Some scientists go into research for basic science, such as finding an enzyme and figuring out its functions and properties. Others like Hsin-Yeh Hsieh, gravitate toward applied sciences where they use what they know to develop technology.
“Because I can use everything I learned in science as building blocks to create a technology that actually can do something and make impacts in real life, that’s what I like about science,” said Hsieh, a research scientist in the George Stewart lab at Bond LSC.
Hsieh began her science journey in Taiwan, where she went to school for several years, studying molecular and microbiology as well as medical technology. Her love of science began in high school, she explained, “I was very curious about what happens in human health.”
After completing her Bachelor of Science at Kaohsiung Medical University in Taiwan, Hsieh worked in the Cancer Research group of Institute of Biomedical Sciences in Acdemia Sinica in Taiwan, before she came to the U.S. for her graduate study in the Pathology and Anatomical Department of MU Medical School. For her Ph.D. study, she had the opportunity to work on a research in blood banking and transfusion, which focused on enzymatically converting type A and type B blood into universal blood type O. This is where she really found her niche, “I am in the field I enjoy,” she said. However, her biggest challenge for the blood type conversion is how to remove the enzyme from the converted red blood cells before transfusion.
After her doctorate, Hsieh joined David Setzer’s lab in Biological Sciences of MU and later, George Stewart offered her a postdoctoral position in his Molecular microbiology lab.
Collaborating with a multidisciplinary team on MU campus including faculty in Veterinary Pathobiology and in the School of Natural Resources, and students in biochemistry and in bioengineering, Hsieh’s team came up with a design for a cartridge that can immobilize enzyme for blood type conversion, and filter enzyme out of the converted red blood cells (RBCs).
“Let’s say, after the blood goes through this cartridge, it gets converted. The converted RBCs flow through the cartridge, but the enzyme is locked behind so it won’t go along with the product,” she explained.
Hsieh and her team actually filed a patent on this process in December 2019 and are currently seeking more funding to continue testing the process in other industrial applications, such as biofuel and special chemical production.
“I can see its potential,” Hsieh said. “We think it can work in biofuel and it can work in pollutant degradation, but we need to prove it with scientific evidence.”
Said Hsieh, “The technology could turn cellulose to sugar, into glucose, that becomes an energy source,”
It can be expanded beyond these applications as well, with potential to help clean the environment.
She explains, “We actually can break down the herbicide pollutants, like atrazine, in the environment using this enzymatic process.”
It took four years to create this cartridge, overcoming several challenges along the way. That did not intimidate Hsieh.
“I like to see if my idea is right on the track or way off, so that’s quite challenging but also fun,” said Hsieh with a chuckle. “You should always explore something new and challenge yourself.”
Day to day, Hsieh is busy working with undergrads in the lab. Their willingness to learn and try anything intrigues Hsieh’s curiosity and expands her horizons in research.
“The students are so open minded. I say, ‘well, let’s take a new approach and do it,’ and they just go ahead and do it. If we get a good result that proves the concept, that’s great. If not, we learn how to troubleshoot and make it work. In the process, students are trained for critical thinking and research strategy” Hsieh explained. “I think that’s a win-win situation, for them to advance in the career path and also for me to move the project forward.”
Expanding her horizons is important not only in the lab, but as well as in her community. When talking about the friends she has made here in Columbia, Hsieh says, “I met most of my friends while playing tennis, and at a point in time, I counted there were people from 46 countries among us because students come to this campus from all over the world.”
Creating a community with different cultures keeps the life interesting and helps her learn of the world, while staying in Columbia all these years. With unfinished tasks in hands, Hsieh is not planning on leaving anytime soon. Describing Columbia, she “enjoys the atmosphere.”
Medical bacteriologist, George Stewart has had a few stops along the way before he got to Bond LSC in 2004. Having done schooling and research at universities from the midwest to the east coast, it has been a long journey filled with many ups and downs, but a rewarding one at that.
Stewart hadn’t always wanted to be a medical bacteriologist though, “When I was going to college, I actually wanted to be a marine biologist,” he says, “And then my sophomore year in college, I took a microbiology course. And that changed that.”
This microbiology course at the University of North Texas was the first course where he was able to do hands on work in a laboratory and he found that, “culturing organisms and identifying organisms was very appealing.”
“The technological advancements are so fast in this discipline that you just have to change and there are easier ways to do things and better ways to do things now,” he states.
