Perhaps the most famous founding doctrine of forensic science was first stated early in the 20th century by French doctor Edmond Locard, a Sherlock Holmes afficionado who realized that physical evidence would be left at virtually every crime scene and would “bear mute witness” against the person committing the crime. The evidence was certainly there, Locard said in developing his Exchange Principle. “Only human failure to find it, study and understand it, can diminish its value.”
For decades investigators have exploited Locard’s Exchange Principle, finding the forensic value of everything from footprints and tool marks to fingerprints and blood spatter. The forensic tools used in the hunt for evidence have improved over the decades as advanced microscopy, spectroscopy, genetic analysis, and rapid forensic database searches have become common.
But one significant advance in modern forensic science came in 2001, with the anthrax attacks in Florida, New York, and Washington, DC that began one week after the September 11 terrorist attacks. Letters containing deadly bacterial spores that caused anthrax were sent to several news media offices and two US senators. Five people died and 17 were infected, triggering one of the largest FBI investigations in history.
That investigation marked the birth of microbial forensics, according to Bruce Budowle, a geneticist with the University of North Texas Health Science Center who was with the FBI at the time. “There was an attack, there was forensic evidence, it was microbial evidence, and we were woefully unprepared for it,” Budowle said. “The technology at the time was limited, so we created the field of microbial forensics. It was dedicated to the analysis of microbial evidence related to a bioterrorist act or a bio-crime. It was very focused on using microbes or their by-products as a weapon.”
The FBI sought assistance from The Institute of Genome Research to sequence the spores for about $250,000 per sample, he said, and the particular strain of the bacteria was, years later, traced to a scientist working in a government lab in Maryland. The scientist committed suicide in 2008 after he became a suspect. A National Academy of Sciences report in 2011 confirmed that the anthrax was the Ames strain, but didn’t conclusively tie it to the scientist’s lab. The FBI said the microbial profile of the spores used in the attacks, plus other evidence, led them to conclude the suspect scientist had indeed been the individual who committed the crime.
With the public focus on the anthrax attacks themselves, few people noticed that the horrific events were creating an entire new field of forensics, one that today has evolved to a point where microbes are on the verge of becoming a major tool not just in bioterrorism, but in forensic investigations on a broad scale.
The advent of massively parallel sequencing and other high throughput technologies promulgated by efforts from the Human Genome Project “opened up microbial forensics to a whole host of other applications,” Budowle said. Those applications go beyond human DNA evidence, expanding the trace evidence humans can leave behind at crime scenes to bacteria, fungi, viruses, and other microbial evidence. The growth of microbial forensics led Budowle to run three NIJ-supported webinars in 2016 on the basics and expansion of the field, as well as conduct NIJ-funded research, completed in 2019, on skin microbes that could be used for human identification.
As with many research areas in science, the microbial forensics techniques developed and implemented over the past 20 years are applied beyond criminal investigations and are the same technologies now being used to identify and limit the spread of the SARS-CoV-2 virus that causes COVID-19 disease. Researchers are finding the techniques that are gaining a foothold in criminal forensics are also useful in detecting the SARS-COV-2 virus.
Rob Knight, Director of the Center for Microbiome Innovation at the University of California, San Diego, conducted early NIJ-supported microbiome research by evaluating the potential of using microbial cells on human skin as trace evidence for criminal investigations. That and similar research has continued, he said, but over the past months he has turned some of the lessons learned in microbial forensics to finding SARS-CoV-2 among the UCSD students.
In addition to a program that directly tests about a thousand people a day for COVID-19 on the UCSD campus, Knight said in December 2020, he has been monitoring the wastewater coming out of dormitories. “We see the viral signal in wastewater about a week before we see clinical signs,” he said. In a small wastewater pilot project, he was able to identify one person with COVID-19 in a dorm of about 500 students. “We were able to figure out where the signal was coming from before anyone had symptoms,” he said. A screening of the dorm found the infected student.
The swabbing techniques he developed while working on an NIJ-supported project on using the microbiome from human skin as trace evidence have proved useful in swabbing classroom floors to find SARS-CoV-2 evidence. “It’s about distinction,” Knight said of searching for the SARS-CoV-2 virus among the other microbes in a classroom. After students leave a classroom, Knight’s researchers speculated that SARS-CoV-2 aerosol floating in the room would eventually settle onto the floor.
They determined that if they swabbed the floor at the center of the room, “you could use this to tell if someone infected with the virus had been in the classroom,” he said. That environmental signal of SARS-CoV-2 is then combined with testing of the students who had been in the room to determine which individual has the virus, he said. The process of finding the virus and then linking it back to a specific person “connects right through to the sorts of interests that led us into the NIJ [microbiome] projects,” he said.
