Environmental Microbial Forensics

Environmental forensics has been previously described as the use of chemical, physical, and statistical techniques to investigate the sources and behavior of contaminants in the environment as a means to determine attribution for legal purposes. Although this definition still holds true, in environmental microbiology, metagenomics is increasingly being used, and the term “microbial forensics” is now used to include the investigation and sources of microorganisms and their genes that could also be used for attribution purposes. The former involves field analytical studies and both data interpretation and modeling connected with the attribution of pollution events to their causes. Microorganisms thrive in contaminated environments and can utilize a wide spectrum of contaminants as nutrients and energy sources. Therefore, the microbial community composition and structure is an exquisite indicator of environmental changes and can be used in forensic studies to trace sources of contamination and reconstruct events, even from a distant past. The latter includes the meta analyses of available data vis-à-vis genes in the environment. Metagenomics has opened up an incredible manner of obtaining useful data, in which sequencing allows us to find millions of genes present in a sample that can be used to reconstruct, e.g., certain parts of a crime scene, to attribute contamination to a specific source, or even to determine cause of death or the diets of ancient ethnic groups.

Environmental microbial forensics and statistics are not always happy bedfellows. Statistical calculations are sometimes inappropriately applied, which can either give the appearance of support for a particular conclusion or fail to support an innately obvious conclusion. This is not due to any intentional deceptiveness by the scientists involved but is a reflection of issues related to dealing with sample sizes, the methodologies involved, and the difficulty of communicating uncertainties.

In a world dominated by microbes, our ancestors evolved alongside an outstandingly diverse ancient microbiota (see Fig. 1 , Table 1 ). Symbiotic relationships between animals, microbes, and viruses have been observed as far back in animal evolutionary history as when hydras made their appearance, and these interactions currently span across all types of life forms ( 1 – 3 ). It’s clear that these ancient relationships have shaped the evolution of both the host and symbionts, although the selective pressures governing these processes remain uncertain ( 4 – 6 ).

Nucleic acids and modern sequencing analyses are the pivot for research in microbiology, as they provide insights into microbial gene pools and essential cellular processes. Particularly, the ability to isolate and sequence ancient DNA (aDNA) is of increasing importance, as it demonstrates gene evolution and also allows us to peek into the possible mechanisms for DNA preservation. aDNA studies have raised both fascination and skepticism, yet information yielded by such studies has served as the basis to understand the origins and evolution of animal and human groups ( 2 – 4 ), culture ( 5 , 6 ), diet ( 7 ), and ancient diseases ( 8 ).

Food production practice has changed substantially in the last few decades in the United States. The number of small farms has decreased, giving way to larger operations. For example, according to U.S. Department of Agriculture (USDA) data, the number of dairy farms with dairy cows fell from 648,000 operations in 1970 to 75,000 in 2006 ( 1 ). While the numbers of farms decreased, the size grew, with herd size rising from 19 to 120 cows per farm in those years. Centralization of food production is becoming a common practice in the United States for many food commodities. In addition, global food trade is also changing the landscape of food production and distribution patterns. With global income level on the rise, there is a growing consumer demand for a variety of food. Facilitated by improved transportation networks, global food trade is expected to increase. Demand for minimally processed food has also been trending up. Minimally processed food is often characterized by high water activity and mild acidic to neutral pH and is not subjected to high-heat treatment. This includes ready-to-eat food, fruits, and vegetables. As a result, a different set of food safety issues emerges: for example, tracking implicated food ingredients in a widely distributed network is increasingly difficult, new foodborne pathogens are discovered from imported food, and common foodborne pathogens are associated with new niches of food products. With new issues and challenges, the regulatory agencies responsible for protecting our food supply have been working hard to ensure public health.

