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Intestinal microbiota and its functions
Digestive and Liver Disease Supplements 3 (2009) 30–34
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Intestinal microbiota and its functions M. Montalto *, F. D’Onofrio, A. Gallo, A. Cazzato, G. Gasbarrini Internal Medicine Department, Gemelli Hospital, Catholic University of Sacred Heart, Rome, Italy
Abstract The digestive tract harbours the largest and most complex microbial community of the human body, the intestinal microbiota, including about 800 different bacteria species. The distribution of this microflora is uneven, with highest concentrations in the colon. Bacterial colonization of human gut by environmental microbes, beginning immediately after birth, becomes more complex with increasing age, with a high degree of variability among human individuals. The gastrointestinal tract is the main site where environmental microorganisms and antigens interact with the host, through intensive cross-talks. Gut microbiota is essential for intestinal development, homeostasis and protection against pathogenic challenge; moreover, gut microbes are involved in metabolic reactions, with harvest of energy ingested but not digested by the host; they have also trophic effects on the intestinal epithelium, by favouring the development of intestinal microvilli, and play a fundamental role in the maturation of the host’s innate and adaptive immune responses. © 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Gut; Intestinal mucosa; Metabolism; Microbiota
1. Introduction The cutaneous and mucosal surfaces of the human body are colonized by microbes deriving from the environment and constituting highly complex ecosystems. In particular, the digestive tract harbours the largest and most complex community of these microorganisms. Being conditions in the various sections of gastrointestinal tract considerably different, the distribution of this microflora is uneven, with low concentrations of bacteria in the stomach and duodenum (up to 103 colony-forming units/ml), increasing concentrations in jejunum and ileum (104 –108 CFU/ml) and the highest concentrations in the colon (up to 1014 CFU/ml), an amount 10 times greater than the total number of human somatic and germ cells [1]. Intestinal microbiota includes about 800 different bacteria species with over 7000 strains [2]. The majority of these species, a large number of which is uncultivable with available media, has been
* Correspondence to: Massimo Montalto, MD, Catholic University of Sacred Heart, Gemelli Hospital, Largo A. Gemelli, 8; 00168 Rome, Italy. Tel.: +39 06 3015 6018, Fax: +39 06 3550 2775. E-mail address: [email protected] (M. Montalto).
identified using molecular methods based on sequencing bacterial ribosomal RNA (16S rRNA) genes and is represented by obligate anaerobes [3,4]. The most common anaerobic genera are Bacteroides, Bifidobacterium, Eubacterium, Fusobacterium, Clostridium and Lactobacillus, whereas aerobes are Gram-negative enteric bacteria (such as Escherichia coli and Salmonella spp.) and Gram-positive cocci (such as Enterococcus, Staphylococcus and Streptococcus); moreover, aerobic fungal species are also present, such as Candida albicans [5]. Bacterial colonization of human gut by environmental microbes begins immediately after birth; the composition of intestinal microbiota, relatively simple in infants, becomes more complex with increasing age, with a high degree of variability among human individuals [6]. However, it maintains a relative stability in the single subjects, in spite of permanent interaction with environmental microbes [7]. Changes in microbiota can be observed as a consequence of diet variations, pathological conditions (such as enteral infections), antibiotic therapy, anti-acid treatment or immunosuppression; a study of adult monogenic twins living apart and their marital partners has emphasized the role of host genotype in determining composition of gut microbiota [7–9].
© 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
M. Montalto et al. / Digestive and Liver Disease Supplements 3 (2009) 30–34
The genomes of all these intestinal microbes form the “microbiome”, representing more than 100 times the human genome; the latter may be considered in association to the microbiome, as the “metagenome” [10,11]. As a consequence, the microbiome provides the human host with additional metabolic functions, described as “metabolome”, allowing the harvest of otherwise inaccessible nutrients.
