Forensic Microbiology

Biocrime or bioterrorism is the threat or use of microorganisms, toxins, pests, prions, or their associated ancillary products to commit acts of crime or terror. Microorganisms can malevolently be used as biological warfare agents, in bioterrorist acts, and in crimes without political intentions. Such actions can be directed against humans and animals and can lead to outbreaks of infections with high morbidity and mortality. In recent years microbial forensics has been established as a new scientific discipline to strengthen the law enforcement response especially in a bioterrorism event. These tools can also be applied to investigate the transmission of pathogenic microorganism caused by sexual abuse and other physical offenses. Several microorganisms are a severe threat to human and/or animal health and a country's agricultural economy. Their malevolent use can have a major socio-economic impact. A number of these pathogens can affect both humans and animals (zoonoses), can contaminate the environment for decades, or may establish new enzootic foci. The World Organisation for Animal Health (OIE) lists several of these agents as diseases of importance to international trade with serious export restrictions for countries where the diseases are endemic.  In the aftermath of the anthrax letters attacks in October 2001 that killed five people it has become evident that biocrimes can only be solved when genomic information can be used to identify the source of an organism. Evidence in a criminal investigation must be collected within the constraints of legal rules to ensure that any prosecution based upon that evidence can withstand judicial review in a court. Therefore, first responders must learn how to secure evidence and preserve the chain of custody. Laboratories that have been officially accredited will be able to provide all relevant documents regarding quality control and assurance, proficiency test results, qualification of laboratory personnel etc. Also case specific material like photographs of gels, bench-notes, validation studies, and controls will usually be adequately documented. Evidence collection, transport, and storage need more attention than is usually needed for clinical routine samples. Test procedure will be according to standard operating procedures (SOPs) and any deviation of protocols will have to be documented. The final report should contain information about the specificity and accuracy of the applied tests and provide an interpretation of the result and its limitations. Nucleic amplification and molecular-epidemiological techniques are essential tools in clinical microbiology for identifying pathogens and in outbreak investigations. Various typing tools have been developed for phylogenetic and phylogeographic studies. In forensic microbiology these methodologies can be used to detect and trace back the spread of microorganisms in the context of a crime. Whole-genome sequencing provides the most comprehensive, reliable and reproducible information about a strain, but until recently this technique was expensive and time consuming. The subsequent annotation of sequences was also a major endeavor. Nowadays this technique has become affordable and reference genomes for all select agents have been sequenced. They can be used to clarify the relationship of suspicious isolates with reference genomes. Centralized reporting and surveillance systems on the national and international level are essential as single cases may be regarded as sporadic although they are part of a larger transboundary outbreak. Surveillance systems have already been established that store and provide DNA fingerprints of microbes being major causes of hospital-acquired or food borne infections. Descriptive epidemiological data have to be analyzed with caution. Natural outbreaks can be difficult to discriminate from intentional use of microorganisms, especially if the organisms are endemic. Only molecular-epidemiological tools can corroborate the chain of infection. Chain-of-custody documentation, secure storage of evidence, tracking of individual items of evidence and their derivatives and all the legal requirements for handling evidence. Chainof-custody protocols document the unbroken chain of records showing who had handled the evidence, where and under which conditions (temperature, time etc.) the material had been stored and whether access to the samples was restricted. Requested by the clinician are appropriate and which tests should be performed. Unfortunately, requests are not always justified by the clinical presentation and the sample matrix is sometimes conflicting. For example a microscopical inspection of sputum samples will indicate, if the quality of the specimens is