Genetic diversity in a Bacillus anthracis historical collection (1954 to 1988)

Bacillus anthracis, the etiologic agent of anthrax, has been widely described as a genetically monomorphic species. We used both multiple-locus variable-number tandem-repeat analysis (MLVA) and pagA gene sequencing to determine the genetic diversity of a historical collection of B. anthracis isolates collected from the 1950s to the 1980s from various geographic locations and sources. We sequenced the pagA gene of 124 diverse B. anthracis isolates and found all previously identified B. anthracis pagA types except type 4. Sixty-three of the 124 B. anthracis strains were identified as pagA type 6, while 44 were pagA type 5, 12 were pagA type 1, and individual isolates were identified for types 2 and 3, respectively. Two new pagA genotypes were discovered in three environmental isolates within the historical collection. Two isolates had the same new genotype, and an additional isolate produced a second new genotype. MLVA detected 22 previously described genotypes in the historical collection. In addition, 33 new MLVA genotypes were found. For 11 isolates, an MLVA genotype could not be assigned because one or more alleles did not amplify. While only two additional B. anthracis pagA types were identified, in two instances, the use of pagA sequencing discriminated isolates with the same MLVA genotype. MLVA revealed that 39 of the 124 isolates were previously undocumented genotypes and that 1 isolate was found to be in the C cluster when it was subtyped by MLVA.