Agriculture Science Today 012 – Phantastic Phage

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http://www.beatricebiologist.com/2010/09/its-probably-just-phage.html

Cartoon rendition of a bacteriophage used with permission of the wonderful Beatrice the Biologist. Check out her blog at: http://www.beatricebiologist.com/2010/09/its-probably-just-phage.html

This week Tim and Steph discuss bacteriophages and their uses in health and food safety.

What is a bacteriophage?

Bacteriophages are viruses that invade bacterial cells and cause the bacterium to lyse, or split open, and subsequently die. Bacteriophages have a very specific relationship with bacteria; they ignore every cell but the strain of bacteria they have evolved to inhabit. That makes them harmless to mammalian cells and harmless even to bacteria they are not specific for. This is different from broad-spectrum antibiotics that can wipe out commensal flora in the intestinal tract along with the infectious bacteria of interest.

When were bacteriophages discovered?

Bacteriophages were discovered twice, independently, by a British and then a French scientist between 1915 and 1917 . The first study that was published regarding the use of phages in treating infectious diseases of humans was in 1921 where scientists were able to use bacteriophages successfully to treat staphylococcal skin infections.

Why aren’t bacteriophages used in medicine anymore?

In 1928 penicillin was discovered which would eventually dominate the way we treat infectious diseases. The miracle antibiotic that is produced from Penicillium chrysogenum fungus pushed  phage therapy out of popular medicine before it became widely used. More and more antibiotics were discovered throughout the 1940’s and 50’s causing interest in phage therapy to decline in western medicine and nowadays it is not often discussed.

Why are we talking about phages today if they’re an old discovery?

In medicine, agriculture and other fields there is an increased interest in phages… this is due to fewer antibiotics being discovered and an alarming increase of multi-drug resistant pathogens. So while phages for medical therapy is not new, the use of phages in food safety is relatively new. In 2006, not that long ago, the FDA and USDA approved the first bacteriophage product that can come in contact with food; Listshield a cocktail of phages that target Listeria monocytogenes. Phage treatments are mixed usually with water and applied as a spray. Once on the food, the phages will inject their DNA material into their targeted bacterial cells, where they replicate until they burst the cell open thus controlling the pathogenic agent.

Recent research on bacteriophage use in E. coli O157:H7 illness prevention

For this episode topic we found a great scientific article from the Journal of Animal Sciences entitled “Development of bacteriophage treatments to reduce E. coli O157:H7 contamination of beef products and produce” by Hong et al., 2014. The paper is no longer open access but if you would like to read it you can find it here. The goal of Hong and colleagues in their paper was to determine whether the use of a phage-based treatments could limit pathogen contamination in food. First, they isolated phages specific for infecting E . coli O157:H7 and characterized them. Of the phages characterized, three potential phages were further tested for their ability to limit E. coli O157:H7 contamination in ground beef, spinach, and cheese. Lastly, they looked at the ability of E.coli to develop resistance to see whether this would be a problem for use of phages like it has been for antibiotics.

How does E. coli get in our food?

Ruminant animals (cows, goats, sheep) are the main reservoirs. 0157:H7 colonizes in the intestinal tract of, for instance, cattle and is passed to the environment through feces and then spread rapidly amongst the herd. It is during the processing step in the slaughterhouse where the pathogen can be transferred from the animal (from its hide, hooves or wherever the feces is located) to meat products. On vegetables such as spinach contamination can happen in field from bacteria in soils or irrigation water. Contamination can also occur during picking or at the packing house due to contaminated equipment or workers.

Selection of phage for use in the study

The environment is absolutely full of phages so you don’t need to look hard to find them but to find specific kinds it’s best to look where there is an abundance of their host bacteria. The researchers collected wastewater from wastewater treatment plants located throughout the state of Indiana and by using a series of laboratory methods (which we won’t bore you with here) isolated 16 different E. coli O157:H7 phages. Each phage was tested for its lytic abilities. What that means is each phage has its own ability to kill or ‘lyse’ bacteria. This is called its ‘lytic ability’. So, an E. coli O157:H7 phage with higher lytic ability would be more effective at killing an E. coli O157:H7 bacterium. What a ‘broad lytic range’ means is that it was able to lyse (or kill) greater than 95% of the bacteria). From this collection of 16 phages, they identified 3 optimal phages based on how well they infect the host and how fast, or slow, they grow (growth kinetics).

