Identification and Characterization of Bacillus anthracis Spores by Flow Cytometry

Rapid and accurate detection of Bacillus anthracis, the causative agent of anthrax, remains an active area of research due to the continued threat of bioterrorist attack. The ability to differentiate Bacillus anthracis spores from spores belonging to other Bacillus species is important for the development of spore-based detection methods. Furthermore, not all Bacillus anthracis strains are fully virulent and the ability to rapidly determine the potential virulence of the spore is also important. Thus far, no spore-based method exists that can simultaneously satisfy both criteria. We conjugated a previously identified synthetic peptide to the fluorescent protein R-phycoerythrin to make a reagent that differentiates among Bacillus species. As expected, the conjugate selectively labeled Bacillus anthracis spores but could not distinguish between spores from fully virulent or minimally virulent strains. In response, our laboratory developed a fluorescent antibody-based assay that can be used to detect protective antigen protein associated with the surfaces of Bacillus anthracis spores. The two methods were combined in a two-color flow cytometric assay capable of simultaneously identifying the spore bacterial species as well as the relative virulence of the spore. This assay is novel in that Bacillus anthracis spores from protective antigen-producing strains can now be distinguished from protective antigen-negative strains. Surface protective antigen was detected in Bacillus anthracis spores that were prepared as long as four years ago; however, prolonged storage of spores was found by transmission electron microscopy to cause degradation of the exosporium and a loss of binding to the species-specific peptide. Based on the results of this study, we classified a set of prototypical flow cytometry dot-plot patterns that can be used to predict the species and relative virulence of unidentified samples of Bacillus spores in as little as one hour. Introduction B. anthracis is a Gram positive, rod-shaped bacterium which forms spores that are resistant to environmental changes such as moderate heating and radiation (Nicholson et al. 2000). B. anthracis, B. cereus, B. thuringiensis, and B. mycoides collectively form the B. cereus group of bacteria, which exist ubiquitously in nature and are genetically related. One of the only distinguishing features among members of the B. cereus group is plasmids that encode for virulence factors (Helgason et al. 2000). The B. anthracis pXO1 plasmid encodes for protective antigen (PA), edema factor (EF), and lethal factor (LF); three proteins which interact synergistically to form edema toxin (PA and EF) and lethal toxin (PA and LF) (Abrami et al. 2005). Fully virulent isolates of B. anthracis also harbor the pXO2 plasmid that encodes for an anti-phagocytic poly-D-glutamic acid capsule (Green et al. 1985). The B. anthracis Ames strain possesses both virulence plasmids, whereas minimally virulent Sterne and Pasteur strains lack pXO2 or pXO1, respectively. Depending on the method of contact with B. anthracis spores, infection can result in cutaneous, gastrointestinal, or pulmonary anthrax. The severity of disease is dependent on the spore’s ability to evade host immune responses, resume vegetative growth, and secrete edema and lethal toxin. Pulmonary anthrax is the form most commonly associated with bioterrorism because spores can be aerosolized and their size is optimal for deposition in the lungs. Once