- Email: [email protected]
Intestinal colonization, microbiota, and probiotics
INTESTINAL COLONIZATION, MICROBIOTA, AND PROBIOTICS SEPPO SALMINEN, PHD,
AND
ERIKA ISOLAURI, MD, PHD
The human intestine is colonized by a large number of microorganisms that inhabit the intestinal tract and support a variety of physiological functions. The stepwise microbial colonization of the intestine begins at birth and continues during the early phases of life to form an intestinal microbiota that is different for each individual subject. This process facilitates the formation of a physical and immunologic barrier between the host and the environment, helping the gastrointestinal tract maintain a disease-free state. Probiotics are viable microbial food supplements that have a beneficial impact on human health. Health-promoting properties have been demonstrated for specific probiotic products. Scientific data are accumulating on these properties, especially in infants; the most significant effects include prevention and treatment of antibiotic-associated diarrhea and rotavirus diarrhea and allergy prevention. Bifidobacteria appear to be the most promising probiotic candidates, followed by defined lactic acid bacteria, which favor specific healthy bifidobacterial growth and species composition. Because viability appears to be important, probiotic properties also should be emphasized to meet this criterion. For future probiotics, the most important requirements include a demonstrated clinical benefit supported by mechanistic understanding of the effect on target population microbiota and immune functions. Genomic information and improved knowledge of microbiotic composition and its aberrancies should serve as a basis for selecting new probiotics for use in specific infant populations. (J Pediatr 2006;149:S115-S120)
evelopment of the microbiota in the newborn gastrointestinal (GI) tract depends on the original inoculum, the immediate living environment, and early feeding practices. The resulting mature intestinal microbiota harbors 10 times more cells than the host. This microbiota provides not only an important barrier between the human host and the environment, but also contact sites between microbes and the developing immune system. The barrier prevents the settlement of unwanted or pathogenic microorganisms and facilitates predigestion of many nutrients. Thus, the barrier function appears to support both the microbiota and the host by creating a protective microecologic environment. The sequencing of the human genome1,2 and specific microbial genomes has demonstrated that human subjects harbor numerous genes in the intestinal microbiota From the Functional Foods Forum and Department of Pediatrics, University of Turku, that influence health by interacting with the host at the mucosal level. The contact Turku, Finland. between the human genome and the commensal or permanent intestinal microorganisms Seppo Salminen is a recipient of a Bristol (microbiome) directly influences the morphology of the gut, providing a basis for the Myers Squibb–Mead Johnson Unrestricted Nutrition Research grant. This is a Nutridisease-free development of the gut.3,4 The human microbiota has been suggested to play tion, Allergy, Mucosal Immunology, and Ina significant nutritional role. By facilitating rapid salvage of energy from many nutrients testinal Microbiota (NAMI) research group report. Mead Johnson sponsored the symand providing diverse metabolic functions, the microbiota enables the host to survive in posium and provided an honorarium for different nutritional environments and to operate without having to adapt or develop all conference attendance, presentation of the 5 article, and submission of a manuscript. The digestive processes. authors are entirely and exclusively responThe early and mature intestinal microbiota are unique to each human being. From sible for its content. birth on, during breast feeding and weaning, the microbiota diversifies and becomes stable Presented as part of a symposium recog6-9 nizing the 25th anniversary of the Bristoland complex, to function as a barrier in the specific conditions of the host. Myers Squibb “Freedom to Discover” NuProbiotics are live microbial food supplements with demonstrated positive effects on trition Grants Program, University of 10,11 Cincinnati, Cincinnati, OH, June 7-8, 2005. host health. Probiotics often act by modifying the process of intestinal microbiota Submitted for publication Apr 14, 2006; development or the composition and activity of developed microbiota. Probiotics also can accepted June 1, 2006. act by direct contact with the mucosal cells, facilitating cross-talk with the immune system Reprint requests: Seppo Salminen, PhD, and microbes. Current probiotics have several demonstrated beneficial effects on infant Functional Foods Forum, University of Turku, 20014 Turku, Finland. E-mail: health, including maintaining healthy intestinal microbiota development and [email protected]. ing deviations observed in gut inflammatory diseases or preceding them. 0022-3476/$ - see front matter
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Gastrointestinal
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Toll-like receptor
Copyright © 2006 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2006.06.062
