This Article Full Text (PDF) Other Versions of this Article: AEM.01179-06v1 72/11/7311    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sadeghifard, N. Articles by Seviour, R. J. Search for Related Content PubMed PubMed Citation Articles by Sadeghifard, N. Articles by Seviour, R. J. Agricola Articles by Sadeghifard, N. Articles by Seviour, R. J.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol. doi:10.1128/AEM.01179-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The mosaic nature of intergenic 16S-23S rRNA spacer regions (ISR) suggests ribosomal RNA operon copy number variation in Clostridium difficile strains

Biotechnology Research Centre, La Trobe University Bendigo, Victoria 3552, Australia; Microbiology Department, Austin Hospital, Studley Road, Heidelberg 3084, Melbourne, Victoria, Australia

^* To whom correspondence should be addressed. Email: Volker.Gurtler{at}austin.org.au,

	Abstract

Clostridium difficile is a major spore forming environmental pathogen causing serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method but fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning and sequencing, the diversity of the ISRs was used as a measure of ribosomal RNA operon copy number. In C. difficile strains 630, ATCC 43593, A and B 11, 11, 7 and 8 ISR length variants respectively were found [containing different combinations of sequence groups (i-xiii)] suggesting 11, 11, 7 and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA^ala gene were found in those that did. The presence of ISR sequence groups (i-xiii) varied between strains with some found in 1, 2, 3, 4 or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity not only among the rrn operons in different strains and different rrn copies in a single strain, but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intra-genic levels than other species when compared by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.