Phage Mediated Biocontrol of Food Borne Bacteria

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Introduction

The [phyllosphere] refers

than 10^{^26} bacteria, Microbes that live in the Phyllosphere are called [Epiphytes]

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].

The need for control of pathogens during the manufacture of food is reflected by the incidence of foodborne bacterial infections. The number of cases of Listeriosis, for example, has been stabilized or is on the rise in many countries, especially in Europe, after having undergone a steep decline in the first part of the last 20 years. Similar trends can be observed for other foodborne infections, and new orally transmitted bacterial diseases are emerging [1]. Studies have also shown evidence that antibiotic resistance traits have entered the microflora of farm animals and the food produced [5].

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.

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 monocytogenes, the bacteria which causes listeriosis is also found on various retail foods and is ubiquitous in the environment [6].

Bacteria to Phage Ratio

"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)

Most food treated is solid and therefore the treatment of phages cannot diffuse to bacterial sites, unlike in a liquid medium. Simply 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. This was made evident by 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^{^4} PFU/mL of phage was added, compared to the 1.9% of cells that survived when 1^{^7} PFU/mL of phage was added [10]. On a solid matrix, the concentration of the bacterial host is not important once the concentration threshold of phage numbers enable it to cover the entire space [1]. Another point to consider is the doubling time of the specific bacteria at a particular environment. 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

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 (1).

Applications in the Food Industry

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.

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

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 (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 to see a photo of phyllospheric bacteria click the link microbial interactions

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 (1).

Key Microorganisms

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 (3).

Listeria monocytogenes

"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)" 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.

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. (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) Delmonte, N., Knief, C., Chaffron, S., 2009. "Community proteogenomics reveals insights into the physiology of phyllosphere." National Academy of Sciences.

(3) Machowicz-Stefaniak, Z., Krol, E. 2006. "Biotic effect of caraway phyllosphere fungi on the pathogenic fungus." Department of Plant Pathology University of Life Science. 2-8

(4)Suslow, T. 2005. "Microbial Food Safety IS Your Responsibility." Vegetable Research Information Center. 1-6.

(5) Howplantswork. 2009. "Life in the Phyllosphere: What Microbes Commonly Dwell on the Surface of Leaves?."

(6) Whipps, J.M. Hand, P. Pink, D. 2008. "Phyllosphere microbiology with special reference to diversity and plant genotype." 2-34

(7) Gardener, B., Frafel, D. 2002. "Biological Control of Plant Pathogens: Research, Commercialization, and Application in the USA." Plant Health Progress. 1-18

(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

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