The first known waterborne pathogen was Vibrio cholerae, the culprit in the London cholera epidemic of 1854 ( 1 ). The discovery that disease could be transmitted from feces to humans via drinking water led to the realization that preventing fecal pollution of water could protect public health, leading to the search for a readily culturable bacteria that was always found at high levels in sewage. Klebsiella and Escherichia coli (first termed Bacillus coli) were identified as indicators of sewage in the 1880s ( 2 – 4 ). In cities teeming with open sewers that discharged directly to water bodies, these intestinal “coliform” bacteria served the purpose of detecting fecal contamination in drinking water and alerting authorities of public health risk. Among the first formal regulations for microbial drinking water quality was the U.S. Public Health Service total coliform standard of 1914 ( 5 ). Among the first regulations for discharges to ambient (surface) water quality was the Federal Water Pollution Control Act, in 1948, which was amended in 1972 to the Clean Water Act. We now understand that fecal or sewage pollution of water by human or animal sources may spread microbial pathogens such as Cryptosporidium parvum, Giardia lamblia, Salmonella spp., E. coli 0157:H7, Campylobacter spp., Shigella spp., and viruses such as hepatitis A, rotaviruses, adenoviruses, enteroviruses, and noroviruses ( 6 – 8 ).

The term “soil forensics” was first coined about 40 years ago, and it may be defined as the in situ study of soil as part of criminal investigations. It includes the study of soil as a material transferred on suspects, victims or objects associated with a crime, with the objective of finding suspects and resolve the crime ( 1 ). Although the term was introduced relatively recently, the examination of sediment for forensic investigations can be traced to the middle 1800s, with Christian Ehrenberg. Ehrenberg analyzed sand to solve the disappearance of coins, ultimately resulting in the resolution of the mystery. An increased interest towards soil forensics was noted with the release of Arthur Conan Doyle stories, but it was Oscar Heinrich and Edmond Locard who proposed the use of soil and sediment in the field of forensic science ( 2 ).

If forensic science is any science used for the purposes of the law (as defined by the American Academy of Forensic Sciences) or for solving a crime, then forensic phytopathology is the application of plant pathology to purposes of the law. In other words, it is the interface between plant pathology and civil and criminal legal affairs. It is a largely unexplored field and is still developing.

Bioterrorism is currently defined as the use of biological agents or pathogens (viruses, bacteria, fungi, parasites, and toxins) for the purpose of attacking and creating havoc in a population considered an enemy. Many of these agents can be isolated naturally from the environment or have been genetically manipulated to become more pathogenic. Additionally, many of these agents of bioterrorism have to be “weaponized” by mixing them with certain chemicals to make them more lethal. The biological agents can then either be used as part of munitions or can be disseminated via air, water, soil, or even food.

The development of paleomicrobiology, with the detection, identification, and characterization of bacteria, viruses, and parasites in ancient remains, is revolutionizing our knowledge of microbial evolution in human history and is laying the foundations for new paleopathological research. Thousands of skeletal individuals, silent for macroscopic analysis, are becoming extremely talkative from a paleopathological perspective. Different techniques can be used in paleomicrobiology, such as light or electron microscopy or immunodetection, but major results have been obtained by PCR-based molecular techniques ( 1 , 2 ) and, in the past few years, mainly by metagenomics approaches, for example, high-throughput sequencing and microarrays ( 3 ).

Microbes are phylogenetically diverse and are comprised of viruses, archaea, bacteria, fungi, protozoa, microalgae, and microscopic metazoa. These organisms are invisible to the naked eye, but all contain unique genome DNA sequences (or RNA in the case of some viruses). The genomes of microbes range in size from ∼13 kb for the smallest RNA viruses and up to several hundred megabases in the case of microscopic planarians. Thus, each genome contains a large amount of sequence information that may allow the classification and characterization of these microbes, which often have few morphological characters to allow them to be distinguished. We are now living in an era whereby complete genome sequences can be generated by individual researchers and may be analyzed using computational techniques, revealing a range of insights into the lifestyle, phylogenetic origin, degree of genetic modification, and pathogenic mechanism of the microbes concerned. Such comparison of the complete genome sequences of microbes is termed comparative microbial genomics.

Microbiome analysis of environmental samples may represent the next frontier in environmental microbial forensics. The microbiome, defined as the sum total of all the genetic material present in a sample, contains evidence of the microbial communities in the sample at the time of collection. As such, it contains clues to past environmental events until the time the sample was collected and processed. This attribute makes the analysis of microbiomes extremely important in identifying and demonstrating the occurrence of an environmental event, be it bioterrorism or a petroleum spill.