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National Institutes of Health Gastrointestinal Microbiota and Advances in Prebiotic and Probiotic Research Conference Summary
Meeting Summary National Institutes of Health Gastrointestinal Microbiota and Advances in Prebiotic and Probiotic Research Conference Summary CRYSTAL MCDADE–NGUTTER,* JAMES VERSALOVIC,‡ WILLIAM ALEXANDER,§ VAN S. HUBBARD,* PAMELA STARKE–REED,* MARGUERITE KLEIN,储 TONSE RAJU,¶ JOHN MILNER,# CINDY DAVIS,# CAROL PONTZER,** JOSE ARENA,‡‡ JON VANDERHOOF,§§ and STEFANIE A. NELSON¶¶ *National Institutes of Health, Division of Nutrition Research Coordination; ‡Texas Children’s Hospital, Baylor College of Medicine; §National Institutes of Health, National Institute of Allergy and Infectious Diseases; 储National Institutes of Health, Office of Dietary Supplements; ¶National Institutes of Health, National Institute of Child Health and Human Development; #National Institutes of Health, National Cancer Institute; **National Institutes of Health, National Center for Complementary and Alternative Medicine; ‡‡National Institutes of Health, Center for Scientific Review; §§Harvard Medical School, University of Nebraska, and Meade Johnson Nutrition; ¶¶ The Scientific Consulting Group, Inc
See related content in the May supplemental issue of Gastroenterology.
T
he National Institutes of Health (NIH) convened the Gastrointestinal Microbiota and Advances in Prebiotic and Probiotic Research (GMAPPR) conference in Rockville, Maryland, on December 11 and 12, 2007. To determine the extent to which the gut microbiota impacts human health and disease, approximately 160 scientists, physicians, and experts from various disciplines gathered at this conference to identify knowledge gaps and discuss future research opportunities with regard to the role of the gut microbiota, prebiotics, and probiotics in health and disease and to support the advancement of prebiotics and probiotics research. Five sessions featured presentations by 24 prominent scientists and physicians. Although this article briefly summarizes selected findings of the GMAPPR conference, a comprehensive summary including all the presentations at this meeting can be found on the NIH Division of Nutrition Research Coordination website: http://dnrc.nih.gov/highlights/gmappr. shtml. Session I, Function of Gut Microbiota, explored the role of intestinal bacteria in the development and modulation of the immune system in humans and animals. Humans are likely to have their first exposure to allergens while in utero, when the fetal immune system is heavily biased toward Th 2 cells. This phenomenon will continue after birth until down-regulation of Th 2-like responses begins during childhood in healthy infants; however, this down-regulation does not occur in children with clinical allergies. A study comparison presented during this session revealed differences in the immune system and allergy development between Estonian and Swedish infants. Estonian infants’ immune systems developed more rapidly than those of Swedish infants because Estonian infants had much higher levels of secretory immunoglob-
ulin (Ig)A, which is indicative of the final phases of immune system maturation. Exposure to pets, farms, daycares, and older siblings was hypothesized to be protective against allergies. Gut microbiota are a prerequisite for the development of oral tolerance. The gut microbes in Estonian infants seem to be more pronounced than those in Swedish infants, which may be attributed to exposure differences between the 2 countries. Likewise, higher levels of Bifidobacteria were observed in healthy infants compared with those with allergies, and similar results continued to be observed until 5 years of age. Throughout the studies, when comparing gut microbiota of allergic and nonallergic infants and children, it is evident that exposure to bacterial species and microbial diversity plays a key role in the development of the immune system. Session II, Defining Normal Gut Ecology, examined and defined normal gut ecology of the healthy human and animal models. The mouth, esophagus, and stomach biota are phylogenetically related, with the mouth being different from the stomach, but both overlapping with the esophagus. Population differences are due to the Firmicutes, which dominate the proximal foregut; however, the stomach harbors a large proportion of gramnegative anaerobes/microaerophils from Bacteroidetes and Proteobacteria. The biota of the esophageal biota 200 species and samples of biota obtained from biopsies can be classified into 2 metagenotypes, I and II, based on 16S rDNA-based genetic distance between samples and a reference range and concentrated in the normal esophagus. Type I is dominated by Streptococcus and associated with the normal esophagus; type II biota are more abundant in gram-negative anaerobic/microaerophilic bacteria and primarily correlated with esophagitis and Barrett esophagus. Type II biota have been connected with gastroesophageal reflux disease, and suggests an altered type I © 2009 by the AGA Institute
0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.03.042 GASTROENTEROLOGY 2009;136:1473–1475
Meeting Summary continued