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SUCCESSION OF MICROBIAL COMMUNITIES IN HEALTH Source of Original Microbiota The basis of the healthy gut microbiota is derived from the mother. The mother’s microbiotic composition and the original inoculum provided at birth depend on poorly understood genetic factors. Diet, environment, and stress factors during pregnancy and birth influence the mother’s fecal microbiota composition and, consequently, the inoculum transferred to the infant at birth.12,13 The microbiota of a newborn develops rapidly after birth and is initially strongly dependent on feeding practices and the newborn infant’s hygienic environment.6,12 Succession of Microbial Communities Establishment of the gut microbiota is characterized by the following steps: early colonization at birth with facultative anaerobes, such as the enterobacteria, coliforms, lactobacilli, and streptococci, first colonizing the intestine, followed in rapid succession by anaerobic genera, such as Bifidobacterium, Bacteroides, Clostridium, and Eubacterium.12,14,15 Molecular studies have indicated that bifidobacteria species in the intestinal tract can range from 60% to 90% of the total fecal microbiota in breast-fed infants and lactic acid-producing bacteria may account for ⬍ 1% of the total microbiota, indicating the significant dominance of bifidobacteria.15 In formula-fed infants, the microbiota often is more complex, but the composition effects depend on the type of formula. The important differences in the microbiota of breast-fed and formula-fed infants do not lie only in the bifidobacterial numbers, but also in the species composition. Bifidobacterium breve, B. infantis, and B. longum species and strains are often found in fecal samples of breast-fed infants, whereas formula-fed infants often also harbor other types of bifidobacteria.6,14 The lactic acid bacteria species composition appears to be rather similar; the most common lactobacilli in breast-fed and formula-fed infant feces constitute Lactobacillus acidophilus group microorganisms, such as L. acidophilus, L. gasseri, and L. johnsonii. The differences in fecal microbiota between breast–fed and formula-fed infants may have decreased due to the development of improved infant formulas.14,16 Impact of Breast-Feeding and Weaning and Gut Microbiota Breast-feeding promotes a strong bifidobacterial presence in the infant gut by providing oligosaccharides that act as favorable substrates for bifidobacteria. Breast-feeding also enhances the bifidogenic effect by providing contact and exposure to the normal microbiota of the mother’s skin and breast milk. Bifidobacterium longum and other intestinal bifidobacteria are adapted to the availability of scanning nutrients in the lower GI tract in infants.17 Such strains rapidly utilize the S116
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Figure. Succession of intestinal microbiota in infants and the influence of probiotics on development.
oligosaccharides from human milk along with intestinal mucins, which are available in the colon of breast-fed infants. Thus, they appear to be designed by nature to reside in the healthy newborn GI tract to assist microbiotic development. Introduction of solid foods, as well as antimicrobial treatment periods, interrupt the constant supply of oligosaccharides and microbes from the mother, introducing new microbial genera and species into the GI tract. This process facilitates the development of adult-type mature microbiota. A schematic view of microbiotic development and factors influencing it is given in Figure 1.
HOST–MICROBE INTERACTION: FROM COMMENSALISM TO MUTUALISM Normal intestinal microbiota are characterized as a complex collection and balance of microorganisms that normally inhabit the healthy GI tract. The indigenous bacteria sometimes have been classified as potentially harmful or health-promoting. Most of them, however, are part of the normal commensal flora. This term indicates a relationship between organisms of 2 or more different species in which 1 species derives benefits from the association while the other(s) remain(s) unharmed or unaffected. Currently, the relationship between intestinal bacteria and the host is referred to as host–microbe cross-talk, implying peaceful coexistence and mutual benefit. Such cross-talk has been recently characterized. Bacteroides thetaiotaomicron, a common gut commensal, contains a large number of genes related to uptake and metabolism of carbohydrates reported for a sequenced bacterium.18 This microorganism has been shown to modulate glycosylation of the intestinal mucus and to induce the production of antimicrobials by the mucosa.19 In this way, the Bacteroides and other intestinal microbes may influence the gut microecology composition and shape the immune system. Bacteroides has been reported to evade detection by the immune system by changing the capsular polysaccharide composition and also surface antigenicity, a cellular modification, The Journal of Pediatrics • November 2006
