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GRK 1482 Jahrbuch 2011-2014

Publications [1] Arumugam M, et. al., Enterotypes of the human gut microbiome. Nature. 2011, 473, 174. [2] Murray E, Arias C, The rise of the Enterococcus: beyond vancomycin resistance. Nature Reviews Microbiology 10. 2012, 266-278. [3] Zwirglmaier K, et. al., Improved fluorescence in situ hybridization of individual microbial cells using polynu- cleotide probes: the network hypothesis. Syst. Appl. Microbiol. 2003, 26: 327-337. [4] ZwirglmaierK,,Improvedmethodforpolynucleotide probebased cell sorting, using DNA-coated microplates. Appl. Envirion. Microbiol. 70. 2004, 494-497. [5] Ludyga NA, Nucleic acid based detection and separation of bacteria causing nosocomial infections using novel types of polynucleotide probes. PhD thesis, Technische Universität München. 2007. ASSOCIATED FELLOWS GRK Progress Report 2011-2014 | Page 63 Aim For in-depth analysis of the metagenomes of selected taxa one needs to establish methods for the targeted enrichment of the metagenomes of certain taxa from complex gut consortia. Since only a specifically targeted part of a complex microbial consortium is extracted and subjected to metagenomic char- acterization, the novel methodology developed and evaluated here, can be used to study certain taxonomic groups in greater depth than is possible with conventional metagenomics. Methods and Results The method mostly focused on in this project is directed to fluorescence in situ hybridization (FISH) taxon-specific cell en- richment, by hybridizing polynucleotide probes to intracellular targets of fixed (permeabilized) cells, while parts of the probes remain in the cells periphery presumably by forming interprobe networks [3]. This can be visualized by FISH as fluorescent halos, surround- ing the individual cells. Cells which react with the polynucle- otide probes are enriched by immobilization on (paramagne- tic) beads or microplate wells via binding of probe labels to appropriate capture components. The standard polynucleo- tide probes are comprised of in vitro transcripts of 23S rRNA domain III, which can be regarded as roughly genus-specific. In its basic features, the taxon-specific cell enrichment method has been developed at the Department of Microbiology in previ- ous work of the group of Dr. W. Ludwig [4, 5]. Prior to working on real (clinical) samples, immobilization of pure cultures of Enterococcus already could be established within this project period. The results were evaluated by 23S rDNA sequencing from these immobilized cells. With respect to immobilization of intestinal relevant bacteria cells, the application to fecal samples needs to be improved. To pre-screen fecal samples for enterococci prior to cell en- richment, an Enterococcus-specific PCR detection system was designed and additionally adapted to fecal samples from mice. Outlook Thus far, immobilization procedures and subsequent target se- quencing of the 16S/23S rDNA and the tuf gene could be suc- cessfully established for cultures of Enterococcus. Future experiments will focus on further enhancing of probe specificity and on modifying this method for spiked samples and real (clinical) fecal samples for subsequent metagenomic analysis. Figure: Fluorescence in situ hybridization with a 23S DIII rDNA targeted polynucleotide probe on pure culture cells of E. faecalis, constitutively resulting in ring shaped Halo signals Supervisors Prof. Dr. Wolfgang Liebl I TUM I Microbiology Dr. Wolfgang Ludwig I TUM I Microbiology Prof. Dr. Dirk Haller I TUM I Nutrition and Immunology Start of project: October 2011 Academic background: Studies of Molecular Microbiology at the University of Innsbruck