2006 Annual Report 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? Why does it matter? Fresh and minimally processed produce are an important part of a healthy diet as they provide needed nutrients, fiber and antioxidants. When two thirds of all Americans are overweight or obese, increasing the consumption of fruits and vegetables is highly desirable in order to fight obesity and associated diseases and lower health costs. Unfortunately, during the last three decades there has been an increase in the number of foodborne outbreaks due to consumption of fresh and fresh-cut fruits and vegetables contaminated with a variety of human bacterial pathogens such as Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes and Shigella spp. There is a need to develop new, more effective intervention strategies. In order to develop such new strategies an in-depth understanding of attachment, biofilm formation and general microbial ecology of pathogens is required. A fundamental understanding of these factors will enhance our ability to develop new, effective intervention strategies. Further, any novel control technology must maintain produce quality at acceptable cost. The overall goal of the research is to reduce the risk of foodborne illness associated with the consumption of fresh or minimally processed produce while maintaining acceptable product quality and shelf-life. The specific objectives of the research program are. 1)Understand pathogen ecology including intrinsic and extrinsic factors affecting attachment, survival and biofilm formation and the interaction of human pathogens with native microorganisms on produce and their effects on decontamination efficacy;. 2)Develop biological-based intervention strategies for pathogen reduction while maintaining product quality making use of the information generated and any antagonistic microbes identified in the first objective; and 3)Develop new effective chemical and physical decontamination interventions and/or improve the performance of current interventions for produce while maintaining product quality. For improved decontamination efficacy and reduced quality loss effective intervention strategies will be tested in various combinations, i.e. hurdle technology. Interventions effective in the laboratory will be scaled up in a Biosafety Level Two (BSL-2) challenge facility. Effective and affordable intervention technologies will be transferred to industry to reduce the risk of produce-related outbreaks of foodborne illness. This research program addresses STP 4.1.2.3 (Food Processes and Products) and 4.2.1.3 (Bacteria and Viruses). Under the 2003-2007 ARS Strategic Plan Objective 3.1, the research addresses 3.1.2 (Food Safety, Postharvest). Under component 1.2 (Post-Harvest Pathogen Reduction) of the NP 108 2006-2010 Action Plan the project specifically addresses problem statements 1.2.3 Production and Processing Ecology by defining pathogen ecology including the importance of biofilm formation, 1.2.4 Processing Intervention Strategies by developing and validating new pathogen intervention strategies, and 1.2.9 Food Security by developing technologies to deactivate/neutralize Select Agent hazards in food and determine the effect of these processes on food quality. The research also addresses the target of Agency Performance Measure 3.1.2 “Develop practices and/or products that reduce postharvest contamination of major animal- and plant-derived food products”. 2.List by year the currently approved milestones (indicators of research progress) FY2006 1. Determine the radiation sensitivity of biofilm-associated bacterial human pathogens (Listeria monocytogenes and E. coli O157:H7) on inert surfaces. 2. Identify and characterize beneficial and deleterious native microflora affecting the safety and quality of salad vegetables using fresh-cut bell pepper and tomato as the model systems. 3. Initiate the evaluation of the use of the biological control agent Pseudomonas fluorescens strain 2-79 for controlling outgrowth of Salmonella from inoculated alfalfa seed during sprouting in glass jars and home-sprouting units. 4. Evaluate the effectiveness of ultrasound in combination with chlorine and aqueous chlorine dioxide for sanitizing alfalfa seed destined for sprouting. 5. Optimize the effectiveness of hot water pasteurization of cantaloupe melons and scale-up the process. 6. Evaluate the effectiveness of chlorine dioxide gas for sanitizing cantaloupe melons FY 2007 7. Determine the effects of postharvest handling including decontamination treatments on the removal of pathogenic, spoilage and antagonistic native microflora from fresh-cut salad vegetables. 