probiotic-supplemented formula [36]. Until now most strains added to formula have been isolated from fermented food or fecal infant microbiota, while strains isolated from breast milk have not been sufficiently investigated. Current Opinion in Biotechnology 2014, 26:141–147
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Administration of bacteria, isolated from breast milk, to the neonate or to the breast-feeding mother provides opportunities for manipulating aberrant microbiota establishment and reducing the risk of disease, [37]. Bifidobacterium and Lactobacillus strains isolated from mother’s milk could serve as probiotic candidates for addition to infant formula. However, other breast milk bacteria could also be targeted, considering their metabolic functions and importance for trophic stability in the gut (Figure 1). For example, strains of Veillonella, as non-carbohydrate fermenting lactate-utilizers (in combination with Propionibacterium spp.), found both in breast milk and corresponding infant feces, may have an important function in the establishment of a balanced trophic chain by metabolizing lactate resulting from lactose fermentation to propionate and acetate [6,38]. These trophic functions may be transferred to the breast-fed neonate and contribute to a healthy colonization and gut maturation process. A number of questions remain to be addressed for the development of new probiotics for infants. Is there a core microbiome, common to all infants, or does the genetic make-up of the individual infant define the ideal composition of its microbiota? Furthermore when is the best
time to give specific probiotics (perinatal or postnatal) [39]? Should probiotics be tailored to the individual for consideration of their effects on intestinal maturation, or can their effect be generalized? Most important for safe applications, however, is to elucidate the mechanisms of probiotics benefits. Figure 2 summarizes the main proposed mechanisms for probiotic activities in infants based on the most recent reviews [33,34,36,39,40,41].
Probiotics for the prevention of infection in infants Adding probiotics to formula represent a main strategy to reduce the incidence and severity of diarrhea in infants. The rationale for the use of probiotics is based on the fact that breast-feeding, in particular exclusive breast-feeding and of long duration, has been shown to provide better protection for infectious diseases, such as gastroenteritis [42]. A broad range of strains, mainly lactic acid bacteria and bifidobacteria, have been investigated in clinical trials, and have shown convincing effects. However, for most strains, the exact mechanisms of probiosis, especially anti-microbial activity or host protection, are still lacking and should be properly investigated before designing clinical trials. Possible mechanisms include direct antagonism by inhibitory substances, competitive
Figure 2
Nutritional Effects
Physiological Effects
• Degradation of human milk oligosaccharides
• Reduction of infant colics
• Increased growth
• Regularization of transit and stool consistence (no clear optimum defined)
• Enhancement of milk composition of the mother through probiotic administration
• Amelioration of intestinal well-being during antibiotherapie • Increased barrier function • Increase in bifidobacteria and lactobacilli populations
Immunostimulating Effects • Protection from viral diarrhea through competition for adhesion sites
Metabolic Factors
• Reduction of infection risk by pH reduction
• Reduction of obesity risk
• Decrease of immune response
• Increased insulin sensitivity
• Balancing of Th1/Th2 response
• Epigenetic effects on the developing intestinal tissue
• Induction of defensin secretion • Competitive inhibition of pathogens Current Opinion in Biotechnology
Summary of main mechanisms of probiotic activities in infants extracted from the most recent reviews on the topic. [33,34,36,39,40,41]. Current Opinion in Biotechnology 2014, 26:141–147
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exclusion and potentially host-mediated effects such as improved barrier function and altered immune response [43]. High prevalence of infectious diseases in infants from developing countries is well known. Probiotics may help reduce diarrhea and antibiotic usage. Decreased incidence of diarrhea was reported in groups treated with Lactobacillus rhamnosus GG [44] and the average duration of diarrheic episodes was lowered from 9.2 d in controls receiving oral rehydration solution (ORS) alone compare to 5.3 d for ORS plus LGG powder [45]. Recent studies performed with breast fed Indonesian infants showed that Lactobacillus reuteri DSM17938 could also reduce the incidence of diarrhea [46] and improve weight gain by about 15% over 6 months [47]. Recent observations also showed positive correlations between population of certain Lactobacillus species, including Lactobacillus reuteri, and BMI [48]. The development of probiotic mixtures combining promising probiotic strains of different complementary effects could protect infants against diarrhea [49] and promote health. Novel probiotics could be based on a combination of strains from breast milk and include the key species in the intestinal trophic chain to reinforce the stability and functionality of the microbiota, which could promote gut health and provide increased protection to infants.
