Progress 04/08/06 to 03/20/11
Outputs
Progress Report Objectives (from AD-416) (1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, and (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks. Approach (from AD-416) We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as �targets� to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life. Polymerase chain reaction (PCR)-based detection assays do not generate live cultures of the contaminating pathogen which are needed to perform the follow-up studies. Alternatives to PCR-based are urgently needed while retaining the benefits of molecular methods. Detection system based on viruses of E. coli O157:H7 was evaluated. This method required less than 12 hours to detect E. coli O157:H7 from beef and generated a live culture the following day. The method is compatible for future high throughput sample analyses requirements. Organic materials from fresh-cut produce can rapidly activate free chlorine in washing solution. A chlorine stabilizer was evaluated for the potential of increasing chlorine efficacy during sanitizing washing of fresh-cut lettuce. While the treatment did not influence significant reduction of bacterial pathogen, it helped prevent cross-contamination of fresh produce in the presence of organic load. The currently recommended produce wash step, described in the model HACCP plan, was not effective. We observed that maintaining sufficient sanitizer level was critical to reduce pathogen survival and its spread. This research over turns many years of industry practices by clearly documenting the risk associated with what was considered safe operating practices. In summary, over the past 5 years of this project, a periplasmic factor from Salmonella and Shigella strains, referred to as osmoregulated periplasmic glucans (OPGs), appears to be needed for optimimal virulence, tissue colonization and competitive growth was identified and partially characterized; the impact of abusive storage temperatures, especially with regards to produce packaged for extended shelf-life, on growth, survival and toxin production by E. albertii strains and E. coli strains obtained from outbreaks was examined; virulence and stress tolerance genes from several outbreak associated strains were examined using molecular traits, such as acid- and heat tolerance, intestinal cell attachment, biofilm formation; the efficacy of recently approved phage cocktails for nonfood contact surfaces was investigated; demonstrated that E. coli O157:H7 can grow significantly on commercially packaged fresh-cut lettuce and baby spinach products despite the presence of large populations of native microflora, countered the common belief that native microflora on fresh-cut vegetables can outcompete pathogens; demonstrated that E. coli O157:H7 can grow significantly on lettuce cored areas during product field-holding and hauling; developed two new prototype lettuce harvest knives with improved food safety features, and an ultrasound- assisted sanitation procedure to enhance lettuce harvest knife disinfection efficacy; examined current tomato post-harvest handling practices with respect to Salmonella contamination and infiltration into tomatoes during packing house operations, and evaluated the sanitizer concentration needed to prevent pathogen survival and cross-contamination during washing. Accomplishments 01 Elucidation of new acid resistance pathway in Shigella. The ability of human pathogens to survive in an acidic environment plays a crucial role in food and water borne diseases. Acidic produce, such as apple cider an apple juice, as well as fresh-cut melons and tomatoes have been implicat in recent outbreaks of infections caused by enteric human pathogens. Analyzing the outbreak-associated strain of Shigella boydii, isolated by the CDC in the year 2000, as well as other Shigella species it was demonstrated that they possess a new acid resistance pathway which was presumed to be absent in all Shigella species. This pathway is operativ in the presence of externally supplied arginine. The availability of oxygen during bacterial growth played an important role in activating ac tolerance pathway of Shigella strains. Understanding the acid tolerance pathways will advance our knowledge of how enteric human pathogens survi on fresh-cut produce. 02 Chlorine stabilizer to improve sanitizing efficiency of chlorine as a produce wash. The produce industry currently faces a major potential foo safety problem, since the chlorine needed to prevent pathogen survival i depleted during commercial wash operations. Working closely with the produce industry, USDA-ARS scientists comprehensively evaluated a novel chlorine stabilizer in maintaining free chlorine efficacy on pathogen survival and cross-contamination during commercial wash operating conditions. We demonstrated that T128 significantly increases the efficacy of chlorine wash against bacterial cross contamination while maintaining the quality of leafy green vegetables under real world fresh cut processing conditions. This research accomplishment was featured at the 2010 Fresh Summit sponsored by the Produce Marketing Association (PM and The Packer. New Leaf Food Safety Solutions Inc is currently using t research findings to optimize the applications of T128. 03 Efficacy of produce re-wash to control pathogen in fresh produce. We investigated the scientific bases for widely operated Hazard Analysis an Critical Control Point (HACCP) program. We demonstrated that produce re- wash, the corrective action currently described in the model HACCP plan not an effective measure to correct for process failure; and maintaining sufficient sanitizer level is critical to reduce pathogen survival and spread. This research over turns many years of industry practices by clearly documenting the risk associated with what heretofore were considered safe operating practices. This research was considered by the produce industry stakeholders as �ground breaking�, and the research publication was recorded as �The Most Read Article� by the International Association for Food Protection.
