Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.
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.


RESULTS
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
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.
Chen
Peter E
PE
Naval Medical Research Center, Biological Defense Research Directorate, Silver Spring, Maryland, United States of America.
Willner
Kristin M
KM
Butani
Amy
A
Dorsey
Shakia
S
George
Matroner
M
Stewart
Andrew
A
Lentz
Shannon M
SM
Cook
Christopher E
CE
Akmal
Arya
A
Price
Lance B
LB
Keim
Paul S
PS
Mateczun
Alfred
A
Brahmbhatt
Trupti N
TN
Bishop-Lilly
Kimberly A
KA
Zwick
Michael E
ME
Read
Timothy D
TD
Sozhamannan
Shanmuga
S
D001408:Bacillus anthracis / Q000187:drug effects / Q000235:genetics* / Q000502:physiology / Q000821:virology; D001435:Bacteriophages / Q000502:physiology; D002939:Ciprofloxacin / Q000494:pharmacology; D019295:Computational Biology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004917:Erythromycin / Q000494:pharmacology; D016680:Genome, Bacterial / Q000235:genetics*; D007753:Laboratories; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
D002939:Ciprofloxacin; D004917:Erythromycin
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