Source: Agricultural Research Service submitted to
POSTHARVEST SURVIVAL STRATEGIES AND BIOCONTROL OF HUMAN PATHOGENS ON FRESH FRUITS AND VEGETABLES
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0405571
Grant No.
(N/A)
Project No.
1275-42000-003-00D
Proposal No.
(N/A)
Multistate No.
S-294
Program Code
(N/A)
Project Start Date
Jun 11, 2002
Project End Date
May 31, 2006
Grant Year
(N/A)
Project Director
BHAGWAT A A
Recipient Organization
Agricultural Research Service
BLDG 003 BARC W RM 331
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011499101040%
7121119110025%
7121499110035%
Goals / Objectives
(1) To evaluate the impact of fresh-cut preparation practices on metabolic processes of fruits and vegetables, microbial survival/growth, and their interactions. (2) To understand survival strategies used by human pathogens on fresh-cut produce, (3) To develop postharvest intervention strategies to limit growth of human pathogens on produce.
Project Methods
To detetermine the effect of industry fresh-cut practices on produce metabolism, we will focus on the washing/sanitizing process since it is a critical control point for HACCP food safety programs. Produce tissue damage, wound response and sensory quality of fresh-cuts prepared using various conditions will be evaluated during storage. Adherence assays for relevant Enterohemorrhagic E. coli have already been developed to study the attachment to lettuce surfaces. Microscopy will be used to examine bacterial adherence patterns using GFP-expressing strains to determine if these adherence patterns suggest particular pathogen reduction schemes. Using gene array analyses, we will identify the gene circuitry involved in attachment-mediated acid tolerance and compare gene expression patterns among attachment-defective S. typhimurium and E. coli mutant strains. Optimization of phage delivery systems, in combination with other factors such as bacteriocins, will be examined for its efficacy to control foodborne pathogens on produce.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Contamination of fresh fruits and vegetables with microorganisms pathogenic to humans has increased in recent years. Due to the increasing consumption of fresh-cut fruits and vegetables, pre-sliced, packaged pieces, the problem only promises to get worse because fresh-cut produce has had its natural barriers to pathogen contamination removed and are often stored in a stressed, sub-optimal condition. There have been many documented cases of foodborne illness due to contamination of fresh fruit and vegetable products with E. coli O157:H7, Salmonella, and Listeria. We need to understand how such contamination occurs, how the human pathogens survive and grow on fresh produce and how we can better devise postharvest systems for eliminating or controlling the growth of human pathogenic microorganisms while maintaining the quality and shelf-life of fresh and fresh-cut produce. The objectives will be achieved through devising better detection methods for pathogens, microbial genome analysis, and developing biocontrol agents. The research undertaken falls under National Program 108- Food Safety and addresses goals 2.1, 2. 2 and 2.5 as described in the National Program Action Plan. 2. List the milestones (indicators of progress) from your Project Plan. Short-term (18 to 24 months): Enumeration and characterization of the major components of microflora of fresh-cut melons, carrots, peppers and lettuce in terms of (a) DNA finger print analysis, (b) extracellular enzyme production and (c) antibiotic resistance patterns. Construction of antibiotic resistant strains of S. poona, EHEC and L. monocytogenes outbreak strains and determination of their growth characteristics on fresh-cut produce. Develop strategies involving phages and/or natural compounds as antagonists to L. monocytogenes and S. poona. Determine the effect of AITC and other natural volatiles on background microflora of fresh-cut honeydew melon and cantaloupe and their effects on the human bacterial pathogens. Determine the contribution of different genes in attachment/survival during acid-challenge and various sanitation agents. Construct a mutant gene bank of EHEC O157:H7 and screen for mutants defective in attachment to lettuce and watermelon rind. Document the effects of current commercial fresh-cut preparation practices on the physiology and biochemistry of produce. Assess various fresh-cut preparation conditions, including washing, cutting, packaging and storage on product shelf-life and foodborne pathogen survival and growth. Identify and characterize wound-healing processes and assess the factors which may control them. Long-term (36 to 44 months): Develop a database of natural microflora of fresh produce using DNA finger printing methods. Determine and characterize the effects of background microflora of fresh-cut produce, and individual components thereof, on the growth and survival of human bacterial pathogens; identify and characterize possible biocontrol agents for human bacterial pathogens. Determine optimal delivery systems for various biocontrol systems previously developed, ie, bacteriophages, AITC, or other volatile natural antimicrobials for fresh-cut honeydew melon and cantaloupe. Isolate and characterize survival mutants identified by gene arrays, TnphoA and STM analysis. Identify the contribution of individual acid- tolerance pathways and attachment mechanisms that exist in enteric pathogens, which may lead to identification of genes which may be good biocontrol agents for sanitation treatments. Evaluate interactions among human pathogens, plant pathogens and fresh- cut produce as influenced by preparation conditions. Assess the impact of wound healing on produce defense or resistance to microbial survival and growth. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Determine the contribution of different genes in attachment/survival during acid-challenge and various sanitation agents. Milestone Fully Met 2. Develop strategies involving phages and/or natural compounds as antagonists to L. monocytogenes and S. poona. Milestone Fully Met 3. Determine the effect of AITC and other natural volatiles on background microflora of fresh-cut honeydew melon and cantaloupe and their effects on human bacterial pathogens. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2006. Determine and characterize the effects of background microflora of fresh-cut produce, and individual components thereof, on the growth and survival of human bacterial pathogens. Determine optimal delivery systems for various biocontrol systems previously developed, ie, bacteriophages, AITC, or other volatile natural antimicrobials for fresh- cut honeydew melon and cantaloupe. Evaluate interactions among human pathogens, plant pathogens and fresh-cut produce as influenced by preparation conditions. Year 2007. Identify the contribution of individual acid-tolerance pathways and attachment mechanisms that exist in enteric pathogens, which may lead to identification of genes which may be good biocontrol agents for sanitation treatments. Identify and characterize possible biocontrol agents for human bacterial pathogens. Assess the impact of wound healing on produce defense or resistance to microbial survival and growth. Year 2008. Complete studies on optimizing DNA preparation methods suitable for real-time PCR from various produce. Identification of fresh- cut processing conditions that influence sanitizer efficacy. Identification of effects of fresh-cut preparation methods on microbial growth. Complete testing efficacy of bacteriophage cocktail against L. monocytogenes isolates. 4a What was the single most significant accomplishment this past year? During the preparation of fresh-cut produce, a large amount of organic matter is released from plant tissues that neutralizes sanitation agents and limits the efficacy of pathogen reduction. We identified a safe and effective new sanitizer (acidified sodium chlorite, or SANOVA) that achieved a 5-log reduction of E. coli 0157:H7, Listeria monocytogenes, and Salmonella serovar poona even in the presence of large organic loads. Furthermore, we optimized sanitation treatment procedures to ensure good quality of shredded carrot and fresh-cut lettuce while maintaining the effective killing power of the sanitizer. These findings are especially useful to the fresh produce industry as they provide practical information in selecting a suitable sanitizer to maintain microbial safety and quality of fruits and vegetables. Action plan component 2.1.1. 4. 4b List other significant accomplishments, if any. The fresh-cut produce industry is growing rapidly with an annual growth rate in the double digits. Various sanitizers, which are effective in reducing foodborne pathogen populations on whole produce, are not as effective on fresh-cut produce. We have shown that a mixture of bacteriophages which specifically target the foodborne pathogen Listeria monocytogenes, when applied at a rate of 10 x 8 PFU/ml within an hour before or after contamination with L. monocytogenes, significantly reduces pathogen populations on honeydew melons. The fresh-cut produce industry can use bacteriophages to implement a successful biocontrol strategy to reduce the potential for outbreaks of foodborne diseases. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Fresh-cut fruits and vegetables have a limited shelf-life, and detection of human pathogens by conventional microbiology procedures is time consuming and delays implementing food safety strategies such as product recalls or epidemiological investigations. During the course of this project, PCR detection methods for three human pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella strains, approved by the Association of Official Analytical Chemists (AOAC), were modified to enable their rapid and simultaneous detection. Real-time detection protocols involving two detection technologies, namely SYBR Green and molecular-beacons, were developed and evaluated on a variety of fresh produce and poultry. Both the fresh produce industry and consumers will benefit from the results of this research. The rapid detection methods will assist in the accurate and economic surveillance of human pathogens in the American food chain. Action plan components 2.5.1.1 and 2.5.2.3.