adequate. In forensic microbiology the same rules apply, but more detailed investigations may be necessary to obtain relevant information about the history of a specimen, environmental conditions, chemical and physical constitution of the matrix, presence of pollen etc. The Centers for Disease Control (CDC) in Atlanta have evaluated the priority of agents according to their relevance for national security due to ease of dissemination and transmission from person to person, high mortality rates, the potential for major public health impact, risk of public panic and social disruption, and the requirement of special action for public health preparedness. Category A includes the most dangerous agents: Variola major virus (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), and viral hemorrhagic fever viruses. Unambiguous identification agent. However, animal models should be avoided for ethical reasons whenever possible. Biological agents can be difficult to cultivate due to sample contamination, low number of bacteria or pretreatment of patients with antibiotics. Some bacteria are fastidious (F.tularensis, Brucella spp.) and require special nutrient media, and some need prolonged cultivation times (Brucella spp.). Phenotypical characteristics such as antibiotic susceptibility and biochemical reaction profiles, susceptibility to specific phages, colony morphology and others are not always reliable. Mutations of agents can be induced or engineered, but naturally occurring atypical strains have also been found e.g. among Bacillus anthracis and Yersinia pestis isolates which can result in misidentification and treatment failure. Commercial biochemical identification systems are not optimized for these agents and can result in misidentification. Multiple antimicrobial resistances can occur through natural horizontal gene transfer or by genetic manipulation. Natural resistance to a multitude of antimicrobials is typical for Burkholderia pseudomallei. Francisella tularensis is naturally resistant to penicillins and cephalosporines. A very dangerous multidrug resistant strain of Yersinia pestis has been isolated from a patient with bubonic plague in Madagascar. This strain carries a self-transmissible plasmid with a genetic backbone also prevalent among Escherichia coli, Klebsiella spp. and Salmonella spp. conferring high-level resistance to streptomycin, tetracyclin, chloramphenicol, and sulfonamides. These facts underline the importance of cultivation and the assessment of antimicrobial susceptibility in addition to more rapid diagnostic tools. A polyphasic approach for identification and typing will help to avoid problems due to atypical genotype and phenotype, inhibition, or lack of specificity or sensitivity of assays. Many real-time PCR assays are highly specific and sensitive and shorten the time required to establish a diagnosis in comparison with conventional PCR protocols, cultivation, and biochemical identification methods. Therefore, real-time PCR assays have been developed for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei and Burkholderia pseudomallei, Francisella tularensis and Yersinia pestis (21). PCR results can be false negative due to inadequate quality of clinical samples, low number of bacteria in samples, DNA degradation, inhibitory substances and inappropriate DNA preparation. Seroconversion may prove the exposure to a certain agent in the past. However, seroconversion can be expected only after several days or weeks and is of little use for rapidly diagnosing infections caused by highly pathogenic agents. It will be difficult to organize serological investigations (including follow-up tests) when a terrorist attack causes mass casualties that need medical treatment or when the situation is complicated by civil unrest, war or natural catastrophes at the same time. Various immunological assays have also been used to identify pathogens in samples of patients and environmental samples. Limitations of these immunological assays are that they are frequently not available commercially, not specific enough, or have not been validated and licensed for use in humans or animals. Moreover, cross-reactions may cause false positives and modified or missing antigenic structures can cause false negatives. Techniques for forensic microbiology can be very similar to those being used for phylogenetic and epidemiological investigations e.g. for food-borne outbreaks. Molecular-epidemiological tools used for genotyping are most promising and have been applied in the past to elucidate the origin of biological agents. Especially whole genome sequencing and bioinformatic tools for comparison of genomes are potent tools, but technical complexity and costs are still prohibitive for routine application.

