Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis

Bacillus anthracis is one of the most genetically homogeneous pathogens des cribed, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving varia ble-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) u ses the combined power of multiple alleles at several marker loci. In our s ystem, fluorescently labeled PCR primers are used to produce PCR amplificat ion products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA seque ncer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two me re 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 f requently identified multiple alleles (from two to nine), with Nei's divers ity values between 0.3 and 0.8. Unweighted pair-group method arithmetic ave rage cluster analysis identified six genetically distinct groups that appea r to be derived from clones. Some of these clones show worldwide distributi on, while others are restricted to particular geographic regions. Human com merce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.