But these changes help Stewart delve deeper in understanding his passion for an organism best known for the white powder derived from its spores and sometimes used in terror attacks. Technically called Bacillus anthracis, the organism causes the disease anthrax. Stewart focuses on the outer spore of the bacteria which first comes in contact with the infected host and is responsible for protein production. He looks at how the spore contributes to anthrax’s ability to persist in soil and how it contributes to resistance properties.
Unfortunately for Stewart, funding for anthrax research has declined over the years, “It’s not considered a high priority organism,” he explains, however, “it still is an organism of concern for bio-threats.” To work around the lack of funding, Stewart has turned in other directions.
“Part of that has actually been the biggest success stories of recent times in my lab,” he said.
The direction? Using Bacillus thuringiensis, a bacterium similar to anthrax, to do research on. One problem Stewart approached using this bacterium was cleaning up Atrazine, a herbicide used by farmers to kill weeds. This herbicide, while helpful to farmers, can contaminate groundwater, potentially causing diseases in humans.
To find a solution, Stewart and Brian Thompson, a postdoc student in his lab, collaborated with Chung-ho Lin, a research associate professor in the MU Center for Agroforestry, “looking at could we possibly have come up with approaches using this basic technology to improve agriculture from a number of different ways to make plants grow faster, better, that sort of thing.”
They found that they could display proteins on Bacillus thuringiensis spore that would break down Atrazine. Farmers would only have to spray their fields with this organism and the soil would be clean of Atrazine.
Taking this a step further, Thompson created the company, Elemental Enzymes, to sell products like this to farmers in order to clean the environment. Located in St. Louis, Elemental Enzymes currently has 11 products in use today on more than 11% of corn acres in the United States in 2019, and that number seems to be increasing.
“It’s kind of interesting in the sense that we started off trying to answer a very basic biological question related to spore biology and involving a significant high consequence pathogen, and we ended up discovering something which has huge benefits for agriculture. So you never know what direction it’s going to take you in these things,” says Stewart.
Stewart hasn’t stopped there. In fact, he was one of the driving forces behind starting a Bachelor of Science degree program in microbiology here at MU. After a microbiology degree program here was discontinued in the early 2000s, Stewart has since found evidence that the degree would be successful here at the university and it was approved by the board of curators and the Missouri Department of Higher Education in 2018. The first students in this program began this past semester and Stewart says, “we’re getting underway.”
Despite these accomplishments, Stewart is getting ready to retire this year, explaining, “part of the reason that I stayed on this year was to make sure this program gets off the ground and help out with that.” While his career is winding down here at MU, he is still keeping busy, “I’m actually teaching two undergraduate courses in microbiology, once called fundamentals of microbiology, it’s an introductory microbiology course. And the other one is public health microbiology, which is more of a junior level type course.”
He also continues to do some work in his lab as he has two graduate students finishing up their degrees this year and recently stepped down as the Department of Veterinary Pathobiology chair in January.
What does Stewart have planned for when he retires? He is not slowing down, that is for sure. Stewart and his wife already have a vacation planned for New Zealand within the next few months, and he plans on spending quality time with his children and grandchildren.
“I won’t have accomplished all the things I wanted to,” he says, “but I think we’ll have gotten the major things done. So that’s satisfying.”
Inter-departmental MU team aims to improve enzyme use and recovery for spectrum of industrial, medical and military applications
By Phillip Sitter | Bond LSC
A mostly-finished cylindrical bio-reactor site sits in a 3D printer after the printing has stopped. With a 3D printer in-house, Chung-Ho Lin said that the inter-departmental team he is part of can generate four or five different prototypes a day to test in their bio-reactor model, instead of having to order from different fabrication companies. A basic printer like this used to cost $8,000, but within the last year prices dropped to only about $1,000. Lin is a research assistant professor at MU’s Center for Agroforestry, and the team and project are coordinated by Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s Bond LSC lab, where the team also does most of its work. | Phillip Sitter, Bond LSC
As Sagar Gupta watched a 3-D printer on a lab countertop construct a jumbo pencil eraser-sized, white plastic cylinder of what looked like a shell holding inter-woven letter Xs, he remarked that the only limitation to what you can print is the size of the printer.
“The timing is perfect, otherwise we wouldn’t have been able to afford it,” Chung-Ho Lin said of the availability of cheaper 3-D printers within the past couple years.
