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Indigenous Flora
4
INDIGENOUS FLORA Jonathan E. Teitelbaum
Researchers have estimated that the human body contains 1014 cells, only 10% of which are not bacteria and belong to the human body proper.1 The mammalian intestinal tract represents a complex, dynamic and diverse ecosystem of interacting aerobic and anaerobic, nonpathologic bacteria. This complex yet stable colony includes more than 400 separate species.2 Within any segment of the gut, some organisms are adherent to the epithelium, while others exist in suspension in the mucus layer overlying the epithelium.3 Binding to the epithelial surface is a highly specific process. For example, certain strains of lactobacilli and coagulase-negative staphylococci adhere to the gastric epithelium of the rat, whereas Escherichia coli and Bacteroides are unable to do so.4 Bacterial adherence is also modulated by the local environment (i.e., pH), surface charge and presence of fibronectin.5 Those unbound bacteria within the lumen of the gut represent those organisms shed from the epithelium or swallowed from the oropharynx. Luminal flora accounts for the majority of organisms within the gut and represents 40% of the weight of feces1; however, the fecal flora found in stool samples does not necessarily represent the important host-microbial symbiosis of the mucosal bound flora.6 Because the majority of indigenous species are obligate anaerobes, their culture, identification, and quantification are technically difficult, and it is estimated that at least half of the indigenous bacteria cannot be cultured by traditional methods.2,7 Limitations of conventional microbiological techniques have confounded a detailed analysis of the enteric flora and led to a shift from traditional culture and phenotyping to genotyping. Modern techniques of ribotyping, pulsed field electrophoresis, plasmid profiles, specific primers, and probes for polymerase chain reaction (PCR) and nucleic acid hybridization and 16S rRNA sequencing have allowed for identification of bacteria without culturing. Furthermore, specific 16S rRNA-based oligonucleotide probes allow detection of bacterial groups by fluorescent in situ hybridization (FISH). Such techniques are limited only by the number of probes developed to date to identify the bacteria of interest. Research efforts analyzing the symbiotic relationship that exists within the human gastrointestinal tract have been aided by studies of two well-described systems: the symbiosis between Rhizobium bacteria and leguminous plants, and the cooperative interaction between Vibrio fischeri and the light-producing organ of the squid. In each host tissue, modifications are made to allow a favorable niche to be established by the symbiont.8 The use of newer microbiological techniques has helped to further elaborate the ways in which bacteria effect change within the host. For example, the use of laser capture microdissection and gene array analysis of germ-free mice colonized with Bacteroides thetaiotaomicron has shown affects on murine genes influencing mucosal barrier function, nutrient absorption, 28
metabolism, angiogenesis, and the development of the enteric nervous system.9 Host activities including processing of nutrients and regulation of the immune system are affected by the genetic potential of the indigenous flora, known as the microbiome.10 The composition of the intestinal microbiome is variable, and its diversity can be affected by alteration in diet and antibiotic use. Genes for specific metabolic pathways, such as amino acid and glycan metabolism, appear to be overrepresented in the microbiome of the distal gut, supporting the notion that human metabolism is an amalgamation of microbial and human processes.11 Of the fungi, only yeasts play a major role in the orointestinal tract, with Candida being the predominant genus. Various strains are commonly, but not always, present in different locations, suggesting that they may only be transient flora. However, some strains of C. albicans can inhabit the gastrointestinal (GI) tract for longer periods of time, as evidenced by the fact that strains isolated from newborns are the same as the mother’s.12 The presence of Candida in the GI tract does not indicate candidiasis. The colony counts of Candida in normal small and large bowel do not exceed 104 colony-forming units (cfu) per milliliter.12 The introduction of Candida into a welldeveloped fecal flora system under continuous-flow culture did not lead to multiplication of the yeast. Thus, normal bacterial flora appears to provide protection against pathologic colonization by yeast. However, if the fecal flora was destroyed by antibiotics, then the yeast would multiply.12,13 The addition of a Lactobacillus species to the system was able to reduce the colony counts of the Candida significantly.13 It has been found that up to 65% of individuals harbor fungi in the stool.14 As opposed to the numerous indigenous bacterial flora and yeast forms, there does not appear to be a normal viral flora.15
UNDERSTANDING THE INDIGENOUS FLORA BY STUDYING GERM-FREE ANIMALS Further understanding of the beneficial effects of developing a normal bacterial flora is achieved by the analysis of germ-free animal models (Table 4-1). Germ-free mice have small intestines that weigh less than those of their normal counterparts. Their intestinal wall is thinner and less cellular; the villi are thinner and more pointed at the tip; and the crypts are shallower, resulting in a reduced mucosal surface area.16 Histologically, the mucosal cells are cuboidal rather than columnar and uniform in size and shape. The stroma has sparse concentrations of inflammatory cells under aseptic conditions with only few lymphocytes and macrophages. Plasma cells are absent, and Peyer’s patches are smaller with fewer germinal centers; consequently, there is little or no IgA expression.17,18 The T-cell component of the lamina propria is largely composed of CD4+ lymphocytes; these