2. Functions of intestinal microbiota The gut represents the natural interface between intestinal microflora and the host; in fact, the mucosal surfaces of the gastrointestinal tract are the main sites where environmental microorganisms and antigens interact with the host, through intensive cross-talks [11]. The relationship between intestinal microbiota and the host is often described as commensal (one partner benefits and the other is apparently unaffected) instead of mutualistic (both partners experience increased fitness); this probably reflects the lack of complete knowledge about all functions of intestinal microbiota and its interactions with the host [9]. It is well documented that gut microbiota is essential for intestinal development, homeostasis and protection against pathogenic challenges, to the point that some investigators have referred to it as an “extra organ” of the host [12]. In particular, gut microbes are involved in metabolic reactions, such as fermentation of non-digestible dietary fiber (resistant starch, some oligosaccharides) with harvest of energy ingested but not digested by the host, biotransformation of conjugated bile acids, degradation of oxalate-based complexes and synthesis of some vitamins (such as B12 and K); they have also trophic effects on the intestinal epithelium, by favouring the development of intestinal microvilli, and play a fundamental role in the maturation of the host’s innate and adaptive immune responses [13,14]. All these properties of gut microbiota have been demonstrated in studies on germ-free rodents, in which the absence of intestinal commensal bacteria is associated to poor growth, requiring higher caloric intake to maintain body weight and supplementation with vitamins K and B, an immature architecture of intestinal mucosa, with low epithelial growth and development, and a higher susceptibility to infections [3,15].
3. Human host/intestinal microbiota interface Homeostasis of the intestinal wall is the result of bacteria–bacteria and bacteria–host interactions. The postnatal bacterial colonization of the gut is essential for the development of the host’s innate and adaptive immune responses, enabling the immune system to develop tolerance to a variety of microbial antigens that result in reduced allergic and inflammatory responses [14,16]. In contrast, germ-free animals have little to no development of gastrointestinal associated lymphoid tissue (GALT), and there
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is marked skewing of the T-helper (Th) cell balance [17]. Moreover, it is known that colonization of intestinal mucosa by a pathogen microorganism leads to a strong inflammatory reaction, whereas commensals induce a self-limiting inflammatory process, without tissue damage, resulting in a state of immunological tolerance. However, the molecular mechanisms of this discrimination between pathogens and commensals are not well defined. It is known that mucosal enterocites are of crucial importance for the interactions of the human host with the intestinal microbiota, other environmental microbes and antigens. Gut epithelial tissue, besides forming an efficient physical barrier preventing the access of environmental pathogens and antigens to the host’s internal milieu, releases chemokines and cytokines that recruit inflammatory and immune cells, involved in the control of these potentially harmful agents [18]. Host–microbe interactions are relevant also in the control of gut microbiota composition [19]. As regards innate effectors, these are involved in the regulation of gut microbiota colonization, such as the production of secretory IgA, which are released in the intestinal lumen, and defensins, antimicrobial peptides acting as natural antibiotics, produced and secreted by mucosal epithelial cells [20]. Defensins have bactericidal activity against Gram-negative and Gram-positive bacteria, and exert activity also against fungi, virus and protozoa [20,21]. Moreover, interactions among bacteria are also crucial for intestinal colonization: in particular, bacterial metabolic activities, such as the production of short-chain fatty acids (SCFA), the modification of potential redox and the synthesis of bacteriocins, can modify intestinal ecology, creating ecosystems that are appropriate for some bacterial genera and hostile for others. Other important mechanisms regulating intestinal colonization are represented by the competition for receptors on the mucus layer or the epithelial cells and communication among bacteria [7]. Interaction among microbes, gut