The basis of Knight’s NIJ projects, along with the microbiome research of several other NIJ-supported scientists over the past 20 years, is the fact that each human carries a distinct microbial signature, a signature that is shed into the environment and left on objects that are touched. The microbiome was portrayed, unintentionally, by Charles Schultz in 1954 when he created the Pig Pen character in the Peanuts comic strip. Pig Pen is a boy who is perpetually surrounded by a cloud of dirt and dust, carrying it with him wherever he goes. Replace the dirt and dust with bacteria, fungi, and viruses, and Schultz seems a visionary. The number of microbes that make up those clouds are enormous but, as they are invisible to the eye, are hard for most people to comprehend.
University of Chicago surgeon and microbial ecologist Jack Gilbert, who has worked with Knight on NIJ-supported microbiome research, noted in a recent University of Chicago Magazine article that “by the time children learn to walk, they are enveloped, inside and out by a massive, invisible kaleidoscope of microorganisms, 100 trillion or so.” The microbes live in mouths, nasal passages, on the skin, and, predominantly, in the digestive system. Most of them, fortunately, are friendly and do such things as help with digestion, build the immune system, and fight off pathogens.
The Human Microbiome Project, a National Institute of Health-supported consortium of universities and research laboratories that worked from 2007 to 2016, found that the microbial communities living “in association” with a human body include eukaryotes, archaea, bacteria, and viruses. The bacteria alone are typically ten times more prevalent on a body than human cells. Those “non-human” microbes have about a thousand times more genes than are present in the entire human genome, and about one to three percent of a person’s body mass is made up of the microbes. For a 200-pound adult, about two or three pounds comes from microorganisms that aren’t actually the person. For most people that means foreign microbes in and on their body weigh about as much as their brain.
“Some scientists have questioned, ‘Are we human or are we a microbiome?’” said Yong Jin Lee, a microbiologist at Albany State University, Albany, Ga. In his NIJ-supported research, “Surveying the Total Microbiome as Trace Evidence for Forensic Identification,” Lee investigated human traces left on items typically found in an office environment, such as cell and desk phones, keyboards, mice, mugs, pens, and staplers. “We looked at the variability between individuals, that we have different microorganisms from person-to-person,” he said. “We thought we could use that individual variability to identify a person. That was the hypothesis we had when we proposed our research to NIJ.”
The objects at seven individuals’ offices were swabbed using five different swabs made of cotton, rayon, polyester, and other material. After analyzing the microbiomes recovered, which included bacteria, fungi, and viruses, the researchers found that the human traces on touched objects “can be linked to the individuals who touched them and, in turn, serve as trace evidence for forensic identification.”
“We swabbed the objects trying to get some microbial signature, not focusing on the human DNA because whenever you touch an object you don’t necessarily leave human cells,” Lee said. “But we do leave our microorganisms on an object, so we swab to get the microbial DNA.” From that, he said, the researchers identified a microbial signature, or profile, that was unique to the individual who touched the object.
In Budowle’s NIJ-supported research on using the human microbiome for individual identification, he noted that the amount of human DNA deposited by touching an object is often very low, below the detection level of typical DNA analysis technologies. His researchers collected assorted bacterial species, as well as fungi and other microbes, from 51 individuals. The researchers developed skin microbiome genetic profiles, focused primarily on microbes on the hand, for human forensic identification. While the human DNA was often low on the samples, the microbial DNA was significantly higher.
“These studies attributed skin microbiome samples collected from the hand to their respective host with up to 100 percent accuracy,” Budowle wrote in a summary of his 2015 NIJ-supported skin colonies report. “The hand is one of the most forensically-relevant sites, regarding touch DNA samples and as such this finding is significant for the potential use of skin microbiome profiling . . . to assist in criminal investigations, such as robberies, homicides, and sexual assaults.”
A realization that came quickly with investigations into touch DNA, whether human or microbe, is that it is difficult to look much beyond the last person who has touched an object. Knight, in one of his NIJ-supported research projects, had multiple people handle objects and then try to sort out how many had touched it, in what order, and identify them. That research serves as a warning to investigators not to touch anything at a crime scene. “Suppose you find something at a crime scene, and someone inadvertently picks it up,” he said. “Can you figure out who touched it before them? I’m not saying it’s impossible but it’s a lot more challenging because whoever is the last person to handle an object tends to be the one leaving trace evidence on it.”
Knight discovered the problem in one of his early microbiome investigations when he examined microbes found on money. “We figured there would be a lot of interesting microbiology on money, and maybe it would depend on the country that the bills were from and the materials, paper or plastic, that the bills were made of.” What they found were a lot of microbes from the researcher who last handled the money, he said, not the people who had used the bills earlier or the country of origin. That work led to his touch investigations for NIJ and the same “last person” rule applied.
