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Journal of Bacteriology, May 2000, p. 2928-2936, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within Bacillus anthracis

P. Keim,1,* L. B. Price,1 A. M. Klevytska,1 K. L. Smith,1,2 J. M. Schupp,1 R. Okinaka,3 P. J. Jackson,3 and M. E. Hugh-Jones2

Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-56401; Department of Epidemiology and Community Health, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803-84042; and Environmental Molecular Biology Group, Los Alamos National Laboratory, Los Alamos, New Mexico 875453

Received 6 December 1999/Accepted 24 February 2000

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC1, vrrC2, vrrB1, vrrB2, and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

* Corresponding author. Mailing address: Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640. Phone: (520) 523-1078. Fax: (520) 523-0639. E-mail: Paul.Keim{at}nau.edu.

Journal of Bacteriology, May 2000, p. 2928-2936, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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