epithelial and immune cells is based on the sensing of microbial signals from the gut lumen through two major pattern-recognition receptor systems, represented by the Toll-like receptors (TLRs), that lie on the surface of epithelial and immune cells, and the nucleotide-binding oligomerization domain/caspase recruitment domain isoforms (NOD/CARD), that lie in cytoplasm [22]. Stimulation of TLRs by microbes initiates a complex signaling cascade, leading to the release of NF-κB, which migrates into the nucleus and binds to genes containing NFκB sites, thus triggering the transcription of a wide variety of cytokines, chemokines, acute-phase proteins and cell adhesion molecules [23]. TLRs also regulate the polarization of CD4+ Th lymphocytes into Th1, Th2 or Th3/Th1 subsets [24]. NODs are a family of cytosolic proteins involved in triggering the signaling cascade that leads to intestinal inflammatory reaction [25]. NOD1 is expressed ubiquitously, and its signaling is required for activation of NF-κB in infections by Gram-negative enteric bacteria able to bypass TLR activation, thus indicating a synergistic relationship between TLRs and NODs [26]. NOD2, expressed by mono-
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M. Montalto et al. / Digestive and Liver Disease Supplements 3 (2009) 30–34
cytes/macrophages and dendritic cells (DCs) and induced in enterocytes by TNF-α, is a sensor for muramyl dipeptide, the smallest bioactive peptidoglycan pattern of Grampositive and Gram-negative bacteria; its activation leads to secretion of proinflammatory cytokines, such as TNF-α and IL-1β, via the NF-κB pathway, and to secretion of IL-6 and IL-12 in monocytes and DCs [25]. DCs and macrophages are the main antigen-presenting cells of the intestinal mucosa. Immature DCs are sentinels continuously sampling the mucosal surface for antigens, through their dendrites. They migrate into the epithelium opening the tight junctions of the upper extremity of enterocytes and, after the contact with microbial products, they activate an endocytosis process and subsequently a migration into draining lymph nodes, where they present the microbials’ antigens processed to the naive B and T lymphocytes [27,28].
4. Intestinal microbiota and metabolism One of most important factors for the persistence of a given bacterium in the gastrointestinal tract is represented by diet, which provides nutrients not only for the host, but also for the bacteria in the gastrointestinal tract [6]. In fact, all dietary components that escape the host’s digestion in the small intestine are potential substrates of bacteria in the colon, thus forming a sort of anaerobic bioreactor. Moreover, differences in diet can lead to variations in the composition of gut microflora. In infancy, it is known that differences in infant-feeding regimens are associated to different gut microflora, consisting mainly of lactic acid bacteria in breast-fed infants, and a mixture of anaerobic and aerobic species in bottle-fed infants [29]. In the elderly people, instead, a reduction in bifidobacteria has been reported [30]. Metabolic additional functions provided by gut microbiota are well shown by Bacteroides thetaiotaomicron, a prominent mutualist of the colon having a great capacity for digestion of otherwise indigestible dietary polysaccharides; in fact, its genome contains the largest ensemble of genes involved in acquiring and metabolizing carbohydrates yet reported for a sequenced bacterium, with two outer membrane proteins that bind and import starch, 226 glycoside hydrolases and 15 polysaccharide lyases. The human genome, by contrast, only contains 98 known or putative glycoside hydrolases and one polysaccharide lyase [31]. The main substrates available to bacteria in the colon are dietary carbohydrates, including resistant starches, dietary fibers (cellulose, hemicellulose, pectin, inulin), unabsorbed sugars and sugar alcohols, dietary proteins, proteins from pancreatic enzymes and gastrointestinal secretions, mucus and sloughed epithelial cells [32,33]. Catabolism of carbohydrates and proteins by intestinal bacteria gives rise to SCFA (acetate, propionate and butyrate) and gases (carbon dioxide, molecular hydrogen and methane); moreover, proteic degradation leads to the formation of branched-chain fatty acids, ammonia, hydrogen sulfide, amines, phenols,
