ABSTRACT
Molecular tools based on small subunit (SSU) rDNA gene sequences offer a powerful and rapid tool for the analysis of complex microbial communities found in the gastrointestinal tracts (GIT) of food animal species. Extensive comparative sequence analysis of SSU rRNA molecules representing a wide diversity of organisms shows that different regions of the molecule vary in sequence conservation. Oligonucleotides complementing regions of universally conversed SSU rRNA sequences are used as universal probes, while those complementing more variable regions of sequence are useful as selective probes targeting species, genus, or phylogenetic groups. Different approaches derive different information and this is highly dependent on the type of target nucleic acid employed and the conceptual and technical basis used for nucleic acid probe design. Generally these approaches can be divided into DNA-based methods employing empirically characterized probes and rRNA-based methods based on comparative sequence analysis for design and interpretation of "rational" probes. Polymerase chain reaction (PCR) based techniques can also be applied to the analysis of microbial communities in the GIT. Direct cloning of SSU rDNA genes amplified from these complex communities can be used to determine the extent of diversity in these GIT communities. Denaturing gradient gel electrophoresis (DGGE) is another powerful tool for profiling microbial diversity of microbial communities in GI tracts. Sequence analysis of the excised DGGE amplicons can then be used to presumptively identify predominant bacterial species. Examples of how these molecular approaches are being used to study the microbial diversity of communities from steers fed different diets, swine fed probiotics, and Atlantic salmon fed aquaculture diets are presented.
ABSTRACT
Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T); (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albensis M384(T); (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.
