title: Genetic barcodes for improved environmental tracking of an anthrax simulant.
abstract: The development of realistic risk models that predict the dissemination, dispersion and persistence of potential biothreat agents have utilized nonpathogenic surrogate organisms such as Bacillus atrophaeus subsp. globigii or commercial products such as Bacillus thuringiensis subsp. kurstaki. Comparison of results from outdoor tests under different conditions requires the use of genetically identical strains; however, the requirement for isogenic strains limits the ability to compare other desirable properties, such as the behavior in the environment of the same strain prepared using different methods. Finally, current methods do not allow long-term studies of persistence or reaerosolization in test sites where simulants are heavily used or in areas where B. thuringiensis subsp. kurstaki is applied as a biopesticide. To create a set of genetically heterogeneous yet phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g., sample preparation) can be varied and the strains can be tested under otherwise identical conditions, we have developed a strategy of introducing small genetic signatures ("barcodes") into neutral regions of the genome. The barcodes are stable over 300 generations and do not impact in vitro growth or sporulation. Each barcode contains common and specific tags that allow differentiation of marked strains from wild-type strains and from each other. Each tag is paired with specific real-time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and from each other. These uniquely barcoded strains will be valuable tools for research into the environmental fate of released organisms by providing specific artificial detection signatures.
authors.affiliation: Biosciences Division, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Buckley
authors.forename: Bryan
authors.initials: B
authors.lastname: Rivers
authors.forename: Sarah
authors.initials: S
authors.lastname: Katoski
authors.forename: Michael H
authors.initials: MH
authors.lastname: Kim
authors.forename: F Joseph
authors.initials: FJ
authors.lastname: Kragl
authors.forename: Stacey
authors.initials: S
authors.lastname: Broomall
authors.forename: Michael
authors.initials: M
authors.lastname: Krepps
authors.forename: Evan W
authors.initials: EW
authors.lastname: Skowronski
authors.forename: C Nicole
authors.initials: CN
authors.lastname: Rosenzweig
authors.forename: Sari
authors.initials: S
authors.lastname: Paikoff
authors.forename: Peter
authors.initials: P
authors.lastname: Emanuel
authors.forename: Henry S
authors.initials: HS
authors.lastname: Gibbons
mesh_terms: D001408:Bacillus anthracis / Q000302:isolation & purification; D001413:Bacillus thuringiensis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004783:Environmental Microbiology; D042822:Genomic Instability; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods