I’m Dr. Tony Kingston, a research scientist eking out a living in the suburbs of Boston. I recently left academia to work in in industry, but I miss teaching so I decided to start writing articles on interesting discoveries in the world of microbiology. My goal is to uncover subjects that are unusual, unique, and might one day have a major impact on our daily lives.

The Necrobiome: What is it and why is it important?

This review is going to highlight necrobiome research, a morbidly... cool niche in the massively popular microbiome field. The microbiome is the collection of microbial organisms (bacteria, fungi, viruses etc.) that live in and around our bodies.

The necrobiome, however, is the collection of microbes that live in and around a corpse. You see, when an animal dies, the microbes living within them continue to live on. This microbial community changes over time as nutrients are consumed and environmental microbes invade. Necrobiome researchers aim to track these changes and hopefully find some useful discoveries from their work.

The necrobiome has garnered interest as a potential tool for forensic analysis. Researchers hope to one day be able to predict what the microbial composition of a corpse will be over time based on environmental conditions. With this knowledge, forensic scientists can analyze the necrobiome sample of a murder victim and be able to discover useful clues concerning the the time and location of death [1].

In other words, 10-20 years from now we can expect the hackneyed conclusion of a CSI episode to hinge on the necrobiome.

Pictured: Not real scientists (source)

The concept of analyzing organisms on a corpse isn’t new. In the past, criminal investigations tracked insects on corpses to determine the time and location of death [2]. However, insects can only provide a limited amount of information.

Microbes, with their endless variety and ubiquity, have the potential to fill in the gaps left by forensic entymology. Necrobiome research has seen rapid growth in recent years as the DNA sequencing technology capable of analyzing microbes en masse becomes cheaper and easier. Despite this growth, the field is relatively new and it’s still has a long way to go before it can actually be used in a criminal investigation. Nevertheless, some of the findings that have been made hold great promise.

How do we study the necrobiome?

Studying the necrobiome is no easy feat! It requires both the messy job of handling corpses and the challenging task of identifying bacteria from an environmental sample. For lab groups working in this field, experiments usually involve three main stages: creation, collection, and analysis.

Creation

Researchers first need to be able to create necrobiome samples in a controlled setting. Unfortunately, that means they need an actual corpse and a place to put it undisturbed. Some labs focus on model animals. One group from Northumbria University in the United Kingdom used still-born piglets [3]. They buried them in metal cages to prevent scavenging and let them decay for 9 months. It seems kind of cruel in a way, but I eat the damn things so who am I to judge.

Other research groups use actual human corpses. I guess they want their necrobiomes to be 100% genuine. This is usually accomplished at a body farm, an enclosed outdoor research facility designed to study decomposition.

This is the least graphic image I could find of an actual body farm. Sorry if anyone is grossed out. The whole point of these body farms is to allow scientist to study human cadavers as they decompose in a natural environment.

Collection

Once a body has begun decomposing, samples need to be collected over time. Since different parts of the human body have different microbiomes, researchers will try to collect samples from the same part of the body. However they have to be careful to minimize disturbing the natural necrobiome while taking samples. I’m no expert on how these samples are taken, so here is a rather dry quote from a recent paper to give you an idea

Immediately before placement, the cadaver’s gastrointestinal tract was sampled: the caecum was swabbed via a small incision, which was re-sealed with standard duct tape as previously described [4].

The whole process feels a bit unceremonious, but I can’t argue with the effectiveness of duct tape.

Many experiments focus on the collection of soil beneath or above the corpse as well. This is because the nutrients from the body leach into the soil causing bacterial blooms that can be tracked over time. In addition, researchers have to measure environmental factors that can affect bacterial growth, such as temperature and humidity. Samples are usually frozen to preserve them until DNA analysis.

Analysis

Once researchers collect their necrobiome samples, they have to identify the bacteria within those samples. This process requires a high level of training and the latest in DNA sequencing technology. In short, scientists first isolate all the bacteria in the sample from the soil or tissue [5]. They then extract DNA from these organisms and amplify their 16s rRNA genes. This gene is found in all bacteria, and its sequence is commonly used to identify an organism. The amplified 16s rRNA genes are sequenced, and a bioinformatician analyzes the results to determine what bacteria were in the original sample.