Probiotics for the premature: preventiontreatments of complications The gut microbiota colonization of premature infants is profoundly different from that of infants born at term. One major difference is the predominance of Firmicutes and proteobacteria in the gut ecosystem, including some potential pathogenic strains belonging to enterobacteria, Klebsiella or Serratia [50]. Colonization of Bifidobacterium, and even Bacteroides, species is also clearly delayed, and the early colonization of the premature infants is very far from the classical healthy pattern. A majority of premature infants have very low birthweights and require long-term hospitalization to avoid complications. Necrotizing enterocolitis (NEC) is probably the most common disease affecting these infants in the neo-natal intensive care unit. Even if the incidence of NEC appears to be low compared with other pediatric diseases (less than 3 infants per 1000 live births), NEC is characterized by a very high risk of mortality, ranging from 10 to 50% [51]. Even though the precise etiology of NEC is not well understood, bacterial colonization could play a key role in the development of this inflammatory intestinal disease. On the basis of the absence of Bifidobacterium species in premature infants, it could be hypothesized that probiotic bifidobacteria supplementation could promote healthy www.sciencedirect.com
colonization of the infant intestine, while preventing growth of predominant proteobacteria. Infant probiotics could also participate in the maturation of the immune system and potentially attenuate inflammation in NEC cases [52]. Lactobacillus strains could also contribute to improved intestinal colonization by producing organic acids and reducing pH leading to an enhanced barrier, and a less favorable environment for gut pathogens. Furthermore, the selection of probiotics exhibiting antimicrobial activities against Citrobacter, an opportunistic infectious species also associated with NEC [50], has potential for NEC prevention or treatment. Reduced incidence of NEC has been observed in premature infants fed with breast milk compared with formula-fed [53]. Breast milk bacteria, with its key trophic roles, could be used to colonize the naive intestines of premature infants with a diverse, resilient and healthy bacterial ecosystem, greatly reducing the occurrence of pathogens. Furthermore, the use of a complex bacterial consortia isolated from breast milk could promote colonization of a balanced healthy microbiota by rapidly establishing a complex ecosystem, protecting the intestine and contributing to immune maturity. According to experimental data and long term use of probiotic-containing formula, probiotics appear to be well tolerated by healthy infants and do not raise safety concerns with respect to growth or adverse effects [33]. However probiotic interventions in premature infants should be carefully evaluated with regard to safety, benefits, and expected risks.
Conclusion During the early years of life, the intestinal microbiota of infants evolves rapidly under the effect of many factors from the host, mother, diet, and environment, until it reaches homeostasis characterized by high biodiversity, resilience and tolerance by the host. This period of early life may be particularly suitable for careful modulation of the composition and activity of the gut microbiota to reach an optimum balance for long lasting and optimum health. After consideration of the functional roles, safety, and technological ability, new strains of Bifidobacterium and Lactobacillus, or even other less common bacteria isolated from breast milk, could pave the way to the next generation of probiotics for successful applications in infant formulas.
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39. Sanz Y: Gut microbiota and probiotics in maternal and infant health. Am J Clin Nutr 2011, 94:2000S-S2005. 40. Fouhy F, Ross RP, Fitzgerald GF, Stanton C, Cotter PD: Composition of the early intestinal microbiota: knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps. Gut Microbes 2013, 3:203-220. Interesting review examining the development of the intestinal microbiota in infants and considering the potential role and opportunities for probiotics modulation. 41. Nauta AJ, Ben Amor K, Knol J, Garssen J, van der Beek EM: Relevance of pre- and postnatal nutrition to development and interplay between the microbiota and metabolic and immune systems. Am J Clin Nutr 2013, 98:586S-593S. 42. Vandenplas Y, De Greef E, Devreker T, Veereman-Wauters G, Hauser B: Probiotics and prebiotics in infants and children. Curr Infect Dis Rep 2013, 15:251-262. 43. Gagnon M, Zihler A, Chassard C, Lacroix C: Ecology of probiotics and enteric protection. In Probiotic Bacteria and Enteric Infections. Edited by Malago JJ, Koninkx J, MarinsekLogar R. Springer Verlag; 2011:65-86.
