The gastrointestinal tract is host to a number of. The Normal Bacterial Flora of the Human Intestine and Its Regulation PRESENTATION

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1 PRESENTATION The Normal Bacterial Flora of the Human Intestine and Its Regulation Balakrishnan S. Ramakrishna, MD, PhD Abstract: The gastrointestinal tract, and the colon in particular, is host to a number of bacteria that reside within its lumen. In health, greater than 90% of this flora is composed of anaerobes, whereas facultative anaerobes and aerobes are present in smaller numbers. The recent development of molecular methods to quantify these bacteria provides powerful tools to study the influence of these organisms on bowel function. These studies indicate that about 75% of fecal bacteria can be characterized, and belong to the 3 dominant groups the Clostridium coccoides-eubacterium rectale group, Clostridium leptum group, and Bacteroides-Prevotella group. The development and maturation of the enteric flora, and also the diversity of the flora, is reviewed. Multiple factors regulate the population number of these bacteria, including gastric acidity, intestinal transit, dietary factors, antibiotic use, and bacterial interactions with other bacteria and with the epithelium. Anaerobic bacteria in the intestine change the redox status of the colon; they also produce molecules such as short chain fatty acids, which influence colonic epithelial and mucosal physiology in many ways. New knowledge suggests that these bacteria exert effects on host immunity, which extend well beyond the intestine. Key Words: intestinal flora, anaerobic bacteria, colon, feces (J Clin Gastroenterol 2007;41:S2 S6) The gastrointestinal tract is host to a number of bacteria that reside within its lumen. It has been estimated that the adult human gastrointestinal tract harbors approximately organisms, 1 which is 10 times the total number of cells within the body. This corresponds to a degree of metabolic activity that is equivalent to that of any organ in the human body, and this is increasingly being recognized. Bacteria in the lumen of the gut also have effects on host immunity that may be important in determining responses in later life. Information on the composition of the bacterial flora of the gut largely came from careful intubation studies of the upper gastrointestinal tract carried out nearly 40 years ago. 2 Bacteria comprise approximately 55% of fecal Received for publication November 19, 2006; accepted November 22, From the Christian Medical College, Vellore, Tamil Nadu, India. Reprints: Prof Balakrishnan S. Ramakrishna, MD, PhD, Christian Medical College, Ida Scudder Road, Vellore , Tamil Nadu, India ( rama@cmcvellore.ac.in). Copyright r 2007 by Lippincott Williams & Wilkins solids, and most studies have focused on the fecal flora as a marker of the intestinal flora. Studies of mucosaassociated flora of the intestine and colon are few, 3,4 and the determinants of mucosal association versus luminal carriage are not clear. The nature of the gastrointestinal flora and the factors that regulate its composition are reviewed here. BACTERIOLOGY OF THE GASTROINTESTINAL TRACT The low luminal ph in the stomach is destructive to most microbes. Helicobacter pylori is the major microbial colonizer of the stomach. Colonization of the bowel by other bacteria is generally noted only from the duodenum onwards. This part of the gastrointestinal tract contains gram-positive bacteria such as streptococci and lactobacilli but may also contain yeasts, usually in counts not exceeding 10 3 organisms/ml of intestinal content. Bacterial overgrowth is diagnosed when the number of bacterial colonies cultured exceeds 10 5 /ml fluid in the upper small intestine, that is 2-log 10 difference compared with the highest number noted in healthy individuals. There are geographic differences in bacterial colonization of the upper gastrointestinal tract. In apparently healthy rural residents of developing countries, jejunal luminal fluid bacterial counts may reach to 10 5 /ml. 5 There is a continuous gradient of increasing numbers of bacteria from upper small bowel to ileum with up to 10 8 bacteria/g contents in the ileum. In the ileum, it is possible to find strict anaerobes in the lumen of the gut. Beyond the ileocecal valve, anaerobes outnumber the aerobes by a factor of 100-fold to 1000-fold, reaching counts of /g feces. The colon of a healthy adult harbors around 400 to 500 different species of bacteria belong to 190 different genera, although a few genera together comprise the major cultivable flora of the feces (Table 1). It is recognized that, because of their fastidiousness, the majority of the fecal anaerobic bacteria are not cultivable. Molecular methods are now available to enumerate and to characterize the fecal anaerobic flora. 5 7 Molecular probing is based on the hybridization of synthetically prepared oligonucleotides to specific target sequences of bacterial DNA. These probes are generally targeted at the bacterial ribosome, predominantly 16S ribosomal DNA, because of the extensive databases of known sequences of this region. Probes for enumerating bacteria are targeted at highly conserved sequences (universal), different variable regions conserved S2 J Clin Gastroenterol Volume 41, Supp. 1, May/June 2007

