Phage Mediated Biocontrol of Food Borne Bacteria: Difference between revisions

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==Introduction==
[[Image:Phyllosphere b gross.png|thumb|400px|right|]]


[[file:phage1.jpg|thumb|300px|right| Bacteriophages infecting a specific bacteria. Image courtesy of http://acdrupal.evergreen.edu/phage/node/14.]]


The [[http://en.wikipedia.org/wiki/Phyllosphere phyllosphere]] refers to the above-ground surfaces of a plant as a habitat for microorganisms, with a heavy emphasis on leaf surfaces. All plants are home to a wide variety of microorganism communities including bacteria, fungi, and yeasts. Some microorganisms benefit the plant, others are plant pathogens and can potentially damage or kill the host plant ([http://ucce.ucdavis.edu/files/filelibrary/5453/6558.PDF 4]). The majority of bacterial colonists on any given plant have no noticeable effect on plant growth or function.
==Introduction==
 
Estimates suggest that the roughly 1 billion square kilometers of worldwide leaf surfaces host more than 10<sup>^26</sup> bacteria, which are the most abundant colonizers of this habitat.  Overall, microbiota in this ecosystem are large enough to have an impact on both global carbon and nitrogen cycles. Further more the phyllosphere mircroorganisms influence their hosts at the level of the individual plants.


With repeated and rapid constant change of environmental conditions occurring on leaf surfaces, the phyllosphere is recognized as a hostile environment to bacteria. Leaf surfaces are often dry and temperatures can reach 40–55°C under intense sunlight. During the night, however, leaves are frequently wet with dew and at cool temperatures (5–10°C) ([http://www.pnas.org/content/106/38/16428.full 2]).
Food borne bacteria exist in all forms of foods humans consume on a daily basis. The control of bacterial pathogens present on fresh fruit and vegetables and ready to eat foods are of major concern since these foods do not generally undergo any further processing or cooking that would kill pathogens before consumption ([http://www.ncbi.nlm.nih.gov/pubmed/19416364 4]). A key reservoir for many human bacterial pathogens is livestock because animals are also subjected to bacterial infections and are contained within relatively enclosed environments ([http://cienciaviva.pt/rede/oceanos/2desafio/16.pdf 2]).


Microbes that live in the Phyllosphere are called [[http://en.wikipedia.org/wiki/Epiphyte Epiphytes]]
[[https://microbewiki.kenyon.edu/index.php/Phage_Therapy Phage therapy]] is the application of bacteriophages to bacterial infections of humans or animals with the goal of reducing bacterial load ([http://cienciaviva.pt/rede/oceanos/2desafio/16.pdf 2]). Bacteriophages are a bacterial parasite, ubiquitous in environment, and can infect over 140 bacterial species ([http://www.ncbi.nlm.nih.gov/pubmed/19416364 4]). They are host specific and can only infect and replicate within specific bacteria. This allows them to target pathogens commonly found in food without reducing the number of commensal bacteria. Phage mediated [[https://microbewiki.kenyon.edu/index.php/Biocontrol biocontrol]] of food borne bacteria is not only an effective means treating pathogenic infections, it is also a solution to the fast-emerging antibiotic resistant bacterial strains ([http://www.springerlink.com/content/hltle2y1cfe2eu3h/fulltext.pdf 5]).


==Physical environment==
==Physical environment==
Line 16: Line 13:
===Distribution of Phages===
===Distribution of Phages===


The leaf surface is exposed to rapidly changing temperature and relative humidity. Also the repeated alternation between presence and absence of moisture due to rain and dew ([http://www.iripz.pl/ftp/09_biotic.pdf 3]). The leaf also provides limited nutrient resources to bacterial colonistsThe rapid and severe changes in the physical conditions of the phyllosphere creates a hostile microbe environment.
Phages are widely distributed in the environment and represent part of the natural microbiological  flora of foods ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612219/ 9]). A study used to identify [[https://microbewiki.kenyon.edu/index.php/Salmonella Salmonella]]-specific phages isolated a total of 232 phages from 26 sampling sites which included broiler farms, poultry abattoirs, and wastewater plants ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]). Bacteriophages which target [[https://microbewiki.kenyon.edu/index.php/Escherichia Escherichia]]. coli are commonly present in sewage, hospital waste water, polluted rivers and fecal samples ([http://mic.sgmjournals.org/content/151/7/2133.full 7])E. coli phages have been recovered from fresh chicken, pork, ground beef, mushrooms, lettuce and other raw vegetables ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). [[https://microbewiki.kenyon.edu/index.php/Listeria Listeria]], the bacteria which causes listeriosis is also found on various retail foods and is ubiquitous in the environment ([http://www.ncbi.nlm.nih.gov/pubmed/11309061 6]).