8. Complete the evaluation of the use of the biological control agent Pseudomonas fluorescens strain 2-79 for controlling outgrowth of Salmonella from inoculated alfalfa seed during sprouting in glass jars and home-sprouting units. 9. Determine the antimicrobial efficacy of cold plasma for eliminating bacterial human pathogens (Salmonella, E. coli O157:H7, L. monocytogenes) inoculated on apple surfaces. 10. Optimize chlorine gas treatments for improving the microbiological safety of and sensory qualities of cantaloupe melons and scale-up the process. FY 2008 11. Determine the radiation sensitivity of biofilm-associated bacterial human pathogens (Salmonella, L. monocytogenes, E. coli O157:H7) on lettuce leaf surfaces. 12. Evaluate the fate of beneficial and deleterious microflora on fresh-cut salad vegetables as affected by storage conditions and packaging materials. 13. Determine the sensitivity of biofilm-associated Salmonella cells on inert surfaces to chlorine dioxide gas. 14. Complete the evaluation of the use of the biological control agent Pseudomonas fluorescens 2-79 for controlling outgrowth of surrogates of bacterial human pathogens in the pilot plant using commercial scale rotary drum or tray systems FY 2009 15. Investigate the antagonist or synergistic interactions between beneficial and deleterious microflora on the safety and quality of fresh-cut salad vegetables. 16. Complete the evaluation the use of mixtures of biological control agents for controlling the outgrowth of Salmonella from alfalfa seed sprouted in glass jars and home-sprouting units. 17. Determine the survival and recovery of Salmonella on tomatoes treated with irradiation under modified atmosphere packaging. 18. Evaluate the effects of hot water treatments on product quality and shelf-life. 19. Optimize chlorine dioxide gas treatments for decontamination of mangoes and almonds. FY 2010 20. Determine the post-irradiation recovery and regeneration of biofilms of biofilm-associated pathogens (Salmonella, L. monocytogenes, E. coli O157:H7) on lettuce leaf surfaces. 21. Determine the optimal storage conditions and packaging methods to minimize the survival and growth of spoilage and human pathogens on fresh-cut salad vegetables. 22. Complete the evaluation of the use of mixtures of biological control agents for controlling the outgrowth of Salmonella from alfalfa seed in the pilot plant using commercial scale rotary drum or tray systems. 23. Evaluate means of reducing undesirable quality effects of antimicrobial interventions (e.g. chlorine dioxide). 24. Evaluate the use of chlorine dioxide gas treatments for decontamination of papayas and oranges. 4a.List the single most significant research accomplishment during FY 2006. Biocontrol for Alfalfa Sprouts: The ability of several plant-associated benign (not pathogenic towards humans, animals or plants) bacteria to inhibit the growth of Salmonella on various microbiological media as well as on growing alfalfa sprouts was established. Results indicated that one strain of the bacterium Pseudomonas fluorescens was very inhibitory towards Salmonella. Application of this bacterium to alfalfa seed inoculated in the laboratory with Salmonella reduced growth of the pathogen by greater than 99.9% during sprouting. The use of this antagonistic bacterium as a biological control agent against Salmonella has great promise for helping to ensure the microbial safety of alfalfa and other types of sprouts for the consumer. NP108 Action Plan Component(s) 1.2.3, 1.2.4, 1.2.8, 2.1.5 4b.List other significant research accomplishment(s), if any. Biocontrol Agents for Fresh Produce: Microorganisms naturally associated with fresh peeled baby carrots have been isolated and characterized. Growth of foodborne human pathogens including Salmonella, Listeria monocytogenes, Yersinia enterocolitica and E. coli O157:H7 was greatly inhibited (from 99% to 99.9%) in carrot tissue homogenates or on green bell pepper slices that were co-inoculated with the native bacteria from fresh peeled baby carrots. Three individual strains useful for controlling the growth of pathogens on fresh produce have been identified. NP108 Action Plan Component(s) 1.2.3, 1.2.4, 1.2.8, 2.1.5 Surface Pasteurization for Cantaloupes: Thermal penetration models and antimicrobial efficacy models were developed for cantaloupe melons treated with a novel hot water system. Results confirmed that 99.999% reduction of Salmonella populations could be obtained and that the edible portions of melons