2 J Clin Gastroenterol Volume 41, Supp. 1, May/June 2007 Normal Bacterial Flora of the Human Intestine TABLE 1. Major Cultivable Genera of Bacteria in the Feces of Adult Humans Type of Bacteria Genus Number Obligate anaerobes Gram-negative bacilli Bacteroides /g feces Fusobacterium 10 9 /g feces Gram-positive bacilli Eubacterium Bifidobacterium Lactobacillus /g feces Cocci (gram-negative and gram-positive) Clostridium Ruminococcus Peptostreptococcus Anaerobic Streptococcus (mainly serogroup D) Facultative anaerobes Enterobacteriaceae E. coli 10 8 /g feces Citrobacter Enterobacter Proteus Klebsiella Pathogens eg, Shigella, Salmonella, Yersinia within a genus (genus-specific), or highly variable regions that characterize individual species (species-specific). Probes aimed at these regions are used to quantitate bacteria using either dot blot hybridization or fluorescence in situ hybridization. Competitive polymerase chain reaction (PCR) can be used to amplify target DNA and compare with competitor DNA (slightly truncated from the target sequence) to provide quantitative information. 8 Quantitative (real-time) PCR using primers for the amplification of target rdna sequences is now increasingly used to quantitate fecal anaerobic bacteria. 9 A recent study of 91 northern European adults using 18 group-specific and species-specific probes detected by fluorescence in situ hybridization revealed that Clostridium coccoides-eubacterium rectale and Clostridium leptum group were the dominant groups of fecal bacteria together accounting for approximately 53% of fecal bacteria, whereas Bacteroides species accounted for 8.5% of fecal bacteria. 10 This contrasted with an earlier study of 27 adult subjects using a set of 6 probes and dot blot hybridization which indicated that Bacteroides was the predominant genus in feces. 11 DEVELOPMENT AND MATURATION OF THE GUT FLORA The gastrointestinal tract is not colonized in utero. The bacterial flora is acquired at the time of vaginal delivery and fecal bacteria number approximately 10 9 /g feces 1 week after birth. Facultative anaerobes appear first, consisting of Enterobacteria, Streptococcus and Staphylococcus. 12,13 In neonates delivered by cesarean section, intestinal colonization tends to be initially with facultative anaerobes and Clostridium species. 14 Breastfed neonates have higher fecal bacterial counts during the first week of life than bottle-fed neonates. 15 Bifidobacterium and Lactobacillus species appear after the first week of life. In breast-fed infants, Bifidobacterium species account for 90% of fecal bacteria, with lesser numbers of Enterobacteriaceae and enterococci. In bottle-fed infants, on the other hand, Streptococcus, Enterobacteriaceae and Bacteroides species tend to predominate. It is suggested that breast milk contains factors that promote the growth of Bifidobacterium bifidum and that formula feeds promote implantation and persistence of Clostridium perfringens. 16 During weaning, the diet changes from a high fat milk-based diet to a diet rich in carbohydrate. Bacterial oxidative metabolism leads to changes in the redox potential of the colon. These changes lead to colonization by strict anaerobes such as Bacteroides and Clostridium species. A longitudinal study of 11 infants using molecular methods showed that bifidobacterial counts and numbers remained relatively unchanged during or after weaning, whereas Ruminococcus and Escherichia coli increased after weaning. 17 By the second year of life, the gut microflora of the child is similar to that of the adult with approximately 400 species of bacteria in the gut. DIVERSITY OF THE GUT FLORA Analysis of diversity (community analysis) of mixed microbial populations is usually performed either by targeting micro-organisms directly (by fluorescence in situ hybridization) or by investigating the composition of the 16S rrna community. Bacterial rrna is amplified by PCR, and examined further by either cloning libraries followed by sequencing, or by subjecting the amplicons to denaturing gradient gel electrophoresis or temporal temperature gradient gel electrophoresis (TTGE or TGGE) with subsequent identification. Diversity has also been analyzed by techniques such as terminal restriction fragment length polymorphism, which is also targeted at rrna. Using amplification and sequencing of bacterial rdna in feces, Suau et al 18 showed that 82 molecular species could be detected within 3 dominant bacterial genera C. coccoides-like, C. leptum-like, and Bacteroides. Study of individual bacterial groups indicates that there is a limited diversity of bacterial groups within an individual. For instance, study of Bifidobacterium species by amplification of genus-specific rdna suggests that approximately half of all people carry just 3 to 4 species in the colon, whereas the other half carries only 1 or 2 species of Bifidobacterium. 