Abstract
Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying anthrax strains during the 2001 bioterrorism-associated outbreak. Because B. anthracis has a low level of genetic variability, only a few subtyping methods, with varying reliability, exist. We initially used multiple-locus variable-number tandem repeat analysis (MLVA) to subtype 135 B. anthracis isolates associated with the outbreak. All isolates were determined to be of genotype 62, the same as the Ames strain used in laboratories. We sequenced the protective antigen gene (pagA) from 42 representative outbreak isolates and determined they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I). MLVA and pagA sequencing were also used on DNA from clinical specimens, making subtyping B. anthracis possible without an isolate. Use of high-resolution molecular subtyping determined that all outbreak isolates were indistinguishable by the methods used and probably originated from a single source. In addition, subtyping rapidly identified laboratory contaminants and non-outbreak–related isolates.
Highlights
Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying strains during the 2001 bioterrorism-associated outbreak
The pagA sequence of all five New England isolates was identified as Protective antigen (PA) genotype VI (Table 2)
During the 2001 anthrax investigation, molecular subtyping of B. anthracis by multiple-locus variablenumber tandem repeat analysis (MLVA) and pagA sequencing was important in linking cases to each other and to contaminated sites and in distinguishing isolates that were not related to this event
Summary
Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying strains during the 2001 bioterrorism-associated outbreak. We initially used multiple-locus variablenumber tandem repeat analysis (MLVA) to subtype 135 B. anthracis isolates associated with the outbreak. We sequenced the protective antigen gene (pagA) from 42 representative outbreak isolates and determined they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I). The recent bioterrorism-associated anthrax outbreak demonstrated the need for rapid molecular subtyping of Bacillus anthracis isolates. Laboratories throughout the United States and around the world received an increased number of specimens to be tested for B. anthracis With such large numbers, occasional contamination or detection of non-outbreak strains was inevitable, and molecular subtyping was used to clarify these situations on several occasions. The recent anthrax outbreak has dramatically illustrated the importance of rapid molecular subtyping during a bioterrorism event
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