Introduction

The human gastrointestinal (GI) tract is the home of a huge microbial assemblage, the microbiota, the vast extent of which is only now being revealed. The number of micro organisms within the intestine greatly exceeds human cells, resulting in one of the most diverse and dynamic microbial ecosystems. Relationships among the microbes, and between the microbiota and the host, have a profound influence on all concerned (1,2). The Gl-tract offers various niches with nutrients, those ingested and generated by the host, and a relatively non-hostile environment to the microbes. The microbiota play essential roles in a wide variety of nutritional, developmental, and immunological processes and therefore significantly contribute to the well being of the host (3-6). During the last decade, specific bacterial isolates, termed "probiotics," have been extensively used in an attempt to modulate the intestinal microbiota to benefit the host. Today, there is persuasive evidence for probiotics in prevention or treatment of a number of intestinal disorders in humans, especially for reducing bouts of diarrhea and providing relief for lactose intolerant individuals (7,8). In order to rationally use probiotics, prebiotics or other functional foods as therapeutic agents, in-depth knowledge of the structure, dynamics, and function of the bacterial populations of the GI-tract microbiota is crucial.

Studying the microbial ecology in the intestine involves determining the abundance and diversity of the microorganisms present, their activity within this niche, and their interactions with each other and their host (symbiosis, commensalism and pathogenicity). Although the human intestinal microbiota have been extensively investigated by culture-based methods more than any other natural ecosystem (9-11), our knowledge about the culturable fraction of this community is limited. This is essentially due to the challenges of obtaining pure cultures of intestinal inhabitants, which are hindered by the largely anaerobic nature of this community, and the paucity of suitable enrichment strategies to simulate intestinal conditions. The advent of molecular techniques based on 16S

ribosomal RNA (rRNA) gene analysis is now allowing a more complete assessment of this complex microbial ecosystem by unraveling the extent of the diversity, abundance and population dynamics of this community (12,13). These techniques have extended our view of those microorganisms that have proven difficult to culture and which play an important role in gut physiology. This huge intestinal microbial reservoir is estimated to contain 1000 bacterial species and as much as 1014 cells (1,14). Besides studying the diversity, it is essential to identify these microbes based upon their eco-physiological traits, i.e., those that are functionally active versus those that are effectively redundant and play little or no role at a particular time or at a given site of the intestinal tract. The latter requires the development of approaches that monitor the activity of these microorganisms at the single cell level in their natural habitat. This chapter initially reviews molecular techniques to study the diversity of the microbiota, and subsequently highlights newly developed molecular methods to study the eco-physiology of the GI-tract.

GI-TRACT MICROBIOTA AS IDENTIFIED BY 16S rRNA GENE ANALYSIS

The human GI-tract microbiota comprise bacteria, archaea and eukarya. It is by far the bacteria that dominate and reach the highest cell density documented for any microbial ecosystem (1). The comparative analysis of environmentally retrieved nucleic acid sequences, most notably of rRNA molecules and the genes encoding them, has become the standard over the last decade for cultivation-independent assessment of bacterial diversity in environmental samples (Fig. 1) (15,16). The 16S rRNA gene comprises highly variable to highly conserved regions, and the differences in sequence are used to distinguish bacteria at different levels from species to domain and determine phylogenetic relationships. rRNA gene fragments are today routinely retrieved without prior cultivation of the microbes by constructing 16S ribosomal DNA (rDNA) libraries. The procedure is based upon polymerase chain reaction (PCR)-mediated amplification of 16S rRNA genes or gene fragments, isolated from the environmental sample, followed by segregation of individual gene copies by cloning into Escherichia coli. In this way a library of community 16S rRNA genes is generated, the composition of which can be estimated by screening clones, full or partial sequence analysis, and comparing them with adequate appropriate reference sequences in databases to infer their phylogenetic affiliation. Large databases of 16S rRNA gene sequence information (>200,000 sequences) for described as well as uncultured microorganisms are available, which provide a high-resolution platform for the assignment of those new sequences obtained in 16S rDNA libraries. Databases harboring 16S rRNA sequences include the ARB software package (17), the Ribosomal Database project (http://rdp.cme.msu.edu/index.jsp) (18) and EMBL (www.embl-heidelberg.de/).

Sequencing of 16S rDNA clone libraries generated from various sites of the GI-tract including terminal ileum, colon, mucosa and feces have confirmed that relevant fractions of gut bacteria were derived from new, as yet undescribed bacterial phylotypes (19-23). Clearly the biases of culturing studies in the 1960s and 1970s such as incomplete knowledge of culture conditions and selectivity had prejudiced the outcome. The new molecular studies revealed that the vast majority of rDNA amplicons generated directly from fecal or biopsy samples of adults, originated from the phyla of the Firmicutes (including the large class of Clostridia and the lactic acid bacteria), Bacteroidetes, Actinobacteria (including Atopobium and Bifidobacterium spp.) and Proteobacteria

DNA/rRNA of intestinal ecosystem

Pregnancy And Childbirth

Pregnancy And Childbirth

If Pregnancy Is Something That Frightens You, It's Time To Convert Your Fear Into Joy. Ready To Give Birth To A Child? Is The New Status Hitting Your State Of Mind? Are You Still Scared To Undergo All The Pain That Your Best Friend Underwent Just A Few Days Back? Not Convinced With The Answers Given By The Experts?

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