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Column: CSI: Everywhere – The Rise of Forensic Microbiology

CSI: Everywhere – The Rise of Forensic Microbiology

In the sixth installment of ‘Just 10% Human,’ Daniel Sprockett explains how your unique microbiome, like your fingerprint, might one day be used for forensic investigation. As usual, there’s a bunch of other crazy facts contained in there as well. Read on for your shot of science for the day.

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Last week, I discussed some of the ways that microbes residing on our bodies continually shape and are shaped by the other microbial ecosystems that they come in contact with. These interactions can have profound effects on our health and behaviour, but can also reveal patterns in our world normally hidden from our view. Forensic microbiologists are now able to leverage these microbial interactions to answer questions about the assignment of blame or the establishment of innocence in a legal context. The tools currently being developed in this emerging field could one day be added to the analytical toolbox that investigators use to solve crimes.

For example, we know that in addition to our actual fingerprints, we also leave bacterial imprints on objects we touch and interact with. Some estimate that we shed over 15 million bacteria every single hour. In theory, this bacterial signature could be used to place a suspect at the scene of a crime, but only if that signature has two essential characteristics.

First, in order to discriminate between multiple suspects, a person’s microbial signature must be highly individualistic. If microbes found at the scene of a crime have a high probability of coming from two or more people, then they would be of no more use to forensic scientists than a shoe imprint or an abandoned article of clothing. Their presence is consistent with the person being at the scene of the crime, but not sufficient for prosecution.

As it turns out, most studies of the human microbiome has found it to be highly specific to the individual, with samples from the same person being far more similar to each other than samples taken from different people. But this only holds true if you look at certain components of the microbial community. While most of our body-associated microbes are pulled from a limited pool, we also harbor small amounts of unique bacteria that we’ve acquired over the course of our own particular history of environmental exposures. As a result, the microbial differences that allow us to discriminate between individuals are only visible if we use highly sensitive techniques that can detect rare microbes that are present at very low abundances.

The second characteristic necessary for the microbiome to be useful in forensic contexts is that it must be stable and resilient to change. If your microbial signature is unique but can change abruptly, you can’t say with any certainty that what it looked like at the time of the crime or event in question.

In this case, the literature is far more nuanced. A study published just last week in the journal Science showed that around 60% of microbial strains in the gut stayed stable over the course of five years. They also estimated that this could extend over decades, but studies confirming this haven’t been completed yet. In general, most studies find that samples of microbial communities taken closer in time are more similar to each other. In other words, the microbiome changes, but it changes relatively slowly. In normal, healthy adults. At some body sites. As long as they don’t take any antibiotics.

But instead of theorizing, a group of researchers at the University of Colorado, Boulder did what scientists do best: they put it to the test. As part of a proof-of-concept study, they devised a scheme where they would sample the microbes on hands of volunteers, as well as objects that they commonly touch. They swabbed keyboards used by three individuals, as well as their fingertips, and found that they were able to match all three pairs accurately.

They took this one step further, and swabbed the computer mice of nine volunteers, but instead compared they microbial profile on the mice to a database of microbial profiles from over 270 hands, including the hands of their owners. In all nine cases, the microbial profiles of the computer mice most closely matched the hands of their owners, allowing researchers to correctly identify each pair.

However, this technique only works for objects that are handled regularly. When they tried to isolate microbes from new objects that had only been handled once, they couldn’t isolate enough bacterial DNA for accurate microbial profiling.

In any case, one might argue that it would be better to use the more common, validated method of identifying individuals via their genomic profile, and they’d probably be right. However, there are situations where this or a related approach could prove to be a valuable analytical technique. For example, there are situations where microbial DNA is available when human DNA is sparse or highly degraded, such as in cases where clothing has been left in hot or humid crime scenes. Bacteria thrive in these types of environments, but lone human cells tend to break down. One such investigation used a different microbial profiling technique to match footwear to their owners.

We also know that twins harbor distinct microbial communities. Forensic microbiome analysis could one day to enable investigators to distinguish between genetically identical individuals.

Another potentially powerful application of this technique is in situations where identifying specific types of contact are important, especially in the case of rape or sexual abuse. Different regions of the body harbor distinct combinations of microbes, meaning that the microbial communities found on skin are very different than those found in the oral cavity or genital regions. Human DNA analysis can only identify if contact happened between two people, but microbiome analysis may be able to establish which specific types of contact occurred, and which body parts were involved. A few studies have begun identifying microbial signatures that are capable of reliably indicating their body site of origin, but these types of forensic tests are likely still years away. Similar studies have begun characterizing microbial signatures of water isolated from inside the lungs of bodies recovered from lakes and rivers, which could tell investigators if drowning was the cause of death, or if the body was dumped there afterwards.

