Abstract
Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.
Highlights
Bacillus anthracis is the gram-positive etiologic agent responsible for the potentially fatal infectious disease anthrax
In order to evaluate the ability of genomic platforms to identify and distinguish B. anthracis DNA within a complex nucleic acid mixture, B. anthracis genomic DNA was spiked at six concentrations into either 100 pg aerosol DNA or 1 ng soil DNA
The life cycle and virulence of B. anthracis distinguish it as an organism with a high potential for use as a bioterrorism agent, as well as being problematic as a natural human and animal pathogen in many parts of the world
Summary
Bacillus anthracis is the gram-positive etiologic agent responsible for the potentially fatal infectious disease anthrax This species can form dormant endospores resistant to extreme environmental conditions and can persist for long periods in terrestrial or aquatic environments. Spores function as infectious agents by exiting the dormant state upon contact with a nutrient-rich environment, which, in humans, occurs following respiratory, gastrointestinal, or cutaneous exposure. This germination process subsequently leads to growth in host tissues and, later, expression of toxins responsible for deleterious physiological effects in humans and other mammals [1]. Burkholderia pseudomallei strain K96243, chromosome 1, complete sequence NC_006350
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