Phages, which are harmless viruses that destroy specific bacteria, are becoming useful tools to protect our food supply.
Every year, one in seven people in America comes down with a foodborne illness, typically caused by a bacterial pathogen, including E.Coli, listeria, salmonella, or campylobacter. That adds up to 48 million people, of which 120,000 are hospitalized and 3000 die, according to the Centers for Disease Control. And the variety of foods that can be contaminated with bacterial pathogens is growing too. In the 20th century, E.Coli and listeria lurked primarily within meat. Now they find their way into lettuce, spinach, and other leafy greens, causing periodic consumer scares and product recalls. Onions are the most recent suspected culprit of a nationwide salmonella outbreak.
Some of these incidents are almost inevitable because of how Mother Nature works, explains Divya Jaroni, associate professor of animal and food sciences at Oklahoma State University. These common foodborne pathogens come from the cattle's intestines when the animals shed them in their manure—and then they get washed into rivers and lakes, especially in heavy rains. When this water is later used to irrigate produce farms, the bugs end up on salad greens. Plus, many small farms do both—herd cattle and grow produce.
"Unfortunately for us, these pathogens are part of the microflora of the cows' intestinal tract," Jaroni says. "Some farmers may have an acre or two of cattle pastures, and an acre of a produce farm nearby, so it's easy for this water to contaminate the crops."
Food producers and packagers fight bacteria by potent chemicals, with chlorine being the go-to disinfectant. Cattle carcasses, for example, are typically washed by chlorine solutions as the animals' intestines are removed. Leafy greens are bathed in water with added chlorine solutions. However, because the same "bath" can be used for multiple veggie batches and chlorine evaporates over time, the later rounds may not kill all of the bacteria, sparing some. The natural and organic producers avoid chlorine, substituting it with lactic acid, a more holistic sanitizer, but even with all these efforts, some pathogens survive, sickening consumers and causing food recalls. As we farm more animals and grow more produce, while also striving to use fewer chemicals and more organic growing methods, it will be harder to control bacteria's spread.
"It took us a long time to convince the FDA phages were safe and efficient alternatives. But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
Luckily, bacteria have their own killers. Called bacteriophages, or phages for short, they are viruses that prey on bacteria only. Under the electron microscope, they look like fantasy spaceships, with oblong bodies, spider-like legs and long tails. Much smaller than a bacterium, phages pierce the microbes' cells with their tails, sneak in and begin multiplying inside, eventually bursting the microbes open—and then proceed to infect more of them.
The best part is that these phages are harmless to humans. Moreover, recent research finds that millions of phages dwell on us and in us—in our nose, throat, skin and gut, protecting us from bacterial infections as part of our healthy microbiome. A recent study suggested that we absorb about 30 billion phages into our bodies on a daily basis. Now, ingeniously, they are starting to be deployed as anti-microbial agents in the food industry.
A Maryland-based phage research company called Intralytix is doing just that. Founded by Alexander Sulakvelidze, a microbiologist and epidemiologist who came to the United States from Tbilisi, the capital of Georgia, Intralytix makes and sells five different FDA-approved phage cocktails that work against some of the most notorious food pathogens: ListShield for Listeria, SalmoFresh for Salmonella, ShigaShield for Shigella, another foodborne bug, and EcoShield for E.coli, including the infamous strain that caused the Jack in the Box outbreak in 1993 that killed four children and sickened 732 people across four states. Last year, the FDA granted its approval to yet another Intralytix phage for managing Campylobacter contamination, named CampyShield. "We call it safety by nature," Sulakvelidze says.
Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder."
Photo credit: Living Radiant Photography
Phage preparations are relatively straightforward to make. In nature, phages thrive in any body of water where bacteria live too, including rivers, lakes and bays. "I can dip a bucket into the Chesapeake Bay, and it will be full of all kinds of phages," Sulakvelidze says. "Sewage is another great place to look for specific phages of interest, because it's teeming with all sorts of bacteria—and therefore the viruses that prey on them."
