Phage Invasion Comic Art

Phage Invasion

Pitting Foodborne Bacteria Against Their Own Worst Enemies

By Natalie van Hoose
Illustrations by Tom Kronewitter

A comic-style drawing of Paul EbnerPurdue animal scientist Paul Ebner uses phages to eliminate disease-causing bacteria such as Salmonella and E. coli from meat and produce.

For the past seven years, animal scientist Paul Ebner and his lab have been pelting some of the most infamous pathogens in the food system with a surprising weapon: viruses.

Foodborne bacteria such as Salmonella and E. coli can cause serious, even deadly, illnesses. But pathogens can get sick, too—infected by viruses known as bacteriophages, or phages for short.

Phages are fierce predators, systematically targeting and destroying bacterial cells by hijacking their normal metabolic processes. In the lab, phages carve out visible clearings in petri dishes where bacteria once clouded the glass. It is an impressive feat for a life form too miniscule to be seen with a standard microscope and 10 times smaller than its prey.

Ebner's group focuses on harnessing the antibacterial potency of phages to keep Salmonella out of meat processing facilities and eliminate E. coli from food products such as ground beef and raw spinach. "We're just taking something that happens in nature all the time and concentrating it on certain pathogens," he says.

Paul Ebner discusses how he uses bacteriophages to tackle key foodborne pathogens. (Video by Kelsey Getzin)

Going Viral

Ebner's first stab at using phages was tackling the spread of Salmonella between pigs on their way to the slaughterhouse. Stress levels in pigs rise when they travel, weakening their ability to fend off infectious bacteria in transport trailers and holding pens. A few Salmonella-positive pigs can infect 40 percent of a holding-pen herd in as little as six hours, which increases the risk that disease-causing bacteria will enter the processing facility and contaminate meat products.

But administering drugs to animals shortly before they are slaughtered is not an option. Medications can leave chemical residues in meat, and antibiotics require a longer period to kick in. Ebner saw a situation ripe for phages. "Phages are perfect for the transportation context because they're natural, non-toxic and fast-acting," he says.

Because phages are highly host-specific, Ebner could select a combination that would prove lethal to Salmonella but harmless to other bacteria.

He and his team gave healthy pigs an injection of phages before releasing the animals into a holding pen containing Salmonella-infected pigs and an abundance of their feces. After six hours—the approximate time pigs are corralled together before slaughter—the phage-treated pigs had about 99 percent fewer Salmonella bacteria in their guts than pigs that did not receive any treatment.

Adding microencapsulated phages to livestock feed had a similar success rate, offering a simple, cost-effective treatment option to producers and processors.

In a separate study on immune response, mice had no negative reactions to phage treatment, which Ebner describes as similar to using probiotics.

The research group also found that phages can scrub the vast majority of toxin-producing E. coli from contaminated meat and spinach.

Ebner and graduate students Yingying Hong and Yanying Pan infected fresh spinach leaves and ground beef with about 10 million cells of an especially virulent strain of E. coli to test whether phages could whittle down contamination levels in food products.

After 24 hours, phage treatment had reduced E. coli concentrations in the spinach, stored at room temperature, by more than 99.9 percent. In ground beef stored at room temperature, the phages cleaned up about 99 percent of E. coli bacteria within 24 hours.

"People can get sick from very low concentrations of E. coli," Ebner says. "Applying these kinds of treatments to contaminated foods in the processing plant can make them a lot safer."

Fishing for Phages

Ebner's technique of finding the right phages to unleash on Salmonella and E. coli is simple, if unsavory. He pays a visit to what he describes as "one of the most complex microbiological settings you can find"—the local wastewater treatment facility.

Wastewater is chock-full of bacteria and the phages that feed on them. Ebner scours samples of the water for the phages he needs, using his target bacteria as bait.

He looks for those that kill the bacteria most effectively and replicate the quickest and most efficiently. Once he has isolated multiple candidates, he combines them in concoctions known as phage cocktails. Bacteria can develop defense mechanisms to rebuff phages, and using more than one species of phage reduces the chance that the bacteria can resist the treatment.

The Search-and-Destroy Tactics of Phages

Stuff of Science Fiction

Phages first caught Ebner's attention in a microbiology lecture where he was struck by the virus' appearance: With its 16-sided geometric head, coiled tail and spidery tail fibers, a phage looks like something from the Space Age, not nature.

"In a field where a lot of things look like blobs, a phage resembles the Apollo Lunar Module," Ebner says. "They are really bizarre. Ever since I saw one, I've wanted to work with them."

Adding to their otherworldly oddity are their sophisticated search-and-destroy methods. When a phage detects a host bacterium, it "docks" onto the cell's surface and deploys a syringe-like device that injects its own genetic material into the cell. The bacterium's inner workings come to a halt, and it transforms into a phage-making factory, churning out so many new viruses that it eventually explodes, releasing a swarm of phages to hunt other hosts.

Viral Revival

Possibly the most common life form on the planet, phages thrive in soil systems; on rivers and oceans, where they float in dense microbial mats; and on animals and humans. You can ingest thousands of phages just by licking your lips.

But phages remained a well-kept microbial secret until 1917, when Félix d'Hérelle, a French-Canadian microbiologist, investigated the mysterious disappearance of a bacterial strain he was studying. He deduced that the invisible culprits must be viruses and christened them "bacteriophages," Greek for "bacteria-eaters."

D'Hérelle immediately realized the antibacterial potential of phages and was able to prove their effectiveness as a cure for dysentery. For a few decades, phages were used to treat many kinds of illnesses, including staphylococcal infections, typhoid, cholera and skin diseases.

But phages soon faded from the medical arena, replaced by the discovery of penicillin in 1928 and the subsequent development of antibiotics. Phage treatment has remained a common form of therapy only in Eastern Europe, particularly Georgia, where an institute founded by D'Hérelle houses an extensive library of the viruses and still administers phage therapy.

However, phages are making a steady comeback, aided in part by the high cost of developing antibiotics and the rise of antibiotic-resistant pathogens.

Even though bacteria can also evolve ways of fending off phages, the viruses respond by altering their attack strategies—an arms race among microbes.

Still, Ebner is probing how bacteria become resistant to phages and whether resistance might pose problems for the long-term use of phage therapy.

"Phages are not a substitute for antibiotics," he says. "But when used in the right contexts, they're extremely effective. We're going to see more phage therapy in the future."

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Laser Beams Leave Pathogens No Place to Hide

Food microbiologist Arun Bhunia trains a bright light on pathogens in food products.

With the help of then-Purdue engineer Daniel Hirleman, Bhunia created a laser sensor that can identify many kinds of disease-causing bacteria isolated from food samples in less than 24 hours—about three times faster than conventional detection methods.

Known as BARDOT, the microwave-sized machine uses a laser to scan bacterial colonies on an agar plate. When a colony is illuminated, it produces a black-and-white image called a scatter pattern, a unique arrangement of rings and spokes. Each type of bacterium generates a distinct scatter pattern, serving as a fingerprint by which the researchers can distinguish the bacterium.

Recent studies by the Bhunia lab have shown that BARDOT can identify Salmonella grown from samples of contaminated foods with an accuracy of more than 95 percent. It pinpoints virulent strains of E. coli with an accuracy of more than 90 percent and can also pick out Listeria, Vibrio and Bacillus.

BARDOT is robust enough to classify multiple bacteria with a single scan, offering an intricate portrait of an entire microbial community.

"BARDOT could be used as a screening tool to detect these key pathogens much earlier and more easily than conventional methods, which is crucial to preventing foodborne illnesses," Bhunia says. "We are just now getting a sense of what it can do."

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