In the end, the dynamics of human-associated microbial communities may turn out to be not suitably stable or predictable for needs of the modern justice system. Sophisticated criminals may be able to subvert microbiome analysis with antibiotics, or by taking a bleach bath after perpetrating a crime in an attempt to alter their unique microbial signature. But there are other microbial patterns that can be useful in investigating crimes, and it has its roots in the ecological concept of succession.

Succession is simply the pattern of species changes in ecological communities over time. A tree falling in a forest alters the local environment, and as a result, the ecosystem will change in a predicable way. If you observe that the fallen tree has been replaced with thick bushes, you know that more time has elapsed since the tree fell than if there were only grasses in its place. That is because grasses and weedy species are fast-growing, early colonizers, whereas bushes take more time to become established. Over time, another tree will shoot up above the bushes, blot out their sunlight, and the forest community will erase any evidence that the original tree had ever stood.

Forensic entomology (the study of insects) also relies on ecological succession, and has been around for over 100 years. However, it has only gained traction as an important scientific discipline in the last several decades. Its premise is that you can use the necrophagous (dead-eating) insects found on decaying biological matter to draw inferences about the minimum time since death. This minimum Post-Mortem Interval can be estimated fairly precisely because many insects, especially beetles and flies, have life cycles that progress through highly predictable stages of development, beginning in the larval stage. These are the white, wriggling maggots you see in feces or on dead animals. Larvae progress through several growth stages called “instars” that are separated by molting, or the shedding of their exoskeleton. Each instar takes a very specific amount of time, so if a forensic entomologist can identify both the species of insect and its developmental stage, then they can very precisely estimate the minimal amount of time that has passed since the death occurred. If several weeks have passed, perhaps several waves of insects have already pupated and flown away, but forensic entomologists have methods for quantifying this, as well. “Weedy” insect species tend to arrive first and alter the local environment –changing it in ways that allow other types of insect species flourish.

Blowflies are very useful to forensic entomologists when the body is fresh, because they have very sensitive sense of smell and can quickly find bodies that are several kilometers away. But even in the best of circumstances, there is always a lag between the actual time of death and the time it takes insects to find the body and lay eggs. Sometimes this lag can be difficult to estimate, especially in variable weather conditions, or if the body is found partially covered or submerged.

This is why investigators have turned to the human microbiome. Microbes are already present on the body, so there is no time lag following death. The immune system is in constant communication with microbes, so when those signals stop after death, the microbiome rapidly begins to shift states. Microbes begin colonizing areas of the body that the immune system normally cordons off. Since oxygen rich blood is no longer being pumped into tissues, microbes that thrive in low oxygen environments begin to bloom. Some microbes produce enzymes and/or gasses that make the local environment more habitable for other microbes. These stages occur with the regularity of a ticking clock, and scientists are currently working hard to identify which changes to specific body regions are most predictable and useful to forensic microbiologists for determining the time of death.

One small, early study of decaying pig carcasses (a common stand-in for humans) showed that bacterial taxa became more diverse over the course of decay, and that certain groups of microbes were significantly enriched at certain time points. However, many more validation studies are needed to establish how these changes are affected by factors like temperature and humidity, as well as characteristics of the victim.

Investigators have also begun using highly sensitive microbial identification methods to detect changes in the soil microbiome caused by the disposition of corpses in clandestine graves. Such techniques have the potential to identify areas where bodies have been buried, giving police and investigators a much more targeted approach for locating missing remains.

When most people hear the words “forensic microbiology,” their mind usually turns to the latest anthrax scare. But as you can see, the tools of modern microbiology have far more applications than bioterrorism. This burgeoning discipline has a long way to go before its methods are robust enough to stand up to cross examination in a court of law, but their promise is tantalizing. DNA sequencing gets cheaper and easier every year, and statistical modeling of microbial communities continues to become increasingly sophisticated. I imagine it won’t be long before you begin seeing these techniques applied in high-profile criminal cases, or at the very least, on the an episode of CSI: Crime Scene Investigation.

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Daniel Sprockett is a researcher at the Case Western Reserve University School of Medicine in Cleveland, Ohio. He currently resides in Double Bay with his wife, Andrea, while she completes a Master’s of International Public Health at the University of Sydney. Dan will return to the United States in September, when he begins his PhD in Microbiology and Immunology at Stanford University.

@DanielSprockett
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