47. Agustina R, Bovee-Oudenhoven IMJ, Lukito W, Fahmida U, van de Rest O, Zimmermann MB, Firmansyah A, Wulanti R, Albers R, van den Heuvel EGHM et al.: Probiotics Lactobacillus reuteri DSM 17938 and Lactobacillus casei CRL 431 modestly increase growth, but not iron and zinc status, among Indonesian children aged 1-6 years. J Nutr 2013, 143:1184-1193. 48. Million M, Angelakis E, Maraninchi M, Henry M, Giorgi R, Valero R, Vialettes B, Raoult D: Correlation between body mass index and gut concentrations of Lactobacillus reuteri, Bifidobacterium animalis, Methanobrevibacter smithii and Escherichia coli. Int J Obes (Lond) 2013, 37:1460-1466. 49. Chapman CMC, Gibson GR, Rowland I: Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr 2011, 50:1-17. 50. Morowitz MJ, Denef VJ, Costello EK, Thomas BC, Poroyko V, Relman DA, Banfield JF: Strain-resolved community genomic analysis of gut microbial colonization in a premature infant. Proc Natl Acad Sci U S A 2011, 108:1128-1133. An excellent paper describing gut microbiota composition in a premature infant at strain level resolution during the first three weeks of life, and highlighting the early colonization and variations in populations of Citrobacter strains.
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46. Agustina R, Kok FJ, van de Rest O, Fahmida U, Firmansyah A, Lukito W, Feskens EJ, van den Heuvel EG, Albers R, BoveeOudenhoven IM: Randomized trial of probiotics and calcium on diarrhea and respiratory tract infections in Indonesian children. Pediatrics 2012, 129(64):41.
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53. Schanler RJ, Shulman RJ, Lau C: Feeding strategies for premature infants: beneficial outcomes of feeding fortified human milk versus preterm formula. Pediatrics 1999, 103:1150-1157.
Current Opinion in Biotechnology 2014, 26:141–147
ScienceDirect Probiotics tailored to the infant: a window of opportunity Christophe Chassard, Tomas de Wouters and Christophe Lacroix Initial neonatal gut colonization is a crucial stage for developing a healthy physiology, beneficially influenced by breast-feeding. Breast milk has been shown not only to provide nutrients and bioactive immunological compounds, but also commensal bacteria, including gut-associated anaerobic bacteria such as Bifidobacterium species. Infant formulas are increasingly supplemented with probiotic bacteria despite uncertainties regarding their efficacy, and lack of mechanistic understanding. Breast milk may be a valuable source of such bacteria which, upon validation of their mechanism of action, might open a window of opportunity for developing probioticsupplemented infant formula with proven efficacy. Addresses Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zu¨rich, Schmelzbergstrasse 7, Zu¨rich CH-8092, Switzerland Corresponding author: Lacroix, Christophe ([email protected])
Current Opinion in Biotechnology 2014, 26:141–147 This review comes from a themed issue on Food biotechnology Edited by Mattheos AG Koffas and Jan Marienhagen
Available online XXX 0958-1669/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.12.012
Introduction The human gastrointestinal tract harbors one of the densest bacterial ecosystems found in nature, conferring its host with increased digestive capacity, beneficial metabolites and vitamins, and protection against pathogens. In fact the collective microbial activity in the colon resembles that of an additional human organ, which is why it has been referred to as ‘the forgotten organ’ or ‘virtual organ within an organ’ [1,2]. The mutualistic interactions between the enteric microbiota and the host are essential for health [3,4]. Imbalances in host–microbe communication can lead to aberrant responses towards the microbiota, resulting in immune-related disorders, of which the most studied are inflammatory bowel diseases (IBD). Establishment of the neonatal gut microbiota starts from birth. It has been suggested that the first contacts with pioneer bacteria could be deterministic for subsequent gut maturation, metabolic and immunologic programming, and consequently for short-term and long-term health. www.sciencedirect.com
