B-195. Evaluation of Virulence Plasmid Genetic Markers in Bacillus anthracis

K. J. Stupec, S. W. Jones, M. L. McKee;
BEI Resources, ATCC, Manassas, VA.

Background: Bacillus anthracis is a gram-positive aerobic spore-forming bacterium that was made infamous when it was sent as a spore-powder through the US Postal System in 2001. As a result, it became necessary to develop PCR based tests for detection of genetic markers associated with virulence genes encoded on the plasmids of Bacillus anthracis, pXO1 and pXO2, to differentiate isolates. It was the goal of this study to create a coordinate set of primers for identifying plasmid-based virulence markers including toxin subunits and capsular genes in order to characterize B. anthracis strains in the BEI Resources as to their virulence potential. Methods: This study involved the selection of primers from previously published sources and assay optimization of those primers to create a panel of eight B. anthracis plasmid-associated genetic markers. These markers include two variable repeat loci: pXO1aat, pXO2at; three genetic loci on pXO1: lef (lethal factor), cya (edema factor) and pag (protective antigen); and three genetic loci on the pXO2 plasmid: the capsular genes A, B and C (capA, capB and capC). B. anthracis strains were propagated and characterized using polyphasic methods including morphological colony characterization via growth on Sheep Blood Agar, microscopic examination, genetic 16S ribosomal gene DNA sequencing, and API biochemical analyses. Results: DNA was extracted, using an in-house mechanical lysis method, from multiple strains of B. anthracis, characterized by depositors as containing varying plasmids. Each DNA was evaluated for the presence or absence of all eight markers, using PCR. The assays were optimized and used to screen 15 strains of B. anthracis. 100% concordance was observed. Conclusions: This study clearly demonstrated the genetic content of virulence genes on the plasmids associated with B. anthracis. Characteristic profiles of the genetic markers detected in the strains of B. anthracis demonstrated the potentiality for full plasmid-associated virulence at the genetic level.