Bacteria underpin much of our world, acting behind the scenes to affect the health and behavior of animals and plants. They help produce food, provide oxygen, and even reshape the environment through a vast array of biological processes. They come in a phenomenal number of strains—many still unknown—and thrive in different ecological and environmental niches all over the world.
But while their diverse behaviors make them essential to life, bacteria can also be deadly. This threat only grows as greater global travel brings people into contact with new places, foods, and animals, dramatically increasing the chances of exposure to dangerous microbial species known as pathogens. New pathogens, both naturally occurring and adversary-engineered, are increasingly likely to emerge due to changes in the environment, rising global population, and the wide availability of genetic engineering tools to both state and non-state actors.
These factors, coupled with faster potential dispersal due to increasing global travel and population density, have significantly increased the danger posed by bacterial pathogens. A teaspoon of soil can contain as many as 1 billion bacteria, but most of them are completely harmless, or even beneficial. That also means that if someone with nefarious intentions adds dangerous bacteria to the soil, it can be difficult to sort out the good from the bad.
How can the Department of Defense—whose forces, deployed around the world, constantly come into contact with new bacteria—discriminate between harmless and virulent strains to prevent a disease outbreak that threatens military readiness?
In an age where bioterrorism is a very real concern, DARPA is working on new ways to detect potential human-introduced pathogens in the environment—before people become ill. In Feb 2018 DARPA’s Biological Technologies Office launched Friend or Foe, a new program that proposes to develop a platform technology that rapidly screens unfamiliar bacteria to establish their pathogenicity and even discover unknown pathogenic traits, necessary first steps for designing effective biosurveillance and countermeasures.
“Trends such as rising global population, changes in the environment, and the growing accessibility of tools for genetic engineering mean that our armed forces are increasingly likely to face new bacterial pathogens, whether they occur naturally or are engineered by adversaries,” said Paul Sheehan, the program manager for Friend or Foe. “Our existing biosurveillance strategies don’t work on previously undiscovered bacteria or on bacteria that have been specifically designed to evade detection by current tests. We need new screening tools that can quickly characterize the threat to enable a rapid response.”
Friend or Foe program
Existing forensic technologies for identifying bacteria are limited in their application and fall primarily into two categories: rapid diagnostic microbiology, which is confined to approximately 350 known strains and requires cultured bacteria; and metagenomics, which only inventories are previously known bacteria present in a sample. Both technologies take 36 hours or longer to deliver results, and neither is capable of quickly evaluating previously unknown bacteria, especially strains that cannot be cultured in a laboratory. This means that, at present, the vast majority of bacteria species cannot be readily evaluated for risk to humans.
Yet within this diversity of bacteria—at least 10 exp (7) to 10 exp (9) species—there lies a large pool of unknown traits that could contribute to future pathogenicity. And, since bacteria can transfer traits between species fairly rapidly, individual strains can acquire new capabilities to help them evade the body’s innate immune response or to resist antibiotics.
Although new genetic sequencing tools are being developed that can quickly read a bacterium’s genotype—its genetic makeup—sequencing alone will unlikely solve the challenge of assessing risk. That’s because simply knowing genotype is not the same as knowing phenotype—how that bacterium’s genetic code leads to function. The sheer sequence of a bacterial genome does not indicate whether or not the bacterium is pathogenic in humans.
To directly and efficiently test for pathogenicity, Friend or Foe aims to build a portable platform that screens many unfamiliar strains of bacteria at once to reveal their phenotypes. Developing such a platform will require overcoming numerous engineering challenges. First, without killing the bacteria, the technology must extract and isolate them from complex environments such as soil, runoff, sewage, biofilms, and medical samples, where numerous strains of bacteria live together.
Second, the system must sustain the bacteria in simulated host environments long enough to conduct testing. And third, it must run and evaluate a gauntlet of physical and chemical tests on the bacteria—the biological equivalent of the game “Twenty Questions”—to determine their pathogenicity.
The Friend or Foe system will test for three traits of pathogenicity. First, can the bacteria survive and establish a niche in a host organism? Does it, for instance, adhere to the host’s cell membranes? Second, can the bacteria harm its host? For example, does it secrete toxins or have flagella that could disrupt the host’s mucosal tissue? And third, can the bacterium protect itself? Does it inactivate the host’s protective antibodies or resist antibiotics?
Dangerous bacteria would be flagged for genetic sequencing to map the newly discovered pathogenic trait to specific genes, leading to simpler biochemical tests for that pathogen in the future. A side benefit of the program would be speeding up all future efforts to identify new bacterial traits and the genes that provide them, which would support research ranging from antibiotic production to the degradation of pollutants.
