BIOCONTROL OF FOODBORNE PATHOGENS ON FRESH PRODUCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1000648
• Project No.: OHO01311
• Project Start Date: Oct 1, 2013
• Project End Date: Sep 30, 2018

Project Director
Yousef, AH, EL.

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Food Science & Technology

Non Technical Summary
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.

Animal Health Component

0%

Research Effort Categories

Basic

20%

Applied

50%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4099	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

fresh

porduce.

pathogens.

intervention.

bacteriophage.

biocontrol.

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

Project Methods
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).

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:- Fresh produce industry - Safety-conscious consumers Changes/Problems:Changes were made to make the research more applicable to the food industry. For example, we discovered that when we inoculate pathogens on produce to study its decontamination with ozone,the size of inoculum affected the degree of decontamination. In fact, realistic inoculum that mimicked natural contamination produced the best results. What opportunities for training and professional development has the project provided?Training a grdauate student (Mustafa Yesil) who obtained his Ph. D. in 2017 How have the results been disseminated to communities of interest?Publications were shared with selected fresh produce processors What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? - Developed new approach to treat fresh produce with ozone - Discovered a new bacteriophage that can be applied to fresh produce to control Escherichia coli O157:H7

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: 153. Yesil, M., Huang, E., Yang, X., and Yousef, A.E. 2017. Complete genome sequence of Escherichia phage OSYSP. Genome Announc. 5:e00880-17.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: 154. Yesil, M., Kasler, D.R., Huang, E., and Yousef, A.E. 2017. Efficacy of gaseous ozone application during vacuum cooling against Escherichia coli O157:H7 on spinach leaves as influenced by bacterium population size. J. Food Prot. 80:1066-1071.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:- Fresh Produce Industry - Scientific community interested in fresh produce safety - USDA NIFA proposal reviewers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two students and a technician have been trained to decontaminate fresh produce. How have the results been disseminated to communities of interest?- Publications listed earlier. -Nonthermal technology workshops/symposia(May, 2017, Chicago, IL and October 2017, Connell) What do you plan to do during the next reporting period to accomplish the goals?Achieve the stated objectives and finalize the project.

Impacts
What was accomplished under these goals? 1. A new bacteriophage (Escherichia phage OSYSP) has been fully characterized and its utility as a biocontrol agent has been confirmed. Detail of the genome of our phage can be found at this link:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646388/ 2. Developing an effective method for decontamination of fresh produce by combination of bacteriophage and ozone gas: Treatment of spinach leaves with gaseous ozone and bacteriophage alone or in combination significantly (p < 0.05) reduced the viable E. coli O157:H7 populations on produce. Microbial reductions by gaseous ozone and bacteriophage only treatments were ranged from 1.8 to 3.4 log CFU/g and 1.7 to 3.4 log CFU/g, respectively. Sequential treatments of ozone followed by bacteriophage produced a synergy and reduced E. coli O157:H7 by 5.2 log CFU/g on spinach leaves. When the antimicrobial application order was reversed, additive inactivation responses were observed. Irrespective to the antibacterial application order in sequential treatments, no E. coli O157:H7 survivors were detected (p < 0.05) at lower inoculum sizes tested. The potent pathogen intervention techniques of this study could be easily integrated into conventional fresh produce processing with minimal changes as post-harvest chlorine spray and industrial chlorine-wash could be replaced with bacteriophage application, and combined with a gaseous ozone treatment during vacuum cooling.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yesil, M., Kasler, D.R., Huang, E., and Yousef, A.E. 2017. Efficacy of gaseous ozone application during vacuum cooling against Escherichia coli O157:H7 on spinach leaves as influenced by bacterium population size. J. Food Prot. 80:1066-1071.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Fresh produce Industry. Consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergrauate student has been trained and qualified to join a graduate program at the Ph. D. level How have the results been disseminated to communities of interest?Published in a scientific journal What do you plan to do during the next reporting period to accomplish the goals?Optimize the process developed in the previous phase

Impacts
What was accomplished under these goals? Developing and Optimizing Bacteriophage Treatment to Control Enterohemorrhagic Escherichia coli on Fresh Produce: Bacteriophages are potentially useful in controlling foodborne pathogens on minimally processed products since phage application is a non-destructive treatment. The purpose of this study was to evaluate the efficacy of a newly isolated environmental bacteriophage against enterohemorrhagic Escherichia coli on fresh produce, ?