Impacts
(N/A)

Publications

  • Kim, J., Luo, Y., Saftner, R.A., Gross, K.C. 2005. Effect of delayed packaging and modified atmosphere packaging on quality of fresh-cut romaine lettuce. Journal of the American Society for Horticultural Science. 130:116-123.
  • Leverentz, B., Conway, W.S., Janisiewicz, W.J., Camp, M.J. 2004. Optimizing concentration and timing of a phage spray application to reduce Listeria monocytogenes on fruit tissue. Journal of Food Protection. 67(8) :1682-1686.
  • Liming, S.H., Bhagwat, A.A. 2004. Application of molecular beacon -- real- time PCR technology to detect Salmonella species contaminating fruits and vegetables. International Journal of Food Microbiology. 95:177-187.
  • Liming, S.H., Zhang, Y., Meng, J., Bhagwat, A.A. 2005. Detection of listeria monocytogenes in fresh produce using molecular beacon - real-time pcr technology. Journal of Food Science. 69:240-245.
  • Patel, J.R., Bhagwat, A.A., Sanglay, G.C., Solomon, M.B. 2005. Rapid detection of Salmonella from hydrodynamic pressure-treated poultry using molecular beacon real-time PCR. Food Microbiology. 23(1):39-46. Available: http://authors.elsevier.com/sd/article/S0740002005000213
  • Wang, H., Feng, H., Luo, Y. 2004. Microbial reduction and storage quality of fresh-cut cilantro washed with acidic electrolyzed water and aqueous ozone. Food Research International. 37:949-956.
  • Wang, H., Feng, H., Luo, Y. 2004. Browning control and pathogen reduction on cut surfaces of apple slices with acidic electrolyzed water[abstract]. American Society of Agricultural Engineers Special Meetings and Conferences Papers. Abstract number 046053.
  • Chan, L., Han, R., Low, S., Tan, J., Bhagwat, A.A. 2005. Environmental adaptive mutations in diarrheagenic escherichia coli and their effect on infection dose. Proceedings of the US-Japan Cooperative Program in National Resources, Cherry, J. P. and Pavlath, A. E. (Eds.). p. 370-374.
  • Gonzalez, R.J., Luo, Y., Ruiz-Cruz, S., Mcevoy, J.L. 2004. The efficacy of sanitizers on the reduction of e. coli o157:h7 from shredded carrots under simulated fresh-cut processing conditions. Journal of Food Protection. 67(11):2375-2380.
  • Izumi, H., Luo, Y., Rodov, V., Watada, A. 2005. Technologies for maintaining quality of fresh-cut produce. In: New Enviromentally Friendly Technologies to Prevent Spoilage and Maintain Quality of Agricultural Produce, (Ben-Yehoshua, ed). CRC Press, Boca Raton, Florida. p. 149-203.


Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Contamination of fresh fruits and vegetables with microorganisms pathogenic to humans has increased in recent years. Due to the increasing consumption of fresh-cut fruits and vegetables, pre-sliced, packaged pieces, the problem only promises to get worse because fresh-cut produce has had its natural barriers to pathogen contamination removed and are often stored in a stressed, sub-optimal condition. There have been many documented cases of foodborne illness due to contamination of fresh fruit and vegetable products with E. coli O157:H7, Salmonella, and Listeria. We need to understand how such contamination occurs, how the human pathogens survive and grow on fresh produce and how we can better devise postharvest systems for eliminating or controlling the growth of human pathogenic microorganisms while maintaining the quality and shelf-life of fresh and fresh-cut produce. The objectives will be achieved through devising better detection methods for pathogens, microbial genome analysis, and developing biocontrol agents. The research undertaken falls under National Program 108- Food Safety and addresses goals 2.1, 2. 2 and 2.5 as described in the National Program Action Plan. 2. List the milestones (indicators of progress) from your Project Plan. Short-term (18 to 24 months): Enumeration and characterization of the major components of microflora of fresh-cut melons, carrots, peppers and lettuce in terms of (a) DNA finger print analysis, (b) extracellular enzyme production and (c) antibiotic resistance patterns. Construction of antibiotic resistant strains of Salmonella poona, EHEC and L. monocytogenes outbreak strains and determination of their growth characteristics on fresh-cut produce. Develop strategies involving phages and/or natural compounds as antagonists to L. monocytogenes and S. poona. Determine the effect of AITC and other natural volatiles on background microflora of fresh-cut honeydew melon and cantaloupe and their effects on the human bacterial pathogens. Determine the contribution of different genes in attachment/survival during acid-challenge and various sanitation agents. Construct a mutant gene bank of EHEC O157:H7 and screen for mutants defective in attachment to lettuce and watermelon rind. Document effect of current commercial fresh-cut preparation practices on the physiology and biochemistry of produce. Assess various fresh-cut preparation conditions, including washing, cutting, packaging and storage on product shelf-life and foodborne pathogen survival and growth. Identify and characterize wound-healing processes and assess the factors which may control them. Long-term (36 to 44 months): Develop a database of natural microflora of fresh produce using DNA finger printing methods. Determine and characterize the effects of background microflora of fresh-cut produce, and individual components thereof, on the growth and survival of human bacterial pathogens; identify and characterize possible biocontrol agents for human bacterial pathogens. Determine optimal delivery systems for various biocontrol systems previously developed, ie, bacteriophages, AITC, or other volatile natural antimicrobials for fresh-cut honeydew melon and cantaloupe. Isolate and characterize survival mutants identified by gene arrays, TnphoA and STM analysis. Identify the contribution of individual acid- tolerance pathways and attachment mechanisms that exist in enteric pathogens, which may lead to identification of genes which may be good biocontrol agents for sanitation treatments. Evaluate interactions among human pathogens, plant pathogens and fresh- cut produce as influenced by preparation conditions. Assess the impact of wound healing on produce defense or resistance to microbial survival and growth. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Milestones listed below were scheduled to be completed under year 2. All the milestones were completed. Determine the contribution of different genes of diarrheagenic E. coli for it's survival during acid-challenge. Construction of antibiotic resistant strains of S. poona, EHEC and L. monocytogenes outbreak strains and determination of their growth characteristics on fresh-cut produce. Develop rapid methods for enumeration and characterization of human pathogens from fresh and fresh-cut produce. Document effect of current commercial fresh-cut preparation practices on the physiology and biochemistry of produce. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2005. Determine the contribution of different genes in attachment/survival during acid-challenge and various sanitation agents. Develop strategies involving phages and/or natural compounds as antagonists to L. monocytogenes and S. poona. Determine the effect of AITC and other natural volatiles on background microflora of fresh-cut honeydew melon and cantaloupe and their effects on human bacterial pathogens. Year 2006. Determine and characterize the effects of background microflora of fresh- cut produce, and individual components thereof, on the growth and survival of human bacterial pathogens. Determine optimal delivery systems for various biocontrol systems previously developed, i.e., bacteriophages, AITC, or other volatile natural antimicrobials for fresh-cut honeydew melon and cantaloupe. Evaluate interactions among human pathogens, plant pathogens and fresh-cut produce as influenced by preparation conditions. Year 2007. Identify the contribution of individual acid-tolerance pathways and attachment mechanisms that exist in enteric pathogens, which may lead to identification of genes which may be good biocontrol agents for sanitation treatments. Identify and characterize possible biocontrol agents for human bacterial pathogens. Assess the impact of wound healing on produce defense or resistance to microbial survival and growth. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 (one per Research Project): The application of real time PCR technology to detect Salmonella sp. in vegetable rinse water was evaluated. A rapid detection method compatible with high sample volume throughput was designed. The PCR protocol approved by the Association of Official Analytical Chemists (AOAC) was modified to enable real time detection and quantitative analysis of Salmonella contamination in vegetable rinse water. This protocol will ensure rapid, accurate and low-cost surveillance of Salmonella sp. in fresh produce. Both the fresh produce industry and consumers will benefit from the results of this research. B. Other significant accomplishments None C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Developed procedures for a produce wash operation to improve antimicrobial activity. Evaluated the efficacy of ozone on microbial reduction as impacted by the fresh-cut produce wash operation and water quality. Data revealed that the large amount of organic matter released from cut produce neutralizes ozone and limits the efficacy of ozone on pathogen reduction. The fresh-cut fruits also have an active metabolism that can result in a rapid deterioration of tissue if not controlled. A commercial and three experimental wash treatments for fresh-cut apple slices were evaluated for their ability to affect survival of food-borne pathogens and to maintain instrumental and sensory quality characteristics of the slices. In this study it was demonstrated that the experimental wash treatments showed strong antimicrobial activity against E. coli O157:H7, Salmonella spp. and Vibrio spp. During treatment of apple slices, the wash solutions became progressively adulterated with fruit juice and they lost their antibacterial activity. Regarding microbial safety, wash solutions should not be reused on multiple batches of sliced apples. Instead, alternative washing strategies that maintain the antimicrobial properties of the wash solutions need to be developed for fresh-cut apple slices. Developing wash treatments that both control browning and provide microbial safety will safeguard the consumer. This information will benefit the produce industry in developing a safe and efficacious anti-microbial agent to ensure safety and quality of fresh-cut products. This accomplishment provides the necessary understanding of major factors influencing the microbial status within the fresh-cut produce processing ecosystem, and also provides the foundational information for the future development of strategies to control microbial growth and improve food quality and safety. Action plan components 2.5.1.1 and 2.5.2.3 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Invited presentations were made at the International Fresh-cut Produce Association Annual Meeting and to fruit processors and an equipment manufacturer regarding alternative methods to maintain quality and food safety of fresh-cut fruit products. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Bhagwat, A. A., Lauer, W. 2004. Foodborne outbreaks in raw produce can be prevented. Food Qual. v11: 62-63. Scientific Publications:

Impacts
(N/A)