Effects of phage treatment on contaminated ground beef

Researchers gathered ground beef samples from local retail outlets and then inoculated them with the E. coli O157:H7. The three phages were mixed together in a cocktail in equal amounts and then applied to the meat. Phage treatment of E. coli O157:H7 contaminated beef was tested under 3 conditions (1) room temperature (2) refrigeration and (3) undercooking. Ground beef samples that were contaminated with E. coli O157:H7 but did not receive a phage treatment served as controls (always important in an experiment). After 24 hours of their testing conditions, the authors measured the amount of E. coli on the meat. Concentrations of E. coli O157:H7 in phage-treated ground beef were significantly less than those in untreated ground beef for both room temperature and refrigeration conditions. When contaminated beef samples were undercooked (internal temperature of 46°C), concentrations of E. coli O157:H7 in phage-treated ground beef were significantly less than those found in untreated, undercooked ground beef. However, there was no significant difference in E. coli O157:H7 concentrations between phage-treated and non-treated groups when contaminated beef was cooked to an internal temperature of 63°C.

Effect of phage treatment on contaminated spinach

Researchers choose three spinach leaves of similar sizes. Then they inoculated the leaves with E. coli by dropping a the E. coli mixture onto the leaves. There, of course, was a control group that was inoculated but did not receive any phage application. They then incubated the leaves at room temperature and measured the concentrations of E. coli at 24 h, 48 h, and 72 h post-treatment. They found that the application of the phage cocktail significantly reduced the concentration of viable E. coli O157:H7 on spinach surfaces and this was true for all three timepoints measured, after storage at room temperature for 24 h, 48 h and 72 h. So, phage treatment could potentially be very effective at reducing E. coli levels in our spinach.

Effect of phage treatment on cheese

When they did this same experiment as spinach only instead using E. coli contaminated cheese, there were no significant difference in concentrations of E. coli between cheese that had and had not been treated with the phage cocktail. We wondered if there was an antibacterial effect of cheese pH or possibly an effect on the phages.

Can bacteria become resistant to phages?

One of the concerns with any antimicrobial technology is the possibility for a bacteria, like E. coli, to develop resistance. While scientists hypothesize that phage resistance might be easier to overcome than resistance to antibiotics, it is still a concern and raises serious safety questions that must be researched. In previous studies, phage-resistant mutants of bacteria were detected within hours to days, especially when a single phage was used in an experiment. Hong and colleagues tested for phage resistance in their study. After a series of ‘spot inoculations’ and running other microbiological and biochemical assays, they did find resistance to some degree for all three phages. The mechanisms of phage resistance can be grouped into four major categories: adsorption prevention (the bacteria doesn’t allow the phage to attach), prevention of phage DNA entry (the bacteria doesn’t let phage DNA get in), degradation of phage nucleic acid (the bacteria degrades the phage DNA once it gets in), and abortive infection systems (the bacteria and phage both die). In this study, the authors determined that adsorption prevention was one of the main ways that bacteria were becoming resistant to the phage, however, it wasn’t the sole reason. Further studies are needed to determine the exact mechanisms of phage resistance.

Another concept that the authors investigated regarding phage resistance was something called Fitness Cost. Fitness is a central idea in evolution and describes the ability of, in this case bacteria, to survive and reproduce. Fitness cost is when some genetic change in the bacteria can cause decrease fitness. In this case, the question is, do bacteria that are resistant to phages have decreased fitness in the absence of phages compared to bacteria that do not have resistance. This phenomenon is also talked about a lot in regards to antibiotic resistant bacteria. Previous studies showed that when an antibiotic is taken out of the bacterial environment, the previously resistant bacteria have slow growth rates and more quickly to die compared to non-resistant bacteria; suggesting whatever made these bacteria resistant also decreases their overall fitness. So, the authors wanted to know ‘Do our phage-resistant mutants that we found have decreased fitness compared to the non-resistant mutants’. This is an important question because if phage treatment becomes mainstream in food safety and the issue of phage resistance becomes a problem, simply removing the phage treatment for a period of time may not solve the problem. The authors found that their phage-resistant mutants did not have decreased fitness compared to their non-resistant counterparts suggesting that if resistance developed, simply removing the phage treatment may not reverse resistance.

That’s all folks!

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P.S. If you’re interested in the mucus surfaces and phage science Tim mentioned you can read about it here.


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