Engineering researchers have developed a new method to kill deadly pathogenic bacteria, including listeria, in food handling and packaging. This innovation represents an alternative to the use of antibiotics or chemical decontamination in food supply systems. Using nature as their inspiration, the researchers from Rensselaer Polytechnic Institute in Troy, New York State, have successfully attached cell lytic enzymes to food-safe silica nanoparticles, and created a coating with the demonstrated ability to selectively kill listeria. Listeria monocytogenes is the bacterium that causes the infection listeriosis. Severe cases can develop septicaemia or meningitis. Although a rare infection, its public health importance lies in its high case fatality rate, with a wide range of groups at increased risk of infection and harm – particularly pregnant women, those with a weakened immune system and older people. Between 10 and and 20 cases of listeria infection are reported to Ireland’s Health Protection Surveillance Centre every year. The foodborne bacteria causes an estimated 500 deaths every year in the United States. “Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces,” wrote the authors in their paper entitled ‘Enzyme-based Listericidal Nanocomposites’, which has been published in Scientific Reports from the Nature Publishing Group. The engineers have developed a coating that kills listeria on contact, even at high concentrations, within a few minutes and without affecting other bacteria. The lytic enzymes can also be attached to starch nanoparticles commonly used in food packaging. MODULAR METHOD This new method is modular, and by using different lytic enzymes, could be engineered to create surfaces that selectively target other deadly bacteria such as anthrax, said Prof Jonathan Dordick, vice president for research at Rensselaer, who helped lead the study. This research, which combined the expertise of chemical engineers and material scientists, took place in the Rensselaer Center for Biotechnology and Interdisciplinary Studies and the Rensselaer Nanoscale Science and Engineering Center for the Directed Assembly of Nanostructures. Collaborating with Prof Dordick were colleagues Ravi Kane, the P.K. Lashmet professor of chemical and biological engineering, and Linda Schadler, associate dean for academic affairs for the Rensselaer School of Engineering. “In this study, we have identified a new strategy for selectively killing specific types of bacteria. Stable enzyme-based coatings or sprays could be used in food supply infrastructure—from picking equipment to packaging to preparation—to kill listeria before anyone has a chance to get sick from it,” Prof Kane said. “What’s most exciting is that we can adapt this technology for all different kinds of harmful or deadly bacteria.” This most recent study builds upon the research team’s success in 2010 of creating a coating for killing methicillin-resistant Staphylococcus aureus (MRSA), the bacteria responsible for antibiotic resistant infections. While the previous coating was intended for use on surgical equipment and hospital walls, the development of a listeria-killing coating had the extra challenge of needing to be food-safe. Prof Dordick and the research team found their answer in lytic enzymes. Viruses that affect bacteria, called phages, inject their genetic material into healthy cells. The phage takes over a healthy cell, and in effect transforms the host cell into a little factory that creates more phages. Near the end of its life cycle, the original phage creates and releases lytic enzymes, which break down and make holes in cell walls of the infected bacteria. The manufactured phages escape through these holes and go on to infect other healthy cells. LYTIC ENZYMES Nature used lytic enzymes to break out of bacterial cells, Prof Dordick said, and the researchers worked for years to exploit the same lytic enzymes to break into bacteria such as MRSA and listeria. To stabilise the listeria bacteriophage endolysin Ply500, the engineers attached them to US Food and Drug Administration-approved silica nanoparticles to create an ultra-thin poly(hydroxyethyl methacrylate) film. The researchers also used maltose binding protein to affinity bind Ply500 to edible crosslinked starch nanoparticles commonly used in food packaging, via construction of a maltose-binding protein fusion. Both Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) within 24 hours at concentrations as high as 100,000 bacteria per milliliter — a significantly higher concentration than normally found in food contamination situations – in non-growth sustaining phosphate-buffered saline as well as under growth conditions on lettuce. “Starch is an inexpensive, edible material often sprayed into the packaging as a powder layer on meat product. We took advantage of the natural affinity of a maltose binding protein fused to Ply500, and biologically bound Ply500 to starch as a non-antibiotic, non-chemical agent for reducing the threat of listeria to our food supply,” Schadler said. Looking forward, the research team plans to continue investigating new methods for harnessing the power of lytic enzymes to selectively kill harmful bacteria. “This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure,” they concluded in the research paper. Along with Prof Dordick, Prof Kane and Schadler, co-authors on the paper are Rensselaer postdoctoral researchers Kusum Solanki, Naveep Grover, Elena Paskaleva, and Lillian Lee, and Rensselaer graduate students Patrick Downs, and Krunal Mehta. This research was supported with funding from Sealed Air Corporation.