Molecular Subtyping of Bacillus anthracis and the 2001 Bioterrorism-Associated Anthrax Outbreak, United States - Bioterrorism-Related Anthrax

Emerging Infectious Diseases, Oct, 2002 by Alex R. Hoffmaster, Collette C. Fitzgerald, Efrain Ribot, Leonard W. Mayer, Tanja Popovic

Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying 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 nonoutbreak-related isolates.

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The recent bioterrorism-associated anthrax outbreak demonstrated the need for rapid molecular subtyping of Bacillus anthracis isolates. Numerous methods, including multiple-locus enzyme electrophoresis (MEE) and multiple-locus sequence typing (MLST), have shown the lack of genetic diversity of B. anthracis (1-4, unpub, data). Despite this low diversity, methods have been developed that can detect differences between B. anthracis isolates. Amplified fragment length polymorphism (AFLP) analysis has been used to detect differences between B. anthracis isolates and to examine phylogenetic relationships between B. anthracis and its close relatives, B. cereus and B. thuringiensis (4,5). Keim et al. (6) reported on multiple-locus variable-number tandem repeat analysis (MLVA) for subtyping B. anthracis, which unlike AFLP is designed to subtype B. anthracis specifically and cannot be used to address phylogenetic relationships between Bacillus species. MLVA determines the copy number of variable-number tandem repeats (VNTR) at eight genetic loci (six chromosomal and one on each of the two plasmids). Recently, MLVA has been used to differentiate 426 B. anthracis isolates into 89 distinct genotypes and to study the ecology of anthrax (6,7). MLVA is relatively simple, has excellent reproducibility, can subtype multiple strains on a single gel, and gives results in <8 hours.

Protective antigen (PA) is one of the three anthrax toxin proteins and is key to developing immunity to anthrax. Sequencing the gene that encodes PA (pagA) has been used to subtype 26 diverse B. anthracis isolates into six PA genotypes (8). Although sequencing of pagA results in limited numbers of subtypes, it does have the added benefit of determining if the pagA gene has been altered or engineered.

During the 2001 bioterrorism-associated anthrax outbreak, we used MLVA to subtype isolates from patients, the environment, and powders. Subtyping of B. anthracis allowed anthrax cases to be linked to environmental specimens and powders and provided information about potential sources. Sequencing of pagA was also performed on a subset of these B. anthracis isolates, and we confirmed that the pagA sequence was not altered. In addition, we used these methods on DNA extracted from select clinical specimens to detect and subtype B. anthracis directly from clinical specimens. During the outbreak, 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. Overall, the recent anthrax outbreak has dramatically illustrated the importance of rapid molecular subtyping during a bioterrorism event.

Materials and Methods

During the 2001 anthrax outbreak investigation, 135 B. anthracis isolates were subtyped. The identity of all strains was confirmed with standard microbiologic procedures and the Laboratory Response Network (LRN) testing algorithm (9,10). Isolates were obtained from patients with laboratory-confirmed anthrax (n=10), powders (n=4), and environmental specimens (n=121). For comparison purposes, five B. anthracis isolates originating from New England in the 1960s and 1970s, the Ames strain, and the Pasteur strain were included.

DNA extractions of 28 clinical specimens from six patients with confirmed inhalational anthrax were used for molecular subtyping. These specimens included blood, pleural fluid, blood cultures, serum, cerebrospinal fluid (CSF), lung tissue, and lymph node tissue.

DNA from all strains was prepared with a heat lysis method. Isolates were streaked onto trypticase soy agar containing 5% sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD) and incubated overnight at 37[degrees]C. A single colony was transferred and dispersed into 0.22-gm centrifugal filter units (Millipore, Bedford, MA) containing 200 [micro]L 10 mM Tris-HCl (pH 8.0). The suspension was heated at 95[degrees]C for 20 min and cooled to room temperature. The filter units were then centrifuged in a microfuge at 6,000 x g for 2 min and the filter discarded. The resulting lysate was stored at -20[degrees]C until use. DNA from clinical specimens was extracted with a Qiagen DNA Mini Kit per manufacturer's instructions (Qiagen Inc., Valencia, CA).