Impacts
(N/A)
Publications
- Luo, Y., He, Q., Mcevoy, J.L. 2010. Effect of storage temperature and duration on the behavior of Escherichia coli O157:H7 on packaged fresh-cut salad containing Romaine and Iceberg lettuce. Journal of Food Science. 75(7):M390-M397.
- Kannan, P., Yong, H., Reiman, L., Cleaver, C., Patel, P., Bhagwat, A.A. 2010. Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef. Foodborne Pathogens and Disease. 12:1151-1158.
- Goh, K., Chua, D., Beck, B., Mckee, M., Bhagwat, A.A. 2010. Arginine- dependent acid-resistance pathway in Shigella boydii. Archives Of Microbiology. 193(3):179-185.
- Luo, Y., Nou, X., Yang, Y., Turner, E.R., Alegre, I., Alegre, M., Feng, H., Conway, W.S. 2011. Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7 cross-contamination during fresh-cut produce wash. Journal of Food Protection. 74(3):352-358.
- Nou, X., Luo, Y., Hollar, L.A., Yang, Y., Feng, H., Millner, P.D., Shelton, D.R. 2011. Chlorine Stabilizer T-128 enhances efficacy of chlorine against cross contamination by E. coli O157:H7 and Salmonella in fresh-cut lettuce processing. Journal of Food Science. 76(3):M218-M224.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) (1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, and (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks. Approach (from AD-416) We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as �targets� to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life. Food-borne illness outbreaks associated with consumption of Salmonella spp. contaminated tomatoes have negatively impacted public health, consumer confidence in eating fresh produce, and the U.S. economy. Information is critically needed on how to prevent pathogen contamination during tomato harvesting and post-harvest handling. ARS scientists examined in detail the current tomato post-harvest handling practices on Salmonella contamination and infiltration in relation to tomato packing house operations. We also evaluated the sanitizer concentration needed to prevent pathogen survival and cross-contamination during washing. Information generated will help to establish science-based tomato handling standards to prevent pathogen contamination and internalization. Food-borne illness outbreaks associated with consumption of produce contaminated with Shigella spp. is a significant, on-going problem. These outbreaks have negatively impacted public health, consumer confidence in fresh-cut produce and the U.S. economy. The need to improve sanitation during fresh-cut preparations is both urgent and critical. USDA-ARS scientists determined that Shigella spp. synthesize unique glucose polymers when exposed to low nutrient conditions such as in vegetable wash waters. The polymers enable cells to maintain integrity and prevent them from bursting in low nutrient conditions. Human colonic cells were grown in vitro and the ability of Shigella cells to infect these cells was determined. Our data indicated that in vitro infection assays were not sufficient to differentiate the wild type and the mutant lacking the ability to synthesize periplasmic glucose polymers. Although specific requirement of glucose polymer for growth was demonstrated in low nutrient media, an animal model with a primate host will be needed to perform virulence analysis of Shigella spp. Accomplishments 01 Recent studies have shown that harvesting knives used for lettuce field- coring can serve as vehicles to transfer human pathogens like E. coli O157:H7 from soil to lettuce where it can further grow and proliferate. However, specific scientific data that are suitable for risk assessments are not available, but are urgently needed. Specific information and practical solutions to prevent/reduce pathogen contamination are also lacking. ARS researchers at Beltsville, MD investigated the potential pathways associated with pathogen transference from contaminated harvest knives to lettuce and the factors that aggravate or minimize pathogen contamination during harvesting. In collaborating with a scientist at t University of Illinois via a grant from the Center for Produce Safety an California Leafy Green Research Board, we further prototyped two new lettuce harvest knives with improved food safety features, and developed an ultrasound-assisted sanitation procedure to enhance lettuce harvest knife disinfection efficacy. Research findings provide specific scientific information for the industry and regulatory agencies to perfo risk assessment under realistic field conditions, and also provide the needed tools for the industry to minimize pathogen contamination cost- effectively. 