As part of the research, Budowle’s investigators also looked at a DNA phenomenon known as shedding. Some people naturally shed a lot of human DNA, while others shed very little. Most people are assumed to be somewhere in between. Budowle’s work found that there appears to be some slight correlation between the level of human DNA shedding and microbial DNA shedding, something that might be helpful to future investigations.
Beyond matching a microbiome trace to an individual as part of criminal investigations, how revealing can the millions of particles left on a surface be? “There is evidence that suggests the environment obviously will dictate to some degree the microbes you have,” Budowle said. And the stressors a person has experienced can also impact results. “Antibiotics can have an effect on a microbiome, as can disease.” Some microbes can be tougher, more stable and persistent than their counterpart human cells in the environment, he said. “So, you’re likely to be able to get that signature for longer, especially in a more degraded sample.”
For forensic microbes to become an important part of crime lab investigations, both Budowle and Knight said, gathering and analyzing samples will have to become inexpensive and routine. “If it’s $500,000 bucks a sample they’re not going to do it,” Knight said. “If it’s $5 a sample, then it could be routine.”
The largest obstacle now, he said, is the complexity of the data analysis. “One of the problems they are trying to solve is how do you put [microbiome evidence] into an easy-to-use interface,” he said. “You have these incredibly complex underlying data sets. So, what we need to do for microbes is the same sort of thing we do for sending photos or videos by phone. How do we take that noisy, highly technical data and do data processing on it so that people get a clear picture they can interpret?”
Budowle noted that crime labs are known for being resistant to change and that is often an appropriate reaction. “They have limited resources, they don’t have time to do research, and it takes a lot of effort and resources to have a technology validated and put into operations.”
However, “there is a portion of the forensic community that looks to the future,” he said. Advances are typically driven by need and support of the federal government, he said. The work on microbial forensics developed and advanced rapidly in response to the anthrax attacks, he said. In the process, expertise and standards of practice were developed, and that carried into further work on the microbiome, although originally as a tool to prepare for terrorist attacks.
“You really have to think of it on a five to 20-year kind of schedule because we have technologies that are coming out now that have been in the works for more than a decade,” Budowle said. “Rapid DNA (automated DNA processing) is an example of work that was started 15 years or more ago, and NIJ and DOD and Homeland Security funded some of these things. And we’re just seeing the benefits of that work.”
The advances in DNA sequencing technology have made much of microbial forensics possible,” he said, and more than a decade after it was first developed, “we are just starting to see the inroads that might go into crime labs today.”
Budowle doesn’t believe microbial DNA will supplant human DNA in the foreseeable future, “but with advances in sequencing and in bioinformatics, there may be more signature information out there that makes it appealing.” Microbiome research might progress faster, he noted, if focused centers of excellence were established across the country. The work is expensive, he said, and trying to do the work in individual labs slows progress.
As for all of the swabs for detecting microbes, many developed in NIJ-supported research, Knight said thousands of them were recently flown to the International Space Station (ISS) where they are being used to create a microbial map of the entire interior of the space station. “The idea is to look at the total microbiology of the ISS and see how much of that we can track to particular sources.” Of particular interest are microbes already found on the ISS that haven’t been seen on Earth. Knight said he believes they are not alien, but instead evolved from earlier microbes on the space station. “We can’t guarantee that they don’t exist on Earth,” he said, “but we can say that we haven’t seen them in even the very large data sets like the entire Earth microbiome project.”
Budowle recently looked at DNA profiles he generated back in the early 1980s. “We thought they were spectacular,” he said of those early profiles. “We’d perfected the process and they looked good. I look at them today and say, ’Oh, these are ugly.’”
The microbiome research is progressing and much of the analysis today is quite impressive, he said. “That might turn into a functional operation by the routine [crime] laboratory in the next handful of years.”
But based on his long experience, Budowle’s sure that 20 years from now, when microbiome researchers look back, “we’re going to say we were in the stone ages in 2020. It’s just the way things are.”
[note 1] Bruce Budowle, “Human Microbiome Species and Genes for Human Identification,” Final report to the National Institute of Justice, grant number 2015-NE-BX-K006, May 2019, NCJ 252942.
[note 2] Rob Knight et al., “Evaluating the Skin Microbiome as Trace Evidence,” Final report to the National Institute of Justice, grant number 2014-R2-CX-K411, June 2018, NCJ 251647.
[note 3] University of Chicago Magazine, Winter/21, Vol. 113, #2.
[note 4] Budowle, “Human Microbiome Species and Genes for Human Identification.”