indoles and mercaptanes [33]. Among SCFA, butyrate is considered the preferred fuel of the colon epithelial cells, that derive 70% of their energy from the oxidation of this substrate; SCFA irrigation for 2 to 3 weeks improves macroscopic and histological lesions of inflammation resulting from diversion colitis, arising after complete diversion of fecal stream [34,35]. Butyrate decreases the expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α), TNF-β, interleukin-6 (IL-6) and IL-1β by inhibiting the activation of nuclear factor κB (NF-κB) in lamina propria mononuclear cells of patients affected by inflammatory bowel diseases [36,37]. Another member of intestinal microbiota, Oxalobacter formigenes, is involved in the metabolism of oxalate, and the absence of this microbe is a risk factor for urolithiasis by calcium oxalate stones [38,39]. Moreover, gut microflora takes part in the transformation of a large variety of plant-derived non-nutritive substances, like lignans and flavonoids, with formation of active or inactive metabolites. Lignans and flavonoids, also known as phytoestrogens, have been proposed as chemoprotective substances; in particular, isoflavones have been studied for their action of prevention of hormone-related cancers (such as breast cancer), atherosclerosis, osteoporosis and alleviation of menopausal symptoms [40,41]. It is known that metabolic diseases, like obesity and type 2 diabetes mellitus, result from a variable combination of genetic and environmental factors (such as excessive energy intake and reduction of physical activity) [42]. However, it can be observed that when a population is subjected to the same nutritional stress, some individuals are less susceptible to diet-induced weight-gain and hyperglycemia: a possibile explanation of this is based on the above-mentioned role of intestinal microbiota in the energy metabolism [43–45]. In a recent study on rodents, a lower food intake in mice with intestinal microbiota causes a 40% higher body fat content and 47% higher gonadal fat content in comparison to germ-free mice; moreover, germ-free mice colonized with microbes of mice with gut microflora increased their fat mass and developed insulin resistance [46]. Mechanisms of these changes are represented by an increase in glucose intestinal absorption, energy extraction from non-digestible food components with consequent higher glycemia and insulinemia, favouring lipogenesis, through an increase in the activity of the enzyme lipoprotein lipase (LPL) and the suppression of the fasting-induced adipose factor, that normally inhibits LPL. Other studies, in rodents and humans, showed that obesity is associated to alterations in the composition of gut microflora, with a 50% reduction of Bacteroidetes and a proportional increase in Firmicutes, supporting the hypothesis that differences in populations of intestinal microbiota may lead to differences in metabolism of substrates and energy harvest [45,47]. Metabolic activities of gut microbiota, in addition to providing the above-mentioned effects, can also result in the production of harmful substances, involved in pathogenesis
M. Montalto et al. / Digestive and Liver Disease Supplements 3 (2009) 30–34
of cancer: degradation of proteins can lead to formation of ammonia, high concentrations of which have been shown to act as tumor promoters, and catabolism of aminoacids cysteine and methionine by sulphate-reducing bacteria results in the formation of hydrogen sulphide, a highly toxic compound involved in impairment of cytochrome oxidase, butyrate utilization, synthesis of mucus and methylation of DNA [48,49]. Decarboxylation of amino acids leads to the formation of amines, that may react with nitrites derived from metabolism of intestinal bacteria to form highly carcinogenic nitrosamines [50]. Anaerobic fermentation of the aromatic aminoacids tyrosine and tryptophan gives rise to phenols and indoles, that act as procarcinogens in colon cancer [51]. All these findings show why a meat-rich diet, typical of westernized countries, is associated to a higher risk of cancer [52]. Also metabolism of bile acids by colonic microflora may be involved in carcinogenesis: in facts, a fraction of cholic acid reaching the colon is converted in deoxycholic acid, a secondary bile acid with a well recognized co-carcinogen and lithogenic properties, high fecal and plasma levels of which are observed in patients affected by colon cancer [53,54]. At last, other compounds (such as dietary components, pharmaceuticals and endogenous metabolites) that are excreted in bile after hepatic hydroxylation and conjugation with either glucuronic acid, sulphate or glutathione, are deconjugated by intestinal microbes, entering the enterohepatic circulation and thus being reabsorbed [55].