The two men were acutely aware, as the printer continued its methodical manufacture, that they may be architects of the first steps in a bio-chemical revolution.
It’s a revolution that could be hugely profitable financially and may help to save lives on battlefields, clean up some kinds of pollution and enable humans to venture further into space for a cheaper cost, among other things.
To understand how this cross-disciplinary team working in George Stewart’s lab at the Bond Life Sciences Center got there, we have to back up a little bit.
Sagar Gupta holds a vial of carbon solution. Most of the team’s prototype designs for bio-reaction sites are made of carbon, and some are even bio-degradable. | Phillip Sitter, Bond LSC
From a bottleneck to a bioreactor
Their work began three years ago with a project to develop technology to reduce the cost of converting cellulose into glucose for biofuels — essentially the process by which raw plant fiber from wood or leaves is turned into a sugar that can be more efficiently burned to produce energy.
“That has been the bottleneck for the biofuel industry,” said Lin.
The team — consisting of Lin, a research assistant professor at MU’s Center for Agroforestry; Stewart, Hsinyeh Hsieh and several undergraduate and recently graduated students including Gupta — already developed E. coli bacteria that can mass-produce engineered enzymes to convert cellulose into glucose.
These enzymes speed up the reactions and reduce the cost because they have linkers attached to them — protein hooks that let them be recovered after a single use as catalysts in biological reactions, rather than having to throw them out. Hsieh said she developed this with Stewart’s input, and the assistance of a recently graduated student, Che-Min Su.
However, the team needed a platform for the linkers to hook onto — something they could continuously use to reel in their catch.
The answer in their search for the correct platform arrived when affordable 3-D printing technology came onto the market. With their own 3-D printer in-house, they custom-designed different platforms for their experiments and completely bypassed having to shop around with different fabrication companies.
All of the ingredients were there with that plastic cylinder Gupta and Lin watched print. The team now had a cheap way to mass produce and repeatedly recover enzymes. With this capability, they could produce a more efficient bioreactor — a controlled, isolated system in which desired reactions can take place with higher outputs of quantity and quality of a desired product.
It’s much like the more familiar concept of a nuclear reactor, which controls and isolates a nuclear chain reaction to harvest the most energy possible. The catalysts in that reaction are radioactive particles that give off heat as they decay. In a physical reaction, the heat released boils liquid water into gaseous steam, and the steam turns a turbine generator that makes electricity.
But in the team’s bioreactors, catalysts are enzymes that chemically react with cellulose and transform it into glucose instead of electricity. The glucose can be fermented further into butanol that can ultimately be used for liquid fuels to power vehicles.
A bio-reactor column stands packed with carbon fibers submerged in enzymes. If the column were hooked up a continous flow system, substrate would be pumped through it to spur bio-chemical reactions on the surface of the carbon fibers, or whatever other type of site is packed inside. | Phillip Sitter, Bond LSC
Money and blood
While only at a bench-top, proof-of-concept scale, the team’s first bioreactor has lasted more than four months. With prospects to increase its size, they “could be saving at least $10 to $12 million per year on an industrial scale,” said Gupta. Gupta graduated in May from MU with an MBA, and now works for Lin.
That estimate is just for one individual bioreactor. Begin to multiply it, and the cost-savings add up very quick.
“Nowadays, probably a majority of pharmaceutical companies have already switched their manufacturing process into the enzymatic process. One thing nice about the enzymatic process is that it can eliminate [the need for] a lot of hazardous chemicals. They also tend to have a better yield,” Lin explained.
Lin added that there is a bonus of complexity within this kind of 3-D platform system. Individual enzymes have different linkers, and this allows for multiple enzymes to catalyze reactions and be recovered on the platform at the same time. This is especially cost-saving because the conversion of cellulose into glucose requires three different kinds of enzymes.
“Because of this high specificity, we don’t need any enzymatic purification process,” he said.
Once the enzymes hooked to a platform start to naturally decay, the team can simply remove the decayed enzymes by a hot water bath and soak it in a new batch of enzymes, just like swapping out an empty printer cartridge for a full one with fresh ink.
While their primary focus is on biofuels, they are very aware that more efficient and cheaper bioreactors could have huge implications for a broad spectrum of industries.
One use they are developing could effectively transform one blood type into another using enzymes.
“This is not a completely new technology, but in the past, I would say back in the 90’s, some people tried some clinical trials and they ran into a problem, because a lot of times after the conversion, [loose] enzymes would get into the recipients’ bloodstream and cause an auto-immune reaction,” said Lin.