INDIGENOUS FLORA Jonathan E. Teitelbaum
Researchers have estimated that the human body contains 1014 cells, only 10% of which are not bacteria and belong to the human body proper.1 The mammalian intestinal tract represents a complex, dynamic and diverse ecosystem of interacting aerobic and anaerobic, nonpathologic bacteria. This complex yet stable colony includes more than 400 separate species.2 Within any segment of the gut, some organisms are adherent to the epithelium, while others exist in suspension in the mucus layer overlying the epithelium.3 Binding to the epithelial surface is a highly specific process. For example, certain strains of lactobacilli and coagulase-negative staphylococci adhere to the gastric epithelium of the rat, whereas Escherichia coli and Bacteroides are unable to do so.4 Bacterial adherence is also modulated by the local environment (i.e., pH), surface charge and presence of fibronectin.5 Those unbound bacteria within the lumen of the gut represent those organisms shed from the epithelium or swallowed from the oropharynx. Luminal flora accounts for the majority of organisms within the gut and represents 40% of the weight of feces1; however, the fecal flora found in stool samples does not necessarily represent the important host-microbial symbiosis of the mucosal bound flora.6 Because the majority of indigenous species are obligate anaerobes, their culture, identification, and quantification are technically difficult, and it is estimated that at least half of the indigenous bacteria cannot be cultured by traditional methods.2,7 Limitations of conventional microbiological techniques have confounded a detailed analysis of the enteric flora and led to a shift from traditional culture and phenotyping to genotyping. Modern techniques of ribotyping, pulsed field electrophoresis, plasmid profiles, specific primers, and probes for polymerase chain reaction (PCR) and nucleic acid hybridization and 16S rRNA sequencing have allowed for identification of bacteria without culturing. Furthermore, specific 16S rRNA-based oligonucleotide probes allow detection of bacterial groups by fluorescent in situ hybridization (FISH). Such techniques are limited only by the number of probes developed to date to identify the bacteria of interest. Research efforts analyzing the symbiotic relationship that exists within the human gastrointestinal tract have been aided by studies of two well-described systems: the symbiosis between Rhizobium bacteria and leguminous plants, and the cooperative interaction between Vibrio fischeri and the light-producing organ of the squid. In each host tissue, modifications are made to allow a favorable niche to be established by the symbiont.8 The use of newer microbiological techniques has helped to further elaborate the ways in which bacteria effect change within the host. For example, the use of laser capture microdissection and gene array analysis of germ-free mice colonized with Bacteroides thetaiotaomicron has shown affects on murine genes influencing mucosal barrier function, nutrient absorption, 28
metabolism, angiogenesis, and the development of the enteric nervous system.9 Host activities including processing of nutrients and regulation of the immune system are affected by the genetic potential of the indigenous flora, known as the microbiome.10 The composition of the intestinal microbiome is variable, and its diversity can be affected by alteration in diet and antibiotic use. Genes for specific metabolic pathways, such as amino acid and glycan metabolism, appear to be overrepresented in the microbiome of the distal gut, supporting the notion that human metabolism is an amalgamation of microbial and human processes.11 Of the fungi, only yeasts play a major role in the orointestinal tract, with Candida being the predominant genus. Various strains are commonly, but not always, present in different locations, suggesting that they may only be transient flora. However, some strains of C. albicans can inhabit the gastrointestinal (GI) tract for longer periods of time, as evidenced by the fact that strains isolated from newborns are the same as the mother’s.12 The presence of Candida in the GI tract does not indicate candidiasis. The colony counts of Candida in normal small and large bowel do not exceed 104 colony-forming units (cfu) per milliliter.12 The introduction of Candida into a welldeveloped fecal flora system under continuous-flow culture did not lead to multiplication of the yeast. Thus, normal bacterial flora appears to provide protection against pathologic colonization by yeast. However, if the fecal flora was destroyed by antibiotics, then the yeast would multiply.12,13 The addition of a Lactobacillus species to the system was able to reduce the colony counts of the Candida significantly.13 It has been found that up to 65% of individuals harbor fungi in the stool.14 As opposed to the numerous indigenous bacterial flora and yeast forms, there does not appear to be a normal viral flora.15
UNDERSTANDING THE INDIGENOUS FLORA BY STUDYING GERM-FREE ANIMALS Further understanding of the beneficial effects of developing a normal bacterial flora is achieved by the analysis of germ-free animal models (Table 4-1). Germ-free mice have small intestines that weigh less than those of their normal counterparts. Their intestinal wall is thinner and less cellular; the villi are thinner and more pointed at the tip; and the crypts are shallower, resulting in a reduced mucosal surface area.16 Histologically, the mucosal cells are cuboidal rather than columnar and uniform in size and shape. The stroma has sparse concentrations of inflammatory cells under aseptic conditions with only few lymphocytes and macrophages. Plasma cells are absent, and Peyer’s patches are smaller with fewer germinal centers; consequently, there is little or no IgA expression.17,18 The T-cell component of the lamina propria is largely composed of CD4+ lymphocytes; these