The infant microbiome is partly inherited from the mother and the environment during the first 2 years of life. Recent research has indicated that breast milk also contains a broad diversity of microbes present in the maternal gut and are transmitted to the baby [5,6,7]. A recent hypothesis suggests a novel way of mother– neonate communication, in which maternal gut bacteria reach breast milk via intestinal translocation and blood carriage, describing an internal entero-mammary pathway to influence neonatal gut colonization and maturation of the immune system. This novel pathway of bacterial transfer would support the addition of carefully selected bacteria from mother’s breast milk to formula and opens new windows of opportunity for designing probiotics tailored to the infant. Probiotics are defined as live organisms, which when administered in sufficient amounts, can have a beneficial effect on the host’s health [8]. This review will discuss the current knowledge of the infant gut colonization considering both phylogenetic and functional diversity in the establishment of a balanced trophic chain. The potential of ‘traditional probiotics’ in shaping the infant gut microbiota and influencing intestinal functions and host health over the long term will be addressed. Opportunities to develop efficient probiotic strategies based on increased understanding of the roles and functions of the gut microbiota and on mechanisms of probiotic activity that support development and health of the infant will also be discussed.
The enteric microbiota, a complex and adaptive organ The human gastrointestinal tract (GIT) is colonized by a highly diverse, commensal microbiota, comprising mainly bacteria, which with a total number of 1014 cells form one of the densest known ecosystems and outnumber the cells and gene repertoire of their host by an estimated factor of 10 and 150, respectively [9]. Using advanced molecular tools, it has been estimated that each individual harbors at least 100–200 different bacterial species and that the collective human gut microbiota encompasses over 1800 genera, 16 000 phylotypes at the species-level and 35 000 phylotypes at the strain-level [10,11]. More than 99% of the gut microbiota are obligate anaerobes, most belonging to the Clostridia class within the Firmicutes phylum, as well as to the Bacteroidetes phylum. The gut microbiota is involved in the regulation of multiple host physiological pathways, connecting the gastrointestinal tract, liver, immune system, adipose tissue, muscle, and brain [12,13]. Current Opinion in Biotechnology 2014, 26:141–147
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Establishment of the gastrointestinal microbiota in the infant It is generally accepted that the fetal GIT is sterile and that the establishment of the gut microbiota is immediately initiated at birth and characterized by a dynamic succession of bacterial populations until a homeostatic adult-like microbiota is established by the age of 2–3. The diversity of the early microbiota is relatively low, and inter-individual variations in diversity and functionality are higher as compared to adults [14]. The neonatal gut microbiota establishment is non-random, as opposed to chaotic, and follows a smooth temporal gradient with gradually increasing diversity [15,16]. Early culture-dependent research performed in the second half of the 20th century led to the still widely accepted classical colonization dogma, according to which facultative anaerobic bacteria, mainly Staphylococcus, Enterococcus, Streptococcus spp. and members of the Enterobacteriaceae, act as pioneer bacteria to reach high densities within the first days of life. By depletion of oxygen these facultative anaerobes create a reduced environment, which enables the successive establishment of obligate anaerobic populations, such as Bifidobacterium, and members of the Bacteroidetes and Clostridia [17]. Culture-independent studies confirmed these findings. As such butyrate-producing members of the Clostridia (e.g. Roseburia and Faecalibacterium spp.) are essential for adult colonic health but are not usually detected in neonates [6,18]. Using both anaerobic culture-dependent (i.e. culture, isolation and 16S rRNA gene sequencing) and culture-independent methods (i.e. qPCR and pyrosequencing), Jost et al. [5] recently demonstrated that obligate anaerobes, such as members of the Bacteroidetes, may reach adult-like population densities already within the first week of life and thus earlier than previously assumed. These populations have never been extensively studied in neonates. The fact that other strict anaerobes are able to colonize early suggests that their establishment is rather directed by metabolic cross-feeding between members of the microbiota than by oxygen pressure, as generally assumed.
