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Applied and Environmental Microbiology, June 2009, p. 4079-4088, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02729-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of Vibrio parahaemolyticus Isolates by Using Capillary Electrophoresis{triangledown} ,{dagger}

Erika Harth-Chu,1 Romilio T. Espejo,2 Richard Christen,3 Carlos A. Guzmán,1 and Manfred G. Höfle1*

Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, D-38124 Braunschweig, Germany,1 Instituto de Nutrición y Tecnología de Alimentos (INTA), Universidad de Chile, El Líbano 5524, Macul, Santiago 6903625, Chile,2 Virtual Biology Lab, Centre for Biochemistry, University of Nice Sophia-Antipolis and CNRS, Campus Valrose, 06108 Nice, France3

Received 29 November 2008/ Accepted 7 April 2009

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.

* Corresponding author. Mailing address: Department of Vaccinology and Applied Microbiology, Helmholtz Centre of Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. Phone: 49-531-6181-4234. Fax: 49-531-6181-4699. E-mail: mho{at}gbf.de

{triangledown} Published ahead of print on 17 April 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2009, p. 4079-4088, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02729-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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