Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

Abstract

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.

Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011

“…Together with other recent work (17)(18)(19), this study argues strongly for multiple genome sequences to understand patterns of transmission within an outbreak. Such analyses can already be conducted in a matter of days, and technological advances will only improve our ability to perform them in real time.…”

“…Highly accurate sequencing and SNP identification can overcome the noise from sequencing error and discern phylogenetic signal, which may, as in this case, depend on a small number of nucleotides. As demonstrated by the multiple independent sequencing efforts related to this E. coli O104:H4 outbreak (9, 10, 13, 15), and also epidemiological investigations of other infectious diseases (17)(18)(19), genomic epidemiology is likely to become the standard strategy in molecular epidemiology as the cost of sequencing continues to decline and technology becomes more widely accessible.…”

Insights from genomic comparisons of genetically monomorphic bacterial pathogens

“…And their recent origins allow easier reconstructions of their history than is possible with most other microbes. The recent recognition that some lineages have high mutation rates, almost as high as those of some RNA viruses, means that real time evolution can be used for epidemiological [77] and forensic [78] tracking. I anticipate a veritable flood of publications using comparative genomics for such purposes in the near future because the technological challenges are now relatively minor.…”

Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios

“…Wholegenome based approaches also were used for the bioterrorism investigation of the anthrax letters in the Amerithrax case. 28 The whole-genome methodology involves the isolation of genetic material from the sample, enrichment toward the target, enrichment using nonspecific amplification strategies, and finally deep sequencing of the sample. Strategies for sample preparation (extraction of genomic material and enrichment of target-specific material) and amplification of material vary greatly between groups.…”