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United States Department of Agriculture

Agricultural Research Service

2011 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.

3.Progress Report
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.

1. 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 and apple juice, as well as fresh-cut melons and tomatoes have been implicated 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 operative in the presence of externally supplied arginine. The availability of oxygen during bacterial growth played an important role in activating acid-tolerance pathway of Shigella strains. Understanding the acid tolerance pathways will advance our knowledge of how enteric human pathogens survive on fresh-cut produce.

2. Chlorine stabilizer to improve sanitizing efficiency of chlorine as a produce wash. The produce industry currently faces a major potential food safety problem, since the chlorine needed to prevent pathogen survival is 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 (PMA), and The Packer. New Leaf Food Safety Solutions Inc is currently using the research findings to optimize the applications of T128.

3. Efficacy of produce re-wash to control pathogen in fresh produce. We investigated the scientific bases for widely operated Hazard Analysis and Critical Control Point (HACCP) program. We demonstrated that produce re-wash, the corrective action currently described in the model HACCP plan is 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.

Review 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.

Last Modified: 11/26/2015
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