Caister Academic Press

Molecular Methods for Probiotics Research

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Molecular Methods for Probiotics Research. A review of current methods and protocols for microbial ecology.

Molecular Methods for Probiotics Research

Adapted from Erwin G. Zoetendal and Roderick I. Mackie in Probiotics and Prebiotics: Scientific Aspects
Molecular Methods for Probiotics Research: It is now generally accepted in microbial ecology that cultivation-based approaches provide an incomplete picture of microbial diversity in the gastrointestinal (GI) tract because only a minority of microbes can be obtained in culture. Therefore, the application of molecular approaches, especially those focused on 16S ribosomal RNA (rRNA) sequence diversity, have become popular as they enable researchers to bypass the cultivation step. These approaches have provided considerable information about microbial ecosystems, including the GI tract. Chapter 1 in Probiotics and Prebiotics: Scientific Aspects summarizes the different approaches and their impact on our knowledge of the ecology of the GI tract and provide guidelines for future research directions with a focus on pre- and probiotics.

Microbial ecology of the GI tract ecosystem involves several areas which include 1) the investigation of the microbes present, 2) their in vivo activity, and 3) their relationship with each other and the host animal. This and the fact that the most GI tract locations are intractable to sample, indicates that studying the microbial ecology of the GI tract is very complex. During the last decades most of our knowledge concerning the ecology of the GI tract has been derived from the development of anaerobic culture techniques, the use of rodent and other animal models, and the development of gnotobiotic technology by which germ-free or animal models with a defined microbial community structure could be derived and maintained. Nowadays, Molecular Microbial Ecology can be seen as the fourth major source of knowledge, which offers great promise for the future.

The first question ecologists have to answer is "which bacteria are present in the GI tract" and this is already a complicated task. For many ecosystems it is estimated that only a few percent of microbes can be obtained in culture. With estimates between 10 and 50% the estimate of culturability of GI tract bacteria is relatively high, although it should be noted that this estimate is based on numbers and not diversity. Nevertheless, it is still a minority. For decades the difference between total microscopic counts and colony forming unit counts was explained by the number of dead cells in the sample since cultivation of GI tract bacteria requires strict anoxic procedures. Using viability and dead stains it has been shown that indeed one third of the total bacterial community detected in feces may be derived from dead cells. However, after the discovery that ribosomal RNA (rRNA) is present in every cell and that its nucleotide sequence can be used for phylogenetic classification, the introduction of the so-called "16S rRNA approach" has demonstrated that a majority of GI tract bacteria are phylogenetically different from those described in culture. Therefore, it can be concluded that many bacteria escape cultivation procedures. Besides the difficulty of applying strict anoxic procedures, additional reasons for this cultivation anomaly may include the selectivity of the media that are used, unknown growth requirements, and exposure of stress to the microbes during the cultivation procedures. In addition, it is also reasonable to assume that bacteria have adapted to GI tract conditions and as a result need specific interactions with other microbes and host cells. Therefore, the circumvention of these limitations in detecting and identifying GI tract microbes, and unraveling their function requires the application of several culture-independent approaches.

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