Conflict of interest statement None declared.
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Intestinal microbiota and its functions M. Montalto *, F. D’Onofrio, A. Gallo, A. Cazzato, G. Gasbarrini Internal Medicine Department, Gemelli Hospital, Catholic University of Sacred Heart, Rome, Italy
Abstract The digestive tract harbours the largest and most complex microbial community of the human body, the intestinal microbiota, including about 800 different bacteria species. The distribution of this microflora is uneven, with highest concentrations in the colon. Bacterial colonization of human gut by environmental microbes, beginning immediately after birth, becomes more complex with increasing age, with a high degree of variability among human individuals. The gastrointestinal tract is the main site where environmental microorganisms and antigens interact with the host, through intensive cross-talks. Gut microbiota is essential for intestinal development, homeostasis and protection against pathogenic challenge; moreover, gut microbes are involved in metabolic reactions, with harvest of energy ingested but not digested by the host; they have also trophic effects on the intestinal epithelium, by favouring the development of intestinal microvilli, and play a fundamental role in the maturation of the host’s innate and adaptive immune responses. © 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Gut; Intestinal mucosa; Metabolism; Microbiota
1. Introduction The cutaneous and mucosal surfaces of the human body are colonized by microbes deriving from the environment and constituting highly complex ecosystems. In particular, the digestive tract harbours the largest and most complex community of these microorganisms. Being conditions in the various sections of gastrointestinal tract considerably different, the distribution of this microflora is uneven, with low concentrations of bacteria in the stomach and duodenum (up to 103 colony-forming units/ml), increasing concentrations in jejunum and ileum (104 –108 CFU/ml) and the highest concentrations in the colon (up to 1014 CFU/ml), an amount 10 times greater than the total number of human somatic and germ cells [1]. Intestinal microbiota includes about 800 different bacteria species with over 7000 strains [2]. The majority of these species, a large number of which is uncultivable with available media, has been
* Correspondence to: Massimo Montalto, MD, Catholic University of Sacred Heart, Gemelli Hospital, Largo A. Gemelli, 8; 00168 Rome, Italy. Tel.: +39 06 3015 6018, Fax: +39 06 3550 2775. E-mail address: [email protected] (M. Montalto).
identified using molecular methods based on sequencing bacterial ribosomal RNA (16S rRNA) genes and is represented by obligate anaerobes [3,4]. The most common anaerobic genera are Bacteroides, Bifidobacterium, Eubacterium, Fusobacterium, Clostridium and Lactobacillus, whereas aerobes are Gram-negative enteric bacteria (such as Escherichia coli and Salmonella spp.) and Gram-positive cocci (such as Enterococcus, Staphylococcus and Streptococcus); moreover, aerobic fungal species are also present, such as Candida albicans [5]. Bacterial colonization of human gut by environmental microbes begins immediately after birth; the composition of intestinal microbiota, relatively simple in infants, becomes more complex with increasing age, with a high degree of variability among human individuals [6]. However, it maintains a relative stability in the single subjects, in spite of permanent interaction with environmental microbes [7]. Changes in microbiota can be observed as a consequence of diet variations, pathological conditions (such as enteral infections), antibiotic therapy, anti-acid treatment or immunosuppression; a study of adult monogenic twins living apart and their marital partners has emphasized the role of host genotype in determining composition of gut microbiota [7–9].
© 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
M. Montalto et al. / Digestive and Liver Disease Supplements 3 (2009) 30–34
The genomes of all these intestinal microbes form the “microbiome”, representing more than 100 times the human genome; the latter may be considered in association to the microbiome, as the “metagenome” [10,11]. As a consequence, the microbiome provides the human host with additional metabolic functions, described as “metabolome”, allowing the harvest of otherwise inaccessible nutrients.