Several factors may influence the microhabitat experienced by bacteria on leaves. First, the leaf itself is surrounded by a very thin laminar layer in which moisture emitted through stomata may be sequestered, alleviating the water that stress epiphytes ([http://ucce.ucdavis.edu/files/filelibrary/5453/6558.PDF 4]).
[[file:wikitable.jpg|thumb|450px|right| Table indicating the % of surviving cells when a certain concentration of phage is added, over a time interval. ([http://www.ncbi.nlm.nih.gov/pubmed/19076235 10])]]


===Bacteria to Phage Ratio===
===Bacteria to Phage Ratio===


Second, some cells in a leaf bacterial population, particularly in [[http://en.wikipedia.org/wiki/Plant_pathology plant-pathogenic]] populations locally invade the interior of the leaf, avoiding the stresses on the exterior of the leaf by residing in substomatal chambers or other interior locationsWhile some phytopathogens have the option of avoiding stresses, most epiphytes must tolerate them in some way (Lindow, S).
Based on kinetics, thermal motion-driven particle diffusion and mixing due to fluid flow or bacterial swimming will result in a higher rate of phage and bacterium encounters ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). When treating solid food, a sufficiently high number of phages is required to infect low numbers of bacteria due to the unlikelihood that they will come into contact.
[[Image:Leaf surface1.jpg|thumb|300px|right| "Under the microscope, aerial plant leaves resemble eerie landscapes, with deep gorges, tall peaks and gaping pits that riddle the waxy surface." -Leveau, J. (2009)]]
 
Epidermal cells produce hills and valleys that will determine the shape and size of low areas on the surface, which will influence the shape and spread of water droplets on the plant. The first contact between immigrating bacteria and a leaf normally occurs at the plant cuticle. The waxy layer, which has different three-dimensional crystalline structures on different plant species and can change as leaves age. These modifications limit passive diffusion of nutrients and water vapor from the plant's interior onto the surface and defines the hydrophobicity of the leaf. Thick waxy cuticles have thus been thought to interfere with bacterial colonization of plants by limiting diffusion of nutrients and inhibiting the wetting of the leaf surface (Lindow, S).
The concentration threshold of phage numbers is also a factor in reducing the number of bacteria. A study which showed the mean % of surviving Salmonella cells in a nutrient broth decreased significantly with an increase in phage concentration. After 120 minutes, 90.3 % of cells remained when 1.8x10<sup>^4</sup> PFU/mL of phage was added, compared to the 1.9% of cells that survived when 1.1x10<sup>^7</sup> PFU/mL of phage was added ([http://www.ncbi.nlm.nih.gov/pubmed/19076235 10]). However, if the number of target bacteria falls below a minimum number, the large number of phage required may render phage therapy impractical ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]).


===Interaction Surfaces===
===Interaction Surfaces===


Because the Phyllosphere is a hostile environment for the residing microorganisms  physical parameters contribute to stressful conditions, such as UV radiation, temperature shifts, and the presence of reactive oxygen species. Adaptation to stressful conditions was reflected by the detection of various proteins, assigned to diverse bacterial genera and detected in all analyzed samples. Among these proteins were superoxide dismutase, catalase, DNA protection proteins, chaperones, and proteins involved in the formation of the osmoprotectant trehalose ([http://aem.asm.org/cgi/content/full/69/4/1875#F1 1]).
Phages must be applied and retained near the treated surfaces in order to avoid wasteful phage washing and potential inactivation of virulence particles, particularly from wash fluids that clean processing areas ([http://www.ncbi.nlm.nih.gov/pubmed/21803890 8]). Phages need to be immobilized in the right orientation in order to obtain high capture efficacy of bacteria and reduce binding to non-specific bacteria. Through electrostatic interactions, studies have shown that negatively charged phages immobilized to positively charged cellulose membranes resulted in a lowered bacterial count for both Listeria and E. coli O157:H7 studies ([http://www.ncbi.nlm.nih.gov/pubmed/21803890 8]).


