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Folia Microbiol (2012) 57:325330 DOI 10.1007/s12223-012-0135-4
PCR detection of uncultured rumen bacteria
Jaime A. Rosero & Lenka trosov & Jakub Mrzek &
Kateina Fliegerov & Jan Kopen
Received: 9 December 2011 /Accepted: 4 January 2012 /Published online: 14 April 2012 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012
Abstract 16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.
Introduction
Animal digestive tract harbors a diversified microbial consortium with complex interactions inside this community as well as interactions between microbes and host animal. The most advanced cooperative animalmicrobe relationship has evolved in ruminants (Mackie 2002). Many significant representatives of the rumen microbial population have already been described; however the cultured microorganisms represent only a minor part of this ecosystem. Methods of molecular biology based mostly on sequence analysis of 16S RNA ribosomal gene (Kong et al. 2010) have revealed in recent years a vast number of anaerobic microorganisms,
which are unknown or have not yet been cultivated (Tasse et al. 2010). The uncultured species in the rumen are associated with the liquid phase, feed particles as well as rumen wall and can achieve up to 80 % (Koike et al. 2003; Sadet-Bourgeteau et al. 2010). Properties of these species might have a significant impact on the metabolic activities as indicated by latest metagenomic studies (Vieites et al. 2009; Hess et al. 2011). The phylogenetic analysis of ruminal fiber-associated community disclosed three unidentified groups of bacteria (Koike et al. 2003), which were connected with a different type of diet. Through the enrichment process, the relative abundance of one of these groups was significantly increased...