A review of gastrointestinal microbiology with special emphasis on molecular microbial ecology approaches

All animals, including humans, are adapted to life in a microbial world. Large populations of micro-organisms inhabit the gastrointestinal tract of all animals and form a closely integrated ecological unit with the host. This complex, mixed, microbial culture can be considered the most metabolically adaptable and rapidly renewable organ of the body, which plays a vital role in the normal nutritional, physiological, immunological and protective functions of the host animal. Bacteria have traditionally been classified mainly on the basis of phenotypic properties. Despite the vast amount of knowledge generated for ruminal and other intestinal ecosystems using traditional techniques, the basic requisites for ecological studies, namely, enumeration and identification of all community members, have limitations. The two major problems faced by microbial ecologists are bias introduced by culture-based enumeration and characterization techniques, and the lack of a phylogenetically-based classification scheme. Modem molecular ecology techniques based on sequence comparisons of nucleic acids (DNA or RNA) can be used to provide molecular characterization while at the same time providing a classification scheme that predicts natural evolutionary relationships. These molecular methods provide results that are independent of growth conditions and media used. Also, using these techniques, bacteria can be classified and identified before they can be grown in pure culture. These nucleic acid-based techniques will enable gut microbiologists to answer the most difficult question in microbial ecology: namely, describing the exact role or function a specific bacterium plays in its natural environment and its quantitative contribution to the whole. However, rather than replacing the classical culture-based system, the new molecular-based techniques can be used in combination with the classical approach to improve cultivation, speciation and evaluation of diversity. The study of microbial ecology in gut ecosystems involves investigation of the organisms present (abundance and diversity), their activity (usually determined in vitro, but in vivo activity or expression of activity is really required), and their relationship with each other and the host animal (synergistic and competitive interactions). This entails the study and measurement of many types of interactions, both beneficial and competitive. Traditionally, media for isolation of bacteria from natural environments are basically of two types: those that simulate the habitat in broad terns, i.e. habitat-simulating and non-selective media; and those designed to enumerate and isolate bacteria of a particular type or from a specific biochemical niche, i.e. niche-simulating or functional or nutritional group analysis. A third type, less important in the ecological sense, does not simulate the habitat, is often highly selective and is used to isolate specific bacterial groups. Specific nutritional types of bacteria may be isolated by the use of enrichment media. This type of medium, basically a refinement of the habitat-simulating medium, is widely used in environmental microbiology and has been applied with some success to the gut ecosystem. Although estimates of microbial number rely on culture techniques, microscopic examination is a most useful technique for evaluating the efficacy of other enumeration approaches. The combination of microscopy with specific phylogenetic stains or fluorescent antibodies enables bacteria to be specifically detected and enumerated in mixed populations. The introduction of genetic-based technologies, and in particular those relating to 16S rRNA typing, are rapidly replacing conventional detection and enumeration methods in studies of the mammalian intestinal tract. Although molecular techniques promise a fuller and more accurate description of the true diversity, structure and dynamics of complex microbial communities than the present culturing studies, each technique suffers from its own experimental bias and selectivity. Different methods used for direc t molecular detection are reviewed in the paper. Attention is given to molecular characterization of complex communities; in particular, the application of molecular fingerprinting techniques to examine diversity and community structure in complex gut bacterial communities. Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) methods have been successfully applied to the analysis of human, pig, cattle, dog and rodent intestinal populations. In future, genomics technologies will provide gut microbial ecologists with their best opportunity for a complete or global analysis of the molecular mechanisms involved in metabolism and regulation within the bacterial community and between host and resident microbes. Molecular microbial techniques have been widely and successfully used to study microbial diversity in environmental microbiology but have had limited application in the gastrointestinal ecosystem. (author)