==Applications in the Food Industry==
==Applications in the Food Industry==
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===Treatment Methods===
===Treatment Methods===


There is evidence that bacteria form large and heterogeneous aggregates on plant surfaces. Microscopic examinations of colonized leaves show that plant surfaces have many epiphytes occuring on them in large mixed-bacterial-species aggregates that also harbor fungi. While large numbers of solitary bacterial cells occur on plants, a few large masses of apparently mixed bacterial species can be found.  
US Food and Drug Administration have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods ([http://www.ncbi.nlm.nih.gov/pubmed/19416364 4]). The actual application strategies can include dipping, spraying, or adding phages as a liquid to a larger volume of food material ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). The timing of phage application should also be considered with regards to the relative point of slaughter and/or packaging ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).


Such aggregates can constitute between 30 and 80% of the total bacterial population on certain plant species. These assemblages, with an extent and structure similar to those of biofilms that develop in aquatic habitats, are probably found only on long-lived leaves in moist climates such as the tropics or wet temperate regions such as the Pacific Northwest.
===Benefits===


===Benefits===
Compared to antibiotic treatment, phage mediated biocontrol only infect the targeted subset of bacteria, avoiding the imbalance of commensal flora often caused by broad-spectrum antibiotics. Phages are also self-limiting because they only replicate as long as the targeted bacterium is present ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Phages are also constantly evolving to circumvent their host’s defenses and resistant bacteria are often less fit than their phage-sensitive counterparts ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]).


The conglomerates on most other plants, while still sizable, are best known as aggregates. The formation of aggregates by bacteria on plants has major implications for the ability of these microbes to colonize and survive the harsh environment of the phyllosphere ([http://aem.asm.org/cgi/content/full/69/4/1875#F1 1]); it may provide them with a means to modify their immediate environment in the habitat . The production of extracellular polysaccharide (EPS), which is considered to form a major part of the bacterial aggregate matrix, may benefit epiphytes in the phyllosphere
[[file:bacteriophagecycle.gif|thumb|450px|right| "In other infected cells, phage development is repressed and phage DNA integrates into the bacterial chromosome. The resulting lysogenic cell can replicate indefinitely, but can be induced to return to the lytic cycle with the excision of phage DNA from the chromosome." Image courtesy of http://www.nature.com/nrg/journal/v4/n6/fig_tab/nrg1089_F1.html]]
to see a photo of phyllospheric bacteria click the link [http://aem.asm.org/cgi/content/full/69/4/1875/F1 microbial interactions]


===Safety===
===Safety===


Water availability is one of the most highly fluctuating factors on leaf surfaces. The heavy EPS slime within aggregates can shield the bacteria from desiccation stress by buffering the matric and osmotic potentials of their surroundings. Furthermore, EPS helps to protect plant-associated bacteria from reactive oxygen species, which are often encountered on plants. It has been demonstrated that aggregated bacteria resist oxidative stress better than planktonic bacteria ([http://aem.asm.org/cgi/content/full/69/4/1875#F1 1]).
Studies have show that E. coli phages have no significant effect on the E. coli found in the gut of mice and humans. A reason for this may be due to the fact that commensal E. coli found in test subjects live in niches not easily accessible to environmental phages. Because phages are present in a wide variety of foods consumed every day, it is clear that they are non-toxic to humans ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).  
There are concerns regarding temperate (lysogenic) phages that do not kill their hosts immediately, and can alter the host’s phenotype through genome integration ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Most phage genes should be silent, however certain phage virulence toxin genes can otherwise transform harmless bacteria into pathogens ([http://cienciaviva.pt/rede/oceanos/2desafio/16.pdf 2]). Therefore, the selection of an appropriate phage for the biocontrol of pathogenic bacteria is crucial.


==Key Microorganisms==
==Key Microorganisms==


===E. coli===
===E. coli===
Certain bacteria in the Phyllosphere can increase the saturation of leaves via production of compounds with surfactant properties. This ability occurred in 50% of the Pseudomonas strains tested. Because of the hydrophobic nature of the cuticle, it is likely that increased saturation of these habitats allows diffusion of substrates, making them more readily available to epiphytic bacteria (Lindow, S). Biosurfactants facilitate the moving of bacteria on the phylloplane, this phenomena was suggested for tolaasin, a toxin produced by Pseudomonas tolaasi. The water film created by the surfactant could spread the bacteria across the leaf surface to areas where nutrients are more abundant. The production of biosurfactants may be one trait which bacteria can alter their habitat to exploit it more efficiently ([http://www.iripz.pl/ftp/09_biotic.pdf 3]).  
 