02 Enteric foodborne pathogens such as Salmonella, Shigella, and Escherichi coli O157:H7 have been studied extensively in the laboratory media and i their animal hosts. However, information on their growth and survival in the vegetable washing and packaging environment is most urgently needed develop effective hygiene aids which will eliminate these pathogens from raw produce. A small sugar polymer was recently identified within the membranes of Salmonella spp. called periplasmic glucans. The periplasmi glucans were needed to optimize pathogen�s motility and virulence (teste in mice model). Using genetic analysis we demonstrated that enteric pathogens have secondary genetic circuits to induce motility even in the absence of periplasmic glucans. However such motile Salmonella strains which were still lacking in periplasmic glucans synthesis remained less virulent in mice compared to their wild type counter parts. The data indicate a more direct role for periplasmic glucans in virulence and disease causing ability of Salmonella strains. Identification of genes regulating periplasmic glucans mediated motility and survival are crucia in designing specific sanitation agents. The development of better cleaning agents to eliminate Salmonella spp. in food would be extremely beneficial to the food processing industry.
Impacts
(N/A)
Publications
- Allende, A., Mcevoy, J.L., Tao, Y., Luo, Y. 2008. Antimicrobial effect of acidified sodium chlorite, sodium chlorite, sodium hypochlorite, and citric acid on Escherichia coli O157:H7 and natural microflora of fresh- cut cilantro. Food Control. 20:230-234.
- Kannan, P., Dharne, M., Smith, A.D., Karns, J.S., Bhagwat, A.A. 2009. Motility revertants of opgGH mutants of Salmonella enterica serovar Typhimurium remain defective in mice virulence. Current Microbiology. 59(6) :641-645.
- Kou, L., Luo, Y., Ding, W., Liu, X., Conway, W.S. 2009. Hot water treatment in combination with rachis removal and modified atmosphere packaging maintains quality of table grapes. HortScience. 44:1947-1952.
- Luo, Y., He, Q., Mcevoy, J.L., Conway, W.S. 2009. Fate of Escherichia coli O157:H7 in the presence of indigenous microorganisms on commercially packaged baby spinach as impacted by storage temperature and time. Journal of Food Protection. 72(10):2038-2045.
- Zhou, B., Feng, H., Luo, Y. 2009. Ultrasound enhanced sanitizer efficacy in reduction of Escherichia coli O157:H7 population on spinach leaves. Journal of Food Science. 74(6):M308-313.
- Liu, L., Dharne, M., Kannan, P., Smith, A.D., Meng, J., Fan, M., Boren, T., Ranallo, R., Bhagwat, A.A. 2010. Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri. Archives Of Microbiology. 192(3):167-174.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) (1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, and (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks. Approach (from AD-416) We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as �targets� to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life. Significant Activities that Support Special Target Populations A mucuous-like substance (biofilm) produced by Salmonella enterica serovar Typhimurium strains that allows for survival under low nutrient conditions such as in vegetable wash waters was discovered. In addition, the small molecular weight sugar compound in Salmonella spp. that is essential for biofilm formation was identified. Genetic mutation in the corresponding gene rendered Salmonella strains incapable of forming biofilms. The utility of using several software programs to interpret the genetic information obtained from several genome sequence projects was demonstrated. Genetic analysis revealed new pathways in several Shigella spp. that could aid the pathogen to overcome the human