Publications

  • Allende, A., Luo, Y., Mcevoy, J.L., Artes, F., Wang, C.Y. Microbial and quality changes in fresh-cut baby spinach stored under map and super atmospheric oxygen conditions. Postharvest Biology and Technology 33 (2004) 51-59
  • Bhagwat, A.A. 2003. Rapid detection of Salmonella from vegetable rinse- water using a real-time PCR. Food Microbiology. 21:73-78.
  • Bhagwat, A.A., Bhagwat, M. 2004. Comparative analysis of transcriptional regulatory elements of glutamate-dependent acid-resistance systems of Shigella flexneri and Escherichia coli O157:H7. Federation of European Microbiological Societies Microbiol Letters. 234:139-147.
  • Bhagwat, A.A., Saftner, R.A., Abbott, J.A. 2004. Evaluation of wash treatments for survival of foodborne pathogens and maintenance of instrumental and sensory characteristics of fresh-cut apple slices. Food Microbiology. 21(3):319-326.
  • Yingru, C., Liming, S.H., Bhagwat, A.A. 2004. Occurrence of inhibitory compounds in spent growth media that interfere with acid-tolerance of enteric pathogens. International Journal of Food Microbiology. 91:175-183.
  • Gonzalez, R.J., Mcevoy, J.L., Luo, Y. 2004. Efficacy of sanitizers on pathogen reduction of fresh-cut carrots under simulated processing conditions. Annual Meeting of the Institute of Food Technologists. Abstract#114 C-5.
  • Kim, J., Luo, Y., Gross, K.C. 2003. Effect of packaging film in the quality of fresh-cut salad savoy. Postharvest Biology and Technology. 32(2004)99-107
  • LIAO, C., MCEVOY, J.L., SMITH, J.L. 2003. CONTROL OF BACTERIAL SOFT ROT AND FOODBORNE HUMAN PATHOGENS ON FRESH FRUITS AND VEGETABLES. IN: Research Signpost, Kerala, India. Advances in Plant Disease Management. P. 165-193.
  • Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladze, T., Saftner, R.A., Sulakvelidze, A. 2003. Biocontrol of Listeria monocytogenes on fresh-cut produce by combining bacteriophages and a bacteriocin. Applied and Environmental Microbiology. 69(8):4519-4526.
  • Luo, Y., Mcevoy, J.L., Wachtel, M.R., Kim, J., Huang, Y. 2003. Package film oxygen transmission rate affects postharvest biology and quality of fresh-cut cilantro leaves. American Society for Horticultural Science. HortScience Vol.39(3)June 2004, pg 567-570
  • Fraser, K., Tuite, N., Bhagwat, A.A., O'Bryne, C. 2004. Investigating the basis of homocysteine toxicity in escherichia coli. In: Proceedings of Bionet Conference, November 2003, Galway, Ireland.
  • Bhagwat, A.A., Lynn, C., Shufen, R.H., Moghe, G., Phadke, R., Fernalld, R. T., Cregan, P.B. 2004. Role of response regulatory genes, rpos and gade (formerly yhie) in acid-resistance systems of foodborne outbreak- associated pathogenic escherichia coli strains. In: Proceedings of EURESCO Conference, Bacterial Neural Networks, May 8-14, 2004, San Feliu, Spain, p. 43.
  • Saftner, R.A., Abbott, J.A., Bhagwat, A.A. 2004. Quality measurement of intact and fresh-cut slices of 'fuji', 'goldrush' 'granny smith', and 'pink lady' apples. Food Technologists Institute. Abstract #83, D-30.
  • Kim, J., Luo, Y., Gross, K.C. 2003. Effect of storage temperature on keeping quality of salad savoy. Proceedings of International Congress of Refrigeration. CD-ROM. Washington, D.C.
  • Luo, Y., Yang, T. 2003. Determining tissue damage of fresh-cut vegetables using image technology. In: Acta Horticulture Proceedings 628:97-102.


Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Contamination of fresh fruits and vegetables with microorganisms pathogenic to humans has increased in recent years. We need to understand how such contamination occurs, how the human pathogens survive and grow on fresh produce and how we can better devise postharvest systems for eliminating or controlling the growth of human pathogenic microorganisms while maintaining the quality and shelf-life of fresh and fresh-cut produce. The objectives will be achieved through devising better detection methods for pathogens, microbial genome analysis, and developing biocontrol agents. 2. How serious is the problem? Why does it matter? Contamination of fresh fruits and vegetables with human pathogens is critical to the American public. There is an increasing incidence of sickness due to contamination of fresh fruits and vegetables with foodborne pathogens. Due to the increasing consumption of fresh-cut fruits and vegetables, pre-sliced, packaged pieces, slices or chunks, the problem only promises to get worse because fresh-cut produce has had its natural barriers to pathogen contamination removed and are often stored in a stressed, sub-optimal condition. There have been many documented cases of foodborne illness due to contamination of fresh fruit and vegetable products with E. coli O157:H7, Salmonella, and Listeria. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety of Animal and Plant Products. This program allows scientists to focus on microbiological safety of fresh fruit and vegetables. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: Considering the limited shelf life of produce, detection of human pathogens from fresh produce is a crucial step in implementing food safety. In this study, PCR detection methods for three human pathogens, Escherichia coli O157:H7, Listeria monocytegenes, and Salmonella strains, approved by the Association of Official Analytical Chemists (AOAC), have been modified to enable near-instantaneous detection and quantitative analysis. This protocol will ensure accurate and economic surveillance of human pathogens in the American food chain. Both the fresh produce industry and consumers will benefit from the results of this research. B. Other Significant Accomplishment(s), if any: Several recent E. coli O157:H7 outbreaks have occurred due to the consumption of raw produce that had presumably been cross-contaminated from meat in a food preparation setting. We examined cross-contamination of E. coli O157:H7 from beef patties to hands, cutting-boards and lettuce. The contact of lettuce with a contaminated cutting board resulted in the transfer of E. coli O157:H7 from the board to the lettuce and warm water rinses of cutting boards were not effective at removing bacteria. Our results will be helpful to commercial food handlers and consumers, since they have documented that the significant contamination of lettuce, and therefore possibly other fresh produce, can occur by cross- contamination from other sources in a kitchen or other food preparation environment. C. Significant Accomplishments/Activities that Support Special Target Populations. None. D. Progress Report: Various sanitizers, which are effective in reducing foodborne pathogen populations on whole produce, are not as effective on fresh-cut produce. Naturally occurring bacteriophages, or viruses of bacteria, may be viable alternatives to sanitizers. In cooperation with scientists at Intralytix, a commercial bacteriophage-producing company in Baltimore, MD, and an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virgina, ARS scientists at Beltsville, Maryland, have shown that a mixture of bacteriophages, which specifically target the foodborne pathogen Listeria monocytogenes, reduces populations of this pathogen on fresh-cut honeydew melons. Application of bacteriophage mixture when applied within one hour of the produce being processed was most effective in reducing the populations of the L. monocytogenes. The fresh-cut produce industry could use bacteriophages to implement a successful biocontrol strategy to reduce the potential for outbreaks of foodborne diseases. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Salmonellae are the most frequently reported cause of food-borne outbreaks of gastroenteritis in the United States. In clinical trials the minimum number of salmonellae required to cause disease in healthy volunteers is estimated to be approximately one million cells. However, recent reports from various outbreaks associated with tomatoes and melons involving Salmonella species, fewer numbers of cells (approximately 100 cells) were sufficient to cause the disease. Acidic conditions in the stomach of humans is our first line of defense against invading pathogens. Therefore, we wanted to determine if the acidic nature of some of the fresh-cut produce (for example, tomatoes and apples) could have a role in making salmonellae resistant to the stomach acidity. Our data indicated that produce acidity played no role in increasing acid tolerance of salmonellae. However, it provided a suitable surface for the pathogen to attach. Mere attachment to fresh-cut produce surfaces as well as to inert surfaces, such as nylon and tissue paper, helped salmonellae endure acidity much better. The prevention of contamination caused by human pathogens during harvesting, washing, cutting, slicing, packaging and transporting is of considerable importance to public health and, consequently, is a major issue for the fresh-cut produce industry. The results from this research will help the produce industry design effective control measures to eliminate surface-attached pathogens during fresh-cut produce processing. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 2004: Develop cost effective postharvest treatments, which will ensure microbiological safety while maintaining product quality, and to provide technical support for regulatory approval of such postharvest system treatments. Year 2005: Identify gene sets of enteropathogens which are crucial for their survival on fresh and fresh-cut produce and evaluate them as targets in order to develop new produce washing and storage protocols. Year 2006: Determine the role of interactions among human pathogens, plant pathogens, and plant metabolites with reference to microbial food safety of fresh and fresh-cut produce. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Initiated a Cooperative Research and Development Agreement (A. Bhagwat, #58-1275-3-0102) with a small business to determine the efficacy of a rapid and real-time polymerase chain reaction-based methods to detect Salmonella sp. and Listeria monocytogenes on fresh and fresh-cut produce.

