For the past four years, scientists around the world have descended on subway stations from Buenos Aires to Beijing to swab a variety of surfaces.
Working their way from the ticket machines at the entrance, to handrails and elevator buttons, to the benches on the platforms, they collect microbiome samples that passengers have left behind to learn what secrets may be revealed.
You may have already heard something about the microbiome. Dove has even promoted their newest body wash as being "microbiome friendly". But what does that mean? What is the microbiome?
The human body is made up of more than just cells. As Gabriella Mason-Buck, a PhD student at Kings College in London stated at the 30th International Symposium on Human Identification, humans are, in fact, “walking petri dishes”. The newest studies reveal that we may have as much as three times more bacterial cells than human cells in our body!
Simply put, the microbiome constitutes all the non-human organisms that are found within the human body and on the skin, including bacteria and viruses. Some of these components are helpful, and others may cause harm.
As we continue to look at the world through a smaller lens, scientists like Gabriella have begun to wonder if the composition of the microbiome has implications for forensic science. Can we learn more about a perpetrator by analyzing all that they leave behind and not just their DNA?
In her presentation, Gabriella described the ways in which forensic metagenomics can assist in solving crimes:
Bioterrorism/Biocrime
Seven days after the terrorist attacks of September 11, 2001, anonymous letters containing deadly anthrax spores were mailed to media companies and congressional offices. In the following months, these letters were responsible for the deaths of five individuals and the infection of 17 others. When microbial forensics revealed that the anthrax used in the letters originated from a particular sample in Bruce Ivins’ lab, he was arrested for the crimes.
Postmortem Interval (PMI)
The National Institute of Justice (NIJ) has funded several projects focused on the forensic applications of microbiome analysis. One focus involves the necrobiome, the community of organisms found on or around decomposing remains. These microbes could be used as an indicator of PMI when investigating human remains. Recent research published in PLOS ONE examined the bacterial communities found in human ears and noses after death and how they changed over time.
The researchers were interested in developing an algorithm using the data they collected to estimate of time of death. Using the full data set of nose and ear bacterial DNA sequences, Johnson et al. were able to develop a model through computer learning from an algorithm that can identify time of death plus or minus 55 acumulated degree days (ADD) or about two days in the Tennessee summer.
Human Identification
Also, at ISHI 30, Allison Sherier, a student ambassador from the University of North Texas Health Science Center, presented her PhD thesis work, in which she hopes to use the human skin microbiome as an alternative source of DNA to identify evidence from crime scenes.
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Cause of Death
Forensic metagenomics can help identify poisons or other substances ingested into the body.
Animal Identification
When it comes to studying the microbiome, Gabriella mused, “We’re not just looking at microbes or viruses, but all of the DNA. Given that, can [analysts] work out whether or not an individual [suspect] had an animal, like a dog?”
Body Fluid and Body Site ID
In a paper published last year, Akos Dobay et al., of the Zurich Institute of Forensic Medicine, examined whether using bacterial markers would be an appropriate approach for forensic body fluid and tissue identification. He stated, “Bacteria are not only ubiquitous throughout the human body, but also, bacterial community structures are distinct across body sites.” Their findings suggest that bacterial markers can be used in the identification of saliva, skin, peripheral blood, and semen samples, but the microbial signatures of vaginal and menstrual samples are too similar to differentiate from one another.
Geolocation
Is it possible to determine where in the world, or within a city, a sample has come from?
The MetaSUB Project
Approximately 55% of the world’s population is centralized in urban areas. Each day, millions of people around the world commute by mass transit. In large cities and small, busses, trains, subway systems, and more, shuttle passengers from one location to another. Studies have suggested that urban environments, and mass transit systems in particular, have geospatially unique metagenomic profiles.
The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium has an interesting origin. Dr. Christopher Mason of the Weill Cornell Medical School was traveling on the New York City subway with his daughter, when she licked the subway pole. While some of us may have had a different reaction, Dr. Mason’s was to wonder what exactly was on the pole that she’d just licked. Alongside co-founder Ebrahim Afshinnekoo, who serves as the Director of Metagenomics, and with funding assistance from the Bill and Melinda Gates Foundation, the consortium was created with a goal of examining the unique metagenomic profiles recovered from mass transit systems.
In total, there are currently 61 cities across the globe actively involved in the consortium, with numerous scientists participating. They have collected hundreds of samples from turnstiles, emergency exits, ticket kiosks, benches, handrails, garbage cans, elevators and more, in each city.
Gabriella Mason-Buck participates in the London chapter, where she and her colleagues sampled 272 different stations within the city. She describes her experience in the video below.
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After the samples are collected, they are brought back to the lab where DNA extraction is performed. Next, a DNA sequencing library is prepared and results are gathered through massively parallel sequencing. After a bioinformatic analysis is performed, the data are presented in geospatial metagenomic and forensic genetic maps like the one below. These maps will help researchers identify and track antimicrobial resistance markers (AMRs) in urban environments and will assist in the identification of novel biosynthetic gene clusters (BGCs) for drug discovery.