So what does the necrobiome look like?

The graph below is from a 2015 paper analyzing soil necrobiome samples underneath human cadavers at various stages of decomposition [4].

I don’t have the desire to describe details of each decomposition stage, so use your imagination for terms like “bloat” or “advanced II.” The authors also include a control sample (soil without any nearby bodies), and a sample of the human gut microbiome for easy reference. The control, gut, and various decomposition stages are each represented by a single column of a bar graph with multiple colored layers. Each layer refers to a different type of bacteria (classified by phyla).

As you can see, the bacterial composition of the soil changes as the bodies decay. Most notably, the Acidobacteria disappear over time as the Firmicutes become prevalent. The authors hypothesized that these changes were due to the nutrients released by the body during decomposition. Acidobacteria tend to prefer soil with low nutrient availability and were probably overgrown by species that proliferated. The Firmicutes were presumably released from the human gut into the nutrient rich soil and flourished.

Since human-derived firmicutes bloomed underneath cadavers, the authors were surprised that Bacteriodes, another major component of the human gut microbiome, didn’t seem to flourish as well. Thus, they went back to the sequencing data and focused on the different Bacteriodes species they identified. They found that if the separated human-associated Bacteriodes from non-human associated Bacteriodes, they were able to see a change in soil samples over the different stages of decomposition.

This chart shows the abundance of human-associated Bacteroides in soils below decomposing cadavers. It shows that human-associated Bacteriodes do invade the soil underneath cadavers and replace the non-human associated Bacteriodes over time. However, these human-associated species disappear shortly after the body is removed.

How can we use this information?

Papers like the one described above are a small piece of a much larger puzzle. Scientists ultimately want to know exactly how a necrobiome develops in and around a human corpse over time.

Here we see this development, but only for a specific climate, temperature, and setting. We can imagine that one day, a detective might be able to identify human-associated Bacteriodes in a soil sample to confirm that a body was recently in a location. However, the reproducibility of these results needs to be validated, and this kind of analysis would need to be much more practical to be a viable forensic tool.

With any luck, this field will continue to grow. Labs will continue to publish on this subject and further complete the puzzle of the human necrobiome. As DNA sequencing becomes easier and cheaper over time, forensic labs may be able to incorporate necrobiome analysis into their investigations. Thus, an invaluable tool for forensic investigations (and terrible CSI episodes) will be born.

Pictured: Again, not real scientists (source)

References

[1] Pechal JL, Crippen TL, Benbow ME, Tarone AM, Dowd S, Tomberlin JK. The potential use of bacterial community succession in forensics as described by high throughput metagenomic sequencing. Int J Legal Med. 2014 Jan;128(1):193-205. doi: 10.1007/s00414-013-0872-1. Epub 2013 Jun 10.

[2] Joseph I, Mathew DG, Sathyan P, Vargheese G. The use of insects in forensic investigations: An overview on the scope of forensic entomology. J Forensic Dent Sci. 2011 Jul;3(2):89-91. doi: 10.4103/0975-1475.92154.

[3] Olakanye AO, Nelson A, Ralebitso-Senior TK. A comparative in situ decomposition study using still born piglets and leaf litter from a deciduous forest. Forensic Sci Int. 2017 Jul;276:85-92. doi: 10.1016/j.forsciint.2017.04.024. Epub 2017 May 6.

[4] Cobaugh KL, Schaeffer SM, DeBruyn JM. PLoS One. 2015 Jun 12;10(6):e0130201. Functional and Structural Succession of Soil Microbial Communities below Decomposing Human Cadavers. doi: 10.1371/journal.pone.0130201. eCollection 2015.

[5] Finley SJ, Benbow EM, Javan GT. Potential applications of soil microbial ecology and next-generation sequencing in criminal investigations. Applied Soil Ecology. 2015 Apr;88:69-78.