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Journal of Clinical Microbiology, March 2007, p. 1024-1028, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.02023-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Typing and Subtyping of Clostridium difficile Isolates by Using Multiple-Locus Variable-Number Tandem-Repeat Analysis{triangledown}

Renate J. van den Berg,1 Inge Schaap,1 Kate E. Templeton,1,{dagger} Corné H. W. Klaassen,2 and Ed J. Kuijper1*

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden,1 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands2

Received 1 October 2006/ Returned for modification 8 November 2006/ Accepted 5 December 2006

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.

* Corresponding author. Mailing address: Department of Medical Microbiology, E4-67, Centre of Infectious Diseases, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31-71-5263931. Fax: 31-71-5248148. E-mail: e.j.kuijper{at}lumc.nl.

{triangledown} Published ahead of print on 13 December 2006.

{dagger} Present address: Specialist Virology Centre, Royal Infirmary Hospital, Edinburgh, United Kingdom.

Journal of Clinical Microbiology, March 2007, p. 1024-1028, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.02023-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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