In lab settings, Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder." Once phages multiply enough, they are harvested, dispensed into containers and shipped to food producers who have adopted this disinfecting practice into their preparation process. Typically, it's done by computer-controlled sprayer systems that disperse mist-like phage preparations onto the food.
Unlike chemicals like chlorine or antibiotics, which kill a wide spectrum of bacteria, phages are more specialized, each feeding on specific microbial species. A phage that targets salmonella will not prey on listeria and vice versa. So food producers may sometimes use a combo of different phage preparations. Intralytix is continuously researching and testing new phages. With a contract from the National Institutes of Health, Intralytix is expanding its automated high-throughput robot that tests which phages work best against which bacteria, speeding up the development of the new phage cocktails.
Phages have other "talents." In her recent study, Jaroni found that phages have the ability to destroy bacterial biofilms—colonies of microorganisms that tend to grow on surfaces of the food processing equipment, surrounding themselves with protective coating that even very harsh chemicals can't crack.
"Phages are very clever," Jaroni says. "They produce enzymes that target the biofilms, and once they break through, they can reach the bacteria."
Convincing the FDA that phages were safe to use on food products was no easy feat, Sulakvelidze says. In his home country of Georgia, phages have been used as antimicrobial remedies for over a century, but the FDA was leery of using viruses as food safety agents. "It took us a long time to convince the FDA phages were safe and efficient alternatives," Sulakvelidze says. "But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
The agency had granted Intralytix its first approval in 2006, and over the past 10 years, the company's sales increased by over 15-fold. "We currently sell to about 40 companies and are in discussions with several other large food producers," Sulakvelidze says. One indicator that the industry now understands and appreciates the science of phages was that his company was ranked as Top Food Safety Provider in 2021 by Food and Beverage Technology Review, he adds. Notably, phage sprays are kosher, halal and organic-certified.
Intralytix's phage cocktails to safeguard food from bacteria are approved for consumers in addition to food producers, but currently the company sells to food producers only. Selling retail requires different packaging like easy-to-use spray bottles and different marketing that would inform people about phages' antimicrobial qualities. But ultimately, giving people the ability to remove pathogens from their food with probiotic phage sprays is the goal, Sulakvelidze says.
It's not the company's only goal. Now Intralytix is going a step further, investigating phages' probiotic and therapeutic abilities. Because phages are highly specialized in the bacteria they target, they can be used to treat infections caused by specific pathogens while leaving the beneficial species of our microbiome intact. In an ongoing clinical trial with Mount Sinai, Intralytix is now investigating a potential phage treatment against a certain type of E. coli for patients with Crohn's disease, and is about to start another clinical trial for treating bacterial dysentery.
"Now that we have proved that phages are safe and effective against foodborne bacteria," Sulakvelidze says, "we are going to demonstrate their potential in therapeutic applications."
This article was first published by Leaps.org on October 27, 2021.
Alida de Flamingh and her team are collecting elephant dung. It holds a trove of information about animal health, diet and genetic diversity.
Approximately 27 percent of mammals and 28 percent of all assessed species are close to dying out. The IUCN Red List of threatened species, simply called the Red List, is the world’s most comprehensive record of animals’ risk of extinction status. The more information scientists gather, the better their chances of reducing those risks. In Africa, populations of vertebrates declined 69 percent between 1970 and 2022, according to the World Wildlife Fund (WWF).
“We put on sterile gloves and use a sterile swab to collect wet mucus and materials from the outside of the dung ball,” says Alida de Flamingh, a postdoctoral researcher at the University of Illinois Urbana-Champaign.
“When people talk about species, they often talk about ecosystems, but they often overlook genetic diversity,” says Christina Hvilsom, senior geneticist at the Copenhagen Zoo. “It’s easy to count (individuals) to assess whether the population size is increasing or decreasing, but diversity isn’t something we can see with our bare eyes. Yet, it’s actually the foundation for the species and populations.” DNA analysis can provide this critical information.
Assessing elephants’ health
“Africa’s elephant populations are facing unprecedented threats,” says de Flamingh, the postdoc, who has studied them since 2009. Challenges include ivory poaching, habitat destruction and smaller, more fragmented habitats that result in smaller mating pools with less genetic diversity. Additionally, de Flamingh studies the microbial communities living on and in elephants – their microbiomes – looking for parasites or dangerous microbes.