The novel capability developed under FoF will provide detailed, high-throughput phenotype-based characterization of unknown bacteria through the identification of pathogenic traits. Specifically, the technology should reliably extract representative samples of bacteria from complex environments, maintain their viability while they are repeatedly interrogated to identify virulence factors, and then analyze them using an omics approach that leverages external pathogen and gene databases. Ultimately, this technology will detect bacterial pathogens as, or even before, they emerge as a threat to the public.
“There are millions of species of unidentified bacteria in the world, and we now have no quick way of knowing which of those might endanger our troops,” Sheehan said. “If we’re successful in creating a tool for rapid evaluation of bacterial phenotype, we’ll deliver the Defense Department a powerful new capability for force protection and a powerful deterrent to the development of engineered bio-threats
The performing teams for Friend or Foe will develop complete end-to-end systems that will probe the phenotypes of bacteria to assess their pathogenic potential. The first step will be the extraction and isolation of potentially unknown and/or unculturable bacteria from complex matrices such as laboratory swabs, sewage/run-off, and biofilms.
This will be followed by interrogation of the extracted bacteria with a series of non-destructive biochemical and biophysical assays. In the initial phase, interrogation of small populations (<100) of bacteria is acceptable; however, the ultimate goal will be the interrogation of a single bacterium.
Bacteria identified as pathogenic should be processed for omics analyses to enable the mapping of phenotype to genotype. For the first two phases of the program, phenotype discovery will be demonstrated on contrived samples composed of known bacteria.The complexity of these samples will increase as the program progresses, ending with real-world samples in Phase III. These real-world samples will only be examined in conjunction with a Government partner specifically tasked to prevent bacterial outbreaks.
DARPA envisions Friend or Foe as a four-year, fundamental research program. Once the program begins, a separate independent verification and validation (IV&V) team contracted by DARPA will work with performer teams to provide standardized biological samples that simulate different environments and include mixtures of known bacteria. The IV&V team will also evaluate the effectiveness of the performers’ systems following demonstrations.
Researchers supporting Friend or Foe must adhere to all applicable guidelines for biosecurity. DARPA has structured the program so that potential discovery of new pathogens can only take place under the guidance and supervision of federal agencies tasked with preventing the spread of disease.
Defense Advanced Research Projects Agency (DARPA) has awarded a $14.2 million grant to a team led by professors at Texas A&M
Defense Advanced Research Projects Agency (DARPA) has awarded a $14.2 million grant to a team led by professors at Texas A&M to develop a way to quickly detect which bacterial pathogens are present in a soil or water sample.
Samuel, along with Arum Han, PhD, professor in the Department of Electrical and Computer Engineering, and Paul deFigueiredo, PhD, associate professor, and Erin Van Schaik, PhD, research assistant professor, both in the Department of Microbial Pathogenesis and Immunology, and other colleagues at Texas A&M, will try to create a device to rapidly and efficiently characterize the harmfulness of bacteria in the environment—essentially “culturing the unculturable,” Samuel said. Raytheon is also developing a portable device to evaluate bacteria and their potential to cause harm—information that could support the future development of medical countermeasures and improved screening tools.
Samuel said a limited number of available microbiology techniques such as advanced DNA isolation techniques can identify bacteria and their genetic makeup, but they are not high throughput enough and are not phenotype-based identification systems. High throughput technology means screening done very rapidly for many samples. An organism’s phenotype is its observable physical properties or functions, determined by genotype and environmental factors.
“DARPA decided to expand the universe of ways that people can detect bacteria,” Samuel said. “The agency is trying to take high throughput technologies and merge them with novel phenotypic assay systems that have not yet been created as high throughput tools.” Phenotypic assay systems measure the presence or activity of an organism’s properties, for example, the ability of bacteria to invade a host cell or activate or repress host detection of the bacteria.
Researchers in the microbial pathogenesis and immunology department are providing guidance on understanding the host-pathogen interactions, answering questions such as: Are the bacteria producing toxins? Are they going inside a host cell? Do they survive inside the host? And do they have drug resistance? Answering these questions at a single cell level, Samuel said, will enable the team to determine the bacteria’s phenotype and, with bioinformatic prediction tools, determine if each bacterial cell is predicted to be pathogenic.
Han said his team, based on these biological assays, “is developing tools that actually allow us to measure that host-pathogen interaction one cell at a time and allow us to do that measurement on millions and millions of cells within a very short time period.” To do this, they will build a unique microfluidic lab-on-a-chip device that can rapidly test millions of individual cells, an essential component of the platform DARPA hopes to develop.
They will do this by screening each microorganism in the sample to see if it has the properties of a pathogen. “There are unique qualities that pathogens have that non-pathogens do not,” Samuel said. “For example, pathogens have the ability to kill specifically eukaryotic cells types, including human cells, as part of their pathogenic strategy.” But because no one can screen millions of cells by hand, the team is working on building a microfluidic device to do so automatically.