which may help original colonizers permanently settle in the intestine.18,20 The intestinal mucosa and its immune system are adapted to the proximity and to the constant microbial challenge. The GI epithelium is equipped with pattern recognition receptors, including toll-like receptors (TLRs), which recognize specific conserved pathogen-associated molecular patterns. Nonpathogenic microbes share these structures; consequently, TLRs cannot distinguish between pathogens and commensals.21 Intracellular signalling pathways of TLRs result in production of proinflammatory cytokines through activation of the transcription nuclear factor B. These gut microbiota impact healthy immunophysiologic regulation, contributing to the anti-inflammatory tone in the gut. Indeed, several characteristics of the gut epithelium have been thought to prevent inappropriate immune responses toward indigenous gut microbiota. These include a relatively sparse expression of both certain TLRs and their essential co-receptors on the intestinal epithelium, as well as intracellular location of a few TLRs. Abundant immunoglobulin A antibody production at mucosal surfaces contributes to the intestinal barrier function by binding to and excluding antigens. Maturation of dendritic cells carrying commensals and subsequent secretion of cytokines and chemokines then influence the polarization of T-helper cells and thereby the adaptive immune responses. This type of immune response has been suggested to prevent commensals from breaching the gut mucosal barrier, whereas pathogenic bacteria preferably destroy it.22 In experimental studies in mice deficient in MyD88, an adaptor molecule essential for the TLR-mediated induction of inflammatory cytokines, demonstrated that TLR signalling pathways control the homeostasis of the epithelium and appear to be critical for protecting the host against gut injury by controlling cytoprotective factors and epithelial cell proliferation.23 Important functions of resident bacteria on the host’s physiology include metabolic activities, trophic effects on the intestinal epithelium, and protection against the overgrowth of potential pathogens in the GI tract. Along with these effects, specific strains of the gut microbiota elicit anti-inflammatory responses in the intestinal epithelial cells, thereby strengthening intestinal homeostasis. The importance of host–microbe interaction is most vital in the neonatal period, when the establishment of a normal microbiota provides the host with its most substantial antigen challenge, a strong stimulatory effect for the maturation of the gut-associated lymphoid tissue.24 On this basis, the aims of probiotic therapy are to avert deviant microbiota development, impaired gut barrier function, abnormal immune responsiveness, and immunoinflammatory disease. Indeed, probiotics in the diet may exert clinical effects beyond the nutritional impact of food. Future research should provide the necessary tools, such as DNA microarrays, to unravel the in vivo functions of gut microbes25 or to monitor the effect of diet on microbiota and the host’s genes and their expression.26 The goals are to correctly interpret the complex messages provided by the Intestinal Colonization, Microbiota, and Probiotics
multitude of microarray data and to further develop a bioinformatic approach.
ROLE OF PROBIOTICS Probiotics have been defined by the International Life Sciences Institute Europe as “viable microbial food supplement[s] which when taken in the right dose beneficially influence the health of the host.”10 Practically the same definition of probiotics is used by the Food and Agriculture Organization of the United Nations and the World Health Organization.11 These definitions require that safety and efficacy be demonstrated scientifically for each strain and each product.
Probiotics and Intestinal Colonization Specific probiotics have been assessed for their ability to attach to human intestinal mucosa, but such attachment is of a temporary nature. For example, administering Lactobacillus GG to infants may result in a 2- to 12-week fecal recovery of the administered strain in feces, indicating the potential to multiply and survive in the normal intestinal tract as well as during rotavirus diarrhea.27-29 Fecal recovery is not necessary for demonstrating probiotic health effects, which depend on the interaction of the strain at the target site. Importance of Viability Few studies have assessed nonviable probiotics in humans. However, viability appears to be an important factor in probiotics to facilitate health effects. Kirjavainen et al30 reported that only viable probiotics in extremely sensitive infants were able to alleviate symptoms of atopic dermatitis, as reported by Isolauri et al.31 It is clearly of high interest to continue such studies not only for probiotics, but also for characterizing the viability of the residing intestinal microbiota. Genomic Information on Probiotics Many members of the Lactobacillus and Bifidobacterium genera are commonly used as probiotics. In 1995, the genome of the first free-living organism, the bacterium Haemophilus influenzae, was sequenced.32 Since then, more than 200 bacterial genomes, mainly pathogenic microorganisms, have been sequenced. The first genome of a lactic acid bacterium was completed in 1999.33 Recently, the genomes of many other lactic acid bacteria,34 bifidobacteria,17 and other intestinal microorganisms18,20,35 have been sequenced.36 However, genomic data on 2 of the most widely studied probiotics (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-12) are not publicly available. Genomic information on B. longum,17 L. plantarum,34 L. johnsonii,37 or L. acidophilus,38,39 all established probiotics, provide insight into the adhesive mechanisms present in these microorganisms, which provide the basis for both settlement into the gut during different age phases and communication of regulatory signals to specific areas and sites of the gut S117
mucosa. A eukaryotic-type serine protease inhibitor has been identified in the genome of B. longum that may contribute to the immunomodulatory activity of Bifidobacterium. Operons coding for bacteriocins have been identified in L. johnsonii and L. acidophilus that may modify the succession of microbiota in humans over time.
