Microbial Forensics: A Powerful Tool for Pursuing Bioterrorism Perpetrators and the Need for an International Database

Ruifu Yang^1* and Paul Keim²

¹Beijing Institute of Microbiology and Epidemiology, No.20, Dongdajie, Fengtai, Beijing 100071, P.R. of China

²Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, USA

*Corresponding Author:

Ruifu Yang
Beijing Institute of Microbiology and Epidemiology
No.20, Dongdajie, Fengtai, Beijing 100071, P.R. of China
E-mail: ruifuyang@gmail.com

Received Date: November 04, 2011; Accepted Date: January 07, 2012; Published Date: January 13, 2012

Citation: Yang R, Keim P (2012) Microbial Forensics: A Powerful Tool for Pursuing Bioterrorism Perpetrators and the Need for an International Database. J Bioterr Biodef S3:007. doi: 10.4172/2157-2526.S3-007

Copyright: © 2012 Yang R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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The Real Threat of Bioterrorism

Bioterrorism has long been recognized as a threat to human beings, animals and agriculture [1]. The world was awakened with a real case following September 11th 2001 in the U.S.A. with the anthrax-letter attacks. Bioterrorism is the deliberate use of living organisms or their byproducts (toxins) to inflict harm to susceptible humans, animals, plants, the environment or material by individuals, state-sponsored groups, insurgent/rebel, Doomsday/cult-type group, nonaligned terrorists, splinter groups, or lone offenders, in order to cause societal panic or menace societal safety and stability. The motives behind bioterrorism can be political, ideological, religious, or criminal, and sometimes only for personal reprisal. It is different from biological warfare, which is also the use of living organisms or their byproducts (toxins) to inflict harm, but it has been used by armies for military purposes [2]. Both bioterrorism and biological warfare can have devastating effects on all kinds of biodiversity, including humans, animals, plants, and other life. They can also be directed against resources, for example water or food supplies [3] and, in modern applications, against natural and manufactured materials [4]. Other expressions, including biocrime (biological crime), bioattack (biological attack), biological incidence, biothreat (biological threat) and biowar (biological war) have also been used to describe bioterrorism or biological warfare.

Bioterrorism or biological warfare is not a new concept because history offers several, if not many, examples of the intentional use of biological agents or diseased cadavers as weapons for inflicting harm upon enemies. We can trace examples back to the 14th century’s siege of Kaffa (now in Ukraine) for the earliest recorded biological war, which occurred between the Tatars and Kaffa. The Tatar forces catapulted plague corpses into the besieged city for spreading this disease. A plague outbreak was documented in Kaffa and it has been postulated that the fleeing citizens (as well as rats) of Kaffa who escaped via ship to various Mediterranean ports aided in the development of the second plague pandemic in the mid-1300s [5]. Another example of using disease as a biological weapon was the distribution of blankets or handkerchiefs used by the British smallpox victims as gifts to the hostile Native Americans by British troops in the 18th century [6]. Although there was indeed an outbreak of smallpox among these Native American tribes, it is difficult to know if these outbreaks were due to the British troop’s activities or a natural consequence of susceptibility. Biological warfare has occurred more recently during World War I and II and was supported by the biological weapons research programs of German, American, British, USSR and Japanese governments for military use [6]. Particularly notable was the Japanese Army’s notorious Unit 731, whose military medical doctors and specialists, under the command and direction of Lt. Gen. Ishii Shiro carried out diabolical experiments in Pingfan, Harbin, China on humans for the purpose of developing effective biological warfare weapons [7].

Modern examples of biological weapon and bioterrorism activity include: incidents in former USSR’s Biopreparat [8,9]; Iraq’s biological weapons program [8]; Salmonella contamination of the salad bars in The Dalles, Oregon, by the Rajneeshee commune for religious and political purposes [10]; anthrax hoaxes in the late 1990s in the United States [11]; bioterrorist attacks and development of botulinum toxin, anthrax, cholera, and Q fever for bioterrorist use in Japan by Aum Shinrikyo [12]; assassination of two Bulgarian traitors, Vladimir Kostov and Georgi Markov, by ricin ammunition in a small bullet shot by a modified umbrella [6]; anthrax spore letter attack in the United State in 2001 [13]; ricin bioterrorism attack in U.K. and several other documented bioterrorism hoaxes and non-succeed bioterrorist attacks [14].