gastric barrier. DNA-based analysis, in combination with antibiotic resistance profiles, were used to classify different Salmonella isolates obtained from retail foods. DNA-finger print patterns of eight isolates matched with the CDC/FDA data base of previous outbreak and clinical isolates indicating their potential to cause disease. With the exception of isolates obtained from alligator meat (tetracycline resistant) and orange juice (chloramphenicol and sulfisoxazole resistant), the remainder of the isolates were susceptible to the panel of 15 antimicrobials tested. The characterization of Salmonella isolates with the proposed scheme enabled us to determine the potential risk associated with individual strains. Studies indicate that Escherichia coli O157:H7 can be transferred from contaminated coring knives to the edible portions of lettuce during lettuce coring-in-field harvesting, and that E. coli O157:H7 can grow significantly on lettuce cored areas during product field-holding and hauling. Risk mitigation approaches were identified, including frequent sanitation of the coring knives, separating coring from cutting, and rapid cooling postcoring/harvesting. Studies were conducted to determine the microbial profile of fresh tomatoes, to investigate pathogen internalization pathways, and to assess the practicality and efficacy of consumer stem scar removal in reducing food safety risks. The vast majority of microorganisms are located on the stem-scars and in the underlying vascular bundle tissues within 15 mm of the stem-scar. The vascular bundles connecting the stem-scar and internal tissues play a critical role in pathogen internalization. Washing tomatoes with chlorinated water effectively inactivates surface attached microorganisms, but not internalized pathogens. Physical removal of stem-scar and underlying tissues, along with surface washing, significantly reduces both surface attached and internalized microorganisms. Technology Transfer Number of New CRADAS: 1
Impacts
(N/A)
Publications
- Bhagwat, A.A., Jun, W., Liu, L., Porteen, K., Dharne, M., Pheh, B., Tall, B.D., Kothary, M.H., Gross, K.C., Angle, S., Jianghong, M., Smith, A.D. 2009. Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice. Microbiology. 155:229-237.
- Liu, L., Tan, S., Jun, W., Smith, A.D., Meng, J., Bhagwat, A.A. 2008. Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy- green vegetable wash-waters and colonization in mice. FEMS Microbiology Letters. 292:13-20.
- Xia, X., Zhao, S., Smith, A.D., Mcevoy, J.L., Jianghong, M., Bhagwat, A.A. 2009. Characterization of Salmonella isolates from retail foods based on serotyping, pulse field gel electrophoresis, antibiotic resistance and other phenotypic properties. International Journal of Food Microbiology. 129:93-98.
- Chua, T., Bhagwat, A.A. 2009. A rapid and simple DNA extraction procedure to detect Salmonella spp. and Listeria monocytogenes from fresh produce using real-time PCR. Journal of Food Analytical Methods. 2:96-101.
- Fratamico, P.M., Bhagwat, A.A., Injaian, L., Cray, P.J. 2008. Characterization of shiga toxin-producing escherichia coli strains isolated from swine feces. Journal of Food Protection. 5(6):827-838.
- Bhagwat, A.A., Bhagwat, M. 2008. Methods and tools for comparative genomics of food-borne pathogens. Foodborne Pathogens and Disease. 5(4) :487-497.
- Wang, H., Feng, H., Liang, W., Luo, Y., Malyarchuk, V. 2009. Effect of surface roughness on retention and removal of Escherichia coli O157:H7 on surfaces of selected fruits. Journal of Food Science. 74(1):E8-E15.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) (1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks. Approach (from AD-416) We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as �targets� to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts, and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life. Significant Activities that Support Special Target Populations An increasing number of food-borne illness outbreaks associated with the consumption of packaged fresh-cut products calls for attention for maintaining food safety. Produce grows in the natural environment and undergoes much handling on its journey from farm