Factors influencing the infant microbiota The composition and activity of the gut microbiota codevelop with the host starting from birth, and are subjected to a complex interplay that depends on the hosts’ genetics, nutrition, and life-style. Natural delivery and feeding mode (i.e. vaginal delivery at term and exclusive breast-feeding) have been associated with beneficial health effects related to gut colonization and development, such as protection against infection, reduced infant morbidity and mortality, and low incidence of immunological disorders [19,20,21]. Natural delivery and breast feeding provide early contact with the maternal microbiota and are thought to be crucial for gut maturation, metabolic and immunologic programming, host–microbe homeostasis establishment [22]. The major potential Current Opinion in Biotechnology 2014, 26:141–147
sources of pioneer bacteria and factors that may influence neonatal gut microbiota composition include transmission to the fetal gut in utero [23], delivery mode, environmental factors such as geographical location [24], familial environment, sanitary conditions, mode of feeding, and medical treatment of the mother or the infant [19]. Human breast milk is a complex nutrient shaped by millions of years of evolutionary adaptation. It provides tailored nutritional, protective, immunological and developmental functions to suit all the needs of the developing neonate in an age-adapted manner while sustaining a healthy microbiota establishment [25]. This makes mother’s milk the undisputable gold standard for early nutrition that no formula milk can compete with [26]. Breast milk itself has been identified as a continuous source of live commensal maternal bacterial, including strict anaerobes, able to colonize the neonatal gut, and thus to influence early host-microbe interactions and neonatal development [5,27,28,29]. The way of entry of bacteria into the mammary gland and breast milk remains unclear to date. Bacteria may enter the mammary gland by contamination from outside, and/or from within, via a bacterial entero-mammary pathway [7]. In a recent study, using a combination of culture and culture-independent methods, Jost et al. [5] showed that gut-associated obligate anaerobic genera, such as Bifidobacterium, Bacteroides, Parabacteroides, and members of the Clostridia (Blautia, Clostridium, Collinsella and Veillonella) were shared between maternal feces, breast milk and neonatal feces. Furthermore, the same strain of Bifidobacterium breve could be isolated in all three ecosystems within one mother–neonate pair. Several butyrate-producing members of the Clostridia (Coprococcus, Faecalibacterium, Roseburia and Subdoligranulum) were shared between maternal feces and breast milk but remained undetected in neonate feces. Despite this considerable diversity of breast milk bacteria [6], their impact on neonatal gut microbiota establishment, gut maturation, immunity development and consequences on later health status, remain largely unknown.
Establishment of a healthy trophic equilibrium in the gut Major sources of carbohydrates for the infant colonic microbiota are human milk oligosaccharides (breast milk), prebiotics (formula), endogenous glycans (mucines) of host origin, undigested monosaccharides and lactose [30]. Complex carbohydrates are mainly degraded by Bifidobacterium, Lactobacillus and Bacteroides (human milk oligosaccharides and mucin degraders) into small sugars. Simple sugars are further metabolized together with lactose by numerous glycolytic microbes, including the predominant lactate producing bacteria such as Streptococcus, Staphylococcus, Enterococcus and enterobacteria (Figure 1). Large amounts of lactate are produced by these species during infant gut fermentation. Lactate can www.sciencedirect.com
Probiotics tailored to the infant Chassard, de Wouters and Lacroix 143
Figure 1
Primary Substrates
Secondary Substrates
Final Products
Lactate utilizers
Source of Dietary Carbohydrates
Lactobacillus* Propionibacteria* Veillonella *
Lactate
Lactose
Propionate
Lactose utilisers Staphylococcus Streptococcus
Lactobacillus*
Breast milk
Propionibacterium* Bifidobacterium* Staphylococcus Streptococcus ...
Propionibacterium* Bacteroides
H2 Glucose
Host metabolism
Other mono- and oligosaccharides
Clostridium
CO2
HMO HMO degraders Acetate producers
Lactobacillus* Bacteroides* Bifidobacterium*
Bacteroides
Butyrate Clostridium ?