“Testing millions of cells to see whether they are potentially harmful or not, one cell at a time, cannot be achieved within a reasonable time frame even with the most advanced robotic liquid handling system,” Han said. “This is where the beauty of microfluidic devices comes in, where it can be used to test and probe every one of the millions of cells for their pathogenic traits with single-cell resolution.”
Han said a microfluidic device can handle very small volumes of liquid or cells. It has many micrometer-sized fluidic channels that allow millions of single cells to flow through and be controlled very accurately. “The lab-on-a-chip device is a device where a series of high-precision cell and liquid assay steps can be integrated together to conduct a complex assay on a single chip format, thus the term lab-on-a-chip,” Han said. He said conventional laboratory methods for collecting and measuring cells using pipettes will not work when trying to measure the properties of millions of cells.
First, it will create water-in-oil emulsion droplets, each encapsulating a single bacterial cell. These droplets will each have eukaryotic host cells with the potential pathogen. “You need to see what happens when they’re together,” Samuel said. That’s the next step: high-throughput phenotyping, in which each bacterial cells can be characterized according to certain characteristics.
The final step is metagenomic analysis on each particle. Although this might seem to be the easy part, most of the time genetic sequencing is done on samples that have thousands (if not millions) of cells. Sequencing a single cell, on the other hand, is very new technology, and in this case, that information has to be referenced back to each individual bead.
“You have to label the DNA from each cell, uniquely, so you can backtrack,” Samuel said. The goal is for the device to then take all of this data—the genomics and the phenotypes of each droplet—and put it all into an algorithm to create a readout that will predict whether pathogens are present in the sample, and if so, which ones they are. “You commonly hear about big data…this is big data,” Samuel said. “You have to relate all of this information back to each particle in a massive database.”
Applying to real-world situations
The group is starting with bacteria from soil samples from an environmental microbiology group from the University of Oklahoma, one of its project partners, but Han said they will eventually apply the technique to water samples. Other project partners include other researchers from Texas A&M, the University of California San Francisco, the University of Virginia and Argonne National Lab.
“Dirt is not simple,” said James Samuel, PhD, Regents Professor and head of the Department of Microbial Pathogenesis and Immunology at the Texas A&M College of Medicine and principal investigator of this project. “In the past, we’ve only known about the bacteria that are able to be grown on a petri dish in the lab, but that’s only a fraction of what’s really there, whether we’re talking about in the soil or in your stomach. It’s not a trivial problem.”
Their goal is to have a lot of good data within the first 18 months of the project. “We may not be able to measure everything, but we should be able to measure a good number of characteristics of unknown microorganisms,” Han said.
To help with different aspects of this project, the Texas A&M team, which also includes Arul Jayaraman, PhD, the Ray B. Nesbitt Professor of Chemical Engineering, and Tony Provin, PhD, professor and extension specialist of the soil and crop sciences, is collaborating with other groups at the University of Oklahoma, University of California San Francisco, the University of Virginia, and the Argonne National Lab.
This information can then be used for a variety of applications, from knowing which antibiotic to give to someone exposed or—in the case of purposefully created bacteria—a way to track it back to its source. “Until you know what pathogens are in the environment, you can’t know how to counter them,” Samuel said. “That’s what this project aims to find out.”
Raytheon developing portable “Friend or Foe” system to identify harmful bacteria before they cause harm
Under the DARPA Friend or Foe program, Raytheon is developing biosurveillance technology that detects bacterial pathogens as soon as or before they threaten the military and citizens. Current biosurveillance strategies are not effective on undiscovered bacterial strains or on bacteria engineered to evade detection. To overcome this problem, Friend or Foe will characterize bacteria quickly by examining its behavior.
“Population growth, global travel, climate change—all of these factors increase the risk of exposure to unfamiliar bacteria,” said Aaron Adler, Ph.D. and principal investigator for the Friend or Foe program at Raytheon BBN Technologies. “Most of those bacteria are harmless or even beneficial, but our goal is to develop a system that lets people know quickly when they are not as a cue to take mitigating action.”
The screening process begins with collecting and isolating a single bacterium in a tiny cube with a porous membrane. Sensor arrays in the cube make initial measurements on respiration, consumption of specific nutrients and metabolite production. Suspect bacteria is then extracted and exposed to synthetic substances that mimic human tissues to test for pathogenicity.
“To get a reliable risk assessment, we need to understand not just the bacteria’s genetic makeup, or genotype, but how it functions – its phenotype,” said Adler. “We’re looking at ways to subject the bacteria to a gauntlet of behavior screenings so we can determine its ability to cause disease.”