Approximately 415,000 elephants inhabit Africa today, but de Flamingh says the number would be four times higher without these challenges. The IUCN Red List reports African savannah elephants are endangered and African forest elephants are critically endangered. Elephants support ecosystem biodiversity by clearing paths that help other species travel. Their very footprints create small puddles that can host smaller organisms such as tadpoles. Elephants are often described as ecosystems’ engineers, so if they disappear, the rest of the ecosystem will suffer too.
There’s a process to collecting elephant feces. “We put on sterile gloves (which we change for each sample) and use a sterile swab to collect wet mucus and materials from the outside of the dung ball,” says de Flamingh. They rub a sample about the size of a U.S. quarter onto a paper card embedded with DNA preservation technology. Each card is air dried and stored in a packet of desiccant to prevent mold growth. This way, samples can be stored at room temperature indefinitely without the DNA degrading.
Earlier methods required collecting dung in bags, which needed either refrigeration or the addition of preservatives, or the riskier alternative of tranquilizing the animals before approaching them to draw blood samples. The ability to collect and sequence the DNA made things much easier and safer.
“Our research provides a way to assess elephant health without having to physically interact with elephants,” de Flamingh emphasizes. “We also keep track of the GPS coordinates of each sample so that we can create a map of the sampling locations,” she adds. That helps researchers correlate elephants’ health with geographic areas and their conditions.
Although de Flamingh works with elephants in the wild, the contributions of zoos in the United States and collaborations in South Africa (notably the late Professor Rudi van Aarde and the Conservation Ecology Research Unit at the University of Pretoria) were key in studying this method to ensure it worked, she points out.
Protecting chimpanzees
Genetic work with chimpanzees began about a decade ago. Hvilsom and her group at the Copenhagen Zoo analyzed DNA from nearly 1,000 fecal samples collected between 2003 and 2018 by a team of international researchers. The goal was to assess the status of the West African subspecies, which is critically endangered after rapid population declines. Of the four subspecies of chimpanzees, the West African subspecies is considered the most at-risk.
In total, the WWF estimates the numbers of chimpanzees inhabiting Africa’s forests and savannah woodlands at between 173,000 and 300,000. Poaching, disease and human-caused changes to their lands are their major risks.
By analyzing genetics obtained from fecal samples, Hvilsom estimated the chimpanzees’ population, ascertained their family relationships and mapped their migration routes.
“One of the threats is mining near the Nimba Mountains in Guinea,” a stronghold for the West African subspecies, Hvilsom says. The Nimba Mountains are a UNESCO World Heritage Site, but they are rich in iron ore, which is used to make the steel that is vital to the Asian construction boom. As she and colleagues wrote in a recent paper, “Many extractive industries are currently developing projects in chimpanzee habitat.”
Analyzing DNA allows researchers to identify individual chimpanzees more accurately than simply observing them, she says. Normally, field researchers would install cameras and manually inspect each picture to determine how many chimpanzees were in an area. But, Hvilsom says, “That’s very tricky. Chimpanzees move a lot and are fast, so it’s difficult to get clear pictures. Often, they find and destroy the cameras. Also, they live in large areas, so you need a lot of cameras.”
By analyzing genetics obtained from fecal samples, Hvilsom estimated the chimpanzees’ population, ascertained their family relationships and mapped their migration routes based upon DNA comparisons with other chimpanzee groups. The mining companies and builders are using this information to locate future roads where they won’t disrupt migration – a more effective solution than trying to build artificial corridors for wildlife.
“The current route cuts off communities of chimpanzees,” Hvilsom elaborates. That effectively prevents young adult chimps from joining other groups when the time comes, eventually reducing the currently-high levels of genetic diversity.
“The mining company helped pay for the genetics work,” Hvilsom says, “as part of its obligation to assess and monitor biodiversity and the effect of the mining in the area.”
Of 50 toucan subspecies, 11 are threatened or near-threatened with extinction because of deforestation and poaching.