The World Health Organization estimates that 5 million children die each year as a consequence of acute diarrhea, with E. coli being the cause of a third of the cases in developing countries.  E. coli O157:H7 is found in the feces of 7% of cattle at slaughter, and this becomes the source for meat contamination ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1])([http://mic.sgmjournals.org/content/151/7/2133.full 7]). Thus, phage therapy will be most effective in the biocontrol of meat production if used prior to slaughter.
 
Studies have shown that when is beef artificially contaminated with E. coli with a cocktail of three different phages, no viable bacteria were found in 7 out of 9 samples. Another study has shown the elimination of 94 - 100% of E.coli at higher phage doses on a variety of food products with hard surfaces ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).


===Listeria monocytogenes===
===Listeria monocytogenes===
[[image:Bio_interatcions.gif‎|thumb|350px|right|
 
"Schematic diagram representing various hypothetical bacterial-habitat modifications in the phyllosphere, such as the release of nutrients from plant cells and bacterial cell dispersal effected by the production of syringomycin, which may act both as a phytotoxin and as a surfactant (A); the release of saccharides from the plant cell wall, caused by bacterial secretion of auxin (B); and protection from environmental stresses via production of EPS in bacterial aggregates(C)"
L. monocytogenes is an opportunistic food borne pathogen that affects the young, the old, immunocompromised and the pregnant women population ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). It causes a serious disease called Listeriosis, with ill-effects ranging from gastroenteritis to septicaemia ([http://www.ncbi.nlm.nih.gov/pubmed/20002687 11]). It can tolerate high levels of salt content, can grow at acidic pH values and can still replicate at temperatures below 1 degrees celsius ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612219/ 9]). L. monocytogenes is usually killed during pasteurization or other heat treatments ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]), however it has been isolated from ready-to-eat foods such as milk, cheese, cold-cut meats, smoked fish, seafood and vegetables ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612219/ 9]). No clear reservoir has been identified, but contaminations can occur via ingredients, factory workers and equipment ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).
Communities in the phyllosphere are thought to be limited by carbon availability, and it may be expected that access to carbon compounds on leaves is a major determinant of epiphytic colonization. There is evidence that small amounts of nutrients, such as simple sugars (glucose, fructose, and sucrose) leach from the interior of the plant.]]
 
Studies have shown that cocktails of different L. monocytogenes phages caused a reduction of 2 to 4.6 logs of bacteria on apple slices ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). The addition of broad-host-range phages such as A511 and P100 to liquid foods caused bacterial counts to drop rapidly below levels of detection. On solid foods, these phages reduced L. monocytogene counts by up to 5 log units ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). In 2006, the US Food and Drug Administration approved the use of phage cocktail for use in ready-to-eat meats to prevent L. monocytogenes contamination ([http://www.ncbi.nlm.nih.gov/pubmed/19416364 4]).


===Salmonella===
===Salmonella===
Communities in the phyllosphere are thought to be limited by carbon availability, and it may be expected that access to carbon compounds on leaves is a major determinant of epiphytic colonization. There is evidence that small amounts of nutrients, such as simple sugars (glucose, fructose, and sucrose) leach from the interior of the plant. ([http://aem.asm.org/cgi/content/full/69/4/1875#F1 1]).


Salmonella causes diarrhea and is one of the principal causes of food-borne illness on a global scale ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Birds, particularly chicken remains to be the main reservoir by which this bacteria enters the food production system, however it is not the only reservoir, thus other food products can be contaminated ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1])[3]. Phage therapy would not introduce new biological agent into the food chain since the Salmonella phage is readily isolated from poultry ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]). Because contamination of livestock is the main cause of Salmonella diseases, phage intervention should occur at the pre-slaughter level and post-slaughter level ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).
Studies have shown that bacteriophages can reduce cecal colonization of S. enterica in commercial broiler chickens ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]). In post-slaughter treatment, the Salmonella phage Felix O1 caused a 2 log reduction of bacteria in hot dogs ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). The US Environmental Protection Agency approved use of a Salmonella phage product to be sprayed or used as a wash on chicken prior to slaughter ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).