The knowledge needed for developing biological weapons is accessible to individuals through the open literature and the Internet. Many bioterrorism-associated pathogens exist in host animals or vectors in their natural foci; bulk production of biological agents is cheap, with delivery and release not difficult to accomplish. The availability of information and ease of operation makes bioterrorism a method that could be quickly applied to many different nefarious purposes.

With advances of biotechnology, it is feared that bacteria and viruses could be modified in various ways to turn them into more powerful bioweapons [15]. The traditional biological agents, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella spp., etc., can be engineered with new features, such as antibiotic resistance, vaccineavoidance, decontaminant insensitive, environmentally stable and many others. Some have even speculated that advanced biotechnologies could be used by bioterrorists for creating engineered agents that target specific racial groups, a stealth virus that could incorporate itself into the DNA of the target population, and engineered virus with genes that control human behaviors and affect whole populations. These advanced technologies have been demonstrated through the creation of a novel virus by recombinating IL4 gene into mousepox virus [16], and the in vitro recreation of the 1918 Spanish flu virus [17] and polio virus [18]. Scientists in different fields that are paying attention to bioterrorism and biotechnology advances are increasingly worried about the misuse of biotechnology [4,15,19-22].

Microbial Forensics

Microorganisms have been used as means to perpetrate criminal acts, most recently highlighted by the terrorist attack on the U.S.A. using anthrax spore-containing letters in the fall of 2001. There are a number of issues for us to answer: such as the first responders’ safety and safety of the public, suspected site investigation, sample collection, sample transfer, pathogen’s isolation and identification, and the source-tracing of the bioagent. Source-tracing of a pathogen is of paramount importance for supporting criminal investigations and prosecuting the perpetrator(s) [23]. A number of questions needed to be addressed during the anthrax attacks about the relationship and the cause of cases in the four states, the relationship and origin of the anthrax bacteria isolated in different states, and the identity of the domestic or international perpetrator who released the pathogen [23]. All these questions can be answered within the purview of microbial forensics, which focuses on the individual or group who perpetrated the threat. During a bioterrorism criminal investigation, microbial forensics will employ traditional investigative methodologies, established molecular techniques, and newer advanced methods that still may be just under development for the identification and source-tracing of the pathogen [23]. As early as 1995, Dr. Randal S. Murch suggested that the investigation and solution of bioterrorism events would rely heavily on forensic science [24]. The laboratory analyses used for microbial forensics may include molecular sequencing, microbial cultures, biochemistry, electron microscopy, crystallography, and mass spectrometry. These analyses go well beyond those used for medical diagnoses and epidemiologic investigations [25].

The Federal Bureau of Investigation (FBI) of the United States created the Scientific Working Group on Microbial Genetics and Forensics, assembled from leading experts in the relevant fields, U.S. government agencies, professional organizations, and nonprofit and academic institutions, in order to develop, establish, and validate analytical methods, protocols and informatics, standards, training and certification programs, quality assurance programs, and research priorities for enhancing the capabilities of microbial forensics. The National Bioforensics Analysis Center was established recently in the United States by the Department of Homeland Security and it will be operated in partnership with the FBI for providing a central facility to conduct analysis of evidentiary material.