Identifying toucan families
Feces aren't the only substance researchers draw DNA samples from. Jeffrey Coleman, a Ph.D. candidate at the University of Texas at Austin relies on blood tests for studying the genetic diversity of toucans---birds species native to Central America and nearby regions. They live in the jungles, where they hop among branches, snip fruit from trees, toss it in the air and catch it with their large beaks. “Toucans are beautiful, charismatic birds that are really important to the ecosystem,” says Coleman.
Of their 50 subspecies, 11 are threatened or near-threatened with extinction because of deforestation and poaching. “When people see these aesthetically pleasing birds, they’re motivated to care about conservation practices,” he points out.
Coleman works with the Dallas World Aquarium and its partner zoos to analyze DNA from blood draws, using it to identify which toucans are related and how closely. His goal is to use science to improve the genetic diversity among toucan offspring.
Specifically, he’s looking at sections of the genome of captive birds in which the nucleotides repeat multiple times, such as AGATAGATAGAT. Called microsatellites, these consecutively-repeating sections can be passed from parents to children, helping scientists identify parent-child and sibling-sibling relationships. “That allows you to make strategic decisions about how to pair (captive) individuals for mating...to avoid inbreeding,” Coleman says.
Jeffrey Coleman is studying the microsatellites inside the toucan genomes.
Courtesy Jeffrey Coleman
The alternative is to use a type of analysis that looks for a single DNA building block – a nucleotide – that differs in a given sequence. Called single nucleotide polymorphisms (SNPs, pronounced “snips”), they are very common and very accurate. Coleman says they are better than microsatellites for some uses. But scientists have already developed a large body of microsatellite data from multiple species, so microsatellites can shed more insights on relations.
Regardless of whether conservation programs use SNPs or microsatellites to guide captive breeding efforts, the goal is to help them build genetically diverse populations that eventually may supplement endangered populations in the wild. “The hope is that the ecosystem will be stable enough and that the populations (once reintroduced into the wild) will be able to survive and thrive,” says Coleman. History knows some good examples of captive breeding success.
The California condor, which had a total population of 27 in 1987, when the last wild birds were captured, is one of them. A captive breeding program boosted their numbers to 561 by the end of 2022. Of those, 347 of those are in the wild, according to the National Park Service.
Conservationists hope that their work on animals’ genetic diversity will help preserve and restore endangered species in captivity and the wild. DNA analysis is crucial to both types of efforts. The ability to apply genome sequencing to wildlife conservation brings a new level of accuracy that helps protect species and gives fresh insights that observation alone can’t provide.
“A lot of species are threatened,” Coleman says. “I hope this research will be a resource people can use to get more information on longer-term genealogies and different populations.”
The test tubes contain tiny DNA/enzyme-based circuits, which comprise TRUMPET, a new type of electronic device, smaller than a cell.
While a computer gives these inputs in binary code or "bits," such as a 0 or 1, biocomputing uses DNA strands as inputs, whether double or single-stranded, and often uses fluorescent RNA as an output.
Adamala’s research focuses on developing such biocomputing systems using DNA, RNA, proteins, and lipids. Using these molecules in the biocomputing systems allows the latter to be biocompatible with the human body, resulting in a natural healing process. In a recent Nature Communications study, Adamala and her team created a new biocomputing platform called TRUMPET (Transcriptional RNA Universal Multi-Purpose GatE PlaTform) which acts like a DNA-powered computer chip. “These biological systems can heal if you design them correctly,” adds Adamala. “So you can imagine a computer that will eventually heal itself.”
The basics of biocomputing
Biocomputing and regular computing have many similarities. Like regular computing, biocomputing works by running information through a series of gates, usually logic gates. A logic gate works as a fork in the road for an electronic circuit. The input will travel one way or another, giving two different outputs. An example logic gate is the AND gate, which has two inputs (A and B) and two different results. If both A and B are 1, the AND gate output will be 1. If only A is 1 and B is 0, the output will be 0 and vice versa. If both A and B are 0, the result will be 0. While a computer gives these inputs in binary code or "bits," such as a 0 or 1, biocomputing uses DNA strands as inputs, whether double or single-stranded, and often uses fluorescent RNA as an output. In this case, the DNA enters the logic gate as a single or double strand.