ABERRANT GUT MICROBIOTA: CLINICAL CONSEQUENCES Specific imbalances or deviations in the intestinal microbiota may make humans more vulnerable to intestinal inflammatory diseases and systemic diseases beyond the intestinal environment. Specific Bifidobacterium species in the healthy infant gut are the most predominant and metabolically active organisms; specific clostridia also are often present. Changes in their quantitative and qualitative composition appear to serve as useful indicators of deviations from the balanced microbiota. Other specific microbial biomarkers for health remain to be defined among the developing intestinal microbiota. Specific deviations in intestinal microbiota, including decreased numbers, an atypical composition of bifidobacteria, and aberrancies in clostridia content and composition, may predispose infants to allergic disease.40,41 Similarly, deviations from the normal microbiota are associated with antibiotic side effects. Aberrant microbiota during childhood may predispose a person to both inflammatory gut disease and diarrhea.42,43 Consequently, understanding the quantitative and qualitative microbiota composition will help provide targets for probiotic intervention. Numerous human intervention studies have assessed the efficacy of specific probiotics in the treatment and risk reduction of infectious diarrhea.44,45 The effect has been explained by a reduction in the duration of rotavirus shedding, normalization of the gut, increased permeability caused by rotavirus infection, and increased numbers of IgA-secreting cells against rotavirus. Moreover, the ability of specific probiotics to increase the expression of mucins may contribute to the barrier effect and the inhibition of rotavirus replication. Particularly, the immunomodulatory potential of specific probiotics has introduced new potential therapeutic strategies for combating allergic and inflammatory conditions.24 Probiotic bacteria may counteract the inflammatory process by stabilizing the gut’s microbial environment and the intestine’s permeability barrier, as well as by enhancing the degradation of enteral antigens and altering their immunogenicity. Another explanation for the gut-stabilizing effect could be improvement of the intestine’s immunologic barrier, particularly the intestinal IgA responses. Probiotic effects also may be mediated by controlling the balance between proinflammatory and anti-inflammatory cytokines. These effects, which are exemplified in the reduced disease activity and increased intestinal permeability, have been achieved in pediatric patients with Crohn’s disease and atopic eczema.31,46-49 In clinical trials in adults, results from preparations containing 4 strains of lactobacilli (L. casei, L. plantarum, L. acidophilus, and L. delS118
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brückii subsp. bulgaricus) and 3 bifidobacteria strains (B. longum, B. breve, and B. infantis), together with Streptococcus salivarius subsp. thermophilus, are encouraging for preventing relapses of chronic pouchitis.50 The preventive potential of probiotics in atopic diseases has been demonstrated in a double-blind, placebo-controlled study.50-52 Probiotics administered prenatally and postnatally for 6 months to children at high risk for atopic disease reduced the prevalence of atopic eczema to half that in infants receiving placebos, and the effect appears to extend beyond infancy.
PERSPECTIVES Advancing genomic research will provide data on host– microbe interactions to identify key processes of microbiotic development and maintenance. These include nutrient–microbe interactions and a detailed knowledge of the transfer of microbial communities from parent to infant. Such data should provide the basis for the development of new probiotics.53 Probiotic genome analysis will predict their properties and interactions in human use, allowing their application to human studies of specific target populations. Development of genetic tools aids analysis of the functionality of these strains,54,55 thereby facilitating the development of a new generation of probiotics that are more site-specific and target disorder-specific and safe.
CONCLUSION The healthy infant microbiota acts as an organ to utilize nutrients and also as a defense mechanism against harmful environmental exposures. Deviations in composition can be related to multiple disease states within the intestine but also beyond it. Similarly, components of the human intestinal microbiota or organisms entering the intestine may have both harmful and beneficial effects on human health. The current available information focuses mostly on the role of infant microbiota and the first colonization steps in later health. Bifidobacteria play a key role in this process; clostridia also may serve as biomarkers of changing environmental conditions. Maternal–infant contact, breast milk oligosaccharides, and breast milk microbes exert significant effects on initial microbiotic development. The initial inoculum at birth is promoted through prebiotic galacto-oligosaccharides in breast milk and introduces environmental bacteria through maternal skin and other contact with the infant, thus providing a means to guide the development of individually optimized microbiota under existing environmental conditions. A future goal is to apply the knowledge of microbiotic composition and aberrancies to selecting the right probiotics for a particular target population. Another goal is to ensure that probiotics and prebiotics administered to infants are safe in terms of both long-term use and effects beyond infancy.