The task for microbial forensics includes sample identification, collection, handling and preservation, analytical method selection, casework analysis, and result’s interpretation, validation and quality assurance. Hence, the new discipline of microbial forensics is a conglomeration of an array of well-established fields, such as microbial genomics, phylogenetics, forensic information, bioinformatics, computer science, and traditional microbiology and epidemiology. Microbial forensics will be most effective if there is sufficient basic scientific information concerning genetics, evolution, physiology and ecology of the biological agents. Molecular techniques have been used for years to trace outbreaks of microbial diseases, a practice called molecular epidemiology. PulseNet USA was established in 1996 by the United States Center for Disease Control for molecular surveillance of foodborne infections [26]. It has been successfully used for detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. The PulseNet network is now being replicated in different ways in Canada, Europe, the Asia Pacific region, and Latin America [27]. These independent networks work together in PulseNet International allowing public health officials and laboratorians to share molecular epidemiologic information in realtime and enabling rapid recognition and investigation of multi-national foodborne disease outbreaks [27]. It represents a good example on how to establish a platform to be used as a primary tool in preparedness and response to bioterrorism. The types of genetic markers and features that can impact statistical inferences of microbial forensic evidence include: single nucleotide polymorphisms (SNPs), repetitive sequences, insertions and deletions, mobile elements, pathogenicity islands, virulence and resistance genes, housekeeping genes, structural genes, whole genome sequences, asexual and sexual reproduction, horizontal gene transfer, conjugation, transduction, lysogeny, gene conversion, recombination, gene duplication, rearrangements, and mutational hotspots [28]. Nucleic acid based typing technologies include multilocus sequence typing (MLST) [29], Multilocus variablenumber tandem-repeat analysis (MLVA) [30], clustered regularly interspaced short palindromic repeats (CRISPR) [31], whole genome sequencing [32], and microarrays [33-39]. Some of these techniques have been used successfully for tracking B. anthracis used in the “white powder letter” biocrimes in 2001 [32] and release of aerosolized B. anthracis spores over Kameido, Japan by the Aum Shinrikyo cult [40]. Other potential molecular markers used for microbial forensics include protein and chemical markers of lipid polysaccharide and sugar etc. The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced [41-45]. It has been employed for predicting the delta(18)O and deltaD values of spores produced in nutritionally identical media and local water sources for five different locations around the United States [44]. No matter which techniques are used, a basic reference database for these markers is of paramount importance. Next, we will show the development of a genetic polymorphism database for Yersinia pestis and an example of tracking B. anthracis used in the 2001 anthraxletter biocrime in the United States by different techniques.

Genetic Polymorphism Database for Source-Tracing of Bioterrorism-Associated Agents

As mentioned above, different techniques can be employed for establishing basic reference databases for microbial forensics analysis. Genetic, protein and chemical markers have been investigated for Yersinia pestis, the causative agent of bubonic and pneumonic plagues. It is thought to be one of the most dangerous bacterial pathogens in the world. Credible estimates of the number of people killed by this bacterium during the course of history approach 200 million. Plague is maintained in natural disease foci in wild rodents through transmission between rodents by their flea ectoparasites. The striking lethality is a consequence of vector-borne transmission peculiarities for this pathogen in nature as infected fleas have to leave their dead hosts in the search of a blood meal and, thus, carry the infection to a new victim, new host. Y. pseudotuberculosis is believed to be the ancestor of Y. pestis [46-48]. Their evolutionary divergence occurred 2,600-28,000 years ago [46]. This speciation and further intraspecific modifications resulted in the wide spectrum of Y. pestis intraspecies groups (biovars, subspecies, ecotypes, plasmidovars, genotypes, etc.) differing in their host range and lethality. Currently, the microevolution of Y. pestis accommodates the adaptation of this pathogen to more than 200 species of wild rodents inhabiting natural plague foci on all of the continents except for Australia and Antarctica. Plague transmission is mediated by at least 80 different species of fleas. Studying Y. pestis microevolution may help solve basic medical microbiological questions such as the origin and evolution of animal and human infectious diseases, as well as, improve the methods of microbial forensics, agent identification, geno- and genomotyping of Y. pestis

In the past few years, and owing in part to the availability of whole genome sequence data from many bacterial species including different strains from the same species, a high number of polymorphism sources, and consequently typing methods, have emerged. For Yersinia pestis these include: MLST [49], SNPs [50], MLVA [51,52], different region (DFR) typing [53] and analysis [31]. These new methods (SNPs, MLVA, DFR and CRISPRs) are likely to replace the previous pattern-comparison methods (such as insertion sequence (IS) typing by southern blotting, ribotyping or pulsed-field gel electrophoresis) which are more expensive and not fully appropriate to the creation of international databases [54-58]. As sequencing technologies advance, bacterial whole genome sequence analysis is becoming relatively easy for even conventional laboratories [59,60]. Using genome-wide variations of a pathogen for source tracing analysis will become popular because it will give us a comprehensive view of genetic variation from a specific pathogen and, hence, relatively definitive conclusions.