However, by being able to immobilize enzymes with their linkers on this 3-D device, they should be able to get around that problem, he said.
“I think there’s great potential for the soldier on the battlefield,” Lin cited as an application for the technology. A field doctor or medic wouldn’t have to worry about waiting on a certain type of blood for a transfusion, because they could convert another batch of blood into a universal-donor type.
Another team member, Hien Huynh explained that the more enzyme you add in ratio to the substrate, in this instance blood, the faster the conversion process will go — “maybe just 30 minutes.”
Hsieh wrote that “Blood type conversion would be the ultimate challenge for our bioreactor, because it has so many clinical aspects to be concerned [about] and conquered. It is a challenge but our [multi-disciplinary] team is willing to take it on and make it work.”
Lin said that the team has already submitted a letter of intent to the U.S. Department of Defense, “hopefully to secure some support for the blood-conversion application.”
Hien Huynh packs a bio-reactor column with carbon fiber bio-reactor sites that look like feathers. The sites are coated in enzyme before being packed into the column. | Phillip Sitter, Bond LSC
Enzymes in action
There are other potentially massive implications for the battlefields of the future.
“You can immobilize anti-microbial, anti-fungal and anti-inflammatory enzymes on a surface to use as a wound-healing patch,” Lin said, noting that such a patch could be used on the battlefield, as well as for cosmetic surgery recovery.
But the applications don’t stop there. Other uses could use enzymes to clean up TNT residues leeching out of unexploded ordinance like cluster bomblets, mortars, rocket-propelled grenades and landmines buried in the ground before the toxic residues contaminate groundwater.
Even within the confines of biofuels, there’s a strong military market. By 2020, the Navy wants 50 percent of its total energy consumption to come from alternative sources as opposed to petroleum-based fuels — part of a broader strategy to go green. The U.S. military in the near future wants to reduce the cost of its energy consumption and secure a stable domestic supply of energy.
According to the U.S. Government Accountability Office, from fiscal years 2007 to 2014, the Department of Defense bought 32 billion gallons of petroleum-based fuels at a cost of $107.2 billion.
Away from the military sphere, Lin detailed other uses for cheaper, higher quality enzymes. It could purify and recycle urine into clean water on space flights on for astronauts or convert waste into energy with an ammonia fuel cell that’s already available.
Mass-produced enzymes can be used for water treatment on earth, too. Pollutants like dioxin and herbicides like atrazine that contaminate soil can be bio-remediated in the same way that TNT residues can be cleaned up.
The food industry already uses enzymes as flavor removers to remove strong tastes from products like beer.
Minh Ma simulates the end result of a successful operation of the bio-reactor. She extracts and separates samples of real glucose product produced by the reactions in the column. A stronger yellow color in the solution indicates a higher concentration of glucose. | Phillip Sitter, Bond LSC
A bright bioreactor future
To call the team’s work revolutionary might be a bit premature.
There is a whole process ahead of them, including patent filing and university reviews, before the team can approach investors with the assurance their discoveries are legally protected. And, future investors will ultimately help determine how the technology is used.
But, Lin and the others might just have found themselves in the right place at the right time to make major breakthroughs, and that’s not all due to just advancements in technology.
“We have identified new directions and found a new niche to be competitive. I think the most important resource we have is people, and their brains,” Lin said.
Hsieh wrote that “To assemble a successful team is to put the right talent in the proper position and to inspire them to challenge themselves. I was lucky to come across so many young, talented students who are eager to learn and work hard for their bright future on MU’s campus.”
Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s Bond LSC lab, coordinates this project. Hsieh is an expert in gene fusion, enzyme production and characterization and enzymatic blood type conversion. Stewart is a medical bacteriologist, McKee Professor of Microbial Pathogenesis and chair of Veterinary Pathobiology at MU.
Lin works with Stewart and Hsieh to develop concepts, design prototypes and assemble the rest of the team — students and recent graduates — that optimizes the enzymatic reactions and the physical and chemical aspects of their bioreactor system. Minh Ma is a junior studying bio-chemistry. Mason Schellenberg studies bio-engineering, will be a senior and worked to find the most efficient platform design that the team’s 3-D printer could produce. Hien Huynh is a recent graduate who works on immobilizing enzymes. In addition to his MBA, Gupta also has a background that includes nano-technology, molecular engineering and financing. He concentrates on the feasibility and market potential of the team’s work.
