Bacterial Diversity in the Human Gut
Introduction
The human digestive tract is colonized by microorganisms to varying degrees throughout its length. Because of acid pH, and the short retention time of gastric contents, numbers of viable bacterial cells in the stomach are usually <102 per ml, with aciduric Gram-positive species such as lactobacilli and streptococci predominating, although numbers can increase 1000-fold postprandially. While the rapid passage of digestive materials through the upper small bowel does not allow time for significant bacterial growth to occur, their numbers increase considerably to >108 per ml in the distal ileum (Macfarlane and Cummings, 1991). The rate of movement of intestinal contents slows markedly in the large bowel, which facilitates the development of large complex bacterial communities (Cummings 1978, Cummings 1993). Indeed, the vast majority of cells associated with the human body (eukaryotic and prokaryotic) are anaerobic bacteria growing in the colon (Savage, 1977). Bacteria comprise 40–45% of fecal material on a dry weight basis (Stephen and Cummings, 1980), which equates to about 18 grams of bacterial dry matter, or a total bacterial mass in the colon of about 90 grams (Macfarlane and Cummings, 1991).
The large bowel is the main area of permanent microbial colonization of the human gastrointestinal tract, and several hundred bacterial strains and species have been isolated from this complex ecosystem, where viable counts in feces typically reach 1011–1012 per gram (Finegold 1983, Moore 1974), with anaerobic bacteria predominating (Finegold 1974, Hentges 1993, Holdeman 1976, Moore 1974). Microbial cell population densities increase progressively from the cecum to the distal large intestine (Fig. 1). Bacterial colonization of the gut is affected by a wide variety of host, microbiological, environmental and dietary factors, as indicated in Table I.
Until recently, our knowledge of bacterial diversity in the large intestine was derived by using classical viable counting techniques; however, the advent of molecular methods of analysis has led to renewed interest in the structure and composition of the gut ecosystem.
Section snippets
Culturing Studies
Selective and non-selective culture methods have been the standard techniques used to quantitate bacterial populations in feces; however, for various reasons, not all bacteria can be cultivated. This can result in underestimation of bacterial population sizes and microbiota diversity (O'Sullivan 1999, Wilson 1996), although culturing techniques have been thought to overestimate numbers of some important groups of gut bacteria, such as bacteroides and bifidobacteria (Dore 1998, Sghir 2000).
PCR-Based Molecular Techniques
Polymerase chain reaction (PCR) amplification is a rapid, accurate and sensitive method of analysis that can be used to detect bacteria in complex communities. It has the ability to amplify DNA fragments from a background of other genomes by the use of genus- or species-specific primers (Matsuki 1999, Wang 1996) and can identify the bacteria without the need for culture. PCR can reduce the selection bias introduced by traditional culture methods (Head et al., 1998), but DNA⧸RNA may not be
PCR-Independent Molecular Methods
Two molecular methods can be used to quantitate bacteria by using 16S rRNA targeted oligonucleotide probes, dot-blot hybridization, and fluorescent in situ hybrization (FISH).
Real-Time PCR
Coventional PCR of microbial populations determines only that bacteria are present in a community and not their relative abundances. However, with the advent of real-time PCR, 16S rRNA gene copy numbers can be determined by using genus- or species-specific primers, with external standards, with the incorporation of a fluorescent dye such as SYBR green into double-stranded DNA. Real-time PCR has been used to study intestinal populations (Huijsdens et al., 2002), while Malinen et al. (2003)
The Developing Microflora in Infants
Babies are colonized with bacteria from the mother and environment at birth. Whether the infant is breast or bottle-fed has an important effect on the types of bacteria that grow in the gut. While some workers have found no differences in fecal microfloras in breast and bottle-fed infants (Heavey 1999, Lundequist 1985), the majority of studies have shown that there are major differences in bacterial community structure (Beerens 1980, Benno 1984, Harmsen 2000a, Martin 2000).
Using culture-based
The Gut Microflora in Adults
Studies investigating the stability of the adult fecal microflora by using culturing methods indicate that while bacterial groups such as lactobacilli, bacteroides, and bifidobacteria seem to remain relatively constant (Bornside 1978, Finegold 1983, Holdeman 1976), there can be considerable changes in microbiota composition at the species level (Holdeman et al., 1976). For example, in 10 volunteers studied over 12 months, numbers of Bacteroides fragilis were found to vary as much as a
Bacterial Colonization in Different Regions of the Large Bowel
Studies on bacterial diversity in the gastrointestinal tract are restricted to a large extent by the inaccessibility of parts of the gut. Consequently, the vast majority of studies have been made by using fecal material, which only provides information on microbial populations occurring in the lumen of the distal large intestine. This does not necessarily reflect the composition of bacterial communities in the proximal colon or on mucosal surfaces lining the bowel.
Conclusions
Early studies on the composition of the human colonic microbiota depended on culturing techniques. These investigations demonstrated that the large intestine was a highly complex microbial ecosystem containing vast numbers of bacteria that could be assigned to several hundred bacterial species. However, there have been great developments in molecular methods of population analysis over the last decade, which have indicated that the colonic microbiota contains greater bacterial diversity than
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