Genetic subtyping of Bacillus anthracis using multiple-locus variable-number tandem repeat analysis and single nucleotide polymorphisms

Anthrax, caused by the bacterium Bacillus anthracis , is a disease of historical and current importance with a natural transmission cycle involving wildlife, livestock, and humans. Although B. anthracis occurs worldwide, the global transmission patterns and world-wide diversity of this important pathogen has remained cryptic. In addition to being a naturally occurring global health risk, B. anthracis has received notoriety for its use as a biowarfare and bioterrorism agent. The effectiveness of B. anthracis as a bioweapon and the limited knowledge of this pathogen's global genetic population structure, underscores the need to: (1) develop novel molecular approaches for detecting this pathogen; (2) improve and augment existing molecular subtyping systems and; (3) accurately define its global diversity, transmission patterns and evolution.;This dissertation describes; the development of methods for detecting and subtyping B. anthracis using single nucleotide polymorphisms (SNPs) and variable number of tandem repeat (VNTRs) markers, the application of these markers to subtype a worldwide isolate collection, and the elucidation of the pathogen's global population genetic structure. In chapter 2, we show a SNP in the plcR gene of B. anthracis is ubiquitous in a globally and genetically diverse panel of B. anthracis strains. Significantly, this SNP is species-specific for B. anthracis since it is not found in genetic near neighbors, such as B. cereus and B. thuringeinsis.;Chapter 3 describes the application of a molecular genetic assay, termed TaqMan Mismatch Amplification Mutation Assay (TaqMAMA), to selectively amplify and detect a range of B. anthracis DNA template levels.;In chapter 4 we describe the use of mass spectrometry to accurately characterize both VNTR and SNP loci on the same platform. The application of this technology to subtyping B. anthracis represents a major advance in the field, since it does not require time-consuming and expensive methods such as fluorescence-based capillary electrophoresis.;The 5th chapter of the dissertation describes the discovery of SNPs defining clonal lineages in B. anthracis and the application of these SNPs, in conjunction with VNTRs, to examine a collection of 1,033 B. anthracis isolates from 42 countries. This combination of markers permitted the analysis of older phylogenetic relationships and younger population-level structure in this pathogen and to document its evolutionary progression and global transmission patterns.;The 6th chapter of the dissertation capitalizes on the global database described in the chapter 5 to focus SNP discovery efforts for the Ames strain of B. anthracis; the strain used in the 2001 anthrax letter attacks. (Abstract shortened by UMI.).