Endogenous
Mucins
Mucin degraders Bacteroides Bifidobacterium*
Acetate
*Viable bacteria found in breast milk HMO = human milk oligosaccharides
Current Opinion in Biotechnology
Trophic chain and corresponding functional microbial groups involved in intestinal carbohydrate fermentation in infants.
be subsequently converted into propionate by Veillonella and propionibacteria species (lactate-utilizing bacteria) or butyrate, while lactate accumulation may have detrimental effects on gut health. Although not indicative of potential local effects, fecal lactate concentrations can be considered as a marker of metabolic dysbiosis which has been associated with diseases or antibiotic treatments [31,32]. Butyrate concentrations found in infant feces are usually very low and correlate with the low population of Clostridium species colonizing the gut in the first weeks of life. Small amounts of H2 are also produced and potentially used by gut microbes or excreted in expired breath (Figure 1). Optimal colonization of the infant gut is thus characterized by a balance of metabolic activities of the gut microbiota, emphasizing the importance of considering the trophic contributions of microbial groups when modulating infant microbiota with probiotics.
Tailored probiotics towards infant development and long life health In infants, health and well-being are tightly linked to the development of the intestine and its digestive and immune capacities. Manipulation of the microbiota at this early stage could therefore have a high and www.sciencedirect.com
long-lasting impact. Because a dynamic succession of colonizers, influenced by host and environmental effects, takes place during colonization of the infant’s gut, the timing of administration of probiotics could be important to maximize effectiveness. Furthermore, tailoring probiotics for infants would require the identification of key colonizers for each step of this succession, and methods to promote their establishment. Most studies conducted with probiotics in pediatrics were carried out with strains of Bifidobacterium and Lactobacillus isolated from infants, and to a limited extent Propionibacterium and Streptococcus [33,34]. Even though some positive effects with probiotics supplemented infant formula were observed for the treatment and prevention of short-term diseases, such as antibiotic-associated and infectious diarrhea, there is only scarce but promising evidence on growth, host metabolism, atopic disease, allergy, and intestinal inflammation [35]. The ESPGHAN Committee on Nutrition concluded in 2011 that there is insufficient data to recommend the routine use of probiotic-supplemented formula [36]. Until now most strains added to formula have been isolated from fermented food or fecal infant microbiota, while strains isolated from breast milk have not been sufficiently investigated. Current Opinion in Biotechnology 2014, 26:141–147
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Administration of bacteria, isolated from breast milk, to the neonate or to the breast-feeding mother provides opportunities for manipulating aberrant microbiota establishment and reducing the risk of disease, [37]. Bifidobacterium and Lactobacillus strains isolated from mother’s milk could serve as probiotic candidates for addition to infant formula. However, other breast milk bacteria could also be targeted, considering their metabolic functions and importance for trophic stability in the gut (Figure 1). For example, strains of Veillonella, as non-carbohydrate fermenting lactate-utilizers (in combination with Propionibacterium spp.), found both in breast milk and corresponding infant feces, may have an important function in the establishment of a balanced trophic chain by metabolizing lactate resulting from lactose fermentation to propionate and acetate [6,38]. These trophic functions may be transferred to the breast-fed neonate and contribute to a healthy colonization and gut maturation process. A number of questions remain to be addressed for the development of new probiotics for infants. Is there a core microbiome, common to all infants, or does the genetic make-up of the individual infant define the ideal composition of its microbiota? Furthermore when is the best
time to give specific probiotics (perinatal or postnatal) [39]? Should probiotics be tailored to the individual for consideration of their effects on intestinal maturation, or can their effect be generalized? Most important for safe applications, however, is to elucidate the mechanisms of probiotics benefits. Figure 2 summarizes the main proposed mechanisms for probiotic activities in infants based on the most recent reviews [33,34,36,39,40,41].
