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Journal of Clinical Microbiology, March 2007, p. 736-746, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01895-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats{triangledown} ,{ddagger}

Yael Danin-Poleg,1 Lyora A. Cohen,1 Hanan Gancz,1,{dagger} Yoav Y. Broza,1 Hanoh Goldshmidt,1 Elinor Malul,1 Lea Valinsky,2 Larisa Lerner,2 Meir Broza,3 and Yechezkel Kashi1*

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel,1 Government Central Laboratories, Ministry of Health, Jerusalem 94467, Israel,2 Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim, Tivon 36006, Israel3

Received 12 September 2006/ Returned for modification 30 October 2006/ Accepted 14 December 2006

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

* Corresponding author. Mailing address: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel. Phone: 972-4-8293074. Fax: 972-4-8293399. E-mail: kashi{at}tx.technion.ac.il.

{triangledown} Published ahead of print on 20 December 2006.

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

{dagger} Present address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814.

Journal of Clinical Microbiology, March 2007, p. 736-746, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01895-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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