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Snyder, A.B., Perry, J.J., and Yousef, A.E. 2016. Developing and optimizing bacteriophage treatment to control enterohemorrhagic Escherichia coli on fresh produce. Int. J. Food Microbiol. 236:90-97.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Fruit and vegetable producers and processors; scientists working in food safety; consumers; extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project involved training a graduate student while working toward his Ph.D. degree How have the results been disseminated to communities of interest?The study has been shared during the 2015 annual meeting of the Institute of Food Technologists, held in June Chicago, IL What do you plan to do during the next reporting period to accomplish the goals?Optimize the treatments tested so far, to maximize their effectiveness against pathogens while protecting product quality

Impacts
What was accomplished under these goals? A an effective combination treatment (phage + ozone) can be used by used to decontaminate fresh produce against enterohemorrhagic E. coli Sequential application of bacteriophage and gaseous zone for the inactiviation of escherichia coli O157:H7 on fresh produce. Fresh produce related foodborne outbreaks call for innovative technologies in order to protect the safety of the consumers. These technologies also should prevent economic losses for the fresh produce industry due to contaminated produce. Chlorine is used by industry for decontamination of fresh produce. However, at the permitted levels of use, chlorine does not generate considerable microbial inactivation. Additionally, concerns have been raised due to overuse of chlorine and formation of possible carcinogenic side products of chlorinated water used to wash produce. Ozone and bacteriophage do not have the drawbacks of chlorine, and thus can serve as alternatives to this commonly used sanitizer. The studies that we conducted revealed that an isolated and characterized lytic phage and gaseous ozone treatment individually and when applied sequentially gave significantly higher inactivation of Escherichia coli O157:H7 on spinach leaves when compared to control treatments. In the first study, we investigated the efficacy of gaseous ozone for the inactivation of different contamination levels of E. coli on spinach leaves. The E. coli reductions obtained were 0.2 log CFU/g at the high, and 2.1 log CFU/g at the low inoculum level, when compared to inoculated untreated spinach leaves. Ozone treatment of leaves inoculated at 7.3 log CFU/g significantly (p<0.05) increased the inactivation of E. coli compared to the higher inoculum level. Additionally, spinach leaves were inoculated with 3.2 log CFU/g of E. coli O157:H7 and most probable number technique was employed for the detection of low level of viable cells after ozone treatment. MPN estimates of the target pathogen was <3 MPN/g (i.e., greater than a 3 log reduction). In the second study, we have studied the efficacy of spraying E. coli-specific bacteriophage on produce after harvest and subsequently treating the produce with gaseous ozone during the vacuum cooling for the inactivation of two different inoculum levels of E. coli O157:H7. At the high inoculum level, phage treatment significantly (p<0.05) reduced the target pathogen count by a 2 log CFU/g when compared to inoculated untreated control. The sequential process significantly decreased the E. coli population by a 4 log CFU/g on spinach leaves compared to untreated control. Populations of the likely pathogen contaminants on freshly harvested leaves are expected to be less than the populations used in the aforementioned experiment. When phage and gaseous ozone applied to close to realistic contamination of level of spinach, it reduced the population of pathogen to lower than the detection limit, 1 log CFU/g. The color and texture of the treated pre-bagged spinach leaves were comparable to the inoculated untreated spinach leaves. Regardless of inoculum size for the sequential application of gaseous ozone with another hurdle technology, i.e., phages, promises the enhanced efficacy of gaseous ozone against the contaminants. Due to the superior efficacy of gaseous ozone found at lower inoculum sizes, a lower ozone concentration could be used to achieve better produce quality along with microbiological quality. This studied decontamination method would be improved by using phage cocktails and would be easy to implement into existing practices of the fresh produce industry.

Publications

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: 1. Fresh produce industry 2. Consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student is now pusuing his Ph. D. project which covers this topic. This graduate student submitted a proposal to OARDC Seed Grants, which has been funded. How have the results been disseminated to communities of interest? The 2014 Nonthermal Technology Workshop annual meeting What do you plan to do during the next reporting period to accomplish the goals? Data supporting the hypothesis of the proposal are provided. Data on additional applications of bacteriophage (in combination with ozone) against pathogens on fresh produce

Impacts
What was accomplished under these goals? Bacteriophage are applicable for the control of foodborne pathogens, particularly relevant to the safety of produce and other minimally processed products since phage application is a non-destructive treatment. The purpose of this study was to evaluate an environmental bacteriophage isolate's ability to reduce the population of enterohemorrhagic E. coli on produce. A phage active against Escherichia coli O157:H7 EDL933 was isolated from municipal wastewater and was titered at 108 PFU/ml when replicated with the host in LB broth. Phage treatments were applied to cut green peppers and baby spinach leaves. Cut peppers were treated with UV light to eliminate background biota, then spot inoculated with E. coli O157:H7 EDL933 on cut edges and allowed to dry. Baby spinach leaves were spot inoculated with GFP labeled E. coli O157:H7 and no efforts were made to eliminate background biota. Green pepper pieces were treated with a 5 min rinse in purified phage lysate while baby spinach leaves were rinsed for only 2 min. Treated produce was stored at 4°C, with or without pre-incubation for 4 h at 25°C. A control treatment was applied in the form of a buffer rinse, which reduced E. coli populations by <1 log/g pepper and <1.5 log/g spinach. The phage treatment reduced E. coli populations on peppers and spinach by 2.6 and 3.5 log CFU/g, respectively. These findings suggest the utility of using bacteriophage to selectively control pathogens on fresh produce without incubation at ambient temperatures.

Publications