Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Background: Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA 83 , 83 kDa). Likewise, the edema factor (EF) binds to the PA 83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the fulllength, inactive protein into 63 kDa and 20 kDa antigens (PA 63 and PA 20 ). PA 63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.

Bacillus anthracis causes anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxinproducing ⌬Sterne strain of B. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibitor, while ciprofloxacin is not. Since toxins are produced only by vegetative B. anthracis, the effect of linezolid and ciprofloxacin on toxin production is of interest. The effect of simulated clinical regimens of ciprofloxacin and linezolid on the vegetative and spore populations and on toxin production was examined in an in vitro pharmacodynamic model over 15 days by using the toxin-producing Sterne strain of B. anthracis. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. With ciprofloxacin therapy, the total Sterne population consisted of spores. With linezolid therapy, >90% of the population was vegetative B. anthracis. With ciprofloxacin therapy, toxin was first detectable at 3 h and remained detectable for at least 5 h. Toxin was never detected with linezolid therapy. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. However, the B. anthracis population was primarily spores with ciprofloxacin therapy and was primarily vegetative bacteria with linezolid therapy. Toxin production was detected for at least 5 h with ciprofloxacin therapy but was never detected with linezolid treatment. Linezolid may have an advantage over ciprofloxacin for the treatment of B. anthracis infections.