PCR-DGGE analysis of the autochthonous gut microbiota of grouper Epinephelus coioides following probiotic Bacillus clausii administration : Probiont modulates the gut microbiota of grouper

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development and tissue physiology. In vertebrates, the most populous and complex community of microbes resides in the digestive tract. Here, we describe the establishment of the gut microbiota and its role in digestive tract differentiation in the zebrafish model vertebrate, Danio rerio. We find that in the absence of the microbiota, the gut epithelium is arrested in aspects of its differentiation, as revealed by the lack of brush border intestinal alkaline phosphatase activity, the maintenance of immature patterns of glycan expression and a paucity of goblet and enteroendocrine cells. In addition, germ-free intestines fail to take up protein macromolecules in the distal intestine and exhibit faster motility. Reintroduction of a complex microbiota at later stages of development or mono-association of germ-free larvae with individual constituents of the microbiota reverses all of these germ-free phenotypes. Exposure of germ-free zebrafish to heat-killed preparations of the microbiota or bacterial lipopolysaccharide is sufficient to restore alkaline phosphatase activity but not mature patterns of Gal alpha1,3Gal containing glycans, indicating that the host perceives and responds to its associated microbiota by at least two distinct pathways.