==Current Research==
==Current Research==
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===Bacterial Resistance to Phage Therapy===
===Bacterial Resistance to Phage Therapy===


Bacteria and their bacteriophage are constantly co-evolving. A study showed that E. coli O157, when incubated with phage PP01 for 200 hours, developed a series of mutants which differed in colony morphology, nature of phage receptors OmpC and LPS, and phage susceptibility [7]. The phage responded by evolving a broadened host range [7]. A trade off was observed between resistance to phage and competitiveness with parental strains for resources. For phage resistant strains to be selected for in the wild, they must also compete with many other strains that do not feel this phage pressure (unlike competing again only the phage-susceptible ancestor in the laboratory) [7]. If phage selective pressure is low, such mutants cannot be expected to present any danger in long-term phage based intervention ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Depending on the phage however, many bacteria are favoured in this co-evolutionary arms race (some resistance in certain strains even come without a metabolic cost) [7], thus bacterial resistance may still pose to be a problem in the future.
Bacteria and their bacteriophage are constantly co-evolving. A study showed that E. coli O157, when incubated with phage PP01 for 200 hours, developed a series of mutants which differed in colony morphology, nature of phage receptors OmpC and LPS, and phage susceptibility ([http://mic.sgmjournals.org/content/151/7/2133.full 7]). The phage responded by evolving a broadened host range ([http://mic.sgmjournals.org/content/151/7/2133.full 7]). A trade off was observed between resistance to phage and competitiveness with parental strains for resources. For phage resistant strains to be selected for in the wild, they must also compete with many other strains that do not feel this phage pressure (unlike competing again only the phage-susceptible ancestor in the laboratory) ([http://mic.sgmjournals.org/content/151/7/2133.full 7]). If phage selective pressure is low, such mutants cannot be expected to present any danger in long-term phage based intervention ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Depending on the phage however, many bacteria are favoured in this co-evolutionary arms race (some resistance in certain strains even come without a metabolic cost) ([http://mic.sgmjournals.org/content/151/7/2133.full 7]), thus bacterial resistance may still pose to be a problem in the future.


===Commercial Production of Phages===
===Commercial Production of Phages===


In order for phages to be effective in phage-mediated biocontrol, studies must be tested under conditions which resemble commercial practices. For zoonotic bacteria such as Salmonella, there is need to determine the optimal timing and delivery of bacteriophage in a real-life poultry industry setting [3]. In order to have this intervention be scaled up for commercial production, cost-effectiveness vs. efficacy in real-life application will need to be assessed ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Market acceptance by the food industry and the consumer will need to occur before it can be considered an ideal antibacterial agent ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).
In order for phages to be effective in phage-mediated biocontrol, studies must be tested under conditions which resemble commercial practices. For zoonotic bacteria such as Salmonella, there is need to determine the optimal timing and delivery of bacteriophage in a real-life poultry industry setting ([http://www.ncbi.nlm.nih.gov/pubmed/17526794 3]). In order to have this intervention be scaled up for commercial production, cost-effectiveness vs. efficacy in real-life application will need to be assessed ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]). Market acceptance by the food industry and the consumer will need to occur before it can be considered an ideal antibacterial agent ([http://www.ncbi.nlm.nih.gov/pubmed/20214608 1]).


==References==
==References==
Line 75: Line 78:
(1) Hagens, S., Loessner, M.J. “Bacteriophage for Biocontrol of Foodborne Pathogens: Calculations and Considerations.” Current Pharmaceutical Biotechnology 2010, 11, 58-68
(1) Hagens, S., Loessner, M.J. “Bacteriophage for Biocontrol of Foodborne Pathogens: Calculations and Considerations.” Current Pharmaceutical Biotechnology 2010, 11, 58-68


(2) Delmonte, N., Knief, C., Chaffron, S., 2009. "Community proteogenomics reveals insights into the physiology of phyllosphere." National Academy of Sciences.
(2) Goodridge, L., Abedon, S.T. “Bacteriophage biocontrol and bioprocessing: Application of phage therapy to industry.” Cienciava.pt 2003, 53 (6), 254-262
 
(3) Atterbury, R.J., Van Bergen, M.A.P., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer, A., Wagenaar, J.A., Allen, V.M., Barrow, P.A. “Bacteriophage Therapy to Reduce Salmonella Colonization of Broiler Chickens.” Appl. Environ. Microbiol 2007, 73 (14), 4543-4549
 
(4) O’Flaherty, S., Paul Ross, R., Coffey, A. “Bacteriophage and their lysins for elimination of infectious bacteria.” Federation of European Microbiological Societies 2009, 33, 801-819
 
(5) Teuber, M. “Spread of antibiotic resistance with food-borne pathogens.” Cellular and Molecular Life Sciences 1999, 56, 755-763