to table, making it vulnerable to human pathogen contamination. Given that there is no effective pathogen killing step during the preparation of packaged fresh- cut products, prevention of pathogen contamination and reduction of the potential for pathogen growth throughout the entire supply chain are critical measures for ensuring produce safety. We demonstrated that storage temperature significantly affects the survival and growth of E. coli O157:H7 at the presence of natural microflora on commercially packaged spinach and lettuce. Storage at 12 C caused rapid growth of E. coli O157:H7 while there was a significantly die off of E.coli O157:H7 when stored at 1 and 5 C. Increasing temperature also accelerated the growth of indigenous microorganism; however, the growth of E.coli O157:H7 is benefited more from increasing in temperature. Refrigeration at 5 C or below can effectively limit the growth of E.coli O157:H7 and promote a general die-off. The information can increase our knowledge of microbial ecology with regard to persistence and proliferation of E.coli O157:H7 during fresh-cut product processing and distribution. The outcome of this research provides avenues of intervention and mitigation of food- borne illnesses on fresh and fresh-cut produce. This research is under National Program 108, Component Ia,b. Producers mainly rely on refrigeration temperatures and, more recently, on modified atmosphere packaging (MAP) to extend shelf-life and to reduce the microbial load. This study examines whether the extension of shelf- life may be creating opportunities for human pathogens to grow, survive, and produce toxins. We examined how the packaging conditions of leafy green vegetables would impact the ability of pathogenic E. coli strains to breach the gastric stomach barrier and to increase the risk of disease. Sub-atmospheric oxygen partial pressures during packaging enabled all 6 O157:H7 isolates to induce acid-resistance over the 8-day storage period if the temperature was > 15 �C. No acid-resistance was induced for MAP- lettuce left at temperatures = 10 �C or for lettuce packed and stored under aerobic conditions. The study highlights some of the potential dangers of abusive storage temperatures, especially regarding produce packaged for extended shelf-life. The results also highlight the biological significance of having multiple acid-resistance pathways and a complex regulatory network of enterohemorrhagic E. coli strains. Analyses of bacterial physiology under packaging conditions will advance our knowledge of how enteric human pathogens survive on fresh-cut produce. The research will benefit the fresh produce industry, as well as increase the microbial food safety of America�s food supply. National Program 108, Component Ia,b.
Impacts
(N/A)
Publications
- Allende, Gonzalez, R.J., Mcevoy, J.L., Luo, Y. 2007. Assessment of sodium hypochlorite and acidified sodium chlorite as antimicrobial agents to inhibit growth of Escherichia coli O157:H7 and natural microflora on shredded carrots. Journal of Vegetable Science. 13:51-63.
- Bhagwat, A.A., Patel, J.R., Chua, T., Chan, A., Cruz, S., Gonzalez, A., Gustavo, A. 2008. Detection of salmonella species in foodstuffs. Methods in Molecular Biology. 439:33-43.
- He, Q., Luo, Y., Chen, P. 2008. Elucidation of the mechanism of enzymatic browning inhibition by sodium chlorite. Journal of Food Chemistry. 110(4) :847-851.
- Kou, L., Luo, Y., Liu, X. 2007. Effects of mild heat treatment on microbial growth and product quality of packaged fresh-cut table grapes. Journal of Food Science. 72(8):S567-573.
- Kim, J., Tao, Y., Luo, Y. 2007. Effect of the combination treatment of 1- methylcyclopropene and acidified sodium chlorite on microbial growth and quality of fresh-cut cilantro. Postharvest Biology and Technology. 144-149.
- Ling, J., Sharma, M., Bhagwat, A.A. 2008. The role of RNA polymerase sigma- factor (RpoS) in induction of glutamate-dependent acid-resistance of Escherichia albertii under anaerobic conditions. FEMS Microbiology Letters. 283:75-82.
- Luo, Y. 2007. Wash operatons affect water quality and packaged fresh-cut romaine lettuce quality and microbial growth. HortScience. 42(6):1413-1419.
