Role of Law Enforcement Response and Microbial Forensics in Investigation of Bioterrorism

Rizici i opasnosti od bioterorizma i biološkog kriminala postali su velika briga i izazov za državne vlade i društvo, jer oni moraju povećati sigurnosne mjere u odnosu na te prijetnje. Primjena zakonskih mjera vrlo je važna za procjenu i istraživanja djelatnosti koje uključuje bioterorizam ili biološki kriminal. Ključ uspješna programa biološke sigurnosti jest i povećanje javne svijesti i rano otkrivanje prijetnji, što treba postići izgradnjom mreže združenih odgovornosti i sposobnosti koje treba razviti u okvirima vlada, sveučilišta, privatnog poduzetništva i javnosti. Mikrobiološka sudska medicina primjenjuje se za analizu i karakterizaciju sudskomedicinskih dokaza, a s ciljem njihova primjerena pripisivanja ili rekonstrukcije kriminalnoga čina. Ovdje se opisuju dva testa koji se zasnivaju na molekularnobiološkoj tehnologiji – C olymerase chain reaction, PCR) i PCR s ponavljajućim elementom – te se pokazuje kako se takvi postupci mogu uporabiti u svrhu istražnoga postupka. Ti molekularnobiološki postupci, na koje se oslanjaju mikrobiološki sudski medicinari, moraju se primjereno vrjednovati, tako da ih svi uključeni razumiju i da se rezultati tumače unutar njihovih ograničenja. Tri vrsti vrjednovanja su prethodno, razvojno i nutarnje. Prvo je potrebno za brz odgovor, kada prijeti neposredna opasnost ili se napad upravo dogodio. Dva potonja rabe se za uvrštenje tih postupaka među rutinske.The risk and threat of bioterrorism and biocrime have become a large concern and challenge for governments and society to enhance biosecurity. Law enforcement plays an important role in assessing and investigating activities involved in an event of bioterrorism or biocrime. Key to a successful biosecurity program is increased awareness and early detection of threats facilitated by an integrated network of responsibilities and capabilities from government, academic, private, and public assets. To support an investigation, microbial forensic sciences are employed to analyze and characterize forensic evidence with the goal of attribution or crime scene reconstruction. Two different molecular biology-based assays – real time polymerase chain reaction (PCR) and repetitive element PCR – are described and demonstrate how molecular biology tools may be utilized to aid in the investigative process. Technologies relied on by microbial forensic scientists need to be properly validated so that the methods used are understood and so that interpretation of results is carried out within the limitations of the assays. The three types of validation are preliminary, developmental, and internal. The first is necessary for rapid response when a threat is imminent or an attack has recently occurred. The latter two apply to implementation of routinely used procedures

Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

Background: In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results: To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare falsepositive SNPs were associated with variable nucleotide tandem repeats. Conclusions: The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution

Pre-columbian origins for North American anthrax

Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ~13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations

Molecular Epidemiology of Anthrax Cases Associated with Recreational Use of Animal Hides and Yarn in the United States

To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins

Single Nucleotide Polymorphism Typing of Bacillus anthracis from Sverdlovsk Tissue

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient’s B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis

Role of Law Enforcement Response and Microbial Forensics in Investigation of Bioterrorism

The risk and threat of bioterrorism and biocrime have become a large concern and challenge for governments and society to enhance biosecurity. Law enforcement plays an important role in assessing and investigating activities involved in an event of bioterrorism or biocrime. Key to a successful biosecurity program is increased awareness and early detection of threats facilitated by an integrated network of responsibilities and capabilities from government, academic, private, and public assets. To support an investigation, microbial forensic sciences are employed to analyze and characterize forensic evidence with the goal of attribution or crime scene reconstruction. Two different molecular biology-based assays – real time polymerase chain reaction (PCR) and repetitive element PCR – are described and demonstrate how molecular biology tools may be utilized to aid in the investigative process. Technologies relied on by microbial forensic scientists need to be properly validated so that the methods used are understood and so that interpretation of results is carried out within the limitations of the assays. The three types of validation are preliminary, developmental, and internal. The first is necessary for rapid response when a threat is imminent or an attack has recently occurred. The latter two apply to implementation of routinely used procedures

Phylogeny of the major groups of <i>B. anthracis</i> after Pearson et al. (2004) and VanErt et al. (2007).

<p>The new branch, “A.Br.011,” is flanked by branches A.Br.008 and A.Br.009. Thus, the group A.Br.008/009 is now subdivided into two groups: A.Br.008/011 and A.Br.011/009. The canSNP signature and assay that defines this new branch is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone-0028274-t003" target="_blank">Table 3</a>.</p

Dendrogram of MLVA-8 analysis of <i>B. anthracis</i> isolates collected from the 1976 California, 2006 New York, 2007 Connecticut anthrax cases, and 2009 New Hampshire case.

<p>All other genotypes are reference genotypes from Keim et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-Keim1" target="_blank">[8]</a>. For the California case, the clinical isolates were GT 76 (n = 4), while environmental isolates were GT 71 (n = 2), GT 72 (n = 2), GT 92 (n = 1), and GT 105 (n = 1). All clinical and environmental isolates from the New York (one clinical, 35 environmental) and Connecticut cases (one clinical specimen, 15 environmental isolates) were GT 1. All clinical (n = 2) and environmental (n = 9) isolates from the NH case were GT 149. Scale bar indicates amount of evolutionary change <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-McDonald1" target="_blank">[12]</a>.</p