(3) Machowicz-Stefaniak, Z., Krol, E. 2006. "Biotic effect of caraway phyllosphere fungi on the pathogenic fungus." Department of Plant Pathology
(6) Walsh, D., Duffy, G., Sheridan, J.J., Blair, I.S., McDowell, D.A. “Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods.” Journal of Applied Microbiology 2001, 90, 517-522
University of Life Science. 2-8


(4)Suslow, T. 2005. "Microbial Food Safety IS Your Responsibility." Vegetable Research Information Center. 1-6.
(7) Brussow, H. “Phage therapy: the Escherichia coli experience.” Microbiology 2005, 151, 2133-2140


(5) Howplantswork. 2009. "Life in the Phyllosphere: What Microbes Commonly Dwell on the Surface of Leaves?.
(8) Anany, H., Chen, W., Pelton, R., Griffiths, M.W. “Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes.” Appl. Environ. Microbiol. 2011, 77 (18), 6379-6387


(6) Whipps, J.M. Hand, P. Pink, D. 2008. "Phyllosphere microbiology with special reference to diversity and plant genotype." 2-34
(9) Guenther, S., Huwyler, D., Richard, S., Loessner, M.J. “Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods.” Appl. Environ. Microbiol. 2008, 75 (1), 93-100


(7) Gardener, B., Frafel, D. 2002. "Biological Control of Plant Pathogens: Research, Commercialization, and Application in the USA." Plant Health Progress. 1-18
(10) Bigwood, T., Hudson, J.A., Billington, C. “Influence of host and bacteriophage concentrations on the inactivation of food-borne pathogenic bacteria by two phage.” Federation of European Microbiological Societies 2009, 291, 59-64


(8) Abell, G., Richter, A. 2008. "Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica." The IMSE journal. 2, 561–570
(11) Allerberger, F., Wagner, M. “Listeriosis: a resurgent foodborne infection.” Austrian Agency for Health and Food Safety 2009, 16, 16-23


Edited by student of Angela Kent at the University of Illinois at Urbana-Champaign.
Edited by student of Angela Kent at the University of Illinois at Urbana-Champaign.


<!-- Do not edit or remove this line -->[[Category:Pages edited by students of Angela Kent at the University of Illinois at Urbana-Champaign]]
<!-- Do not edit or remove this line -->[[Category:Pages edited by students of Angela Kent at the University of Illinois at Urbana-Champaign]]

Latest revision as of 04:13, 27 December 2012

This student page has not been curated.
Bacteriophages infecting a specific bacteria. Image courtesy of http://acdrupal.evergreen.edu/phage/node/14.

Introduction

Food borne bacteria exist in all forms of foods humans consume on a daily basis. The control of bacterial pathogens present on fresh fruit and vegetables and ready to eat foods are of major concern since these foods do not generally undergo any further processing or cooking that would kill pathogens before consumption (4). A key reservoir for many human bacterial pathogens is livestock because animals are also subjected to bacterial infections and are contained within relatively enclosed environments (2).

[Phage therapy] is the application of bacteriophages to bacterial infections of humans or animals with the goal of reducing bacterial load (2). Bacteriophages are a bacterial parasite, ubiquitous in environment, and can infect over 140 bacterial species (4). They are host specific and can only infect and replicate within specific bacteria. This allows them to target pathogens commonly found in food without reducing the number of commensal bacteria. Phage mediated [biocontrol] of food borne bacteria is not only an effective means treating pathogenic infections, it is also a solution to the fast-emerging antibiotic resistant bacterial strains (5).

Physical environment

Distribution of Phages

Phages are widely distributed in the environment and represent part of the natural microbiological flora of foods (9). A study used to identify [Salmonella]-specific phages isolated a total of 232 phages from 26 sampling sites which included broiler farms, poultry abattoirs, and wastewater plants (3). Bacteriophages which target [Escherichia]. coli are commonly present in sewage, hospital waste water, polluted rivers and fecal samples (7). E. coli phages have been recovered from fresh chicken, pork, ground beef, mushrooms, lettuce and other raw vegetables (1). [Listeria], the bacteria which causes listeriosis is also found on various retail foods and is ubiquitous in the environment (6).

Table indicating the % of surviving cells when a certain concentration of phage is added, over a time interval. (10)

Bacteria to Phage Ratio

Based on kinetics, thermal motion-driven particle diffusion and mixing due to fluid flow or bacterial swimming will result in a higher rate of phage and bacterium encounters (1). When treating solid food, a sufficiently high number of phages is required to infect low numbers of bacteria due to the unlikelihood that they will come into contact.

