2008 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, (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.
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.
A close relative of E. coli O157:H7, has been identified as an emerging food-borne pathogen and has been implicated in outbreaks of diarrhea among children. In depth biochemical and physiological characterization of this pathogen is needed for its successful identification. Acid resistance is perceived to be an important property of enteric pathogens, enabling them to survive passage through stomach acidity so that they may then colonize the mammalian gastrointestinal tract. Comparative genetic analyses of E. albertii and E. coli O157:H7 strains were conducted and a unique regulatory pathway was identified in E. albertii, enabling it to withstand gastric acidity. The data indicate that certain E. albertii strains have an alternate RpoS-dependent pathway for acid-resistance under anaerobic growth conditions. Understanding the acid tolerance pathways will advance our knowledge of how enteric human pathogens survive on acidic produce such as apple cider and orange juice. The research will benefit risk analysis of foodborne pathogens to increase the microbial food safety of America’s food supply. National Program 108, Component Ia.
5.Significant Activities that Support Special Target Populations
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.