or an ectopic gastric biota. These findings suggest interesting possibilities for research in the treatment of esophageal disease. By contrast, the analysis of distal gut microbes in adults, which included primate studies from St Louis and San Diego zoos and in the wild, was also covered in this session. It was pointed out that primates’ microbial intestinal diversity is vast containing 70 bacterial divisions, phyla, or deep evolutionary lineages. However, in humans, the number is narrowed to only 2 dominant divisions of bacteria—Bacteroidetes and Firmicutes and 1 member of Archea family. Finally, these primate studies may emphasize also the effect of diet and host genotype on the distal gut microbiota in adults. Session III, Impact of Disease on Gut Microbiota, investigated the impact of diseases, such as necrotizing enterocolitis, irritable bowel disease, and obesity on the gut microbiota (see also accompanying Mini-Review and Perspective by Tilg et al in this issue). Using the SAMP1 mouse as a model of Crohn’s disease, it was suggested that probiotics may help to maintain gut health and homeostasis by stimulating epithelial innate immune responses. Studies with VSL#3 (a mixture of 4 Lactobacilli, 3 Bifidobacteria, and 1 Streptococcus) indicate that VSL#3 may be able to prevent the onset of ileitis and suppress inflammation; however, similar results could not be produced once ileitis had been established. In addition, this study reviewed the well-documented metagenomic studies of the gut microbiome and its role in energy balance. In these studies, a shifting of gut microbiota in response to obesity and diet change was documented. A study examining 12 obese humans demonstrated that Bacteriodetes species increased in the gut as humans lost weight by following either a fat- or carbohydrate-restricted diet. Likewise, similar results could be observed in diet-induced obese mice, which demonstrated an increase in Firmicutes and a decrease in Bacteroidetes when fed a highfat/simple-sugar diet. Current research is investigating the impact of malnutrition on the gut microbiome by analyzing intestinal microbiota of siblings and their mothers in developing countries in an effort to understand why siblings tend to differ in regard to development of kwashiorkor or marasmus. This research may be useful in determining whether gut ecology can predispose individuals to kwashiorkor or marasmus. Session IV, Factors Influencing Gut Microbiota, investigated the association between exposure to probiotics, prebiotics, and antibiotic use and their impact on the microbiome of the normal gut. With regard to environment-imposed alterations to gut microbiota, perhaps no factor is as important as antibiotic use. As the importance of the microbiota in health becomes ever more apparent, the use of antibiotics in pregnant women and infants is a growing concern. Data are emerging that relate early antibiotic use with disease. Increases in resis1474
tant coliform infections and necrotizing enterocolitis have been observed with peripartum use, and suggest that antibiotics given to mothers alter the developing indigenous microbiota, thereby resulting in replacement microbes that may be just as harmful as those intended for eradication. The mother clearly plays an important role in colonization patterns of her infant, not only through initial microbe exposure during birth, but also through breast milk, which is known to be a source of microbes. Aseptically collected breast milk was shown to contain a total microbe concentration of 103 colonyforming units per milliliter. In general, little is known about the impact of antibiotic use by the mother on infant colonization. However, there is emerging evidence of long-term impact on gut microbiota even after antibiotic treatment has ceased. The first colonization of the intestine is one of the most profound immunologic exposures faced by the newborn infant. Cross-talk induces gene expression in both the epithelium and immune system. Niches are formed as part of a potentially longlasting biofilm located within the luminal glycocalyx. The colonizing microbiota impacts Toll-like receptor ligands that are crucial for the intestinal surface to protect and repair itself in the face of infectious or inflammatory insult. Previous studies have established a strong correlation between infection, antibiotic use, and the rise in chronic diseases which support the hypothesis that antibiotic-induced alterations of the gut microbiota may have long-lasting, detrimental effects on infant health. This hypothesis is supported further by animal model studies. Manipulation of the intestinal microbiota in early life with antibiotics may result in significant shortand long-term consequences; “shotgun” approaches to antibiotic use in pregnant or lactating women and their babies should be reconsidered. Important studies needed to address this issue are culture-independent metagenomic analysis of the short- and long-term impact of antibiotics on gut microbiota. The final session, New Developments in Prebiotic and Probiotic Research, summarized recent advances in prebiotics and probiotics research with a description of the classes of probiotics that have different immunomodulatory capacities. Different probiotic strains may either stimulate or suppress immune responses (eg, production of proinflammatory cytokines) in the host, and such differences may have implications for specific applications in disease prevention and treatment. Molecular mechanisms of probiotics were summarized, including effects on human signaling pathways (nuclear factor-B and AP-1) and the nature of secreted “immunomodulins” derived from probiotic lactobacilli. Secreted factors derived from probiotics, such as fatty acids and peptides, may contribute to the ability of microbial communities to regulate mucosal immune functions. In addition, this