The concentration threshold of phage numbers is also a factor in reducing the number of bacteria. A study which showed the mean % of surviving Salmonella cells in a nutrient broth decreased significantly with an increase in phage concentration. After 120 minutes, 90.3 % of cells remained when 1.8x10^4 PFU/mL of phage was added, compared to the 1.9% of cells that survived when 1.1x10^7 PFU/mL of phage was added (10). However, if the number of target bacteria falls below a minimum number, the large number of phage required may render phage therapy impractical (3).

Interaction Surfaces

Phages must be applied and retained near the treated surfaces in order to avoid wasteful phage washing and potential inactivation of virulence particles, particularly from wash fluids that clean processing areas (8). Phages need to be immobilized in the right orientation in order to obtain high capture efficacy of bacteria and reduce binding to non-specific bacteria. Through electrostatic interactions, studies have shown that negatively charged phages immobilized to positively charged cellulose membranes resulted in a lowered bacterial count for both Listeria and E. coli O157:H7 studies (8).

Applications in the Food Industry

Treatment Methods

US Food and Drug Administration have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods (4). The actual application strategies can include dipping, spraying, or adding phages as a liquid to a larger volume of food material (1). The timing of phage application should also be considered with regards to the relative point of slaughter and/or packaging (1).

Benefits

Compared to antibiotic treatment, phage mediated biocontrol only infect the targeted subset of bacteria, avoiding the imbalance of commensal flora often caused by broad-spectrum antibiotics. Phages are also self-limiting because they only replicate as long as the targeted bacterium is present (1). Phages are also constantly evolving to circumvent their host’s defenses and resistant bacteria are often less fit than their phage-sensitive counterparts (3).

"In other infected cells, phage development is repressed and phage DNA integrates into the bacterial chromosome. The resulting lysogenic cell can replicate indefinitely, but can be induced to return to the lytic cycle with the excision of phage DNA from the chromosome." Image courtesy of http://www.nature.com/nrg/journal/v4/n6/fig_tab/nrg1089_F1.html

Safety

Studies have show that E. coli phages have no significant effect on the E. coli found in the gut of mice and humans. A reason for this may be due to the fact that commensal E. coli found in test subjects live in niches not easily accessible to environmental phages. Because phages are present in a wide variety of foods consumed every day, it is clear that they are non-toxic to humans (1). There are concerns regarding temperate (lysogenic) phages that do not kill their hosts immediately, and can alter the host’s phenotype through genome integration (1). Most phage genes should be silent, however certain phage virulence toxin genes can otherwise transform harmless bacteria into pathogens (2). Therefore, the selection of an appropriate phage for the biocontrol of pathogenic bacteria is crucial.

Key Microorganisms

E. coli

The World Health Organization estimates that 5 million children die each year as a consequence of acute diarrhea, with E. coli being the cause of a third of the cases in developing countries. E. coli O157:H7 is found in the feces of 7% of cattle at slaughter, and this becomes the source for meat contamination (1)(7). Thus, phage therapy will be most effective in the biocontrol of meat production if used prior to slaughter.

Studies have shown that when is beef artificially contaminated with E. coli with a cocktail of three different phages, no viable bacteria were found in 7 out of 9 samples. Another study has shown the elimination of 94 - 100% of E.coli at higher phage doses on a variety of food products with hard surfaces (1).

Listeria monocytogenes

L. monocytogenes is an opportunistic food borne pathogen that affects the young, the old, immunocompromised and the pregnant women population (1). It causes a serious disease called Listeriosis, with ill-effects ranging from gastroenteritis to septicaemia (11). It can tolerate high levels of salt content, can grow at acidic pH values and can still replicate at temperatures below 1 degrees celsius (9). L. monocytogenes is usually killed during pasteurization or other heat treatments (1), however it has been isolated from ready-to-eat foods such as milk, cheese, cold-cut meats, smoked fish, seafood and vegetables (9). No clear reservoir has been identified, but contaminations can occur via ingredients, factory workers and equipment (1).

Studies have shown that cocktails of different L. monocytogenes phages caused a reduction of 2 to 4.6 logs of bacteria on apple slices (1). The addition of broad-host-range phages such as A511 and P100 to liquid foods caused bacterial counts to drop rapidly below levels of detection. On solid foods, these phages reduced L. monocytogene counts by up to 5 log units (1). In 2006, the US Food and Drug Administration approved the use of phage cocktail for use in ready-to-eat meats to prevent L. monocytogenes contamination (4).

