Phage 'cocktail' wipes out 99 percent of E. coli in meat, spinach

Paul Ebner  Download Photo

WEST LAFAYETTE, Ind. - Treating food products with select bacteriophages - viruses that target and kill bacteria - could significantly reduce concentrations of E. coli, a Purdue University study shows.

An injection of bacteriophages - also known informally as "phages" - nearly eradicated a toxin-producing strain of E. coli in contaminated spinach and ground beef, in some cases decreasing E. coli concentrations by about 99 percent.

The study suggests that bacteriophage treatment could be an effective tool to help ensure the safety of food products, said Paul Ebner, associate professor of animal sciences.

"Phage treatment is a way of harnessing the natural antibacterial properties of phages to limit E. coli and other important foodborne pathogens," Ebner said. "Applying this kind of therapy to contaminated foods will make them safer."

While most strains of E. coli are harmless, some can cause severe and potentially fatal illnesses. The strain used in Ebner's study - E. coli O157:H7 - caused more than 63,000 illnesses, 2,100 hospitalizations and 20 deaths in the U.S. in 2011. Ingesting as few as 10 colony-forming units of E. coli O157:H7 can result in serious illness.

Most E. coli infections are caused by eating undercooked meat contaminated with the bacteria, but outbreaks associated with fresh produce such as spinach are on the rise.

Ebner and Purdue graduate students Yingying Hong and Yanying Pan infected fresh spinach leaves and ground beef with about 10 million cells of E. coli, a far greater amount than typically found in contaminated food products, Ebner said. The researchers then treated the food with a "phage cocktail," a liquid containing three kinds of phages selected for their ability to quickly and efficiently kill E. coli. Using a variety of phages also helps prevent the bacteria from developing resistance.

After 24 hours, the treatment had reduced E. coli concentrations in the spinach, stored at room temperature, by more than 99.9 percent. E. coli dropped by more than 99.8 percent and about 99.8 percent in spinach after 48 and 72 hours, respectively.

In ground beef stored at room temperature, the phages cleaned up about 99 percent of E. coli bacteria within 24 hours. The number of E. coli in refrigerated and undercooked ground beef shrunk by about 68 percent and 73 percent, respectively.

"Bacteria have viruses just like we do," Ebner said. "We're taking what already exists in nature and concentrating it to have an impact on these bacteria."

The search-and-destroy methods of phages sound like the stuff of science fiction: Spaceship-like phages dock onto the receptor sites of a host bacterium cell and deploy a syringe-like device that penetrates the cell wall. They inject their own DNA into the host cell, transforming it into a phage-making factory. The cell assembles phages until it contains so many it explodes, releasing the next generation of phages to find new hosts. 

"Phages are the most abundant life forms on the planet - if you consider viruses to be alive," Ebner said. "You can eat thousands of phages just by licking your lips."

Ingesting phages does not pose a threat to human health because phages are highly host-specific, only targeting certain types of bacteria, said Ebner.

"Phage therapy is a way of using microbes beneficially, similar to using probiotics in yoghurt," he said.

Interest in using phages as antibacterial treatments has increased with the rise of antibiotic-resistant bacteria. The host specificity of phages can be an advantage over broad-spectrum antibiotics, which can wipe out both pathogenic and beneficial bacteria, Ebner said.

He said phage therapy is not a substitute for antibiotics, but "it can be very effective when used at specific time points and for shorter periods."

The paper was published in the Journal of Animal Science and is available at http://www.journalofanimalscience.org/content/early/2014/02/03/jas.2013-7272.full.pdf+html?sid=e81e1a65-f8f2-4b68-9c16-3335ca254edd 

Writer:  Natalie van Hoose, 765-496-2050, nvanhoos@purdue.edu

Source: Paul Ebner, 765-494-4820, pebner@purdue.edu

VIDEO:

A link to a video of Paul Ebner discussing how he uses bacteriophages to tackle foodborne pathogens is available at http://youtu.be/ZLnfjvZ4sRc 

Escherichia coli O157:H7 remains a foodborne pathogen of concern with infections associated with products ranging from ground beef to produce to processed foods. We previously demonstrated that phage-based technologies could reduce foodborne pathogen colonization in live animals. Here, we examined if a 3-phage cocktail could reduce E. coli O157:H7 in experimentally contaminated ground beef, spinach, and cheese. The 3 phages were chosen from our E. coli O157:H7 phage library based on their distinct origins of isolation, lytic ranges, and rapid growth (40- to 50-min life cycle). Two phages belonged to the Myoviridae family and the other phage belonged to the Siphoviridae family. The phage cocktail was added to ground beef, spinach leaves, and cheese slices contaminated with E. coli O157:H7 (10⁷ cfu) at a multiplicity of infection of 1. Phage treatment reduced (P < 0.05) the concentrations of E. coli O157:H7 by 1.97 log₁₀ cfu/mL in ground beef when stored at room temperature (24°C) for 24 h, 0.48 log₁₀ cfu/mL at refrigeration (4°C), and 0.56 log₁₀ cfu/mL in undercooked condition (internal temperature of 46°C). Likewise, phage treatment reduced (P < 0.05) E. coli O157:H7 by 3.28, 2.88, and 2.77 log₁₀ cfu/mL in spinach when stored at room temperature for 24, 48, and 72 h, respectively. Phage treatment, however, did not reduce E. coli O157:H7 concentrations in contaminated cheese. Additionally, 3 phage- resistant E. coli O157:H7 strains (309-PR [phage resistant] 1, 309-PR4, and 502-PR5) were isolated and characterized to test if phage resistance could limit long-term use of phages as biocontrol agents. Growth kinetics and adsorption assays indicated that phage resistance in strains 309-PR4 and 502-PR5 was mediated, at least in part, by prevention of phage adsorption. Phage resistance in strain 309-PR1 was the result of limited phage proliferation. Phage resistance was stably maintained in vitro throughout a 4-d subculture period in the absence of phage. No significant reductions in bacterial growth or cell adhesion were observed in resistant strains. Taken together, our results provide additional support for the use of phage to control E. coli O157:H7 in food products; however, the emergence of phage-resistant bacteria could limit the efficacy of phage products. Therefore, further studies are needed to develop resistance mitigation strategies to optimize phage-based technologies.  

Ag Communications: (765) 494-2722;
Keith Robinson, robins89@purdue.edu
Agriculture News Page