8 Similarly, a study of preterm infants using cloned 16S rrna and PRC-TTGE indicated that there was a limited diversity of the fecal bacterial flora, with 25 species being identified in 26 infants, all but one of which had a cultivable equivalent. 19 Seven infants were colonized by anaerobes and only 4 by Bifidobacterium species. Studies of diversity using cloning libraries show somewhat divergent results. Hayashi et al 20 reported that the predominant intestinal microbial community consisted of about 130 species or phylotypes, belonging largely to Bacteroides group, Bifidobacterium S3

3 Ramakrishna J Clin Gastroenterol Volume 41, Supp. 1, May/June 2007 group, Streptococcus group, and Clostridium clusters IV, IX, XVIa and XVIII. Approximately, 25% of them belonged to known species, whereas the rest were novel species. More recently, examination of 13,555 rrna sequences from stool or colonic mucosal biopsy detected 395 phylotypes of bacteria, the majority belonging to uncultivable bacteria. 21 These were composed mostly of Firmicutes, 95% belonging to the Clostridia class, and Bacteroides. MUCOSAL-ASSOCIATED FLORA VERSUS LUMINAL FLORA Bacteria in the intestine may be adherent to the mucosa or may remain in the lumen without attachment to the mucosa. Bacteria that attach to the mucosa may exert greater influence on innate immune processes in the intestine. Study of such mucosa-associated flora is limited. Initial studies using culture of mucosal biopsies showed that there was a considerable diversity of the mucosal-associated flora, and the mucosal flora was similar to, or a subset of, the luminal flora. With the availability of molecular methods, this has been reexamined. In a recent study, colonic mucosal biopsies were obtained from 3 individuals, and an effort was made to correlate mucosal-associated flora with the luminal flora. Mucosal bacteria were generally found to be a subset of bacteria within the lumen, and it was eventually concluded that fecal bacteria comprised shed mucosalassociated bacteria plus a separate nonadherent luminal population. 21 Molecular methods using denaturing gradient gel electrophoresis profiling of mucosal-associated bacteria showed little intraindividual variation but considerable variation in the flora between individuals. 22 REGULATION OF THE GUT FLORA Studies using conventional bacteriology indicated that everyone has their own individual pattern of bacterial flora in the intestine, and that this flora remains stable over long periods of time, although there is some increase in species diversity with age. 23,24 The factors that regulate the gut flora are many and not completely elucidated. A number of physiologic factors regulate the gut flora. Important among these are gastric acid and motility. Gastric ph is 1 to 2 in the postabsorptive state and this makes it impossible for many organisms to survive in the stomach. Intestinal transit, by moving the intestinal contents aborally, also regulates the intestinal flora. Because transit is rapid in the upper small intestine, numbers of luminal bacteria are less in this region, whereas slow transit in the colon allows for a large bacterial population in the colon. Within physiologic limits, rapid colonic transit increases fecal bacteria and fecal mass. Thus, hastening colonic transit time from 67 hours to 25 hours increased the fecal weight from 148 to 285 g/d, and increased bacterial dry matter from 16 to 20 g/d. 25 S4 Microbial Factors Interactions between and within populations of bacteria are important in regulating numbers of individual species. The nature of such interactions is listed in Table 2. Dietary Factors These are likely very important factors regulating bacterial composition of the gut. In infancy, mother s milk provides factors that promote the growth of Bifidobacterium. These factors include oligosaccharides present in milk 26 and Lactobacilli that are increased in milk-fed infants. 27 Bacteroides cleave dietary xylans, glucans and pectin, and attack mucin proteins, and preferentially increase when the lumen provides these. In persons who ingest a strictly vegetarian diet, changes noted include a modest reduction in Bacteroides and Bifidobacterium, and an increase in coliforms and enterococci. There has been interest in changing the pattern of the bowel microflora to a predominantly beneficial flora by feeding unabsorbed carbohydrates, predominantly oligosaccharides, as prebiotics to stimulate growth of specific beneficial bacteria. 