SNPs

Due to the close evolutionary relationship between Y. pestis and Y. pseudotuberculosis, they cannot be differentiated by MLST [49]. However, 76 conservative sSNPs within 3,250 orthologous CDSs (coding sequences) were revealed by comparative analysis of three whole genomes of Y. pestis, representing different biovars of this pathogen [50]. A three-branched phylogenetic tree was inferred by using SNP variations and a novel genetic typing scheme was proposed. By using whole genome sequence analysis, more SNPs have been uncovered for genetic typing, which is more discriminating [46]. By using SNP information, the etiology for the Black Death was successfully traced, confirming multiple importation events of the plague to Europe and that the ancient agent was the ancestor of modern Y. pestis biovars Orientalis and Medievalis [61]. By using whole genome sequencing and SNP information, Y. pestis from a specific region could be differentiated [62,63]. In our laboratory we have sequenced more than 100 strains of Y. pestis from different natural plague foci in China and more than 2000 new SNPs were found and used to create a very finely resolved phylogenetic tree (data not published), which will help us to design a more discriminative genotyping method to distinguish Y. pestis isolates.

MLVA

The genomic polymorphism associated with tandem repeats has been instrumental in mammalian genetics for the con¬struction of genetic maps and still is the basis of DNA fin¬gerprinting in forensic applications [64,65]. Tandem repeats are usually classified as satellites sequences (spanning megabases of DNA and associated with heterochromatin), minisatel¬lites (repeat units in the range 6-100 bp and spanning hun¬dreds of basepairs) or microsatellites (repeat units in the range 1-5 bp and spanning a few tens of nucleotides).

Recent studies show that tandem repeat polymorphisms of mini and mi¬crosatellites are likely to be a highly significant source of very informative markers for the identification of patho¬genic bacteria, even for recently emerged, highly monomorphic species [66-73]. Tandem repeats prob¬ably contribute to a pathogen’s adaptation to its host, and they also account for bacterial phenotypic var¬iations depending on their genomic location. For example, if a tandem repeat is lo¬cated within the regulatory region of a gene it can play an on/off switching role in gene expression at the transcriptional level [74]. Similarly, if they are located within coding regions with a repeat unit length of three bases they may cause antigen variation for that bacterium; but if the repeat unit length is not a mul¬tiple of three, they can induce a truncated translation product.

VNTR sequences are common in the Y. pestis genome and occur frequently in gene coding regions. They are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1 [75]. Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Adair et al. [76] identified a tetranucleotide repeat sequence, (CAAA)N, in the genome of Yersinia pestis, and demonstrated that this region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value = 0.82) within a set of 35 diverse strains.

Multiple-locus VNTR analysis (MLVA) has proved to be capable of both distinguishing closely related strains and successfully classifying more distant relationships. Pourcel et al. [52] and Klevytska et al. [75] examined representative strains of Y. pestis by using 25 and 42 VNTR loci, respectively, and vast differences in diversity were observed among these loci. Pourcel et al. [52] grouped 180 Y. pestis into 61 different genotypes with the three biovars distributed correctly into the three main branches. They showed the heterogeneity of biovar Medievalis and proposed that 7 selected VNTR markers would be sufficient for quick characterization of a new strain. Compared to other genotyping methods, MLVA is easily standardized for establishing databases. The fact that Y. pestis is one of the most dangerous bioterrorism agents prevents the international exchange of bacterial strains and limits collaborative research; which creates a barrier for better understanding plague ecology and global evolution. Web-based comparison and identification of VNTR genotypes of Y. pestis capitalizes upon standardized typing systems and minimizes the impact of international transfer barriers.

Le Flèche et al. [74] created a database (https://minisatellites.upsud.fr) of tandem repeats for pathogenic bacteria based on publicly available bacterial genomes. Its wide applicability was illustrated by the characterization of minisatellites from two important human pathogens, Y. pestis and Bacillus anthracis. They found that Y .pestis contains 64 minisatellites with repeat units at least 9 bp long where the unit is repeated at least 7 times.

