Bacteriophages (phages) are viruses with specificity to attack and kill bacteria. This curious name originated from English (bacterium) and Greek (phagein “eat”); thus, defined as a virus that eats bacterium. They do not infect plants, animals, or human cells. Phages are abundant in nature and are part of the natural microflora in humans, plants, and animals.
Image credit: Claudia Narvaez, PhD
Phages have survived for millions of years, which is an indication of their capacity to overcome bacterial resistance mechanisms. An interesting feature of bacteriophages is that they are very specific to the type of bacteria (host) they infect; for example, to kill E. coli O157:H7, particular phages are used that only target E. coli O157:H7, and no other E. coli or other bacteria present within their environment.
Phages were discovered in 1915 and microbiologists began researching the viral nature of phages as well as their strengths and limitations in the field of medicine. However, soon after the discovery of antibiotics in 1928, the antibiotic golden era began and phage therapy research in the western world was virtually forgotten. During this period, new antibiotics were introduced and millions of metric tons of antibiotics have been employed in human medicine and agriculture. Subsequently, in the late 1930s, antibiotic resistance became a clear problem that has continued to grow to this day. In the last 60 years, many bacterial pathogens have evolved into multidrug-resistance (resistance against a variety of antibiotics) forms after antibiotic exposure. These bacteria are known as “superbugs.” Therefore, the use of antibiotics in humans and in agriculture is adding to the problem of antimicrobial resistance worldwide. Consequently, the public is advocating for a decrease or total elimination of the use of antibiotics as growth promoters and even antibiotic use as a prophylactic in livestock.
The search for new alternatives for antibiotics has pushed bacteriophages to the forefront of research. This research is not only increasing in the field of human medicine, but also there is a growing interest in their potential to be used in agriculture. Bacteriophages are being researched for usage as a biocontrol technology to reduce pathogens on vegetables and ready-to-eat foods throughout the production continuum.
Why Vegetables?
These days, consumers are more aware of human behaviors and habits that can impact the environment, including what they eat, where foods come from, how they are processed, and what preservatives have been added. In this context, minimally processed vegetables are ideal for health-conscious consumers. However, fresh produce is a potential source of foodborne illnesses mainly because many vegetables are consumed fresh (raw) and no steps are employed to effectively eliminate pathogens, prior to consumption. A variety of factors can influence the contamination of vegetables with foodborne pathogens. Among these contaminated irrigation water, the use of manure as a fertilizer, contaminated harvesting equipment as well as hygienic practices of workers in the fields, packing houses, and processing plants. The fruit and vegetable industry is very aware of contamination risks, and have dramatically improved food safety procedures in recent years. However, despite all efforts, foodborne outbreaks still occur.
The Shiga-toxigenic E. coli serogroups are among the top foodborne pathogens that have been associated with produce outbreaks. These mainly involve seven E. coli serogroups (O26, O45, O111, O103, O121, O145, and O157:H7). Salmonella and Listeria monocytogenes are other important foodborne pathogens that have been linked to fruit and vegetables.
Vegetable contamination with these pathogenic bacteria begins with their attachment to plant tissue. For example, researchers have shown that E. coli O157, E. coli O157:H7 prefers to attach to cut edges rather than to whole-leaf lettuce, it can attach in a short period of time and can only be partially removed using chlorinated water washes. However, only a few Shiga-toxigenic E. coli cells can cause disease, thus alternative methods to decrease or eliminate these pathogens from fresh produce are needed.
How It Works
Bacteriophages possess attributes to control foodborne pathogens in a unique fashion by infecting bacterial cells, destroying the bacteria, and producing more phages that can repeat the cycle. They also have a history of safe usage and have proven to be effective in reducing Salmonella, E. coli O157:H7, and Listeria monocytogenes in foods. With a research project funded by Ontario Ministry of Agriculture, Food and Rural Affairs, our research team was particularly interested in phages that will reduce Shiga-toxigenic E. coli on fresh lettuce. We tested eight phages. These phages originated from beef cattle manure as the targeted seven pathogenic E. coli serotypes are often found in cattle manure. This is because phages flourish where their targeted bacterial thrive. The phages we used in our laboratory were isolated and purified and then we multiplied the phages to thousands. All eight phages were finally pooled together to produce a phage cocktail. To evaluate the effectiveness of the phage cocktail, we first checked if the phages would be effective in killing bacteria at a refrigerated temperature of (2 degrees Celsius), since this is the temperature used to store fresh vegetables. Then we proceeded to spike lettuce with a cocktail containing all seven E. coli bacteria to simulate contamination.
Our results indicate that STEC bacteriophage mixtures can control some of the six Shiga-toxigenic E. coli on lettuce. The phage cocktail is very effective on lettuce against E. coli O157:H7, O145, and O26, serogroups that are frequently associated with foodborne outbreaks in produce. This phage intervention has the potential to be adopted by industry in order to decrease the foodborne risks associated with fresh produce. A very interesting finding was that these particular phages are more effective at refrigerated temperatures. It is normally reported that bacteriophages are more effective at 25 and 37 degrees Celsius, and it has been pointed out as a disadvantage for phages to be used in refrigerated food. Another disadvantage is that phages could potentially carry virulence or antibiotic resistance genes. Therefore, it is necessary to assure that phages intended for use in foods are not carrying undesirable genes through whole genome sequencing. This is the process used to determine the complete DNA sequence (all of the genes) of an organism’s genome.
Another issue is the development of resistance; however, this problem can be overcome when multiple phages are used in a mixture, as it overwhelms the bacteria with multiple phage attacks. This intervention can be used during lettuce washing and/or packaging steps without altering the flavor, color, or aroma of fresh produce.
Dr. Narvaez-Bravo is the assistant professor at the University of Manitoba, Food Science Department.
She has more than 10 years of experience working on research within the area of microbiology and food safety. Reach her at Claudia.narvaezbravo@ad.umanitoba.ca. Dr. McAllister is principal research scientist with Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, and has been with the organization for the past 25 years. Reach him at Tim.mcallister@agr.gc.a
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