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AND
ERIKA ISOLAURI, MD, PHD
The human intestine is colonized by a large number of microorganisms that inhabit the intestinal tract and support a variety of physiological functions. The stepwise microbial colonization of the intestine begins at birth and continues during the early phases of life to form an intestinal microbiota that is different for each individual subject. This process facilitates the formation of a physical and immunologic barrier between the host and the environment, helping the gastrointestinal tract maintain a disease-free state. Probiotics are viable microbial food supplements that have a beneficial impact on human health. Health-promoting properties have been demonstrated for specific probiotic products. Scientific data are accumulating on these properties, especially in infants; the most significant effects include prevention and treatment of antibiotic-associated diarrhea and rotavirus diarrhea and allergy prevention. Bifidobacteria appear to be the most promising probiotic candidates, followed by defined lactic acid bacteria, which favor specific healthy bifidobacterial growth and species composition. Because viability appears to be important, probiotic properties also should be emphasized to meet this criterion. For future probiotics, the most important requirements include a demonstrated clinical benefit supported by mechanistic understanding of the effect on target population microbiota and immune functions. Genomic information and improved knowledge of microbiotic composition and its aberrancies should serve as a basis for selecting new probiotics for use in specific infant populations. (J Pediatr 2006;149:S115-S120)
evelopment of the microbiota in the newborn gastrointestinal (GI) tract depends on the original inoculum, the immediate living environment, and early feeding practices. The resulting mature intestinal microbiota harbors 10 times more cells than the host. This microbiota provides not only an important barrier between the human host and the environment, but also contact sites between microbes and the developing immune system. The barrier prevents the settlement of unwanted or pathogenic microorganisms and facilitates predigestion of many nutrients. Thus, the barrier function appears to support both the microbiota and the host by creating a protective microecologic environment. The sequencing of the human genome1,2 and specific microbial genomes has demonstrated that human subjects harbor numerous genes in the intestinal microbiota From the Functional Foods Forum and Department of Pediatrics, University of Turku, that influence health by interacting with the host at the mucosal level. The contact Turku, Finland. between the human genome and the commensal or permanent intestinal microorganisms Seppo Salminen is a recipient of a Bristol (microbiome) directly influences the morphology of the gut, providing a basis for the Myers Squibb–Mead Johnson Unrestricted Nutrition Research grant. This is a Nutridisease-free development of the gut.3,4 The human microbiota has been suggested to play tion, Allergy, Mucosal Immunology, and Ina significant nutritional role. By facilitating rapid salvage of energy from many nutrients testinal Microbiota (NAMI) research group report. Mead Johnson sponsored the symand providing diverse metabolic functions, the microbiota enables the host to survive in posium and provided an honorarium for different nutritional environments and to operate without having to adapt or develop all conference attendance, presentation of the 5 article, and submission of a manuscript. The digestive processes. authors are entirely and exclusively responThe early and mature intestinal microbiota are unique to each human being. From sible for its content. birth on, during breast feeding and weaning, the microbiota diversifies and becomes stable Presented as part of a symposium recog6-9 nizing the 25th anniversary of the Bristoland complex, to function as a barrier in the specific conditions of the host. Myers Squibb “Freedom to Discover” NuProbiotics are live microbial food supplements with demonstrated positive effects on trition Grants Program, University of 10,11 Cincinnati, Cincinnati, OH, June 7-8, 2005. host health. Probiotics often act by modifying the process of intestinal microbiota Submitted for publication Apr 14, 2006; development or the composition and activity of developed microbiota. Probiotics also can accepted June 1, 2006. act by direct contact with the mucosal cells, facilitating cross-talk with the immune system Reprint requests: Seppo Salminen, PhD, and microbes. Current probiotics have several demonstrated beneficial effects on infant Functional Foods Forum, University of Turku, 20014 Turku, Finland. E-mail: health, including maintaining healthy intestinal microbiota development and [email protected]. ing deviations observed in gut inflammatory diseases or preceding them. 0022-3476/$ - see front matter