2. Functions of intestinal microbiota The gut represents the natural interface between intestinal microflora and the host; in fact, the mucosal surfaces of the gastrointestinal tract are the main sites where environmental microorganisms and antigens interact with the host, through intensive cross-talks [11]. The relationship between intestinal microbiota and the host is often described as commensal (one partner benefits and the other is apparently unaffected) instead of mutualistic (both partners experience increased fitness); this probably reflects the lack of complete knowledge about all functions of intestinal microbiota and its interactions with the host [9]. It is well documented that gut microbiota is essential for intestinal development, homeostasis and protection against pathogenic challenges, to the point that some investigators have referred to it as an “extra organ” of the host [12]. In particular, gut microbes are involved in metabolic reactions, such as fermentation of non-digestible dietary fiber (resistant starch, some oligosaccharides) with harvest of energy ingested but not digested by the host, biotransformation of conjugated bile acids, degradation of oxalate-based complexes and synthesis of some vitamins (such as B12 and K); they have also trophic effects on the intestinal epithelium, by favouring the development of intestinal microvilli, and play a fundamental role in the maturation of the host’s innate and adaptive immune responses [13,14]. All these properties of gut microbiota have been demonstrated in studies on germ-free rodents, in which the absence of intestinal commensal bacteria is associated to poor growth, requiring higher caloric intake to maintain body weight and supplementation with vitamins K and B, an immature architecture of intestinal mucosa, with low epithelial growth and development, and a higher susceptibility to infections [3,15].
3. Human host/intestinal microbiota interface Homeostasis of the intestinal wall is the result of bacteria–bacteria and bacteria–host interactions. The postnatal bacterial colonization of the gut is essential for the development of the host’s innate and adaptive immune responses, enabling the immune system to develop tolerance to a variety of microbial antigens that result in reduced allergic and inflammatory responses [14,16]. In contrast, germ-free animals have little to no development of gastrointestinal associated lymphoid tissue (GALT), and there
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is marked skewing of the T-helper (Th) cell balance [17]. Moreover, it is known that colonization of intestinal mucosa by a pathogen microorganism leads to a strong inflammatory reaction, whereas commensals induce a self-limiting inflammatory process, without tissue damage, resulting in a state of immunological tolerance. However, the molecular mechanisms of this discrimination between pathogens and commensals are not well defined. It is known that mucosal enterocites are of crucial importance for the interactions of the human host with the intestinal microbiota, other environmental microbes and antigens. Gut epithelial tissue, besides forming an efficient physical barrier preventing the access of environmental pathogens and antigens to the host’s internal milieu, releases chemokines and cytokines that recruit inflammatory and immune cells, involved in the control of these potentially harmful agents [18]. Host–microbe interactions are relevant also in the control of gut microbiota composition [19]. As regards innate effectors, these are involved in the regulation of gut microbiota colonization, such as the production of secretory IgA, which are released in the intestinal lumen, and defensins, antimicrobial peptides acting as natural antibiotics, produced and secreted by mucosal epithelial cells [20]. Defensins have bactericidal activity against Gram-negative and Gram-positive bacteria, and exert activity also against fungi, virus and protozoa [20,21]. Moreover, interactions among bacteria are also crucial for intestinal colonization: in particular, bacterial metabolic activities, such as the production of short-chain fatty acids (SCFA), the modification of potential redox and the synthesis of bacteriocins, can modify intestinal ecology, creating ecosystems that are appropriate for some bacterial genera and hostile for others. Other important mechanisms regulating intestinal colonization are represented by the competition for receptors on the mucus layer or the epithelial cells and communication among bacteria [7]. Interaction among microbes, gut