The inter-individual variability of the postmortem microbiome has prompted research into whether microbiome analyses may be useful to forensic scientists, who apply scientific methods to collect and examine evidence for criminal investigations. Is it possible for investigators to identify suspects and determine when, where and how someone died based on their death microbiome? Many studies tip the answer in favor of a (highly tentative) yes. Still, there are limitations that must be overcome before postmortem microbiome analyses become an accepted and routine part of forensic investigations.
Death and Decay: The Role of Microbes in Decomposition
Immediately following death, the immune system shuts down. Without the constant surveillance of the host immune response, microbes in the gut begin to proliferate and spread to other organs. This process of microbial migration is relatively quick-one study in mice demonstrated that intestinal bacteria (e.g., Lactobacillus spp., Enterococcus spp. and Escherichia coli) were found in peripheral organs, including the spleen, liver and kidney, as soon as 5 minutes after death.
Over time, cell oxygen levels decrease to promote a depletion of aerobic microbes and proliferation of anaerobic varietiessuch as Clostridium spp., which are thought to break down lipids and complex carbohydrates that make up human tissues. The lack of oxygen also causes tissues to lyse and release a diverse repertoire of nutrients that further support growth of anaerobic bacteria. As they feast, these organisms produce gases that cause the body to swell and, eventually, rupture. Atmospheric oxygen floods the body and, once again, aerobic microbes reign supreme.
Eventually soft tissues are completely degraded, and only bones are left behind. However, bones are not microbe-free-they harbor their own microbial signature. In a recent study, scientists compared bacterial populations on skeletal remains to those in surrounding soils and the human gut. They found that bone-dwelling microbes were distinct from those in the soil and gut, though maintained similarities with each source. The degree of similarity depended on whether a bone rested above or below ground. Bacterial communities on surface bone, for instance, were more like soil communities and had higher abundances of species belonging to the Chloroflexi, Chlamydiae and Deinococcus-Thermus phyla. Buried bones had a similar microbial profile to gut samples (e.g., more anaerobic organisms), perhaps because of lower oxygen concentrations underground. These findings suggest that human and soil-associated bacteria coalesce to create a unique bone microbial profile after death. Moreover, they highlight that environmental conditions, such as how far underground remains are, influence postmortem microbiome composition.
To that end, numerous factors influence each phase of decomposition and the microbes that regulate them. Age, disease, whether a body is outside or inside, temperature and humidity all shape postmortem microbiome structure. As such, there can be considerable variation between individuals' microbiomes in death, just as in life.
The Postmortem Microbial "Fingerprint" and Forensic Science
The use of microbes in forensics is nothing new. Founded in the wake of the 2001 anthrax attacks in Washington, D.C, microbial forensics has become an important discipline within the forensics field. It centers on "applying scientific methods for analyzing evidence from a bioterrorism attack, biocrime, hoax, or inadvertent release of a biological agent or toxin," with attribution (i.e., determining where a biothreat came from, and who is responsible for its distribution) being the "ultimate goal." Microbial forensics has also been applied in investigations of sexual assault and intentional pathogen exposure, among others.
Historically, microbial forensics has focused on individual microbial taxa isolated from pure or homogenous samples. Thanks to advancements in DNA sequencing technologies, like metagenomics, scientists can now analyze complex microbial communities in their entirety, including those associated with deceased individuals and/or scenes of death. This has expanded the list of potential applications for microbes in forensic science.
One application that has gained particular attention is using the postmortem microbiome to estimate the time elapsed since a person has died, known as the postmortem interval (PMI). Forensic investigators estimate PMI based on pathological and physiological indicators, such as wound condition, body temperature and the presence and succession of invertebrate species. Capitalizing on successional variations in postmortem microbiome structure (i.e., the ebb and flow of aerobic and anaerobic bacteria throughout decomposition) may complement traditional PMI techniques.
Several studies in animal models, and with humans, have yielded intriguing results. For example, researchers used computational modeling to successfully estimate the PMI of mice within 3.3 days based on the bacteria on their body and head skin. In another study, scientists collected samples from the oral cavity of mice 24, 144 and 244 hours postmortem. They identified a strong correlation between PMI and the abundance of Gammaproteobacteria and Proteus spp., perhaps pointing to these bacteria as useful PMI indicators.
In the context of human remains, analysis of bacterial consortia on human rib bones predicted PMI with an accuracy of +/- 34 days over 1-9 months after death. The researchers noted that anthropologists give PMI estimates with errors ranging from months to years, highlighting how their techniques narrow this window. How the findings relate to investigations that necessitate shorter timescales is less clear. Still, another group sampled intestinal contents from deceased individuals over 20 days to show that the relative abundance of 2 common gut genera, Lactobacillus and Bacteroides, significantly decreased over time. These results suggest these genera could serve as microbial markers for PMI assessments.
Beyond PMI estimations, the postmortem microbiome has other potential uses. For example, it may be possible to determine where and how an individual died based on the microbes in, on or around the body. Several studies have shown, for instance, that looking for bacteria associated with aquatic environments may be indicators for death by drowning. Investigators may also be able to use the postmortem microbiome to identify a victim and/or suspect. This includes communities on a decedent, as well as those occupying trace evidence at a scene of death. Indeed, when researchers used 16S rRNA amplicon sequencing to examine bacterial populations on objects sampled from 16 scenes of death, the object could be traced to the deceased individual's postmortem skin microbiome 75% of the time, on average. Some objects were more well-suited for microbial "fingerprinting" than others (e.g., medical devices and smoking pipes versus car keys), suggesting the usefulness of these methods may vary on a case-by-case basis.
Limitations and Next Steps
Though promising, there are several factors hindering the routine adoption of postmortem microbiome analyses in forensic investigations. For one, most studies have been small and vary in their experimental methods (e.g., DNA isolation protocol, sequencing platforms and reference databases). This lack of standardization makes interpreting data between studies challenging. It also prevents microbiome analytics from becoming reliable, trustworthy tools within the legal system.
Temporal variations in the postmortem microbiome can also complicate investigations. The composition of our microbiome changes regularly-circadian oscillations in bacterial community structure have been demonstrated in the gut and saliva. Therefore, samples from a scene of death provide a "snapshot" into an individual's microbiome at a given time, but they may not reliably match reference samples. Similarly, samples may contain a mishmash of microbes from various sources (i.e., from a victim, suspect and/or the environment). Determining which microbes belong to whom, and what might be contamination, requires profiling the microbes associated with a particular source, over time and under various conditions-not a small feat. Many crime laboratories do not currently have the resources and expertise for these types of analyses.
As is true for the microbiome field overall, most studies of the postmortem microbiome examine which microbes are present, but not the functional potential of the community. This is critical to understand the succession of microbial species throughout decomposition, and how they regulate the process. For instance, using 16S rRNA sequencing coupled with metabolomics, researchers determined that Psuedomonas spp. and microbial phosphate solubilization likely play a role in degradation of human bone samples. By combining sequencing methods with those that probe mechanisms of degradation, forensic scientists could better understand why certain microbes are present in specific circumstances of death.
Additionally, most studies to date have focused on bacterial members of the microbiome-a small sampling of the microbes present within the community. Honing in on populations of other organisms, like fungi, would paint a more comprehensive picture of the postmortem microbial populations.
The relationship between forensic science and the postmortem microbiome is young; its growth depends on maturation and innovation of the technologies used to investigate complex microbial communities. Yet, in time, microbiome analyses could become valuable, complementary tools to established investigative techniques.
Over the past decade, the microbiome has become a hot topic in the microbial sciences. But where did the concept come from? Its history goes back further than you think.