Denoeud et al. [77] then presented an Internet-based resource to help develop and perform tandem repeats based bacterial strain typing. These tools are also accessible through the above Web link. There are four parts to the web page: 1) The “Tandem Repeats Database” enables the identification of tandem repeats across entire genomes. 2) The “Strain Comparison Page” identifies tandem repeats differing between different genome sequences from the same species. 3) The “Blast in the Tandem Repeats Database” facilitates the search for a known tandem repeat and the prediction of PCR product sizes using primers containing the tandem repeat. 4) The “Bacterial Genotyping Page” is a service for strain identification at the subspecies level.

MLVA has been used by American scientists to establish a database for more than 1000 Y. pestis strains [78]. The 43 markers used are relatively shorter VNTRs while the Europe scientists employed relatively longer repeats for database establishment [52]. We also employed these markers for successfully tracing the source of a primary pneumonic plague outbreak in 2009 [79]. If an internationally accepted database is developed, the method employed needs to be standardized.

DFR

Bacteria can adapt themselves to a new niche by gene loss and/ or acquisition. These events can be analyzed by using whole genome sequencing or subtractive hybridization, but they are too expensive and laborious to be applied to a large number of bacterial strains. Whole genome-based DNA microarray is an ideal technique for analyzing genomic fragment differences among large numbers of bacterial strains. Zhou et al. employed a whole genome DNA microarray, comprising 4005 ORFs (genes) from Y. pestis 91001 and CO92, to screen the differences between 43 strains of Y. pestis and 7 strains of Y. pseudotuberculosis [53]. They found twenty-two DFRs among these strains. Next, one or more genes were chosen from each DFR to represent the genomic difference. PCR amplification of the selected genes was performed on more than 900 isolates of Y. pestis, isolated from different natural plague foci and different years, to screen for the distribution of DFRs in these strains. The DFR profiles of these isolates could be assigned into fourteen genomovar. The DFRs represent the dynamic regions of the Y. pestis genome in natural populations; suggesting their features of acquisition or deletion have a role in the adaptive evolution of Y. pestis. The parallel microevolution of the Y. pestis genome in natural populations was examined and revealed evidence of genome content flux through the loss/acquisition of plasmids and chromosomal segments.

The microevolution of the genomovars is perfectly consistent with the expansion of plague foci [53,80]. Each of the genomovars is confined to a specific geographic region, namely a plague focus or part of a focus with a unique set of environments, reservoirs and vectors. Most of the geographic regions with different primary reservoirs have their own unique genomovars [80]. Sometimes, there is more than one genomovar in a single focus and within a single primary reservoir, but each of the genomovars corresponds to a unique natural environment and primary vector(s). Therefore, DFR could be used to roughly locate the origin of an unknown Y. pestis, but more discriminative differentiation of its strains needs the help of other molecular methods such as MLVA or SNPs [81].