epithelial and immune cells is based on the sensing of microbial signals from the gut lumen through two major pattern-recognition receptor systems, represented by the Toll-like receptors (TLRs), that lie on the surface of epithelial and immune cells, and the nucleotide-binding oligomerization domain/caspase recruitment domain isoforms (NOD/CARD), that lie in cytoplasm [22]. Stimulation of TLRs by microbes initiates a complex signaling cascade, leading to the release of NF-κB, which migrates into the nucleus and binds to genes containing NFκB sites, thus triggering the transcription of a wide variety of cytokines, chemokines, acute-phase proteins and cell adhesion molecules [23]. TLRs also regulate the polarization of CD4+ Th lymphocytes into Th1, Th2 or Th3/Th1 subsets [24]. NODs are a family of cytosolic proteins involved in triggering the signaling cascade that leads to intestinal inflammatory reaction [25]. NOD1 is expressed ubiquitously, and its signaling is required for activation of NF-κB in infections by Gram-negative enteric bacteria able to bypass TLR activation, thus indicating a synergistic relationship between TLRs and NODs [26]. NOD2, expressed by mono-
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cytes/macrophages and dendritic cells (DCs) and induced in enterocytes by TNF-α, is a sensor for muramyl dipeptide, the smallest bioactive peptidoglycan pattern of Grampositive and Gram-negative bacteria; its activation leads to secretion of proinflammatory cytokines, such as TNF-α and IL-1β, via the NF-κB pathway, and to secretion of IL-6 and IL-12 in monocytes and DCs [25]. DCs and macrophages are the main antigen-presenting cells of the intestinal mucosa. Immature DCs are sentinels continuously sampling the mucosal surface for antigens, through their dendrites. They migrate into the epithelium opening the tight junctions of the upper extremity of enterocytes and, after the contact with microbial products, they activate an endocytosis process and subsequently a migration into draining lymph nodes, where they present the microbials’ antigens processed to the naive B and T lymphocytes [27,28].
4. Intestinal microbiota and metabolism One of most important factors for the persistence of a given bacterium in the gastrointestinal tract is represented by diet, which provides nutrients not only for the host, but also for the bacteria in the gastrointestinal tract [6]. In fact, all dietary components that escape the host’s digestion in the small intestine are potential substrates of bacteria in the colon, thus forming a sort of anaerobic bioreactor. Moreover, differences in diet can lead to variations in the composition of gut microflora. In infancy, it is known that differences in infant-feeding regimens are associated to different gut microflora, consisting mainly of lactic acid bacteria in breast-fed infants, and a mixture of anaerobic and aerobic species in bottle-fed infants [29]. In the elderly people, instead, a reduction in bifidobacteria has been reported [30]. Metabolic additional functions provided by gut microbiota are well shown by Bacteroides thetaiotaomicron, a prominent mutualist of the colon having a great capacity for digestion of otherwise indigestible dietary polysaccharides; in fact, its genome contains the largest ensemble of genes involved in acquiring and metabolizing carbohydrates yet reported for a sequenced bacterium, with two outer membrane proteins that bind and import starch, 226 glycoside hydrolases and 15 polysaccharide lyases. The human genome, by contrast, only contains 98 known or putative glycoside hydrolases and one polysaccharide lyase [31]. The main substrates available to bacteria in the colon are dietary carbohydrates, including resistant starches, dietary fibers (cellulose, hemicellulose, pectin, inulin), unabsorbed sugars and sugar alcohols, dietary proteins, proteins from pancreatic enzymes and gastrointestinal secretions, mucus and sloughed epithelial cells [32,33]. Catabolism of carbohydrates and proteins by intestinal bacteria gives rise to SCFA (acetate, propionate and butyrate) and gases (carbon dioxide, molecular hydrogen and methane); moreover, proteic degradation leads to the formation of branched-chain fatty acids, ammonia, hydrogen sulfide, amines, phenols,