23,28,29 Antibiotics Antibiotic use has variable and selective influence on the normal enteric bacterial flora fluoroquinolones virtually eliminate aerobic gram-negative bacteria without affecting the anaerobes. Macrolides reduce grampositive cocci and some gram-negative Enterobacteriaceae. Clindamycin and tinidazole affect anaerobes alone. b-lactam drugs reduce all classes of bacteria. A point to remember is that total reduction may be of the order of 99%, yet large numbers of bacteria (eg, ) persist after a course of therapy. Environmental Factors Probiotic administration does not lead to sustained change of the bacterial flora of the intestine. It has been suggested that the sequence in which bacteria settle a niche in the colon owing to chance encounters, nutrient supply, and immune surveillance may well determine the ability of other bacteria to establish residence. 7 If this is true, then administration of probiotic bacteria in the TABLE 2. Types of Interaction Between Bacteria in the Intestine Competition between For growth-limiting nutrients bacteria For adhesion sites on epithelia Commensalism Growth of one species is stimulated by the Amensalism (antagonism) Symbiosis Quorum sensing other Growth of one species is inhibited by the other, eg, by production of short chain fatty acids, hydrogen sulfide, or bacteriocins Two species have an obligate dependence on each other Bacterial sensing of intraspecies numbers is important in regulating the population; role in interspecies signaling is unknown

4 J Clin Gastroenterol Volume 41, Supp. 1, May/June 2007 Normal Bacterial Flora of the Human Intestine neonatal period should influence the composition of the intestinal bacterial flora. This has not been established, that is, that early intervention in the period immediately after birth can lead to sustained change in the bacterial flora. Feeding of Bifidobacterium and Lactobacillus GG to neonates is associated with colonization of the intestine with the organism that is administered, but neither of these studies had long-term follow-up to determine whether there was sustained flora change once the intervention ceased. 31,32 Bacteria-epithelial Cell Interactions Bacteria may induce epithelial cell secretion of microbial nutrients, for example, glycans, which then allow preferential growth of species such as Bacteroides. Bacteria may induce enzymes required for the digestion of ingested foods, and thereby provide nutrients for bacterial growth. They may induce antibacterial peptides which are not effective against that micro-organism but against other competitors. Bacteria also may induce surface receptors for bacterial ligands or for lectinoligosaccharide interactions. FUNCTIONS OF THE GUT FLORA The normal flora of the gut has considerable metabolic activity, which impacts on the human host. Most important is the role of the flora in metabolizing unabsorbed carbohydrate, which leads to salvage of calories from the colon. Unabsorbed dietary carbohydrate includes nonstarch polysaccharides and resistant starch. A significant proportion of the daily starch intake, even up to 20% in some communities, is comprised of resistant starch. Colonic bacteria ferment this to various short chain fatty acids, which are absorbed and result in caloric salvage although at a lower level than absorption of glucose. A number of different anaerobic bacterial groups participate in this fermentative reaction. Short chain fatty acids play multiple roles in colonic physiology including promotion of sodium and chloride absorption, epithelial cell proliferation, epithelial cell differentiation, and epithelial repair. 33 They also have a role in lipid metabolism in the liver. 33 Other important consequences of bacterial metabolism include synthesis of certain vitamins, metabolism of unabsorbed protein leading to production of substances such as phenols, and metabolism of steroid hormones, bile acids and drugs through reactions including hydrolysis, dehydroxylation, decarboxylation, dealkylation, dehalogenation, deamination, and reduction. It is now recognized that the intestinal flora plays important roles in angiogenesis and maintenance of mucosal barrier integrity. 34 Luminal bacteria interact with epithelial cells and other cells of the innate immune system and may alter immune responses to pathogenic bacteria, and regulate inflammation in the gut. 34 The normal intestinal flora is thought to play a role in immune conditioning of the neonatal gut and influence subsequent immune responses of the host, and this is an area that will see further research targeted at preventive intervention. CONCLUSIONS The normal bacterial flora of the human intestine is complex and varied. Molecular methods of analysis are likely to provide new insights into the nature and functions of the flora and alterations in disease. Additional roles for the flora are now being investigated including its role in immune conditioning and inflammation. REFERENCES 1. Luckey TD, Floch MH. Introduction to intestinal microecology. Am J Clin Nutr. 1972;25: Holzapfel WH, Haberer P, Snel J, et al. Overview of gut flora and probiotics. Int J Food Microbiol. 