Meeting Summary continued
session described immunomodulatory effects of prebiotics. In animal models, prebiotics alone or in combination with probiotics enhanced the production of secretory IgA and the anti-inflammatory cytokine interleukin-10, and potentiated natural killer cell-like activities. In a porcine model, prebiotics enhanced the phagocytic activities of neutrophils/monocytes and stimulated proliferative capacities of T lymphocytes. In summary, prebiotics may facilitate shifts in the composition and aggregate functions of intestinal microbial populations to yield fundamental effects on the immune system. Prebiotics may activate specific immune functions while promoting immune homeostasis in the intestinal mucosa. The abilities of a model intestinal commensal bacterium Bacteroides thetaiotamicron to affect carbohydrate utilization patterns in gnotobiotic mouse models were discussed. B thetaiotamicron shifts its gene expression profiles toward different carbohydrate utilization patterns depending on changes in diet and local nutrient availability. Fundamental changes in diet alter the functional capacities of commensal bacteria, and these alterations in bacterial metabolism may determine whether host-derived carbohydrates in the mucus layer, for example, are scavenged effectively by intestinal bacteria. The administration of probiotics may fundamentally alter functional capacities of indigenous bacteria. Each of 3 probiotic strains had a distinct impact on the transcriptome of B thetaiotamicron which suggests that functional effects on the microbiome may
differ dramatically depending on which probiotic strain is administered in vivo. These studies are being extended to examine effects of mixed bacterial species interactions on gene expression profiles of probiotics. This interdisciplinary conference generated stimulating discussion on various aspects of the gastrointestinal microbiota and provided hypotheses for the ways probiotics may function with regard to attachment, regulation of specific mammalian genes, and production of beneficial metabolic products, although more research is needed in this area. It is clear that both probiotic and gut microbiota research are progressing, but more mechanistic studies are needed to understand the interplay of the various factors involved, such as host genotype and composition of the microbial community. Gut microbiota, probiotics, and prebiotics provide a significant opportunity to beneficially affect the health and well-being of the population and should continue to be enthusiastically researched.
Correspondence Address correspondence to: Crystal McDade-Ngutter. e-mail: [email protected] Conflicts of interest The authors disclose the following: James Versalovic has financial arrangements with BIOGAIA (Stockholm, Sweden). The remaining authors disclose no conflicts.
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See related content in the May supplemental issue of Gastroenterology.
T
he National Institutes of Health (NIH) convened the Gastrointestinal Microbiota and Advances in Prebiotic and Probiotic Research (GMAPPR) conference in Rockville, Maryland, on December 11 and 12, 2007. To determine the extent to which the gut microbiota impacts human health and disease, approximately 160 scientists, physicians, and experts from various disciplines gathered at this conference to identify knowledge gaps and discuss future research opportunities with regard to the role of the gut microbiota, prebiotics, and probiotics in health and disease and to support the advancement of prebiotics and probiotics research. Five sessions featured presentations by 24 prominent scientists and physicians. Although this article briefly summarizes selected findings of the GMAPPR conference, a comprehensive summary including all the presentations at this meeting can be found on the NIH Division of Nutrition Research Coordination website: http://dnrc.nih.gov/highlights/gmappr. shtml. Session I, Function of Gut Microbiota, explored the role of intestinal bacteria in the development and modulation of the immune system in humans and animals. Humans are likely to have their first exposure to allergens while in utero, when the fetal immune system is heavily biased toward Th 2 cells. This phenomenon will continue after birth until down-regulation of Th 2-like responses begins during childhood in healthy infants; however, this down-regulation does not occur in children with clinical allergies. A study comparison presented during this session revealed differences in the immune system and allergy development between Estonian and Swedish infants. Estonian infants’ immune systems developed more rapidly than those of Swedish infants because Estonian infants had much higher levels of secretory immunoglob-
