2010 Annual Report
1a.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.
1b.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.
Enteric foodborne pathogens such as Salmonella, Shigella, and Escherichia coli O157:H7 have been studied extensively in the laboratory media and in their animal hosts. However, information on their growth and survival in the vegetable washing and packaging environment is most urgently needed to 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 periplasmic glucans were needed to optimize pathogen’s motility and virulence (tested 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 crucial 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.
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 the University of Illinois via a grant from the Center for Produce Safety and 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 perform risk assessment under realistic field conditions, and also provide the needed tools for the industry to minimize pathogen contamination cost-effectively.
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.
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.