ORIGINAL ARTICLE PCR/DGGE and 16S rRNA gene library analysis of the colonic microbiota of HLA-B27/b2-microglobulin transgenic rats W. McBurney 1 , M. Mangold 1 , K. Munro 1 , M. Schultz 2 , H.C. Rath 2 and G.W. Tannock 1 1 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand 2 Department of Internal Medicine I, University of Regensburg, Regensburg, Germany Introduction Human HLA-B27/b2-microglobulin transgenic (TG) rats have been derived to investigate the influence of the HLA-B27 (a major histocompatibility complex class I molecule) gene on inflammatory disorders of the gut and joints. Germfree TG rats do not exhibit colitis or arthritis, but develop these inflammatory conditions after coloniza- tion by a gut microbiota or by defined mixtures of intes- tinal bacterial species (Taurog et al. 1994; Rath et al. 1996). Treatment with metronidazole attenuates colitis and arthritis, suggesting an important role for anaerobic bacteria in the aetiology of the diseases (Rath et al. 2001). Using PCR/denaturing gradient gel electrophoresis (DGGE), we investigated the composition of the gut microbiota of the colon of TG and NT rats which differed in severity of active disease. Then we compared the results of analysis obtained by PCR/DGGE with that from analy- sis of a 16S ribosomal RNA (rRNA) gene library created from the microbiota of the colon of rats with inflamed Keywords colitis, microbiota, polymerase chain reaction/ denaturing gradient gel electrophoresis, 16S ribosomal RNA gene, transgenic rats. Correspondence G.W. Tannock, Department of Microbiology, University of Otago, PO Box 56, Dunedin, New Zealand. E-mail: gerald.tannock@stonebow.otago.ac.nz Present address: Michael Schultz and Heiko C. Rath, Kreisklinik Wasserburg, Department of Internal Medicine, D-83512 Wasserburg, Germany. 2005/0392: received 13 April 2005, revised 7 June 2005 and accepted 27 June 2005 doi:10.1111/j.1472-765X.2005.01811.x Abstract Aims: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. Methods and Results: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/ DGGE. Six months old TG rats had marked inflammation of the colon com- pared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice’s Similarity Coefficient pro- ximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52–64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. Conclusions: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune sys- tem reacts rather than seek phylogenetic associations. Significance and Impact of the Study: PCR/DGGE can be used as a rapid ini- tial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease. Letters in Applied Microbiology ISSN 0266-8254 ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006) 165–171 165