1998;41: Bhat P, Albert MJ, Rajan D, et al. Bacterial flora of the jejunum. A comparison of luminal aspirate and mucosal biopsy. J Med Microbiol. 1989;13: Bhat P, Shantakumari S, Rajan D, et al. Bacterial flora of the gastrointestinal tract in southern Indian control subjects and patients with tropical sprue. Gastroenterology. 1972;62: Blaut M, Collins MD, Welling GW, et al. Molecular biological methods for studying the gut microbiota: the EU human gut flora project. Br J Nutr. 2002;87(suppl 2):S203 S McCartney AL. Application of molecular biological methods for studying probiotics and the gut flora. Br J Nutr. 2002;88(suppl 1): S29 S Mai V, Glenn Morris J Jr. Colonic bacterial flora: changing understandings in the molecular age. J Nutr. 2004;134: Mangin I, Suau A, Magne F, et al. Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE. FEMS Microbiol Ecol. 2006;55: Matsuki T, Watanabe K, Fujimoto J, et al. Quantitative PCR with 16S rrna-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol. 2004;70: Lay C, Rigottier-Gois L, Holmstrøm K, et al. Colonic microbiota signatures across five northern European countries. Appl Environ Microbiol. 2005;71: Sghir A, Gramet G, Suau A, et al. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl Environ Microbiol. 2000;66: Rotimi VO, Duerden BI. The development of the bacterial flora in normal neonates. J Med Microbiol. 1981;14: Caicedo RA, Schanler RJ, Li N, et al. The developing intestinal ecosystem: implications for the neonate. Pediatr Res. 2005;58: Bezirtzoglou E, Romond MB, Romond C. Modulation of C. perfringens intestinal colonization in infants delivered by Cesarean section. Infection. 1989;17: Bezirtzoglou E. The intestinal microflora during the first weeks of life. Anaerobe. 1997;3: Edwards CA, Parrett AM. Intestinal flora during the first months of life: new perspectives. Br J Nutr. 2002;88(suppl 1):S11 S Magne F, Hachelaf W, Suau A, et al. A longitudinal study of infant fecal microbiota during weaning. FEMS Microbiol Ecol. 2006;58: Suau A, Bonnet R, Sutren M, et al. Direct analysis fo genes encoding 16S rrna from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol. 1999;65: Magne F, Abely M, Boyer F, et al. Low species diversity and high inter-individual variability in faeces of preterm infants as revealed by sequences of 16S rrna genes and PCR-temporal temperature gradient gel electrophoresis profiles. FEMS Microbiol Ecol. 2006;57: Hayashi H, Sakamoto M, Benno Y. Phylogenetic analysis of the human gut microbiota using 16S rdna clone libraries and strictly anaerobic culture-based methods. Microbiol Immunol. 2002;46: S5

5 Ramakrishna J Clin Gastroenterol Volume 41, Supp. 1, May/June Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. Science. 2005;308: Green GL, Brostoff J, Hudspith B, et al. Molecular characterization of the bacteria adherent to human colorectal mucosa. J Appl Microbiol. 2006;100: Blaut M. Relationship of prebiotics and food to intestinal microflora. Eur J Nutr. 2002;41(suppl 1):I11 I Moore WE, Moore LH. Intestinal floras of populations that have a high risk of colon cancer. Appl Environ Microbiol. 1995; 61: Stephen AM, Wiggins HS, Cummings JH. Effect of changing transit time on colonic microbial metabolism in man. Gut. 1987;28: Coppa GV, Bruni S, Morelli L, et al. The first prebiotics in humans: human milk oligosaccharides. J Clin Gastroenterol. 2004;38:S80 S Martin R, Langa S, Reviriego C, et al. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr. 2003;143: Kruse HP, Kleessen B, Blaut M. Effects of inulin on faecal bifidobacteria in human subjects. Br J Nutr. 1999;82: Steer T, Carpenter H, Tuohy K, et al. Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics. Nutr Res Rev. 2000;13: Brismar B, Edlund C, Malmborg AS, et al. Ecological impact of antimicrobial prophylaxis on intestinal microflora in patients undergoing colorectal surgery. Scand J Infect Dis Suppl. 1990; 70: Li Y, Shimizu T, Hosaka A, et al. Effects of Bifidobacterium breve supplementation on intestinal flora of low birth weight infants. Pediatr Int. 2004;46: Petschow BW, Figueroa R, Harris CL, et al. Effects of feeding an infant formula containing Lactobacillus GG on the colonization of the intestine: a dose-response study in healthy infants. J Clin Gastroenterol. 2005;39: Binder HJ, Cummings JH, Soergel KH, eds. Short Chain Fatty Acids. Lancaster: Kluwer Academic; O Hara AM, Shanahan F. The gut flora as a forgotten organ. EMBO Reports. 2006;7: S6