Tiger Energy Solutions, LLC is the team’s industry partner — a spinoff startup from the team’s research project . Their focus in the development of a cheaper and higher quality method of converting cellulose into glucose for biofuels is to produce aviation biofuel. Tiger Energy serves as the interface between the team and industry while the team’s work is scaled-up for commercialization.
From left to right: Minh Ma, Hien Huynh and Sagar Gupta are three of the teams members, standing here in George Stewart’s lab. Ma is junior studying bio-chemistry. Huynh is a recent graduate who works on enzyme immobilization. Gupta is also a recent graduate — he obtained his MBA in May — and he focuses on the financial feasibility and market potential of the team’s work. | Phillip Sitter, Bond LSC
Stewart holds a different colony of anthrax in his lab. Stewart’s work with anthrax and other similar organisms focuses on understanding the tough protein shell of the bacteria’s spores that enable the pathogen to survive in soil for extended periods of time, even hundreds of years. | photo by Phillip Sitter, Bond LSC
By Phillip Sitter | MU Bond Life Sciences Center
For a tiny spore, anthrax holds a lot of danger and promise.
If you found yourself wondering about more than its safety in the lab, we have a answers to a few persistent questions.
What makes anthrax dangerous, and how does it spread? How common are infections of it in nature? If we have antibiotics that already treat it, beyond finding new and better ones why study the organism?
Bovine slayer and bio-weapon
Anthrax bacteria are highly resilient organisms. Like only a few other bacteria in nature, they produce spores with protein-shelled casings that lay dormant in soil for long periods of time, waiting to be taken in by grazing animals like cattle and sheep.
Once they enter the nutrient-rich environment of an animal’s bloodstream — exactly how the bacteria gets there after entering an animal’s mouth is unknown — spores germinate inside the white blood cells that absorb them. As anthrax reproduces rapidly inside its host, it releases toxins that quickly kill the infected animal. That sometimes happens in just a matter of hours according to farmers’ accounts, said George Stewart, Bond Life Sciences Center scientist, medical bacteriologist, McKee Professor of Microbial Pathogenesis and chair of Veterinary Pathobiology at MU.
When infected animals die the bacteria are exposed to oxygen in air that penetrates the decomposing body and new spores escape as the dead host decays. The newly-produced spores are deposited back into the soil, where they wait in a state of suspended animation for as long as it takes to be ingested by another grazing animal, sometimes decades to a hundred years.
“Most of these plates are anthrax, but they’re all non-virulent,” Stewart says of the various bacterial colonies on the counter in his lab. Non-virulent strains are safe to handle with the precautions of a BSL-2 lab: gloves, a lab coat and eye protection. The Bond LSC does not have a BSL-3 or higher facility to study more dangerous pathogens like virulent strains of anthrax. That work is done at the Laboratory for Infectious Disease Research, or LIDR. | photo by Phillip Sitter, Bond LSC
Sporadic, natural outbreaks of anthrax can happen almost anywhere in the world except Antarctica, as the spores have been found to exist worldwide, Stewart said. In the United States, outbreaks in cattle and bison usually happen in the Plains and West in states like Colorado and Wyoming — anywhere that cattle or sheep have been raised or their wildlife equivalents graze, lending to the descriptor from Stewart of “anthrax belt” for the states stretching from Texas to the Dakotas.
During the Cold War, the U.S. and the Soviet Union, among others, were attracted to anthrax for their respective biological weapons programs because of the hardiness of the organism and high lethality rates of untreated gastrointestinal and, especially, pulmonary anthrax infections in humans. Today, Americans’ are probably most familiar with anthrax from the mailing attacks of 2001, when letters containing weaponized anthrax spores were delivered to the offices of media outlets and politicians, infecting 22 people and killing five.
Hero in a half-life shell?
Despite their grim reputation, anthrax spores hold a lot of potential for Stewart.
The outermost layer of the protein-shell structure of a spore holds particular interest — how it’s made, what proteins it’s composed of and the function of those proteins. Studying it could not only help find future anthrax vaccines and therapies, but also be used for other applications.
Scientists have coated spores of a close biological relative of anthrax, Bacillus thuringiensis, with plant growth-promoting and anti-insect enzymes and other proteins to treat crops. Its durable structure in the form of the spore’s protein shell attach to these enzymes and remain longer in the environment, making them more effective, Stewart said.