CRISPRs

CRISPRs are well-defined structures and present in many bacteria and in most archaea, sometimes in multiple copies per genome. The CRISPR structure itself is usually surrounded by CRISPR-associated genes (cas genes). New spacer sequences are not synthesized de novo, but are copied from existing DNA sequences [31]. The vast majority of known spacers lack any similarity to currently available sequences. However, when similarities exist, they most often correspond to short portions of mobile elements such as phages. These observations have led to the suggestion that CRISPRs were a defense-mechanism against genetic invasions [31]. Simple evolution rules have been proposed for CRISPRs which open the way to phylogenetic investigations: (1) new spacers are acquired in a polarized way from one extremity adjacent to the leader sequence which acts as a transcription promoter; (2) losses may occur randomly along the array; (3) the probability of acquisition of the same spacers independently is extremely low. The analysis of CRISPRs have already played an important role in investigating the epidemiology of the major human pathogen, Mycobacterium tuberculosis (the corresponding typing method is called “spoligotyping”). A database containing the typing information from thirty thousand isolates has been built [82]. Although this represents only a very small fraction of TB isolates worldwide, the database is by far the largest existing typing database for a bacterial pathogen. One reason is that the method is sufficiently robust and easy to run at a reasonable cost, such that many laboratories could produce the data and easily share it. Secondly, the resulting data is useful for phylogenetic analysis and has enabled definition of large strain families. A third reason was the relatively simple status of the M. tuberculosis CRISPR locus. The locus is apparently inactive; it does not acquire new spacers, so that a fixed and limited set of relevant spacers could be defined to produce “spoligotyping membranes”. The availability of a similar approach for Y. pestis would be of use for at least two reasons. First, it would allow for the large-scale screening of Yersinia pseudotuberculosis strains to identify Y. pestis’s closest neighbors, as well as providing a systematic and routine typing approach for current Y. pestis collections and new isolates. Secondly, CRISPRs represent a potentially very interesting tool for the investigation of ancient DNA (aDNA). The nature of the early plague pandemics is still controversial, and one reason for this may be the lack of appropriate genetic targets for Y. pestis aDNA investigation. Y. pestis CRISPR loci are still active31 and able to acquire new spacers (in contrast with the M. tuberculosis CRISPRs). Therefore, it is necessary to list the repertoire of existing CRISPR spacers. If this repertoire eventually turns out to be very large, as in some bacteria, then the development of such an assay will necessitate the use of DNA chips, which are able to deal with a larger number of spacers compared to the current spoligotyping assay format. The first repertoire of CRISPR spacers was deduced from Y. pestis isolates that only represent a small part of the global and historical genetic diversity for Y. pestis [31]. Less than 40 spacers were identified, in the three Y. pestis CRISPR loci, suggesting that perhaps the repertoire within a larger collection might remain tractable.

Whole genome sequencing analysis

Whole genome sequencing has been widely used for Y. pestis and used for tracing the source of a laboratory acquired plague [83]. With whole genome sequences, we can identify different kinds of genetic variations, including SNPs, gene rearrangement, gene loss and gain, VNTRs and CRISPRs for a comprehensive understanding of bacterial genome diversity and for a fine source-tracing analysis. When these genome-wide variations are used for typing purpose, it is refered to WGST (Whole Genome Sequence Typing). With the rapid advances of next generation sequencing technologies, we can obtain genome sequences from tens or hundreds of bacterial strains in a single species, providing us an opportunity to build a database of genome diversity for source-tracing analysis. As shown above, the different genetic variation types can be input into databases and then coupled to source-tracing software, rendering a rapid and accurate tracking of the target pathogen.

Other methods

Other methods, including fatty acids analysis and bacterial mass fingerprinting (BMF), are potentially useful for strain discrimination. Song et al. evaluated the possibility of utilizing Cellular Fatty Acids (CFA) information for typing Y. pestis [84]. They used 58 strains representing different ecotypes isolated in different regions of China. Major CFAs of the tested strains are 16:0, cyclo 17:0, 3OH-14:0, ω7c 16:1 and monounsaturated 18 carbon acids, which account for more than 70% of the total CFAs. Dendrogram construction of the tested strains reveals a great homogeneity among these Y. pestis isolates, which suggests that CFA analysis cannot serve as a powerful typing tool for Y. pestis strain differentiation. All the experimental Y. pestis strains were mis-identified as Y. pseudotuberculosis by the Sherlock system. Later, Tan et al. found that these two pathogens could be differentiated by using ratios of certain CFA components as chemical markers [85]. Additionally, Liu et al. developed a universal sample preparation method for BMFanalysis and has demonstrated the discriminatory power of BMF [86]. BMF was obtained by MALDI-TOF MS and it has been shown to be potential for inexpensive and rapid typing of MRSA in infection control [87,88]. MALDI-TOF MS was also successfully employed to identify Legionella spp. with easy operation and high efficiency [89]