- Wang, H., Liang, W., Feng, H., Luo, Y. 2007. Wash Water Hydro-dynamics and Flow Velocity Affect Escherichia coli O157:H7 Population Reductions on Fruit Surfaces. Journal of Food Protection. 70(11):2533-2540.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) (1) Develop rapid and sensitive methods for detection of enteric human pathogens and spoilage bacteria from conventional and organically grown produce, (2) Develop effective postharvest sanitizing procedures providing improved antimicrobial activity while maintaining produce quality and shelf-life, (3) Understand the ecology and mechanisms that allow specific human and spoilage microorganisms to persist on fresh produce and develop control agents to reduce food safety risks. Approach (from AD-416) We will design experiments to optimize rapid, real-time PCR-based microbial detection methods for enteric human pathogens and also assess the application of similar methods for spoilage bacteria on fresh and fresh-cut produce. Because of the high organic load in produce wash water, sanitizer applications approved for fresh and fresh-cut preparation often do not provide effective control of human pathogens or may cause adverse effects on produce quality and shelf-life. Sanitation and wash procedures that are more effective in the presence of a high organic load would be developed. We will identify gene families from human pathogens involved in resistance to sanitation agents as well as stress-tolerance which could be used as �targets� to design novel food sanitation agents. We will develop effective Biocontrol agents such as bacteriophages, yeasts and lactic acid bacteria to enhance food safety by limiting human pathogens on fresh-cut produce without affecting its quality or shelf-life. Significant Activities that Support Special Target Populations Optimization of a DNA preparation method suitable for real-time PCR. Detection of enteric human pathogens on produce at low levels (approaching ~ 1-10 cells per 25 g) can not be accurately and rapidly accomplished using current microbiological methods approved by the USDA. Molecular detection methods such as real-time PCR require high quality DNA preparations which are free of inhibitors of plant origin. We developed a simpler and better DNA isolation protocol in order to free bacterial DNA of plant phenolic and other inhibitory compounds, which facilitated the detection of pathogens at low levels. The protocol was optimized for DNA isolation especially for �in-field� conditions, i.e., minimal laboratory equipment, less need for hands-on manipulation and ambient storage temperatures. Detection of Salmonella spp. and Listeria monocytogenes was possible in a high-throughput manner from a variety of produce such as broccoli, alfalfa sprouts, mixed salad and lettuce. The improved DNA isolation protocol will directly help Hazard Analysis Critical Control Point (HACCP) program development and monitoring human pathogens on fresh produce. Analysis of fresh-cut preparation methods for potential microbial contamination. Coring iceberg lettuce in the field is a new technology that has significantly improved the production yield and reduced shipping cost. However, no scientific studies have been reported on the potential of E.coli O157:H7 contamination during field procedures and the effect of field conditions on the survival and growth of E.coli O157:H7 on pre- cored lettuce. We evaluated the potential for pathogen transfer during coring process and demonstrated that E.coli O157:H7 can be readily transferred from contaminated coring knives to the edible portion of lettuce; also, a 4-hour exposure of cored lettuce to 30 �C ambient environment significantly increased E.coli populations on lettuce. Our results provide critical information for the development and implementation of Good Agricultural Practice (GAP) and Hazardous Analysis and Critical Control Point (HACCP) by the FDA and produce industry. Accomplishments Improved sanitizer efficacy for microbial safety of fresh-cut produce. The control of the growth of disease-causing microorganisms and the enzymatic browning reaction on the cut-surface of apple slices is critical to maintaining the safety and quality of fresh-cut produce. We demonstrated and optimized the utility of a chemical compound, sodium chlorite, that has the potential to control both the browning reaction and pathogen growth for a variety of fresh-cut produce, including apples, pears, and lettuce, etc. This information is very useful for the development of dual control agents and to solve the current challenges facing the industry due to the incompatibility between browning inhibitors (reducing agents) and most sanitizers (oxidants) used commercially. We also found that an ultra sound technology significantly improved the efficacy of various sanitizers, including chlorine and acidified sodium chlorite, on pathogen reduction on spinach. Both the consumers and fresh-cut produce industry will benefit from the results of this research. This research fulfills a top ARS food safety research priority requested by the produce industry. This research is under National Program 108, Section 2.4.1. Technology Transfer Number of New CRADAS and MTAS: 1 Number of Active CRADAS and MTAS: 3 Number of Non-Peer Reviewed Presentations and Proceedings: 7 Number of Newspaper Articles,Presentations for NonScience Audiences: 3
Impacts
(N/A)
Publications
- Zhang, Y., Emily, Y., Cripe, J., Hall, G., Bhagwat, A.A., Meng, J. 2007. Characterization of listeria monocytogenes isolated from retail food. International Journal of Food Microbiology. 113:47-53.
- Sharma, M., Kniel, K., Derevianko, A., Ling, J., Bhagwat, A.A. 2007. Sensitivity of Escherichia albertii, a potential foodborne pathogen, to food preservation treatments. Applied and Environmental Microbiology. 73(13):4351-4353.
- Wang, H., Feng, H., Luo, Y. 2006. The effect of acidic electrolyzed water and peroxyacetic acid on reduction of escherichia coli o157:h7 populations on fresh-cut apples. Journal of Food Safety. 26:335-347.