This long biological half-life even presents potential for bio-remediation work — using natural organisms to cleanse the environment of toxins and pollutants. In the case of B. thuringiensis, this might include cleaning up soil from the herbicide atrazine and the notorious pollutant dioxin, a by-product of various industrial processes including incineration, smelting and the production of paper pulp and some herbicides and pesticides.
According to collective findings of the National Institutes of Health and Environmental Protection Agency and others, atrazine is probably not a human carcinogen but can cause genetic damage in animals and is still acutely toxic to people at high enough levels. According to the World Health Organization (WHO), dioxin causes cancer and other ill human ailments.
Pollutants that contaminate soil can eventually leech into groundwater and also enter the food supply through grazing animals. That makes organisms adapted to living in soil like B. thuringiensis perfect candidates as potential carriers of agents used to clean up soil pollutants like atrazine and dioxin.
However, Stewart said that there is too much public stigma to use anthrax for similar applications, “which is too bad because Bacillus anthracis actually works a little better in these applications.”
Rethinking the anthrax image
So is anthrax a dastardly bio-villain or a misunderstood hero?
The truth is neither. Anthrax bacterium is just another living thing.
As a predator of other living things, needing to feed to survive and reproduce, it goes about its life cycle without any conscious agenda. It has no malice, and unlike a wolf, shark or hawk it doesn’t have a brain so it doesn’t even have instinct, only genetic code to mindlessly live by.
How we interact with anthrax is largely dependent upon us humans.
We have the vaccines to prevent it and antibiotics to treat it if it makes us sick. After that, we can harness and modify anthrax’s natural power for ill as a weapon, or maybe to do some good in the environment. So long as we have the technology to study and manipulate organisms like anthrax, the potential for both scenarios will always be there.
Meanwhile, at MU Stewart will continue to study anthrax, searching for its secrets in order to better understand it, to better serve the rest of us.
George Stewart, McKee Professor of Microbial Pathogenesis and Chair of Veterinary Pathobiology holds up a colony of Bacillus anthracis in his lab. The strain of anthrax he holds is non-virulent, and is therefore safe to handle under BSL-2 precautions as opposed to BSL-3 for virulent strains that cause disease in humans. | photo by Phillip Sitter, Bond LSC
By Phillip Sitter | MU Bond Life Sciences Center
You’ve seen it before in the movies.
Sweaty scientists put on their full-body, spacesuit-like get-ups to stave off a potentially extinction-level outbreak and at least one scientist invariably gets infected with the deadly agent of disease.
While popular culture propagates this sense of peril, in reality bio-containment labs are designed with safety in mind.
“People tend to think bio-containment facilities are dangerous, mostly from movies I think, but the history is actually spectacular,” said George Stewart, a medical bacteriologist, a Bond Life Sciences Center scientist, McKee Professor of Microbial Pathogenesis and Chair of Veterinary Pathobiology.
For Stewart — whose lab works on the basic science behind anthrax — no one is “under the gun because of big outbreaks, not with the pressure you see in movies.”
But one type of pressure is an important part of bio-containment lab safety. Air pressure differences maintain certain labs at lower pressure compared to the rooms and hallways around them, ensuring that air will only flow in toward a lab and not out, keeping any airborne pathogens trapped inside.
“They know if everything is done properly, it’s perfectly safe for them and even safer for everyone else [outside a lab],” Stewart said of the safety features, procedures and systems of bio-containment lab safety in place at facilities like those at the Bond LSC and elsewhere at MU.
Stewart said he is vaccinated against anthrax. “Whether I have protective immunity or not, I don’t know.”
“Almost like you were working underwater”
The powerful capabilities of anthrax and other lethal pathogens call for particular safety precautions for scientists.
Stewart looks like he’s straight out of the movie Contagion when donning the full-body suit for his more dangerous research in a Bio-Safety Level (BSL) 3 facility at MU’s Laboratory for Infectious Disease Research (LIDR). In the trees on the eastern fringes of campus, the specialized building is where he and other researchers study diseases animals can transmit to humans, including plague, Brucella, tularemia and Q fever, and mosquito-borne diseases like dengue, chikungunya and now Zika virus. The safety protocols and systems at a BSL-3 lab like the LIDR facility Stewart described reflect the likely transmission by aerosols of the human pathogens inside.
