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Biocontrol of Foodborne Pathogens on Fresh Produce


GOAL Improving the safety of fresh produce by using bacteriophage against pathogens as an effective biocontrol measure.

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<p>NON-TECHNICAL SUMMARY:<br/> Consumption of fresh produce (fresh fruits and vegetables) can reduced risk of cardiovascular diseases, making these products a necessary component of healthy diet. However, concerns have been raised because of increased disease outbreaks associated with the consumption of contaminated produce. Recently, there has been an increase in the number of disease outbreaks associated with fresh produce contaminated with pathogens such as Escherichia coli O157:H7, Salmonella sp., Listeria monocytogenes, and others. The proposed project aims at developing new and promising control measures to provide safe product to the consumers and to minimize economic losses to the industry. These measures rely of biological agents known to attack and eliminate the pathogens; these are known as bacteriophages.
<p>APPROACH:<br/> PROCEDURE Isolation of anti-Salmonella bacteriophage from the environment and characterization of the isolates for potential control of the pathogen. Bacterial culture: Different serovars of Salmonella enterica will be used as the target microorganisms. Stock cultures, frozen at -80°C, will be transferred to Luria Bertani (LB) broth (Becton, Dickinson and Co., Franklin Lakes, NJ) and incubated overnight at 35°C. These cultures will be transferred again in LB and incubated at 35°C for 12 h with shaking for use in all isolation and purification experiments. Isolation of phage from raw food and environmental samples: Raw vegetables, milk and meat will be screened for presence of anti-Salmonella phage. Additionally, soil, compost, river water, and sewage effluent from the Columbus area will be screened for the presence of phage active against
Salmonella. Isolation method used previously in this laboratory will be applied. Purification of phage candidates: The presence of phage will be confirmed by spotting the isolated phage onto a lawn of the target microorganism on a plate of LB agar supplemented with CaCl2. Following overnight incubation at 35°C, zones of inhibition where the supernatant will be spotted, as indication of the presence of lytic bacteriophages. Positive samples will be further screened to obtain isolates of a single phage. Serial dilutions of the supernatant will be briefly incubated (45 min) at room temperature with the target microorganism then added to of LB soft agar (0.75% agar w/w) supplemented with CaCl2 and poured over a plate of LB agar. The pour plates will be incubated at 35°C for 24 h. Isolated plaque forming units (PFU) will be excised and re-incubated and filter sterilized before again
pour plating to obtain isolated PFUs. The plaques will be excised once more and the process repeated a second time to achieve isolation of a single candidate phage. Phage preparations: Stock lysates for the phage candidates will be prepared by incubation of purified phage with the host in LB broth supplemented with CaCl2 for 48 h at 35°C with shaking. All phage isolates will be incubated with the corresponding target Salmonella serovar. Following incubation, the cultures will be centrifuged (10,000 x g, 10 min) then filter sterilized and the resulting crude lysate will be stored at 4°C. Phage stability during storage: Crude lysates of the phage candidates will be held in storage at 4°C and the titer value for each lysate will be recorded at day 0, 10, 20, and 30 to assess the stability of the phage isolate during storage over time. Cross-reactivity: In addition to the
target Salmonella serovar used for phage isolation, activity of phages on other serovars will be examined. Use of bacteriophage to control Salmonella sp. on leafy greens. Bacterial culture: All Salmonella serovars to be used in these experiments are available in the culture collection of this laboratory. A loop-full of each of the frozen stock cultures, stored at -80°C, will be used to inoculate LB broth (Becton, Dickinson and Co.) and incubated at 35°C for 12 h with shaking. This activated overnight culture will be used to inoculated fresh LB broth and incubated again at 35°C for 12 h for use within experiments. Cells of Salmonella cultures will be harvested by centrifugation (10,000 x g, 3 min). The cells will be re-suspended in phosphate-buffered saline (PBS, pH 7.0) for use in final experiments. Phage preparations: Crude phage lysates will be prepared by incubation of
the phage stock with the host microorganism in LB broth supplemented with CaCl2 for 48 hr at 35°C with shaking. The cultures will be centrifuged (10,000 x g, 10 min) to separate solid matter from the supernatant which contains the phage. The supernatant will be filter-sterilized using a 0.22 µm filter (Millipore, Billerica, MA). The crude phage lysates will be purified by ultracentrifugation (28,000 x g, 3 h) to sediment the phage particles, followed by resuspension of the pellet in PBS. Phage preparations will be titered prior to use to determine its PFU/ml. Fresh produce: Lettuce and spinach will be obtained from a local grocery store (Columbus, OH) and held at 6°C until use. The produce will be cut into suitable size pieces and transferred to Petri dishes. Produce pieces will be treated with UV in a biosafety cabinet for 1 hr to eliminate background biota. The population
of microorganisms prior to UV treatment will be determined. Inoculation of fresh produce samples: Produce will be spot-inoculated with 10 µl of the 12-h bacterial culture in a biosafety cabinet. Inoculated produce will be allowed to dry for 1 h at ambient temperature prior to treatment. Treatments: Cut fresh produce will be dipped in a beaker of lysate with mixing for 2-5 min. Pieces will also be treated with a control treatment to account for reduction in bacterial population due to the action of the rinsing, but not the phage. This control will be a dip for 2-5 min in a beaker of LB broth with mixing. Cut fresh produce will be stored at either 4°C or 25°C. Produce samples will be transferred to stomacher bags for incubation following treatment. Recovery of bacteria: At periodic sampling time points, 0.1% peptone water will be added to produce samples. Samples will be
stomached for 2 min and the fluid will be diluted and spread-plated onto XLD agar plates. All plates will be incubated at 35°C for 24 h. Recovery of phage: Fluid from fresh produce samples will be filter sterilized using a 0.22 µm pore size syringe driven filter unit (Millipore, Billerica, MA) and used as the phage lysate. Serial dilutions of the phage lysates will be prepared and aliquots of these dilutions will be incubated with the host at room temperature for 45 min. The incubated aliquots will be added to molten LB soft agar supplemented with CaCl2 and overlaid on an XLD agar plate. Plates will be incubated at 35°C for 24 h and the resulting PFUs will be quantified to determine phage titer. Statistical analysis: The final, optimized experiments will be run in triplicate for each treatment condition. Salmonella populations and phage titers will be averaged for the
three replicates and converted to logarithmic values prior to statistical analysis. Comparisons between treatments, controls will be made using Statistical Analysis Software (SAS9.2, SAS Institute Inc., Carey, NC).

Yousef, Ahmed
Ohio State University
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