Abstract
BackgroundThe 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 FindingsWe 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/SignificanceSecond 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.
Highlights
The central challenge in the rapid detection, identification and characterization of microbial pathogens lies in the accurate recognition of a trait, or combination of traits, that is unique to a specific bacterial strain [1,2]
We resequenced and annotated eight B. anthracis genomes in order to test the accuracy of 454 whole-genome sequencing (WGS) technology for rapid detection of genetic modifications in this organism
History and subsequent extensive efforts in developing rapid diagnostics and therapeutics to tackle such events some questions still remain: How well are we prepared to face a similar attack? How do we measure progress? One of the ways to address these issues is presented in this article; i.e., can we identify a naturally occurring or genetically engineered biothreat agent in a timely manner
Summary
The central challenge in the rapid detection, identification and characterization of microbial pathogens lies in the accurate recognition of a trait, or combination of traits, that is unique to a specific bacterial strain [1,2]. DNA based assays that detect known genomic signatures have been developed that offer rapid and reliable identification of microbial pathogens [3,4] These approaches can be used with both non-culturable pathogens and when sample quantity is limiting. The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents for epidemiological surveillance of the intentional outbreak and for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. Available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively 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
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