After passing through security access to the building and the labs inside, Stewart enters an ante room off of a hallway. The air pressure in this room and the lab beyond it is such that air will only flow in toward the lab, and not out and away.
Anything that goes into the lab only leaves if it is autoclaved, disinfected in a steel machine using pressurized steam that “essentially kills everything, even heat-resistant spores,” so that means Stewart changes clothes, removes his watch, phone and any other personal items.
Next come layered scrubs and a water-proof Tyvek suit with booties and a hood that cover everything but his hands and face. Two layers of gloves take care of his hands, but shielding his face is a bit more technical.
A plastic face cover with a Tyvek hood shrouds over Stewart’s shoulders. Inside, a pump fills the hood with positively-pressured filtered air – this has the inverse effect of the negative pressure of the rooms and keeps air flowing out away from his face and not toward it.
Everything inside the lab and the building is about redundancies like that. A final safety measure is that all work on pathogens take place in bio-safety hoods – HEPA-filtered cabinets.
Stewart said it can be difficult to hear with the air filter systems blowing, so every move by researchers is calculated and announced. Colleagues take their time in handing off equipment to one another, so as to avoid torn gloves.
It’s “almost like you were working underwater as two divers,” Stewart said about working in the BSL-3 lab with a colleague.
“Everything is orchestrated in a very intentional way.”
Only dangerous when dry
Anthrax isn’t always lethal, so the scene is quite different inside a BSL-2 lab at Bond LSC where Stewart studies non-virulent strains.
BSL-2 labs study infectious agents that can cause disease in humans, but are usually treatable. Researchers only need lab coats, gloves and eye protection in these labs and all waste must be autoclaved. Here anthrax and other colonies of organisms are stacked in covered Petri dishes and handled without any Tyvek or air pumps.
The Anthrax bacterium researched here is missing a specific plasmid, a DNA molecule essential for virulence that protects the anthrax bacteria from white blood cells that attack them.
Stewart holds a colony of anthrax and says that it is impossible to see a visually recognizable colony like this naturally in soil. | photo by Phillip Sitter, Bond LSC
On top of that, all samples in this lab are wet, and anthrax spores are only dangerous as aerosols when dry. Before 2001, Stewart said virulent strains of anthrax were only labeled BSL-2 agents for this reason.
The anthrax letter attacks that year not only changed some of the organism’s lab classifications, but also interest in it. Prior to 2001, Stewart said there was not a lot of funding available for anthrax-focused researchers, a small and tight-knit community. Even though the attacks did spur an increased investment of government money into the field at the time for defense against anthrax as a bio-weapon agent, almost 15 years later Stewart said that funding is more or less back at pre-2001 levels, “perhaps marginally better.”
There are now only a handful of anthrax-dedicated labs in the U.S., Stewart said, trying to name them off as he counted his fingers. The Bond LSC has not had any lab higher than a BSL-2 plus for years now, not since the BSL-3 research moved to LIDR, Stewart said.
Several local residents called when LIDR was under construction and asked questions and voiced concerns about the facility and the work to be done there, Stewart recalled. However, Stewart said he and his colleagues gave the callers honest answers, and he has not heard of any pushback since.
Stewart sees bio-containment labs as positive technological achievements in the study of disease – without them, many advances in treatment would never have been possible. In terms of the work done at facilities at MU and in the Bond LSC, Stewart said “we have the facilities, we have the equipment, we have the training,” to ensure the safety of researchers inside the labs, and even more so everyone else on the outside.
Anthrax is not contagious and responds well to antibiotics, despite concerns in the scientific community Stewart shared that there is a possibility antibiotic resistance could be intentionally engineered into anthrax.
Stewart could only think of a couple of cases when lab workers got infected with the organism through mishaps, and those were at USAMRIID – the United States Army Medical Research Institute for Infectious Disease at Fort Detrick, Maryland – when anthrax was produced there in very large quantities for research of it as a bio-weapon during the Cold War.
When you spend a lot of your time working with potentially lethal pathogens though, what do you tell your doctor when you come in with flu-like symptoms? Stewart said that not only does he and any of his colleagues disclose to their doctor the organisms that they work with, but doctors at MU already know exactly what organisms are being researched inside the LIDR labs. As a precautionary measure for their own well-being in case accidental infection did occur in a lab after all, Stewart or another colleague working with anthrax who turned up sick would receive antibiotics just to be safe – “there are standard operating procedures for everything.”