If the DNA is double-stranded, the system “digests” the DNA or destroys it, which results in non-fluorescence or “0” output. Conversely, if the DNA is single-stranded, it won’t be digested and instead will be copied by several enzymes in the biocomputing system, resulting in fluorescent RNA or a “1” output. And the output for this type of binary system can be expanded beyond fluorescence or not. For example, a “1” output might be the production of the enzyme insulin, while a “0” may be that no insulin is produced. “This kind of synergy between biology and computation is the essence of biocomputing,” says Stephanie Forrest, a professor and the director of the Biodesign Center for Biocomputing, Security and Society at Arizona State University.
Biocomputing circles are made of DNA, RNA, proteins and even bacteria.
Evgeny Katz
The TRUMPET’s promise
Depending on whether the biocomputing system is placed directly inside a cell within the human body, or run in a test-tube, different environmental factors play a role. When an output is produced inside a cell, the cell's natural processes can amplify this output (for example, a specific protein or DNA strand), creating a solid signal. However, these cells can also be very leaky. “You want the cells to do the thing you ask them to do before they finish whatever their businesses, which is to grow, replicate, metabolize,” Adamala explains. “However, often the gate may be triggered without the right inputs, creating a false positive signal. So that's why natural logic gates are often leaky." While biocomputing outside a cell in a test tube can allow for tighter control over the logic gates, the outputs or signals cannot be amplified by a cell and are less potent.
TRUMPET, which is smaller than a cell, taps into both cellular and non-cellular biocomputing benefits. “At its core, it is a nonliving logic gate system,” Adamala states, “It's a DNA-based logic gate system. But because we use enzymes, and the readout is enzymatic [where an enzyme replicates the fluorescent RNA], we end up with signal amplification." This readout means that the output from the TRUMPET system, a fluorescent RNA strand, can be replicated by nearby enzymes in the platform, making the light signal stronger. "So it combines the best of both worlds,” Adamala adds.
These organic-based systems could detect cancer cells or low insulin levels inside a patient’s body.
The TRUMPET biocomputing process is relatively straightforward. “If the DNA [input] shows up as single-stranded, it will not be digested [by the logic gate], and you get this nice fluorescent output as the RNA is made from the single-stranded DNA, and that's a 1,” Adamala explains. "And if the DNA input is double-stranded, it gets digested by the enzymes in the logic gate, and there is no RNA created from the DNA, so there is no fluorescence, and the output is 0." On the story's leading image above, if the tube is "lit" with a purple color, that is a binary 1 signal for computing. If it's "off" it is a 0.
While still in research, TRUMPET and other biocomputing systems promise significant benefits to personalized healthcare and medicine. These organic-based systems could detect cancer cells or low insulin levels inside a patient’s body. The study’s lead author and graduate student Judee Sharon is already beginning to research TRUMPET's ability for earlier cancer diagnoses. Because the inputs for TRUMPET are single or double-stranded DNA, any mutated or cancerous DNA could theoretically be detected from the platform through the biocomputing process. Theoretically, devices like TRUMPET could be used to detect cancer and other diseases earlier.
Adamala sees TRUMPET not only as a detection system but also as a potential cancer drug delivery system. “Ideally, you would like the drug only to turn on when it senses the presence of a cancer cell. And that's how we use the logic gates, which work in response to inputs like cancerous DNA. Then the output can be the production of a small molecule or the release of a small molecule that can then go and kill what needs killing, in this case, a cancer cell. So we would like to develop applications that use this technology to control the logic gate response of a drug’s delivery to a cell.”
Although platforms like TRUMPET are making progress, a lot more work must be done before they can be used commercially. “The process of translating mechanisms and architecture from biology to computing and vice versa is still an art rather than a science,” says Forrest. “It requires deep computer science and biology knowledge,” she adds. “Some people have compared interdisciplinary science to fusion restaurants—not all combinations are successful, but when they are, the results are remarkable.”