remain relatively cool while the rind temperature rapidly rises to a point lethal for Salmonella. These results indicate that surface pasteurization will enhance the microbiological safety of cantaloupes while maintaining the quality of the product. These findings will assist food industry and regulatory agencies in establishing processing guidelines to guard against pathogens, thereby decreasing the incidence of food poisoning outbreaks. NP108 Action Plan Component(s) 1.2.4, 1.2.6 Nalidixic Acid Resistant Bacteria More Sensitive to Irradiation: Resistance to antibiotics has been commonly used as a selective marker for studies of pathogen-inoculated fruits and vegetables. The radiation resistance of three Salmonella isolates known to be sensitive to the antibiotic nalidixic acid (Nal) was compared to that of three isolates known to be resistant to Nal. In phosphate buffer and in orange juice, the Nal-resistant isolates were killed more effectively by ionizing radiation than were the Nal-sensitive isolates. When the Nal-sensitive isolates were made Nal-resistant by repeated exposure to the antibiotic, they became significantly more susceptible to irradiation. These results suggest that native and/or induced resistance to this antibiotic may predispose Salmonella isolates to more effective elimination by ionizing radiation, a result that is influenced by the suspending medium and by the nature of the isolates evaluated. NP108 Action Plan Component(s) 1.2.3, 1.2.4, 1.2.6, 1.2.8 Relating Antibiotic Resistance and Radiation Resistance: Twenty-four clinical isolates of Salmonella were screened for native resistance to multiple concentrations of the antibiotics ampicillin (Amp), chloramphenicol (Chl), or gentamicin (Gm). Each of the 24 isolates (100%) was resistant to Gm and was able to grow when exposed to the highest level of antibiotic. Eight of the 24 isolates (33%) were shown to be resistant to Amp. Seven of the 24 isolates (29%) were shown to be resistant to Chl. In separate experiments, the sensitivities of all of the Salmonella isolates to ionizing radiation were determined. While the radiation sensitivity varied among the isolates, no correlation was found between radiation sensitivity and their antibiotic resistance. Resistance to Amp or Chl is therefore suggested as more appropriate than Nal for use as Salmonella resistance markers in studies of irradiation. The use of validated methods, such as those presented herein, will improve the quality and utility of data obtained in these studies. NP108 Action Plan Component(s) 1.2.3, 1.2.4, 1.2.6, 1.2.8 E. coli Survives Exposure to Negative Air Ions: It has been shown that negative air ions, commonly produced via high voltage discharging, reduce airborne microorganisms. In this study we tested the efficacy of negative air ions for sanitizing mung bean sprout seed and apples. Mung bean seeds, whole “Gala” apples, and apple slices were inoculated with E. coli ATCC 25922 before being exposed to negative air ions for up to 18 h at room temperature. Our results showed that negative air ions had limited effects on E. coli ATCC 25922 on intact apple surfaces and mung bean seed, and had no effect on the bacteria inoculated on apple slices. Furthermore, there was no additive or synergistic effect between negative air ions and acetic acid vapor. The information may be useful for produce industry seeking to adapt new intervention technology to improve the safety of fresh fruits and vegetables. NP108 Action Plan Component(s) 1.2.4 Irradiation Equally Effective on Biofilm and Planktonic E. coli: Three isolates of E. coli O157:H7 were grown in culture in a biofilm reactor for 48h, and irradiated to compare the antimicrobial efficacy of the process against biofilm and planktonic (free floating) pathogen cells. It was determined that irradiation is at least as effective, if not more effective, against biofilm-associated cells as it is against planktonic cells. These results stand in contrast to the reduced efficacy of chemical sanitizers in the same comparison, and highlight the potential for irradiation and other energy-based treatments to eliminate biofilm-associated cells. NP108 Action Plan Component(s) 1.2.3, 1.2.4, 1.2.6, 1.2.8 4c.List significant activities that support special target populations. Significant outreach activities with small, rural sprout growers were undertaken. Many questions concerning the sanitizing of seed and sprouts from growers from around the US and representatives of the International Sprout Growers Association (ISGA) via the telephone or electronic mail were