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SUCCESSION OF MICROBIAL COMMUNITIES IN HEALTH Source of Original Microbiota The basis of the healthy gut microbiota is derived from the mother. The mother’s microbiotic composition and the original inoculum provided at birth depend on poorly understood genetic factors. Diet, environment, and stress factors during pregnancy and birth influence the mother’s fecal microbiota composition and, consequently, the inoculum transferred to the infant at birth.12,13 The microbiota of a newborn develops rapidly after birth and is initially strongly dependent on feeding practices and the newborn infant’s hygienic environment.6,12 Succession of Microbial Communities Establishment of the gut microbiota is characterized by the following steps: early colonization at birth with facultative anaerobes, such as the enterobacteria, coliforms, lactobacilli, and streptococci, first colonizing the intestine, followed in rapid succession by anaerobic genera, such as Bifidobacterium, Bacteroides, Clostridium, and Eubacterium.12,14,15 Molecular studies have indicated that bifidobacteria species in the intestinal tract can range from 60% to 90% of the total fecal microbiota in breast-fed infants and lactic acid-producing bacteria may account for ⬍ 1% of the total microbiota, indicating the significant dominance of bifidobacteria.15 In formula-fed infants, the microbiota often is more complex, but the composition effects depend on the type of formula. The important differences in the microbiota of breast-fed and formula-fed infants do not lie only in the bifidobacterial numbers, but also in the species composition. Bifidobacterium breve, B. infantis, and B. longum species and strains are often found in fecal samples of breast-fed infants, whereas formula-fed infants often also harbor other types of bifidobacteria.6,14 The lactic acid bacteria species composition appears to be rather similar; the most common lactobacilli in breast-fed and formula-fed infant feces constitute Lactobacillus acidophilus group microorganisms, such as L. acidophilus, L. gasseri, and L. johnsonii. The differences in fecal microbiota between breast–fed and formula-fed infants may have decreased due to the development of improved infant formulas.14,16 Impact of Breast-Feeding and Weaning and Gut Microbiota Breast-feeding promotes a strong bifidobacterial presence in the infant gut by providing oligosaccharides that act as favorable substrates for bifidobacteria. Breast-feeding also enhances the bifidogenic effect by providing contact and exposure to the normal microbiota of the mother’s skin and breast milk. Bifidobacterium longum and other intestinal bifidobacteria are adapted to the availability of scanning nutrients in the lower GI tract in infants.17 Such strains rapidly utilize the S116
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Figure. Succession of intestinal microbiota in infants and the influence of probiotics on development.
oligosaccharides from human milk along with intestinal mucins, which are available in the colon of breast-fed infants. Thus, they appear to be designed by nature to reside in the healthy newborn GI tract to assist microbiotic development. Introduction of solid foods, as well as antimicrobial treatment periods, interrupt the constant supply of oligosaccharides and microbes from the mother, introducing new microbial genera and species into the GI tract. This process facilitates the development of adult-type mature microbiota. A schematic view of microbiotic development and factors influencing it is given in Figure 1.
HOST–MICROBE INTERACTION: FROM COMMENSALISM TO MUTUALISM Normal intestinal microbiota are characterized as a complex collection and balance of microorganisms that normally inhabit the healthy GI tract. The indigenous bacteria sometimes have been classified as potentially harmful or health-promoting. Most of them, however, are part of the normal commensal flora. This term indicates a relationship between organisms of 2 or more different species in which 1 species derives benefits from the association while the other(s) remain(s) unharmed or unaffected. Currently, the relationship between intestinal bacteria and the host is referred to as host–microbe cross-talk, implying peaceful coexistence and mutual benefit. Such cross-talk has been recently characterized. Bacteroides thetaiotaomicron, a common gut commensal, contains a large number of genes related to uptake and metabolism of carbohydrates reported for a sequenced bacterium.18 This microorganism has been shown to modulate glycosylation of the intestinal mucus and to induce the production of antimicrobials by the mucosa.19 In this way, the Bacteroides and other intestinal microbes may influence the gut microecology composition and shape the immune system. Bacteroides has been reported to evade detection by the immune system by changing the capsular polysaccharide composition and also surface antigenicity, a cellular modification, The Journal of Pediatrics • November 2006
which may help original colonizers permanently settle in the intestine.18,20 The intestinal mucosa and its immune system are adapted to the proximity and to the constant microbial challenge. The GI epithelium is equipped with pattern recognition receptors, including toll-like receptors (TLRs), which recognize specific conserved pathogen-associated molecular patterns. Nonpathogenic microbes share these structures; consequently, TLRs cannot distinguish between pathogens and commensals.21 Intracellular signalling pathways of TLRs result in production of proinflammatory cytokines through activation of the transcription nuclear factor B. These gut microbiota impact healthy immunophysiologic regulation, contributing to the anti-inflammatory tone in the gut. Indeed, several characteristics of the gut epithelium have been thought to prevent inappropriate immune