Tracking of Bacillus anthracis used in Biocrime of 2001 in the United States

After it was determined that the anthrax cases following September 11 were bioterrorist attacks, the FBI used every investigative technique available to investigate the criminal perpetrator(s). The FBI employed all possible strategies including: round-the-clock surveillances, eavesdropping, searches and hundreds of polygraphs. Altogether, agents conducted 5,000 interviews and served more than 1,700 grand jury subpoenas, which involved 40 of the FBI’s 56 field offices, as well as many of its 44 overseas legal attaches. The Bureau established 112 separate databases to store information about the case, and even offered a reward of $2.5 million for information leading to a conviction. In the end, it was the scientific evidence, including genome sequencing, VNTR analysis and analysis of the extrasporular material in the envelopes, that provided the final results that the B. anthracis attack strain was the Ames strain and “homemade in USA” [90]. Comparison of the wholegenome sequence of Bacillus anthracis isolated from a victim of the bioterrorist anthrax attack with a reference revealed 60 new markers that included single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats [32]. Further molecular subtyping of Bacillus anthracis strains during the 2001 bioterrorismassociated outbreak demonstrated that all isolates were MLVA genotype 62, the same as the Ames strain used in laboratories [13]. Sequence analysis of the protective antigen gene (pagA) from 42 representative outbreak isolates determined that they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I) [13]. Recently, a systematic microbiological analysis of the spores from 2001 letters revealed morphological mutants that are not commonly seen in the environment. The morphological mutant analysis was a novel approach for forensics that combined systematic microbiological analysis with whole-genome sequencing and comparative genomics [91].

Source-Tracing of Vibrio cholera Outbreak in Haiti after Earthquake In 2010

On January 12, 2010 there was a 7.0 magnitude earthquake in the capital of Haiti, Port-au-Prince, and surrounding areas. Some 10 months later there was a devastating outbreak of cholera [92]. Its origin could not be confirmed, but origin of the disease was rumored to be a battalion of Nepalese soldiers serving as United Nations peacekeepers [93,94]. Though the use of WGST, the Haiti outbreak strains were confirmed to be closely linked to Nepalese strains, with only one or two base-pair differences, providing sound evidence that the Haiti outbreak of cholera was caused by a Vibrio cholera strain from Nepal [95]. The importance of this source-tracing analysis is that it shows the rapid global transmission of infectious disease and the power of molecular biological techniques in tracking the agent of infectious disease. This work also strengthens the need for a genetic diversity database based on whole genome sequencing analysis and argues for the sharing of data for all known pathogens though international collaboration. It is only through international data sharing and databases that source-tracing of pathogens will be truly effective.

Database-Building and Perspectives

The new discipline of microbial forensics represents a powerful approach for tracking pathogens associated with bioterrorism or natural disease’s outbreaks. As mentioned above, the key factor for successful development of microbial forensics is database-building for different biomarkers and all available global strains. Given that the transfer of bioterrorism-associated pathogens and their byproducts, even DNAs is frequently restricted or even prohibited by law, it makes databasebuilding for international sharing extremely difficult. In other words, development of such a database is not only a technical challenge for the scientific community, but also a political challenge for the international community. How we use our wisdom to bridge this gap is paramount for the successful development of international microbial forensics. A country-limited database will not be as useful for the investigation of international bioterrorism and international spread of infectious diseases as a global joint effort. Communication between scientists and politicians from different nations, and collaborations between scientists from different countries, will be essential for developing internationally accepted techniques for standardized biomarkers and also critical for establishment of such a database. For example, there are two well-developed MLVA techniques for Y. pestis, but scientists from the U.S.A. employed smaller repeats markers [78], and those from Europe larger ones [52], making these data mostly incompatible. Without the collaboration of scientists from Russia and China, where the most diversified strains of Y. pestis are distributed [48,96,97], the MLVA databases of Y. pestis will not be comprehensive. Therefore, an authoritative database should be built based on the consensus reached by scientists in the field. WGST [95] is now the most applicable technique for such database-building and finely selected MLVA loci could be the second technique of choice because it is relatively easy to standardize [40,79]. Although PFGE has been successfully used for tracing the source of food-borne pathogens, its discriminative power is relatively low and its operation is tedious, rendering this technique unsuitable for forensic database-building. However, the experiences with this technique for database-building should be taken as an example for microbial forensics [26,98-101]. Other kinds of methods, including MLST, DFR and CRISPR analysis, could also be used for a specific case investigation.

In conclusion, development of microbial forensics is critical to the pursuit of biocriminals, and international collaborations and political decisions will play key roles in successful progress of this new discipline.

Acknowledgements

Work in our laboratory has been supported by grants from the National Natural Science Foundation of China (contract no. 30930001) and the State Key Development Program for Basic Research of China (2009CB522600).

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