ulin (Ig)A, which is indicative of the final phases of immune system maturation. Exposure to pets, farms, daycares, and older siblings was hypothesized to be protective against allergies. Gut microbiota are a prerequisite for the development of oral tolerance. The gut microbes in Estonian infants seem to be more pronounced than those in Swedish infants, which may be attributed to exposure differences between the 2 countries. Likewise, higher levels of Bifidobacteria were observed in healthy infants compared with those with allergies, and similar results continued to be observed until 5 years of age. Throughout the studies, when comparing gut microbiota of allergic and nonallergic infants and children, it is evident that exposure to bacterial species and microbial diversity plays a key role in the development of the immune system. Session II, Defining Normal Gut Ecology, examined and defined normal gut ecology of the healthy human and animal models. The mouth, esophagus, and stomach biota are phylogenetically related, with the mouth being different from the stomach, but both overlapping with the esophagus. Population differences are due to the Firmicutes, which dominate the proximal foregut; however, the stomach harbors a large proportion of gramnegative anaerobes/microaerophils from Bacteroidetes and Proteobacteria. The biota of the esophageal biota 200 species and samples of biota obtained from biopsies can be classified into 2 metagenotypes, I and II, based on 16S rDNA-based genetic distance between samples and a reference range and concentrated in the normal esophagus. Type I is dominated by Streptococcus and associated with the normal esophagus; type II biota are more abundant in gram-negative anaerobic/microaerophilic bacteria and primarily correlated with esophagitis and Barrett esophagus. Type II biota have been connected with gastroesophageal reflux disease, and suggests an altered type I © 2009 by the AGA Institute
0016-5085/09/$36.00 doi:10.1053/j.gastro.2009.03.042 GASTROENTEROLOGY 2009;136:1473–1475
Meeting Summary continued
or an ectopic gastric biota. These findings suggest interesting possibilities for research in the treatment of esophageal disease. By contrast, the analysis of distal gut microbes in adults, which included primate studies from St Louis and San Diego zoos and in the wild, was also covered in this session. It was pointed out that primates’ microbial intestinal diversity is vast containing 70 bacterial divisions, phyla, or deep evolutionary lineages. However, in humans, the number is narrowed to only 2 dominant divisions of bacteria—Bacteroidetes and Firmicutes and 1 member of Archea family. Finally, these primate studies may emphasize also the effect of diet and host genotype on the distal gut microbiota in adults. Session III, Impact of Disease on Gut Microbiota, investigated the impact of diseases, such as necrotizing enterocolitis, irritable bowel disease, and obesity on the gut microbiota (see also accompanying Mini-Review and Perspective by Tilg et al in this issue). Using the SAMP1 mouse as a model of Crohn’s disease, it was suggested that probiotics may help to maintain gut health and homeostasis by stimulating epithelial innate immune responses. Studies with VSL#3 (a mixture of 4 Lactobacilli, 3 Bifidobacteria, and 1 Streptococcus) indicate that VSL#3 may be able to prevent the onset of ileitis and suppress inflammation; however, similar results could not be produced once ileitis had been established. In addition, this study reviewed the well-documented metagenomic studies of the gut microbiome and its role in energy balance. In these studies, a shifting of gut microbiota in response to obesity and diet change was documented. A study examining 12 obese humans demonstrated that Bacteriodetes species increased in the gut as humans lost weight by following either a fat- or carbohydrate-restricted diet. Likewise, similar results could be observed in diet-induced obese mice, which demonstrated an increase in Firmicutes and a decrease in Bacteroidetes when fed a highfat/simple-sugar diet. Current research is investigating the impact of malnutrition on the gut microbiome by analyzing intestinal microbiota of siblings and their mothers in developing countries in an effort to understand why siblings tend to differ in regard to development of kwashiorkor or marasmus. This research may be useful in determining whether gut ecology can predispose individuals to kwashiorkor or marasmus. Session IV, Factors Influencing Gut Microbiota, investigated the association between exposure to probiotics, prebiotics, and antibiotic use and their impact on the microbiome of the normal gut. With regard to environment-imposed alterations to gut microbiota, perhaps no factor is as important as antibiotic use. As the importance of the microbiota in health becomes ever more apparent, the use of antibiotics in pregnant women and infants is a growing concern. Data are emerging that relate early antibiotic use with disease. Increases in resis1474