responses toward indigenous gut microbiota. These include a relatively sparse expression of both certain TLRs and their essential co-receptors on the intestinal epithelium, as well as intracellular location of a few TLRs. Abundant immunoglobulin A antibody production at mucosal surfaces contributes to the intestinal barrier function by binding to and excluding antigens. Maturation of dendritic cells carrying commensals and subsequent secretion of cytokines and chemokines then influence the polarization of T-helper cells and thereby the adaptive immune responses. This type of immune response has been suggested to prevent commensals from breaching the gut mucosal barrier, whereas pathogenic bacteria preferably destroy it.22 In experimental studies in mice deficient in MyD88, an adaptor molecule essential for the TLR-mediated induction of inflammatory cytokines, demonstrated that TLR signalling pathways control the homeostasis of the epithelium and appear to be critical for protecting the host against gut injury by controlling cytoprotective factors and epithelial cell proliferation.23 Important functions of resident bacteria on the host’s physiology include metabolic activities, trophic effects on the intestinal epithelium, and protection against the overgrowth of potential pathogens in the GI tract. Along with these effects, specific strains of the gut microbiota elicit anti-inflammatory responses in the intestinal epithelial cells, thereby strengthening intestinal homeostasis. The importance of host–microbe interaction is most vital in the neonatal period, when the establishment of a normal microbiota provides the host with its most substantial antigen challenge, a strong stimulatory effect for the maturation of the gut-associated lymphoid tissue.24 On this basis, the aims of probiotic therapy are to avert deviant microbiota development, impaired gut barrier function, abnormal immune responsiveness, and immunoinflammatory disease. Indeed, probiotics in the diet may exert clinical effects beyond the nutritional impact of food. Future research should provide the necessary tools, such as DNA microarrays, to unravel the in vivo functions of gut microbes25 or to monitor the effect of diet on microbiota and the host’s genes and their expression.26 The goals are to correctly interpret the complex messages provided by the Intestinal Colonization, Microbiota, and Probiotics
multitude of microarray data and to further develop a bioinformatic approach.
ROLE OF PROBIOTICS Probiotics have been defined by the International Life Sciences Institute Europe as “viable microbial food supplement[s] which when taken in the right dose beneficially influence the health of the host.”10 Practically the same definition of probiotics is used by the Food and Agriculture Organization of the United Nations and the World Health Organization.11 These definitions require that safety and efficacy be demonstrated scientifically for each strain and each product.
Probiotics and Intestinal Colonization Specific probiotics have been assessed for their ability to attach to human intestinal mucosa, but such attachment is of a temporary nature. For example, administering Lactobacillus GG to infants may result in a 2- to 12-week fecal recovery of the administered strain in feces, indicating the potential to multiply and survive in the normal intestinal tract as well as during rotavirus diarrhea.27-29 Fecal recovery is not necessary for demonstrating probiotic health effects, which depend on the interaction of the strain at the target site. Importance of Viability Few studies have assessed nonviable probiotics in humans. However, viability appears to be an important factor in probiotics to facilitate health effects. Kirjavainen et al30 reported that only viable probiotics in extremely sensitive infants were able to alleviate symptoms of atopic dermatitis, as reported by Isolauri et al.31 It is clearly of high interest to continue such studies not only for probiotics, but also for characterizing the viability of the residing intestinal microbiota. Genomic Information on Probiotics Many members of the Lactobacillus and Bifidobacterium genera are commonly used as probiotics. In 1995, the genome of the first free-living organism, the bacterium Haemophilus influenzae, was sequenced.32 Since then, more than 200 bacterial genomes, mainly pathogenic microorganisms, have been sequenced. The first genome of a lactic acid bacterium was completed in 1999.33 Recently, the genomes of many other lactic acid bacteria,34 bifidobacteria,17 and other intestinal microorganisms18,20,35 have been sequenced.36 However, genomic data on 2 of the most widely studied probiotics (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-12) are not publicly available. Genomic information on B. longum,17 L. plantarum,34 L. johnsonii,37 or L. acidophilus,38,39 all established probiotics, provide insight into the adhesive mechanisms present in these microorganisms, which provide the basis for both settlement into the gut during different age phases and communication of regulatory signals to specific areas and sites of the gut S117
mucosa. A eukaryotic-type serine protease inhibitor has been identified in the genome of B. longum that may contribute to the immunomodulatory activity of Bifidobacterium. Operons coding for bacteriocins have been identified in L. johnsonii and L. acidophilus that may modify the succession of microbiota in humans over time.