indoles and mercaptanes [33]. Among SCFA, butyrate is considered the preferred fuel of the colon epithelial cells, that derive 70% of their energy from the oxidation of this substrate; SCFA irrigation for 2 to 3 weeks improves macroscopic and histological lesions of inflammation resulting from diversion colitis, arising after complete diversion of fecal stream [34,35]. Butyrate decreases the expression of proinflammatory cytokines tumor necrosis factor-α (TNF-α), TNF-β, interleukin-6 (IL-6) and IL-1β by inhibiting the activation of nuclear factor κB (NF-κB) in lamina propria mononuclear cells of patients affected by inflammatory bowel diseases [36,37]. Another member of intestinal microbiota, Oxalobacter formigenes, is involved in the metabolism of oxalate, and the absence of this microbe is a risk factor for urolithiasis by calcium oxalate stones [38,39]. Moreover, gut microflora takes part in the transformation of a large variety of plant-derived non-nutritive substances, like lignans and flavonoids, with formation of active or inactive metabolites. Lignans and flavonoids, also known as phytoestrogens, have been proposed as chemoprotective substances; in particular, isoflavones have been studied for their action of prevention of hormone-related cancers (such as breast cancer), atherosclerosis, osteoporosis and alleviation of menopausal symptoms [40,41]. It is known that metabolic diseases, like obesity and type 2 diabetes mellitus, result from a variable combination of genetic and environmental factors (such as excessive energy intake and reduction of physical activity) [42]. However, it can be observed that when a population is subjected to the same nutritional stress, some individuals are less susceptible to diet-induced weight-gain and hyperglycemia: a possibile explanation of this is based on the above-mentioned role of intestinal microbiota in the energy metabolism [43–45]. In a recent study on rodents, a lower food intake in mice with intestinal microbiota causes a 40% higher body fat content and 47% higher gonadal fat content in comparison to germ-free mice; moreover, germ-free mice colonized with microbes of mice with gut microflora increased their fat mass and developed insulin resistance [46]. Mechanisms of these changes are represented by an increase in glucose intestinal absorption, energy extraction from non-digestible food components with consequent higher glycemia and insulinemia, favouring lipogenesis, through an increase in the activity of the enzyme lipoprotein lipase (LPL) and the suppression of the fasting-induced adipose factor, that normally inhibits LPL. Other studies, in rodents and humans, showed that obesity is associated to alterations in the composition of gut microflora, with a 50% reduction of Bacteroidetes and a proportional increase in Firmicutes, supporting the hypothesis that differences in populations of intestinal microbiota may lead to differences in metabolism of substrates and energy harvest [45,47]. Metabolic activities of gut microbiota, in addition to providing the above-mentioned effects, can also result in the production of harmful substances, involved in pathogenesis
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of cancer: degradation of proteins can lead to formation of ammonia, high concentrations of which have been shown to act as tumor promoters, and catabolism of aminoacids cysteine and methionine by sulphate-reducing bacteria results in the formation of hydrogen sulphide, a highly toxic compound involved in impairment of cytochrome oxidase, butyrate utilization, synthesis of mucus and methylation of DNA [48,49]. Decarboxylation of amino acids leads to the formation of amines, that may react with nitrites derived from metabolism of intestinal bacteria to form highly carcinogenic nitrosamines [50]. Anaerobic fermentation of the aromatic aminoacids tyrosine and tryptophan gives rise to phenols and indoles, that act as procarcinogens in colon cancer [51]. All these findings show why a meat-rich diet, typical of westernized countries, is associated to a higher risk of cancer [52]. Also metabolism of bile acids by colonic microflora may be involved in carcinogenesis: in facts, a fraction of cholic acid reaching the colon is converted in deoxycholic acid, a secondary bile acid with a well recognized co-carcinogen and lithogenic properties, high fecal and plasma levels of which are observed in patients affected by colon cancer [53,54]. At last, other compounds (such as dietary components, pharmaceuticals and endogenous metabolites) that are excreted in bile after hepatic hydroxylation and conjugation with either glucuronic acid, sulphate or glutathione, are deconjugated by intestinal microbes, entering the enterohepatic circulation and thus being reabsorbed [55].
Conflict of interest statement None declared.
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