A variant of Bacillus thuringiensis subsp. kurstaki containing a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically "barcoded" simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. Barcoded B. thuringiensis subsp. kurstaki spores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcoded B. thuringiensis subsp. kurstaki spores from wildtype spores under field conditions. T he development of sensitive and unequivocal approaches for detecting and tracking highly pathogenic bacteria has traditionally relied upon the use of nonpathogenic spore-producing Bacillus species as model organisms or simulants, whose physical and biochemical properties mimic those of the threat agent. Bacillus anthracis is a proven biothreat agent (5, 14-15, 20, 24) due its high virulence and the ability to form hardy and persistent spores, which can persist for decades in certain environments (21). Historically, nonpathogenic spore-forming bacteria such as Bacillus atrophaeus subsp. globigii have been used as surrogate organisms to simulate B. anthracis (9, 11). The physical properties of Bacillus thuringiensis subsp. kurstaki and its close genetic relatedness to B. anthracis, most notably with regard to the presence of an exosporium, which is absent from B. atrophaeus subsp. globigii, have led to recent preference for the use of B. thuringiensis subsp. kurstaki over B. atrophaeus subsp. globigii (10). However, the use of B. atrophaeus subsp. globigii and B. thuringiensis subsp. kurstaki in test sites is complicated by the fact that both organisms occur naturally in the environment (18; see also the excellent review of environmental B. thuringiensis subsp. kurstaki prevalence by Van Cuyk et al. [27]). B. thuringiensis subsp. kurstaki has a long history of use as a biopesticide, dating back to 1929 studies in the northeastern United States that showed B. thuringiensis to be effective f...
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