ABERRANT GUT MICROBIOTA: CLINICAL CONSEQUENCES Specific imbalances or deviations in the intestinal microbiota may make humans more vulnerable to intestinal inflammatory diseases and systemic diseases beyond the intestinal environment. Specific Bifidobacterium species in the healthy infant gut are the most predominant and metabolically active organisms; specific clostridia also are often present. Changes in their quantitative and qualitative composition appear to serve as useful indicators of deviations from the balanced microbiota. Other specific microbial biomarkers for health remain to be defined among the developing intestinal microbiota. Specific deviations in intestinal microbiota, including decreased numbers, an atypical composition of bifidobacteria, and aberrancies in clostridia content and composition, may predispose infants to allergic disease.40,41 Similarly, deviations from the normal microbiota are associated with antibiotic side effects. Aberrant microbiota during childhood may predispose a person to both inflammatory gut disease and diarrhea.42,43 Consequently, understanding the quantitative and qualitative microbiota composition will help provide targets for probiotic intervention. Numerous human intervention studies have assessed the efficacy of specific probiotics in the treatment and risk reduction of infectious diarrhea.44,45 The effect has been explained by a reduction in the duration of rotavirus shedding, normalization of the gut, increased permeability caused by rotavirus infection, and increased numbers of IgA-secreting cells against rotavirus. Moreover, the ability of specific probiotics to increase the expression of mucins may contribute to the barrier effect and the inhibition of rotavirus replication. Particularly, the immunomodulatory potential of specific probiotics has introduced new potential therapeutic strategies for combating allergic and inflammatory conditions.24 Probiotic bacteria may counteract the inflammatory process by stabilizing the gut’s microbial environment and the intestine’s permeability barrier, as well as by enhancing the degradation of enteral antigens and altering their immunogenicity. Another explanation for the gut-stabilizing effect could be improvement of the intestine’s immunologic barrier, particularly the intestinal IgA responses. Probiotic effects also may be mediated by controlling the balance between proinflammatory and anti-inflammatory cytokines. These effects, which are exemplified in the reduced disease activity and increased intestinal permeability, have been achieved in pediatric patients with Crohn’s disease and atopic eczema.31,46-49 In clinical trials in adults, results from preparations containing 4 strains of lactobacilli (L. casei, L. plantarum, L. acidophilus, and L. delS118
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brückii subsp. bulgaricus) and 3 bifidobacteria strains (B. longum, B. breve, and B. infantis), together with Streptococcus salivarius subsp. thermophilus, are encouraging for preventing relapses of chronic pouchitis.50 The preventive potential of probiotics in atopic diseases has been demonstrated in a double-blind, placebo-controlled study.50-52 Probiotics administered prenatally and postnatally for 6 months to children at high risk for atopic disease reduced the prevalence of atopic eczema to half that in infants receiving placebos, and the effect appears to extend beyond infancy.
PERSPECTIVES Advancing genomic research will provide data on host– microbe interactions to identify key processes of microbiotic development and maintenance. These include nutrient–microbe interactions and a detailed knowledge of the transfer of microbial communities from parent to infant. Such data should provide the basis for the development of new probiotics.53 Probiotic genome analysis will predict their properties and interactions in human use, allowing their application to human studies of specific target populations. Development of genetic tools aids analysis of the functionality of these strains,54,55 thereby facilitating the development of a new generation of probiotics that are more site-specific and target disorder-specific and safe.
CONCLUSION The healthy infant microbiota acts as an organ to utilize nutrients and also as a defense mechanism against harmful environmental exposures. Deviations in composition can be related to multiple disease states within the intestine but also beyond it. Similarly, components of the human intestinal microbiota or organisms entering the intestine may have both harmful and beneficial effects on human health. The current available information focuses mostly on the role of infant microbiota and the first colonization steps in later health. Bifidobacteria play a key role in this process; clostridia also may serve as biomarkers of changing environmental conditions. Maternal–infant contact, breast milk oligosaccharides, and breast milk microbes exert significant effects on initial microbiotic development. The initial inoculum at birth is promoted through prebiotic galacto-oligosaccharides in breast milk and introduces environmental bacteria through maternal skin and other contact with the infant, thus providing a means to guide the development of individually optimized microbiota under existing environmental conditions. A future goal is to apply the knowledge of microbiotic composition and aberrancies to selecting the right probiotics for a particular target population. Another goal is to ensure that probiotics and prebiotics administered to infants are safe in terms of both long-term use and effects beyond infancy.
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al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000;119:305-9. 51. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial. Lancet 2001;357:1076-9. 52. Kalliomäki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet 2003;361:1869-71. 53. Salminen S, Nurmi J, Gueimonde M. The genomics of probiotic intestinal microorganisms. Genome Biol 2005;6:225-7. 54. Boekhorst J, Siezen RJ, Zwahlen MC, Vilanova D, Pridmore RD, Mercenier A, et al. The complete genomes of Lactobacillus plantarum and Lactobacillus johnsonii reveal extensive differences in chromosome organization and gene content. Microbiology 2004;150:3601-11. 55. Callanan M. Mining the probiotic genome: advances strategies, enhanced benefits, perceived obstacles. Curr Pharm Des 2005;11:25-36.
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