tant coliform infections and necrotizing enterocolitis have been observed with peripartum use, and suggest that antibiotics given to mothers alter the developing indigenous microbiota, thereby resulting in replacement microbes that may be just as harmful as those intended for eradication. The mother clearly plays an important role in colonization patterns of her infant, not only through initial microbe exposure during birth, but also through breast milk, which is known to be a source of microbes. Aseptically collected breast milk was shown to contain a total microbe concentration of 103 colonyforming units per milliliter. In general, little is known about the impact of antibiotic use by the mother on infant colonization. However, there is emerging evidence of long-term impact on gut microbiota even after antibiotic treatment has ceased. The first colonization of the intestine is one of the most profound immunologic exposures faced by the newborn infant. Cross-talk induces gene expression in both the epithelium and immune system. Niches are formed as part of a potentially longlasting biofilm located within the luminal glycocalyx. The colonizing microbiota impacts Toll-like receptor ligands that are crucial for the intestinal surface to protect and repair itself in the face of infectious or inflammatory insult. Previous studies have established a strong correlation between infection, antibiotic use, and the rise in chronic diseases which support the hypothesis that antibiotic-induced alterations of the gut microbiota may have long-lasting, detrimental effects on infant health. This hypothesis is supported further by animal model studies. Manipulation of the intestinal microbiota in early life with antibiotics may result in significant shortand long-term consequences; “shotgun” approaches to antibiotic use in pregnant or lactating women and their babies should be reconsidered. Important studies needed to address this issue are culture-independent metagenomic analysis of the short- and long-term impact of antibiotics on gut microbiota. The final session, New Developments in Prebiotic and Probiotic Research, summarized recent advances in prebiotics and probiotics research with a description of the classes of probiotics that have different immunomodulatory capacities. Different probiotic strains may either stimulate or suppress immune responses (eg, production of proinflammatory cytokines) in the host, and such differences may have implications for specific applications in disease prevention and treatment. Molecular mechanisms of probiotics were summarized, including effects on human signaling pathways (nuclear factor-B and AP-1) and the nature of secreted “immunomodulins” derived from probiotic lactobacilli. Secreted factors derived from probiotics, such as fatty acids and peptides, may contribute to the ability of microbial communities to regulate mucosal immune functions. In addition, this
Meeting Summary continued
session described immunomodulatory effects of prebiotics. In animal models, prebiotics alone or in combination with probiotics enhanced the production of secretory IgA and the anti-inflammatory cytokine interleukin-10, and potentiated natural killer cell-like activities. In a porcine model, prebiotics enhanced the phagocytic activities of neutrophils/monocytes and stimulated proliferative capacities of T lymphocytes. In summary, prebiotics may facilitate shifts in the composition and aggregate functions of intestinal microbial populations to yield fundamental effects on the immune system. Prebiotics may activate specific immune functions while promoting immune homeostasis in the intestinal mucosa. The abilities of a model intestinal commensal bacterium Bacteroides thetaiotamicron to affect carbohydrate utilization patterns in gnotobiotic mouse models were discussed. B thetaiotamicron shifts its gene expression profiles toward different carbohydrate utilization patterns depending on changes in diet and local nutrient availability. Fundamental changes in diet alter the functional capacities of commensal bacteria, and these alterations in bacterial metabolism may determine whether host-derived carbohydrates in the mucus layer, for example, are scavenged effectively by intestinal bacteria. The administration of probiotics may fundamentally alter functional capacities of indigenous bacteria. Each of 3 probiotic strains had a distinct impact on the transcriptome of B thetaiotamicron which suggests that functional effects on the microbiome may
differ dramatically depending on which probiotic strain is administered in vivo. These studies are being extended to examine effects of mixed bacterial species interactions on gene expression profiles of probiotics. This interdisciplinary conference generated stimulating discussion on various aspects of the gastrointestinal microbiota and provided hypotheses for the ways probiotics may function with regard to attachment, regulation of specific mammalian genes, and production of beneficial metabolic products, although more research is needed in this area. It is clear that both probiotic and gut microbiota research are progressing, but more mechanistic studies are needed to understand the interplay of the various factors involved, such as host genotype and composition of the microbial community. Gut microbiota, probiotics, and prebiotics provide a significant opportunity to beneficially affect the health and well-being of the population and should continue to be enthusiastically researched.
Correspondence Address correspondence to: Crystal McDade-Ngutter. e-mail: [email protected] Conflicts of interest The authors disclose the following: James Versalovic has financial arrangements with BIOGAIA (Stockholm, Sweden). The remaining authors disclose no conflicts.
1475