received and responded to. 4d.Progress report. Two factors which may limit the efficacy of using a bacterial biocontrol agent (Pseudomonas fluorescens strain 2-79) for controlling the growth of human pathogens such as Salmonella on sprouting seeds in the scale-up experiment have been identified. New strategies for application of strain 2-79 during the sprouting in a glass jar are being developed and tested to avoid the two limiting factors to ensure strain 2-79 as an effective and safe biocontrol agent for reducing the pathogen contamination on seed sprouts. This report serves to document research conducted under a reimbursable cooperative agreement (58-1935-3-325) between ARS and the University of California, Davis, in support of the parent project 1935-41420-011-00D. Previous research in our laboratory has documented the inadequacy of washing processes to inactivate and/or remove microorganisms on cantaloupes, including human pathogens, due to biofilm formation and inaccessibility of microbial attachment sites to washing systems. The development of chlorine dioxide gas treatment capable of reaching and inactivating human pathogens within biofilms or attached to inaccessible sites on cantaloupe surfaces was carried out. Artificially inoculated cantaloupes were fumigated with chlorine dioxide for up to 6 h in a closed chamber that was developed at ERRC, using two different technologies for generating the gas. There was in excess of 5 logs (99.999%) reduction in Salmonella populations following treatment for irrespective of the technology used for generating the gas. The work presented here showed that chlorine dioxide gas treatment of cantaloupes is able to inactivate Salmonella attached to inaccessible sites on the rind or within biofilms. This treatment increased the shelf life of the whole cantaloupe by reducing spoilage microorganism populations on the rind surface, and did not have apparent adverse effects on the quality of this commodity. NP108 Action Plan Component(s) 1.2.4, 1.2.6 This report serves to document research conducted under an Assistance-Type Cooperative Agreement (59-1935-8-047) between ARS and the Pennsylvania State University (PSU), in support of the original parent project 1935-41420-011-00D. During the final year of the Agreement, redesign, fabrication, and installation of conveyor systems for second generation dump tank and dip tank were designed, fabricated, and installed. An inlet pass through system was redesigned, fabricated, installed and tested. Fabrication work is completed on all equipment and systems to overcome deficiencies in performance or to improve safety of operation, and installation is completed. Validation of the BSL-2 containment chamber and the processing equipment using pathogenic and non-pathogenic bacteria were completed. Validation data demonstrated that the containment chamber was fully successful in containing air borne and surface attached bacteria, as well as bacteria suspended in solution. Also, the use of atmospheric steam for decontamination of the BSL-2 chamber and the enclosed equipment following inoculation with pathogenic and non-pathogenic bacteria was shown to be fully successful. Currently, the BSL-2 pilot plant facility is operational for large-scale produce decontamination studies employing human pathogens. NP108 Action Plan Component(s) 1.2.4 This report serves to document research conducted under a specific cooperative agreement (#41420-011-03S) between ARS and Drexel University in support of the parent project 1935-41420-011-00D. Nonthermal plasma (NTP) is a novel technology for sanitizing surfaces which shows promise for treatment of produce. In initial studies using a gliding arc NTP system designed for treatment of inert surfaces, NTP was applied to apples that had been inoculated with the pathogen surrogate Listeria innocua. The application of NTP led to a 90% reduction of the bacterium on the apple surface without altering the appearance of the treated apples during subsequent storage. Data was presented and discussed at international meetings, and based on the insights obtained, Drexel U. collaborators constructed a test prototype of an improved gliding arc NTP system to be installed at ERRC. This prototype was tested on-site at Drexel U. using apples. The pattern of plasma distribution was identified in the prototype, and modifications designed to improved the suitability for NTP application to the surfaces of produce. The NTP unit is complete. A design change has been incorporated into the unit to increase the unit's flexibility to accommodate