Impacts
(N/A)

Publications

  • Bhagwat, A.A. Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. International Journal of Food Microbiology. 2003. v. 84. p. 217-224.
  • Cheng, X., Tao, Y., Chen, Y.R., Luo, Y. NIR/MIR dual-sensor machine vision system for on-line apple stem-end/calyx recognition. Transactions of American Society of Agriculture Engineering. 2003. v. 46. p. 551-558.
  • Gawande, P.V., Bhagwat, A.A. Protective effects of cold temperature and surface-contact on acid tolerance of Salmonella spp. Journal of Applied Microbiology. 2002 v. 93. p. 689-696.
  • Bhagwat, A.A., Chen, Y., Liming, S.H., Saftner, R.A., Abbott, J.A. Evaluation of anti-browning solutions for survival of food-borne pathogens and instrumental and sensory quality characteristics of fresh-cut apple slices. International Symposium on New Functional Ingredients and Foods: Safety, Health and Convenience, Copenhagen, Denmark, April 9-11, 2003. Abstract F05.
  • Luo, Y., McEvoy, J.L., Wachtel, M.R., Kim, J.G., Huang, Y. Package film oxygen transmission rate affects postharvest biology and quality of fresh- cut cilantro leaves. Proceedings Annual Meeting, Institute of Food Technologist (IFT), Chicago, IL, July 12-16, 2003. Abstract p. 265-266.
  • Luo, Y., Li, J., Wang, Y., Shi, X. The effect of negative air ions and ozone on postharvest physiology and microbiology of horticultural crops. CD-ROM. Proceeding of Ozone III Conference-Agriculture Food Processing Applications of Ozone as an Anti-microbial Agent, Fresno, CA, Oct. 28-30, 2002.
  • Wang, H. Feng, H., Luo, Y. Surface treatment of fresh-cut lettuce with acidic electrolyzed water to extend shelf life. Proceedings Annual Meeting, Institute of Food Technologist (IFT), Chicago, IL, July 12-16, 2003. Abstract p. 265.
  • Leverentz, B., Janisiewicz, W.J., Conway, W.S. Biological control of minimally processed fruits and vegetables. Novak, J. S., Sapers, G.M., Juneja, V.K., editors. CRC Press, Boca Raton, FL. Microbial Safety of Minimally Processed Foods. 2003. p. 319-332.
  • Wachtel, M.R., McEvoy, J.L., Luo, Y., Williams Campbell, A.M., Solomon, M. B. 2003. Cross-contamination of lettuce by Escherichia coli O157:H7 via contaminated ground beef. Journal of Food Protection. 66(7):1176-1183.
  • Bhagwat, A.A., Phadke, R.P., Wheeler, D., Kalantre, S., Gudipati, M., Bhagwat, M. Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies. International Symposium on Systeomics: Integrating proteomics, genomics and matabonomics, San Francisco, CA, November 3-6, 2002. Abstract p. 50.


Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Contamination of fresh fruits and vegetables with microorganisms pathogenic to humans has increased in recent years. We need to understand how such contamination occurs, how human pathogens survive and grow on fresh produce and how we can devise postharvest systems for eliminating or controlling the growth of human pathogens while maintaining quality and shelf-life of fresh and fresh-cut produce. The objectives will be achieved through devising better detection methods for pathogens, microbial genome analysis, and developing biocontrol agents. 2. How serious is the problem? Why does it matter? Contamination of fresh fruits and vegetables with human pathogens is a current challenge that is critical to the American public. There is an increasing incidence of sickness due to contamination of fresh fruits and vegetables with foodborne pathogens. Also increasing, is the consumption of fresh-cut, pre-packaged produce. The food safety problem can potentially get worse because fresh-cut produce has had it's natural barriers to pathogen contamination removed. Also, fresh-cut fruit and vegetable products are often stored in a stressed, sub-optimal condition. There have been many documented cases of foodborne illness due to contamination of fresh fruit and vegetable products with E. coli O157:H7, Salmonella, and Listeria. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety of Animal and Plant Products. This program focuses on microbiological safety of fresh fruit and vegetables. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002 year: A number of foodborne outbreaks have linked infection by the enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 to the consumption of contaminated lettuce. Scientists of the Produce Quality and Safety Laboratory at Beltsville examined the association of lettuce seedlings with EHEC O157:H7 strains implicated in lettuce or fruit outbreaks using hydroponic and soil model systems. EHEC strains were able to associate with plants in as few as 3 days in soil, and contamination levels were dose-dependent. This research highlights the necessity for attention to food safety measures in the growth of produce, including the quality of water and other amendments. B. Other Significant Accomplishment(s), if any: Acid tolerance of enteropathogens is a critical issue in determining microbial risk assessment of fresh and fresh-cut produce. Scientists of the Produce Quality and Safety Laboratory at Beltsville examined the survival strategies of E. coli O157:H7 on fresh-cut produce and in the synthetic gastric juice. It was found that availability of glutamate helped E. coli 0157:H7 survive acid-challenge tremendously. This information will be useful in devising food additives for extending the shelf-life of fresh-cut produce while maintaining microbial food safety. C. Significant Activities that Support Special Target Populations: None D. Progress Report: Currently little information is available concerning biocontrol of the human pathogen Salmonella poona on fresh-cut cantaloupe. In collaboration with a fresh-cut produce company, we screened several hundred organisms collected from fresh-cut cantaloupe for antagonistic activity against S. poona. Several yeasts as well as gram positive and gram-negative bacteria were found to negatively impact growth of S. poona on fresh-cut cantaloupe stored at 10DGC. Such organisms, if found to be safe to consume and cause no negative impact on product quality, could be used by fruit processors as a way to safeguard consumers of fresh-cut produce against the occurrence of high numbers of S. poona. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? In cooperation with a bacteriophage producing company, we have conducted cooperative research to determine whether bacteriophage could provide biological control of Listeria monocytogenes on fresh-cut fruits and vegetables. We found that the bacteriophage mixture we used significantly reduced the populations of Listeria monocytogenes on honeydew melon, and this effect was increased by combining nisin with the bacteriophage treatment. This technology will be used by the fresh-cut industry to reduce the possibility for foodborne pathogen outbreaks and decrease their dependency on chemical controls. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 2003: Devise technologies for produce washing and storage to extend shelf-life, devise rapid detection protocols of pathogens and how to limit growth of these microorganisms without affecting the beneficial microorganisms present. Year 2004: Develop cost effective postharvest treatments, which will ensure microbiological safety while maintaining product quality, and to provide technical support for regulatory approval of such postharvest system treatments. Year 2005: Identify gene sets of enteropathogens which are crucial for their survival on fresh and fresh-cut produce and evaluate them as targets in order to develop new produce washing and storage protocols. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Renewed a trust Fund Cooperative Agreement (W. Conway, CRADA #58-3K95-0- 840) with a small business to determine the effect of bacteriophage on foodborne pathogens on fresh-cut fruit and vegetables. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Eberle, U. Heiler aus der jauchegrube. Bild Der Wissenschaft. 2002. v. 4. p. 20-25. McBride, J. Turning the phage on produce pathogens. Agricultural Research. 2001. July. p. 12.

Impacts
(N/A)

Publications

  • 1. Cui, S., Meng, J., Bhagwat, A.A. Availability of glutamate and arginine during acid-challenge determines cell density-dependent survival phenotype of E. coli strains. Applied and Environmental Microbiology. 2001. v 67. p. 4914-4918.
  • 2. Gawande, P.V., Bhagwat, A.A. Inoculation onto solid surfaces protects Salmonella spp. during acid-challenge: a model study using polyethersulfone membranes. Applied and Environmental Microbiology. 2002. v. 68. p. 86-92
  • 3. Leverentz, B., Conway, W.S., Alavidze, Z., Janisiewicz, W.J., Fuchs, Y., Camp, M.J., Chighladze, E., Sulakvelidze, A. Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: A model study. Journal of Food Protection. 2001. v. 64. 1116-1121.
  • 4. Wachtel, M.R., Charkowski, A.O. Cross-contamination of lettuce with Escherichia coli O157:H7. Journal of Food Protection. 2002. v 65. p. 465- 470.
  • 5. Wachtel, M.R., Whitehand, L.C., Mandrell, R.E. Association of Escherichia coli O157:H7 with preharvest leaf lettuce exposure to contaminated irrigation water. Journal of Food Protection. 2002. v 65. p. 18-25.
  • 6. Wachtel, M.R., Whitehand, L.C., Mandrell, R.E. Prevalence of Escherichia coli associated with a cabbage crop inadvertently irrigated with partially treated sewage wastewater. Journal of Food Protection. 2002. v 65. p. 471-475.
  • 7. Wachtel, M.R., Luo, Y., Huang, Y., McEvoy, J.L. Lettuce tissue damage affects the growth and/or survival of Escherichia coli O157:H7. Proceedings Annual Meeting Institute Food Technologists. Anaheim, CA, June 15-19, 2002. Abstract, p. 15-18.
  • 8. Wachtel, M.R., McEvoy, J.L., Luo, Y., Williams-Campbell, A., Solomon, M. B. Cross-contamination of lettuce by Escherichia coli O157:H7 via contaminated ground beef. Journal of Food Protection. 2002. Vol. 65, Supplement A, Abstract, p. 105.