Probiotics for the prevention of infection in infants Adding probiotics to formula represent a main strategy to reduce the incidence and severity of diarrhea in infants. The rationale for the use of probiotics is based on the fact that breast-feeding, in particular exclusive breast-feeding and of long duration, has been shown to provide better protection for infectious diseases, such as gastroenteritis [42]. A broad range of strains, mainly lactic acid bacteria and bifidobacteria, have been investigated in clinical trials, and have shown convincing effects. However, for most strains, the exact mechanisms of probiosis, especially anti-microbial activity or host protection, are still lacking and should be properly investigated before designing clinical trials. Possible mechanisms include direct antagonism by inhibitory substances, competitive
Figure 2
Nutritional Effects
Physiological Effects
• Degradation of human milk oligosaccharides
• Reduction of infant colics
• Increased growth
• Regularization of transit and stool consistence (no clear optimum defined)
• Enhancement of milk composition of the mother through probiotic administration
• Amelioration of intestinal well-being during antibiotherapie • Increased barrier function • Increase in bifidobacteria and lactobacilli populations
Immunostimulating Effects • Protection from viral diarrhea through competition for adhesion sites
Metabolic Factors
• Reduction of infection risk by pH reduction
• Reduction of obesity risk
• Decrease of immune response
• Increased insulin sensitivity
• Balancing of Th1/Th2 response
• Epigenetic effects on the developing intestinal tissue
• Induction of defensin secretion • Competitive inhibition of pathogens Current Opinion in Biotechnology
Summary of main mechanisms of probiotic activities in infants extracted from the most recent reviews on the topic. [33,34,36,39,40,41]. Current Opinion in Biotechnology 2014, 26:141–147
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exclusion and potentially host-mediated effects such as improved barrier function and altered immune response [43]. High prevalence of infectious diseases in infants from developing countries is well known. Probiotics may help reduce diarrhea and antibiotic usage. Decreased incidence of diarrhea was reported in groups treated with Lactobacillus rhamnosus GG [44] and the average duration of diarrheic episodes was lowered from 9.2 d in controls receiving oral rehydration solution (ORS) alone compare to 5.3 d for ORS plus LGG powder [45]. Recent studies performed with breast fed Indonesian infants showed that Lactobacillus reuteri DSM17938 could also reduce the incidence of diarrhea [46] and improve weight gain by about 15% over 6 months [47]. Recent observations also showed positive correlations between population of certain Lactobacillus species, including Lactobacillus reuteri, and BMI [48]. The development of probiotic mixtures combining promising probiotic strains of different complementary effects could protect infants against diarrhea [49] and promote health. Novel probiotics could be based on a combination of strains from breast milk and include the key species in the intestinal trophic chain to reinforce the stability and functionality of the microbiota, which could promote gut health and provide increased protection to infants.
Probiotics for the premature: preventiontreatments of complications The gut microbiota colonization of premature infants is profoundly different from that of infants born at term. One major difference is the predominance of Firmicutes and proteobacteria in the gut ecosystem, including some potential pathogenic strains belonging to enterobacteria, Klebsiella or Serratia [50]. Colonization of Bifidobacterium, and even Bacteroides, species is also clearly delayed, and the early colonization of the premature infants is very far from the classical healthy pattern. A majority of premature infants have very low birthweights and require long-term hospitalization to avoid complications. Necrotizing enterocolitis (NEC) is probably the most common disease affecting these infants in the neo-natal intensive care unit. Even if the incidence of NEC appears to be low compared with other pediatric diseases (less than 3 infants per 1000 live births), NEC is characterized by a very high risk of mortality, ranging from 10 to 50% [51]. Even though the precise etiology of NEC is not well understood, bacterial colonization could play a key role in the development of this inflammatory intestinal disease. On the basis of the absence of Bifidobacterium species in premature infants, it could be hypothesized that probiotic bifidobacteria supplementation could promote healthy www.sciencedirect.com
colonization of the infant intestine, while preventing growth of predominant proteobacteria. Infant probiotics could also participate in the maturation of the immune system and potentially attenuate inflammation in NEC cases [52]. Lactobacillus strains could also contribute to improved intestinal colonization by producing organic acids and reducing pH leading to an enhanced barrier, and a less favorable environment for gut pathogens. Furthermore, the selection of probiotics exhibiting antimicrobial activities against Citrobacter, an opportunistic infectious species also associated with NEC [50], has potential for NEC prevention or treatment. Reduced incidence of NEC has been observed in premature infants fed with breast milk compared with formula-fed [53]. Breast milk bacteria, with its key trophic roles, could be used to colonize the naive intestines of premature infants with a diverse, resilient and healthy bacterial ecosystem, greatly reducing the occurrence of pathogens. Furthermore, the use of a complex bacterial consortia isolated from breast milk could promote colonization of a balanced healthy microbiota by rapidly establishing a complex ecosystem, protecting the intestine and contributing to immune maturity. According to experimental data and long term use of probiotic-containing formula, probiotics appear to be well tolerated by healthy infants and do not raise safety concerns with respect to growth or adverse effects [33]. However probiotic interventions in premature infants should be carefully evaluated with regard to safety, benefits, and expected risks.