future changes in electrode configuration, as needed for testing application to produce with different surfaces. The associated 10kV power supply is nearing completion. NP108 Action Plan Component(s) 1.2.4 This report serves to document research conducted under a new cooperative agreement (#58-1935-6-604N) between ARS and Del Monte Fresh Produce Company in support of the parent project 1935-41420-011-00D. Hot water pasteurization technology for decontamination of cantaloupe melons inoculated with human pathogen and non-pathogenic surrogate was scaled-up from the laboratory to the BSL-2 pilot plant facility. This intervention technology using the new fabricated commercial scale equipment was capable of achieving in excess of 5 logs (99.999%) reduction in the pathogen populations, a significant improvement over conventional technology. The data obtained clearly demonstrate the efficacy of this treatment in maintaining sensory qualities and extending shelf life of fresh and fresh-cut cantaloupes and decreasing the risk of food-borne illnesses. Validation of hot water surface pasteurization using ERRC dip tank under commercial production conditions Cooperator’s processing facility will be conducted by early 2007. NP108 Action Plan Component(s) 1.2.4 5.Describe the major accomplishments to date and their predicted or actual impact. Project 1935-41420-011-00D recently completed the OSQR review and approval process. This project is a replacement for bridge CRIS project 1935-41420-009-00D, which was itself a continuation of two former CRIS Projects (1935-41420-006-00D and 1935-41420-007-00D) which also centered on the development of new intervention technologies to reduce the risk of food borne illness due to contaminated produce. Research under the bridge project was conducted under the proposed milestones for the current project. A new antimicrobial intervention was developed for reducing the risk of food borne illness due to fresh-cut cantaloupe. Fresh-cut cantaloupe has been implicated in several instances of food borne outbreaks due to contamination with Salmonella. To address this issue, research was done in the Food Safety Intervention Technologies Research Unit on the use of hot water and low dose irradiation. Inoculated whole melons were first treated with hot water and then with low dose gamma irradiation of the cut fruit after packaging. The combination treatment was more effective than either treatment alone and had no detrimental effect on product quality. This intervention technology may be useful to the fresh-cut fruit industry for reducing the risk of food borne illness due to contaminated fresh-cut cantaloupe Bacterial regulatory system required for spoilage of carrots identified. Spoilage of produce can lead to large economic losses as well as potentially increasing the risk of foodborne illness. Research carried out in the Food Safety Intervention Technologies Research Unit demonstrated that that a functional two component regulatory system known as GacS/GacA is required for resistance of soft-rotting bacteria of the genus Pseudomonas to commercial sanitizers such as chlorine, hydrogen peroxide, acetic acid and trisodium phosphate. Mutants defective in this regulatory system do not produce extracellular polysaccharides or biofilms. Intervention strategies aimed at disrupting this regulatory system may allow for increased sanitizer effectiveness against these spoilage organisms and reduce the risk of foodborne illness by bacterial human pathogens whose growth and survival are favored in rotted plant tissues. Bacterial antagonists active against the bacterial human pathogens Listeria monocytogenes and Shigella in vitro identified. An alternative intervention to the use of chemicals for elimination of bacterial human pathogens on fresh and fresh-cut produce and sprouting seed may be the use of effective microbial antagonists. In studies done in the Food Safety Intervention Technologies Research Unit, two fluorescent pseudomonads were identified that were highly inhibitory towards L. monocytogenes and Shigella on agar media. These strains may also be effective inhibitors of the two pathogens on produce surfaces including sprouting seed and fresh-cut surfaces. Biological means of decreasing the risk of food borne illness due to produce contaminated with L. monocytogenes or Shigella may be feasible. 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? Information on methods for decontaminating produce and sprouting seed has been disseminated to growers, processors, trade organizations such as the International Sprout Growers Association, regulators and other scientists by means of presentations at meetings, visits to growers and processors, telephone and e-mail correspondence and through peer-reviewed publications. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Annous, B.A., E.B. Solomon and B.A. Niemira. 2006. Biofilms on Fresh Produce and Difficulties in Decontamination. pp.80-85. Food Quality Magazine, April/May 2006. (log # 193933) Core, J. 2005. Washing and sanitizing techniques aim to make produce safer. Agricultural Research. 53(12):17. Niemira, B.A. 2006. Biotechnology: A Look Ahead. IFT Biotechnology Division Newsletter, Spring 2006. (log # 195437) Review Publications Fan, X. 2005. Impact of ionizing radiation and thermal treatments on furan levels in fruit juices. Journal of Food Science. 70(7):E409-E414. Fatemi, P., Knabel, S.J., La Borde, L.F., Patton, J., Sapers, G.M., Annous, B.A. 2006. Influence of punctures, cuts and apple surface morphologies on penetration and growth of escherichia coli o157:h7. Journal of Food Protection. 69(2):267-275. Fett, W.F. 2006. Inhibition of salmonella enterica by plant-associated pseudomonads in vitro and on sprouting alfalfa seed. Journal of Food Protection. 69(4):719-728. Fett, W.F., Tortorello, M., Fu, T. 2005. Seed sprouts: the state of microbiological safety. In: Matthews, K.R., editor. Microbiology of Fresh Produce. Washington, D.C.: ASM Press. p. 167-219. Fett, W.F. 2005. Interventions to ensure the microbial safety of sprouts. In: Sapers, G.M., Gorny, J.R., Yousef, A.E., editors. Microbiology of Fruits and Vegetables. Boca Raton, FL: CRC Press. p. 187-209. Liao, C. 2006. Bacterial soft rot. In: Sapers, G.M., Goprny, J.R., Yousef, A.E., editors. Microbiology of Fruits and Vegetables. CRC Press, Boca Raton, FL. p. 117-134. Liao, C. 2006. Pseudomonas and related genera (xanthomonas, shewanella, etc). In: Blackburn, Clive de W., editor. Food Spoilage Microorganisms. Woodhead Publishing Ltd., Cambridge, England. p. 507-540. Liao, C., Fett, W.F. 2004. Resuscitation of acid-injured salmonella cells in enrichment broth, apple juice, and on cut surfaces of fruits [abstract]. Institutes of Food Technologists. p. 100. Niemira, B.A., Sommers, C.H. 2006. New applications in food irradiation. Heldman, D.R., editor. Encyclopedia of Agricultural, Food, and Biological Engineering. New York, NY: Taylor & Francis Group. p.1-6. Niemira, B.A., Sommers, C.H., Ukuku, D.O. 2005. Mechanisms of microbial spoilage of fruits and vegetables. In: Lamikanra, O., Imam, S.H., Ukuku, D.O., editors. Produce Degradation: Reaction Pathways and their Prevention. New York, NY: Taylor and Francis Group. p. 463-482. Qureshi, N., Annous, B.A., Ezeji, T.C., Karcher, P., Maddox, I.S. 2005. Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reactions rates. Microbial Cell Factories. 4:24. Available: http://www.microbialcellfactories.com/content/4/1/24. Solomon, E.B., Huang, L., Sites, J.E., Annous, B.A. 2006. Thermal inactivation of salmonella on cantaloupes using hot water. Journal of Food Science. 71(2): M25-M30. Annous, B.A., Solomon, E.B., Cooke, P.H. 2005. Biofilm formation by salmonella spp. on cantaloupe surfaces (abstract). International Association for Food Protection. Pager No. P3-39. Niemira, B.A., Alvarez, I., Annous, B.A., Gutsol, A., Fridman, A. 2005. Antimicrobial efficacy of cold atmospheric pressure plasma applied to inoculated food surfaces. IFT-NPD Meeting, September 15-16, 2005, Wyndmoor, PA. Niemira, B.A., Gutsol, A., Fridman, A. 2005. Cold, atmospheric pressure plasma reduces listeria innocua on the surface of apples.Annual Meeting of International Association of Food Protection (IAFP), August 14-17, 2005, Baltimore, MD. p. 2-40. Niemira, B.A. 2005. Nalidixic acid resistance increases sensitivity of salmonella to ionizing radiation in solution and in orange juice. Annual Meeting of International Association of Food Protection (IAFP), August 14-17, 2005, Baltimore, MD. p. 1-33. Niemira, B.A., Solomon, E.B. 2005. Inactivation of planktonic and biofilm-associated salmonella by ionizing radiation. Institute of Food Technologists Annual Meeting, July 17-20, 2005, New Orleans, LA. p. 108-5. Solomon, E.B., Niemira, B.A., Sapers, G.M., Annous, B.A. 2005. Comparison of biofilm formation by salmonella spp. originating from produce, animal, and clinical sources (abstract). International Association for Food Protection. Pager No. P1-48. Solomon, E.B., Cooke, P.H., Burke, A.M., Ukuku, D.O., Annous, B.A. 2005. Biofilm formation by salmonella spp. on cantaloupe surfaces. Journal of Food Safety. 25:276:287.