Salmonella

Salmonella causes diarrhea and is one of the principal causes of food-borne illness on a global scale (1). Birds, particularly chicken remains to be the main reservoir by which this bacteria enters the food production system, however it is not the only reservoir, thus other food products can be contaminated (1)[3]. Phage therapy would not introduce new biological agent into the food chain since the Salmonella phage is readily isolated from poultry (3). Because contamination of livestock is the main cause of Salmonella diseases, phage intervention should occur at the pre-slaughter level and post-slaughter level (1).

Studies have shown that bacteriophages can reduce cecal colonization of S. enterica in commercial broiler chickens (3). In post-slaughter treatment, the Salmonella phage Felix O1 caused a 2 log reduction of bacteria in hot dogs (1). The US Environmental Protection Agency approved use of a Salmonella phage product to be sprayed or used as a wash on chicken prior to slaughter (1).

Current Research

Bacterial Resistance to Phage Therapy

Bacteria and their bacteriophage are constantly co-evolving. A study showed that E. coli O157, when incubated with phage PP01 for 200 hours, developed a series of mutants which differed in colony morphology, nature of phage receptors OmpC and LPS, and phage susceptibility (7). The phage responded by evolving a broadened host range (7). A trade off was observed between resistance to phage and competitiveness with parental strains for resources. For phage resistant strains to be selected for in the wild, they must also compete with many other strains that do not feel this phage pressure (unlike competing again only the phage-susceptible ancestor in the laboratory) (7). If phage selective pressure is low, such mutants cannot be expected to present any danger in long-term phage based intervention (1). Depending on the phage however, many bacteria are favoured in this co-evolutionary arms race (some resistance in certain strains even come without a metabolic cost) (7), thus bacterial resistance may still pose to be a problem in the future.

Commercial Production of Phages

In order for phages to be effective in phage-mediated biocontrol, studies must be tested under conditions which resemble commercial practices. For zoonotic bacteria such as Salmonella, there is need to determine the optimal timing and delivery of bacteriophage in a real-life poultry industry setting (3). In order to have this intervention be scaled up for commercial production, cost-effectiveness vs. efficacy in real-life application will need to be assessed (1). Market acceptance by the food industry and the consumer will need to occur before it can be considered an ideal antibacterial agent (1).

References

(1) Hagens, S., Loessner, M.J. “Bacteriophage for Biocontrol of Foodborne Pathogens: Calculations and Considerations.” Current Pharmaceutical Biotechnology 2010, 11, 58-68

(2) Goodridge, L., Abedon, S.T. “Bacteriophage biocontrol and bioprocessing: Application of phage therapy to industry.” Cienciava.pt 2003, 53 (6), 254-262

(3) Atterbury, R.J., Van Bergen, M.A.P., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer, A., Wagenaar, J.A., Allen, V.M., Barrow, P.A. “Bacteriophage Therapy to Reduce Salmonella Colonization of Broiler Chickens.” Appl. Environ. Microbiol 2007, 73 (14), 4543-4549

(4) O’Flaherty, S., Paul Ross, R., Coffey, A. “Bacteriophage and their lysins for elimination of infectious bacteria.” Federation of European Microbiological Societies 2009, 33, 801-819

(5) Teuber, M. “Spread of antibiotic resistance with food-borne pathogens.” Cellular and Molecular Life Sciences 1999, 56, 755-763

(6) Walsh, D., Duffy, G., Sheridan, J.J., Blair, I.S., McDowell, D.A. “Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods.” Journal of Applied Microbiology 2001, 90, 517-522

(7) Brussow, H. “Phage therapy: the Escherichia coli experience.” Microbiology 2005, 151, 2133-2140

(8) Anany, H., Chen, W., Pelton, R., Griffiths, M.W. “Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes.” Appl. Environ. Microbiol. 2011, 77 (18), 6379-6387

(9) Guenther, S., Huwyler, D., Richard, S., Loessner, M.J. “Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods.” Appl. Environ. Microbiol. 2008, 75 (1), 93-100

(10) Bigwood, T., Hudson, J.A., Billington, C. “Influence of host and bacteriophage concentrations on the inactivation of food-borne pathogenic bacteria by two phage.” Federation of European Microbiological Societies 2009, 291, 59-64

(11) Allerberger, F., Wagner, M. “Listeriosis: a resurgent foodborne infection.” Austrian Agency for Health and Food Safety 2009, 16, 16-23

Edited by student of Angela Kent at the University of Illinois at Urbana-Champaign.