Conclusion During the early years of life, the intestinal microbiota of infants evolves rapidly under the effect of many factors from the host, mother, diet, and environment, until it reaches homeostasis characterized by high biodiversity, resilience and tolerance by the host. This period of early life may be particularly suitable for careful modulation of the composition and activity of the gut microbiota to reach an optimum balance for long lasting and optimum health. After consideration of the functional roles, safety, and technological ability, new strains of Bifidobacterium and Lactobacillus, or even other less common bacteria isolated from breast milk, could pave the way to the next generation of probiotics for successful applications in infant formulas.
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39. Sanz Y: Gut microbiota and probiotics in maternal and infant health. Am J Clin Nutr 2011, 94:2000S-S2005. 40. Fouhy F, Ross RP, Fitzgerald GF, Stanton C, Cotter PD: Composition of the early intestinal microbiota: knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps. Gut Microbes 2013, 3:203-220. Interesting review examining the development of the intestinal microbiota in infants and considering the potential role and opportunities for probiotics modulation. 41. Nauta AJ, Ben Amor K, Knol J, Garssen J, van der Beek EM: Relevance of pre- and postnatal nutrition to development and interplay between the microbiota and metabolic and immune systems. Am J Clin Nutr 2013, 98:586S-593S. 42. Vandenplas Y, De Greef E, Devreker T, Veereman-Wauters G, Hauser B: Probiotics and prebiotics in infants and children. Curr Infect Dis Rep 2013, 15:251-262. 43. Gagnon M, Zihler A, Chassard C, Lacroix C: Ecology of probiotics and enteric protection. In Probiotic Bacteria and Enteric Infections. Edited by Malago JJ, Koninkx J, MarinsekLogar R. Springer Verlag; 2011:65-86.
47. Agustina R, Bovee-Oudenhoven IMJ, Lukito W, Fahmida U, van de Rest O, Zimmermann MB, Firmansyah A, Wulanti R, Albers R, van den Heuvel EGHM et al.: Probiotics Lactobacillus reuteri DSM 17938 and Lactobacillus casei CRL 431 modestly increase growth, but not iron and zinc status, among Indonesian children aged 1-6 years. J Nutr 2013, 143:1184-1193. 48. Million M, Angelakis E, Maraninchi M, Henry M, Giorgi R, Valero R, Vialettes B, Raoult D: Correlation between body mass index and gut concentrations of Lactobacillus reuteri, Bifidobacterium animalis, Methanobrevibacter smithii and Escherichia coli. Int J Obes (Lond) 2013, 37:1460-1466. 49. Chapman CMC, Gibson GR, Rowland I: Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr 2011, 50:1-17. 50. Morowitz MJ, Denef VJ, Costello EK, Thomas BC, Poroyko V, Relman DA, Banfield JF: Strain-resolved community genomic analysis of gut microbial colonization in a premature infant. Proc Natl Acad Sci U S A 2011, 108:1128-1133. An excellent paper describing gut microbiota composition in a premature infant at strain level resolution during the first three weeks of life, and highlighting the early colonization and variations in populations of Citrobacter strains.
44. Basu S, Paul DK, Ganguly S, Chatterjee M, Chandra PK: Efficacy of high-dose Lactobacillus rhamnosus GG in controlling acute watery diarrhea in Indian children: a randomized controlled trial. J Clin Gastroenterol 2009, 43:208-213.
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Current Opinion in Biotechnology 2014, 26:141–147