**Bacillus (B.) anthracis is the causative agent of anthrax and a member of the Bacillus cereu group. This group includes B. anthracis, B. cereus, B. thuringiensis, B. weihenstephanensis, and B. mycoides. These closely related bacteria can be discriminated by using phenotypic characteristics. B. anthracis is typically non-motile, susceptible to penicillin, lysed by the gamma phage, and colonies are non-hemolytic with a typical morphology. However, more than 24 hours are required to assess these characteristics and misidentification can occasionally occur due to variations of the phenotype.

***Plague is caused by the gram-negative bacterium Yersinia pestis and is still endemic in natural foci of Asia, Africa, and America in rural areas. The affected population is mostly poor and is living under deplorable hygienic conditions. Due to the high lethality of plague a rapid and reliable identification of the organism is crucial, but medical services and laboratory facilities are very scarce in the endemic regions of Africa and Central Asia. The validation of diagnostic assays for infectious diseases like plague can be demanding because of very limited access to clinical samples and isolates. None of the previously published real-time PCR assays for diagnosing plague had been clinically validated so far.

****Burkholderia (B.) mallei is a gram-negative bacterium causing glanders and farcy in horses, donkeys, and mules (solipeds) and has been classified by the CDC as a priority category B biological agent. Glanders in horses presents with pneumonia, purulent nasal discharge, and poor general condition, whereas farcy is a chronic cutaneous disease with massively enlarged lymph vessels (“farcy-pipes”) and nodules developing into ulcers. Equines are the only known reservoir for sporadic infections in humans. Humans develop a clinical picture resembling melioidosis which is caused by the closely related bacterium B. pseudomallei. In 2004, an outbreak of glanders in horses was reported to the Office International des Epizooties by the United Arab Emirates. In addition to cultivation and phenotypical identification a new real-time PCR assay was developed for the specific identification of B. mallei, which detected the bacteria in tissues of two horses.

***** Francisella (F.) tularensis is a biological agent of category A and the causative agent of tularemia. The subspecies F. tularensis subsp. holarctica can be found in many regions of the northern hemisphere, but the subspecies F. tularensis subsp. tularensis occurs only in North America. Surprisingly, isolates of F. tularensis subsp. tularensis were recovered repeatedly from fleas and mites captured in the region of the Danube river basin, close to Bratislava in Slovakia. This was extremely unusual and warranted further investigations.

Microbial forensics is a young scientific discipline. Epidemiological tools can be used to trace strains and to clarify the chain of infection, but typing systems have to be especially evaluated for forensic purposes. Classical microbiological techniques are indispensable, but most recent developments including very rapid whole genome sequencing complement the polyphasic approach needed for diagnostics and typing. Only large collections of strains from all over the world and high quality sequence data will provide the basis for meaningful results in microbial forensic investigations.

Acknowledgements:

The Police Department;

www.politie.nl and a Chief Inspector – Mr. Erik Akerboom     ©

Bibliography:

1.    Criminal Investigations – Crime Scene Investigation.2000

2.    Forensic Science.2006

3.    Techniques of Crime Scene Investigation.2012

4.    Forensics Pathology.2001

5.    Pathology.2005 

6.    Forensic DNA Technology (Lewis Publishers,New York, 1991).

7.    The Examination and Typing of Bloodstains in the Crime Laboratory (U.S. Department of Justice, Washington, D.C., 1971).

8.    „A Short History of the Polymerase Chain Reaction". PCR Protocols. Methods in Molecular Biology.

9.    Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press.2001

10.   "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology.1994

11.   Forensic Science Handbook, vol. III (Regents/Prentice Hall, Englewood Cliffs, NJ, 1993).

12.   "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". In Kieleczawa J. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett. 2006

13.   Nielsen B, et al., Acute and adaptive responses in humans to exercise in a warm, humid environment, Eur J Physiol 1997

14.   Molnar GW, Survival of hypothermia by men immersed in the ocean. JAMA 1946

15.   Paton BC, Accidental hypothermia. Pharmacol Ther 1983

16.   Simpson K, Exposure to cold-starvation and neglect, in Simpson K (Ed): Modem Trends in Forensic Medicine. St Louis, MO, Mosby Co, 1953.