He does not want to make light of the dangerous organisms he works with, but inside the BSL-3 facility at LIDR, Stewart said that breathing in HEPA-filtered air all day there does do wonders for his hay fever.
Stewart couldn’t help but share a chuckle with that one. Laughter might be the most un-containable thing in nature.
Anthrax bacteria is a rod-shaped culture. Most common forms of transmission are through abrasions in the skin and inhalation.
Imagine researchers in hazmat suits moving slowly and deliberately through a lab. One of them holds up a beaker. It’s glowing.
This light — or the absence of it — could save millions of dollars for governments and save the lives of anthrax victims.
Scientists at the University of Missouri Laboratory of Infectious Disease Research proved a new method for anthrax detection can identify anthrax quicker than any existing approach.
When the “bioluminescent reporter phage” — an engineered virus — infects anthrax bacteria, it takes on a sci-fi-movie-type glow.
George Stewart, a medical bacteriologist at MU’s Bond Life Sciences Center, and graduate student Krista Spreng, observed the virus against a variety of virulent strains of bacillus anthracis, the bacteria causing anthrax disease.
“For this technique, within a few hours, you’ll have a yes or no answer,” Stewart said.
The research, funded by the USDA, was published in the Journal of Microbiological Methods in Aug. 2013. David Schofield at Guild BioSciences, a biotech company in Charleston, S.C, created the reporter phage.
This new method could save a significant amount of money associated with the decontamination of anthrax from suspected infected areas.
Expensive clean-up from the 2001 “Letter attacks”
With the country on high-alert following Sept. 11, 2001, a slew of bioterrorists mailed anthrax letters, filled with a powder that if inhaled could cause death.
Numerous Post Offices and processing facilities were closed and quarantined.
The clean-up bill for the 2001 Anthrax Letter attacks was $3.2 million, according to a 2012 report in Biosecurity and Bioterrorism: Biodefense Strategy, Practice and Science.
Theoretically, the new detection method would alert of a negative result potentially five hours into clean-up efforts instead of two or three days into expensive decontaminating.
Current methods take anywhere from 24 hours or longer to produce a definitive answer for anthrax contamination.
A five-hour benchmark
Stewart said from contamination levels expected from a bioterrorism threat, a positive answer could be found in five hours. If contamination levels were higher, results would come back much more quickly.
Prior to this bioluminescent reporting phage, experts used techniques that were culture based or PCR (polymerase chain reaction) based. Both methods, require additional time for a definitive answer, a minimum of 24 to 48 hours, Stewart said.
“Normally to identify whether an organisms is present, you have to take the material culture, the organism and all the bacteria that might be present in the sample,” Stewart said. “You have to pick colonies that might be bacillus anthracis and do chemical testing which takes some time.”
From a bio-threat standpoint, breathing in anthrax, is the highest concern for public health and homeland security officials and has the highest fatality rate among forms of anthrax.
“If you have a situation and need a quick yes or no answer, this is a tool that will help that,” Stewart said.
Terrorists have used a powder form of anthrax, which has been slipped into letters of political persons and media. A person is infected when an anthrax spore gets into the blood system, most commonly through inhalation or an abrasion on the body, according to Centers of Disease Control and Prevention.
For low levels of contamination, the bioluminescent reporter phage would still detect the presence of the bacteria, but it would take longer.
“This method will be as quick as any of the others and quicker than most,” Stewart said.
The bioluminescent-detection method can detect low levels of anthrax bacteria and rule out false positives. The added benefit to this reporting system is its ability to show that anthrax bacteria are present and it’s alive, Stewart said.
What’s next?
The next step in the bioluminescent reporter phage is getting it approved so a product can be produced and branded. The agency that would warrant the stamp of approval would depend on the eventual use of the phage — food-related testing would likely go through the Food and Drug Administration, Stewart said.
When that happens, a product would not necessarily require a formal lab — it would need a place where cultures could grow at 37 degrees.
“Samples could be collected, brought back to the state public health lab for example and then the testing could be done within a few hours of the collection of the samples and you would have a result,” Stewart said.
The last anthrax attack was in 2001, but the possibility of one happening again, Stewart said, remains a driver for proactive research.
“In the years since the postal attacks, we haven’t had any bona fide anthrax attacks,” Stewart said. “That doesn’t mean it’s not going to happen — we have to be prepared for when it does occur again.”
