Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

• Authors: Peter E. Chen, Naval Medical Research Center; Kristin M. Willner, Naval Medical Research Center; Amy Butani, Naval Medical Research Center; Shakia Dorsey, Naval Medical Research Center; Matroner George, Naval Medical Research Center; Andrew Stewart, Naval Medical Research Center; Shannon M. Lentz, Naval Medical Research Center; Christopher E. Cook, Naval Medical Research Center; Arya Akmal, Naval Medical Research Center; Lance B. Price, The Translational Genomics Research Institute; Paul S. Keim, The Translational Genomics Research Institute; Alfred Mateczun, Naval Medical Research Center; Trupti N. Brahmbhatt, Naval Medical Research Center; Kimberly A. Bishop-Lilly, Naval Medical Research Center; Michael Zwick, Emory University; Timothy Read, Emory University; Shanmuga Sozhamannan, Naval Medical Research Center
• Journal Title: PLoS ONE
• Volume: Volume 5, Number 8
• Publisher: Public Library of Science | 2010-08-25, Pages e12397-e12397
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1371/journal.pone.0012397
• Subjects: Biology, Genetics; Health Sciences, General; Health Sciences, Immunology
• Keywords: Science & Technology; Multidisciplinary Sciences; Science & Technology - Other Topics; MULTIDISCIPLINARY SCIENCES; INHALATIONAL ANTHRAX; TECHNOLOGIES; CEREUS; BIOTERRORISM; EVOLUTION; STRAIN

Abstract

Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. Methodology/Principal Findings: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. Conclusions/Significance: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.