17.   Fitzgerald FT, Hypoglycemia and accidental hypothermia in an alcoholic population. West J Med 1980

18.   Stoner HB et al., Metabolic aspects of hypothermia in the elderly. Clin Sci 1980

19.   MacGregor DC et al., The effects of ether, ethanol, propanol and butanol on tolerance to deep hypothermia. Dis Chest 1966

20.   Cooper KE, Hunter AR, and Keatinge WR, Accidental hypothermia. Int Anesthesia Clin 1964

21.   Keatinge WR. The effects of subcutaneous fat and of previous exposure to cold on the body temperature, peripheral blood flow and metabolic rate of men in cold water. J Physiol 1960

22.   Sloan REG and Keatinge WR, Cooling rates of young people swimming in cold water. J Appl Physiol 1973

23.   Keatinge WR, Role of cold and immersion accidents. In Adam JM (Ed) Hypothermia – Ashore and Afloat. 1981, Chapter 4, Aberdeen Univ. Press, GB.

24.   Keatinge WR and Evans M, The respiratory and cardiovascular responses to immersion in cold and warm water. QJ Exp Physiol 1961

25.   Keatinge WR and Nadel JA, Immediate respiratory response to sudden cooling of the skin. J Appl Physiol 1965

26.   Golden F. St C. and Hurvey GR, The “After Drop” and death after rescue from immersion in cold water. In Adam JM (Ed). Hypothermia – Ashore and Afloat, Chapter 5, Aberdeen Univ. Press, GB 1981.

27.   Burton AC and Bazett HC, Study of average temperature of tissue, of exchange of heat and vasomotor responses in man by means of bath coloremeter. Am J Physiol 1936

28.   Adam JM, Cold Weather: Its characteristics, dangers and assessment, In Adam JM (Ed). Hypothermia – Ashore and Afloat, Aberdeen Univ. Press, GB1981.

29.   Modell JH and Davis JH, Electrolyte changes in human drowning victims. Anesthesiology 1969

30.   Bolte RG, et al., The use of extracorporeal rewarming in a child submerged for 66 minutes. JAMA 1988

31.   Ornato JP, The resuscitation of near-drowning victims. JAMA 1986

32.   Conn AW and Barker CA: Fresh water drowning and near-drowning — An update.1984;

33.   Reh H, On the early postmortem course of “washerwoman’s skin at the fingertips.” Z Rechtsmed 1984;

34.   Gonzales TA, Vance M, Helpern M, Legal Medicine and Toxicology. New York, Appleton-Century Co, 1937.

35.   Peabody AJ, Diatoms and drowning – A review, Med Sci Law 1980

36.   Foged N, Diatoms and drowning — Once more.Forens Sci Int 1983

37.   "Microscale chaotic advection enables robust convective DNA replication.". Analytical Chemistry. 2013

38.   Sourcebook in Forensic Serology, Immunology, and Biochemistry (U.S. Department of Justice, National Institute of Justice, Washington, D.C.,1983).

39.   C. A. Villee et al., Biology (Saunders College Publishing, Philadelphia, 2nd ed.,1989).

40.   Molecular Biology of the Gene (Benjamin/Cummings Publishing Company, Menlo Park, CA, 4th ed., 1987).

41.   Molecular Evolutionary Genetics (Plenum Press, New York,1985).

42.   Human Physiology. An Integrate. 2016

43.   Dumas JL and Walker N, Bilateral scapular fractures secondary to electrical shock. Arch. Orthopaed & Trauma Surg, 1992; 111(5)

44.   Stueland DT, et al., Bilateral humeral fractures from electrically induced muscular spasm. J. of Emerg. Med. 1989

45.   Shaheen MA and Sabet NA, Bilateral simultaneous fracture of the femoral neck following electrical shock. Injury. 1984

46.   Rajam KH, et al., Fracture of vertebral bodies caused by accidental electric shock. J. Indian Med Assoc. 1976

47.   Wright RK, Broisz HG, and Shuman M, The investigation of electrical injuries and deaths. Presented at the meeting of the American Academy of Forensic Science, Reno, NV, February 2000.