- Wang, H., Feng, H., Luo, Y. 2007. Control of browning and microbial growth on fresh-cut apples by sequential treatment of sanitizers and calcium ascorbate. Journal of Food Science. 72:M1-M7.
- Kim, J., Lim, C., Luo, Y. 2007. Effect of ozonated water and chlorine water wash on the quality and microbial de-contamination of fresh-cut carrot shreds. Korean Journal of Food Preservation. 14(1):54-60.
- Lu, S., Luo, Y., Turner, E.R., Feng, H. 2007. Efficacy of sodium chlorite as an inhibitor of enzymatic browning in apple slices. Journal of Food Chemistry. 104:824-829.
- Zhu, B., Lu, J., Luo, Y., Tao, Y. 2007. Gabor Feature-Based Apple Quality Inspection Using Kernel Principal Component Analysis. Journal of Food Engineering. 81(4):741-749.
- Ruiz-Cruz, S., Luo, Y., Gonzalez, R., Tao, Y., Gonzalez, G. 2006. Effect of acidified sodium chlorite applications on microbial growth and the quality of shredded carrots. Journal of the Science of Food and Agriculture. 86(12):188-1893.
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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? 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 by year the currently approved milestones (indicators of research progress) Year 1 (FY 2007) Complete studies on optimizing DNA preparation methods suitable for real- time PCR from various produce for enteric human pathogens and complete evaluation of primers for amplification of target sequences from produce spoilage bacteria. Identify fresh-cut processing conditions that influence sanitizer efficacy and complete analysis of enteric pathogens with reference to three acid-stress pathways. Identify effects of fresh-cut preparation methods on microbial growth and complete testing
efficacy of bacteriophage cocktail against L. monocytogenes isolates. Year 2 (FY 2008) Complete evaluation of antibody-based detection of L. monocytogenes strains isolated from various foods and complete evaluation of PCR protocols for rapid detection of spoilage bacteria. Complete evaluation of the novel compounds on browning inhibition and pathogen inactivation on apples and Year 1 (FY 2007) Complete studies on optimizing DNA preparation methods suitable for real- time PCR from various produce for enteric human pathogens and complete evaluation of primers for amplification of target sequences from produce spoilage bacteria. Identify fresh-cut processing conditions that influence sanitizer efficacy and complete analysis of enteric pathogens with reference to three acid-stress pathways. Identify effects of fresh-cut preparation methods on microbial growth and complete testing efficacy of bacteriophage cocktail against L. monocytogenes isolates. Complete study on role of glucans in
stress tolerance and virulence of Salmonella spp. Complete evaluation of fresh-cut processing environment on produce metabolism and complete burst size study of bacteriophages provided by Intralytix, Inc. Year 3-4 (FY 2009-2010) Optimize parameters for molecular beacon-based rapid detection methods for Salmonella and Listeria spp. and assays for detection of plant spoilage bacteria. Complete evaluation of the influence of water quality on wash operations and sanitizer efficacy and complete study on functional diversity of rpoS in pathogenic E. coli. Identification of effects of fresh-cut preparation methods on microbial growth and complete testing efficacy of bacteriophage cocktail against L. monocytogenes isolates. Identification of optimal fresh-cut processing for minimal microbial growth and maximum shelf-life and complete study on combining bacteriophages and yeast antagonists on survival of L. monocytogenes. Year 4-5 (FY 2010-2011) Finish studies and transfer technologies on
molecular beacon based rapid detection identification methods and assays for detection of plant spoilage bacteria optimized. Complete evaluation of the novel compound on food quality and safety of fresh-cut pears and lettuce and identify novel gene targets which may have significant role in stress tolerance and survival. Complete studies and transfer technology on optimal fresh-cut processing for minimal microbial growth and maximum shelf life and complete study on combining bacteriophages and yeast antagonists on survival of Salmonella spp. 4a List the single most significant research accomplishment during FY 2006. New project started April 2006, no significant accomplishment this year. The research will benefit the fresh produce industry, as well as increasing the microbial food safety of the Americans food supply. NP 108, component 2.1.1.4. 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). "Fresh-cuts are Popular, any way you slice them: Where Packaging Meet the Product" from the ARS magazine (July 2006) highlighted research done under this project.
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