An example of the maps created by the MetaSUB consortium. Additional maps and details can be viewed on their website: http://metasub.org/
What Have We Learned?
In her presentation, Gabriella said as of 2019 (when there were 58 cities represented), 3,741 samples had been collected. With a 91.4% prediction accuracy of sample to city, 10,928 novel predicted viral species and 4,424 taxa have been identified. Taxonomic diversity was found to decline as distance from the equator increased, and the samples coming from Europe and Africa were the easiest to differentiate. Most of these taxa came from human skin and airways, which is to be expected from touch samples like these. The most common species found is an acne causing bacterium, Cutibacterium acnes. Gabriella stressed that new taxa are being identified every day with more information being obtained about microbial resistance.
So far, 61 core species have been identified and exist within 95% or more of the samples (see a visual from Gabriella’s presentation below). These core species all fall into one of the following phyla:
Actinobacteria
This phylum contains characteristics of both bacteria and fungi and are widely distributed in both terrestrial and aquatic ecosystems (found mainly in the soil). Their functions include the decomposition of complex mixtures of polymers in dead plants, animals, and fungal materials.
Firmicutes
This phylum makes up the largest portion of both the mouse and human gut microbiome. As part of the gut flora, it is involved in energy resorption, and its activity is potentially related to the development of diabetes and obesity.
Proteobacteria
There is such great diversity found within this phylum that it was named after the Greek God of the sea, Proteus, who was able to assume many different shapes. It includes a wide variety of pathogenic genera, such as Escherichia, Salmonella, Vibrio, Helicobacter, Yersinia, and Legionellales. Others are non-parasitic and include many of the bacteria responsible for nitrogen fixation.
In addition to the core species, 1,145 sub-core species were also identified and found to exist in more than 70% of samples. Additionally, 2,466 peripheral species have been identified and exist in less than 25% of samples. Some of these peripheral species may be city-specific. Gabriella also says that approximately 50% of the data remains unclassified, which isn’t surprising as scientists are reliant on databases that may not be all-encompassing or updated regularly.
Visual created by Gabriella Mason-Buck depicting information about the species found.
Challenges and Considerations
Permission from Authorities
In the video interview above, Gabriella mentioned that one of the challenges to obtaining these data started with the sample collection process. Before swabs could be taken, it was necessary for the scientists to explain what they were doing to authorities, so as not to scare the public. That led to many interesting questions and public engagement.
Methodology
Each lab used slightly different methods when obtaining, collecting, and processing the samples, which led to obtaining slightly different data. Gabriella stated in her presentation that this can be an issue when using the data for forensic purposes, when low-level taxa are examined.
Replicates
How many replicate samples should be taken? At the time of her presentation, only one sample was taken from each location. Gabriella suggested that her work showed a need to take at least triplicate samples from each location, because she is unsure if small millimeter or centimeter differences would lead to different taxa presenting themselves.
The "Kitome"
Gabriella coined this term based on the background material or background contamination that showed in her data. She said this contamination could be due to the reagents being used. While free of DNA, they are not free of microbes, so is it possible that microbes are being introduced in the analysis? Additional microbes could be present in the water or consumables used during analysis.
Gabriella stressed that it is important to have both positive and negative controls, mainly for the extraction stage, because it appears this is when the background taxa are being introduced.
Data Analysis
In addition to variation in the methodologies, analysis tools and software also differs between labs. A lab may achieve different results depending on which database their software is linked to. Gabriella also shared concerns about computational capacity. She remarked that the amount of data created is very large and cloud-based storage could be difficult for forensic labs. If this technology were to be implemented for forensic use, questions remain about how these data will be stored and which parts will need to be made available for future reference.
Tracking COVID-19
This year, the rapid spread of the COVID-19 virus has led some cities to move up their sampling day. Beginning in March, scientists in pilot cities in the United States, Europe, Asia, and South America began swabbing their local transit systems. So far, they have collected 1,632 samples, but they anticipate thousands of additional samples to be added in the coming weeks. Though they have not yet analyzed all of the samples collected, Dr. Chris Mason says they’ve not yet found signs of COVID-19 in the New York subway system. In an article with Genetic Engineering & Biotechnology News, he cautions that though no samples have included the virus to date, they have only been swabbing since March 16th and only a small number of samples has been collected.
In this time of uncertainty, it is incredibly difficult to look into the future. Dr. Mason says until recently, the cost of doing routine environmental surveillance has been too high to consider whether we may be able to partition cities into “green zones” which would be safe for people and “red zones” that would be unsafe. Now, current costs may make this a possibility. Given the impact the pandemic has had on large urban areas, it is also necessary to consider the cost of not doing it.