An official website of the United States government.
In this proposal experiments will be conducted to determine the range of bacterial cell surface moieties that induce the plant defense system. Experiments will be conducted with Arabidopsis thaliana and lettuce. <P>Arabidopsis thaliana offers several advantages for the type of research proposed. The entire Arabidopsis thaliana genome has been sequenced. In this study, Arabidopsis plants impaired in plant defense response are used. <P>To test elicitation of plant defenses during endophytic colonization A. thaliana PR1::GUS will be inoculated with E. coli O157:H7 and Salmonella. The plant defense response gene, PR1, is fused to the bacterial uidA (?-glucuronidase) reporter gene. Additionally, the Arabidopsis npr1 mutant line is also used. The DNA binding ability of transcription factors in the plant defense system are regulated by the NPR1 protein.<P> The outcome of experiments may be unexpected in that bacterial growth conditions prior to contact with a plant may stimulate multiple cell surface moieties, some that are recognized by a plant and others that are not. Therefore, initial adherence capabilities may differ significantly from colonization. <P>The interaction of enteric foodborne pathogens with plant tissue is not well understood. The role of key bacterial cell surface moieties in adherence to and colonization of lettuce is investigated. Wildtype bacteria and mutants lacking specific cell surface appendages are used. In addition, bacteria will be exposed to various environments; water, manure, and soil prior to application to the plant. <P>We expect that environmental conditions will influence the extent to which, for example, exopolysaccharide is produced. Limited exopolysaccharide production may actually enhance adherence and colonization depending on how the plants defense system responds. <P>The proposed research is fundamental and mission-linked providing basic knowledge that advances basic and applied research that may be transferred to consumer and industry constituents alike. Internalization will not be addressed since inclusion of such experiments would make the project overly ambitious. <P>The expected impact section should consist of a few paragraphs in non-technical language, which clearly states how the outcomes and results of the proposed project will affect and/or benefit the identified stakeholders. It should be written for a lay audience.<P> The goals of this project are to provide a better understanding of the factors involved in foodborne pathogen adherence to and colonization of plants and the role of plant defense response. <OL> <LI> Determine environmental conditions that influence expression of cell surface moieties by Salmonella and E. coli O157:H7. <LI> Determine change in adherence, colonization and persistence of E. coli O157:H7 and Salmonella, exposed to condition demonstrated to influence cell surface moiety expression, to plant tissue. <LI> Development of lettuce with reduced plant defense system; determine change in adherence, colonization and persistence to lettuce of E. coli O157:H7 and Salmonella exposed to conditions demonstrated to influence cell surface moiety expression.
Non-Technical Summary: This project relates to ensuring food safety. In the past five years a wealth of research has been published on the interaction of foodborne pathogens with plant tissue, but many pieces of the puzzle remain missing. The lack of knowledge of foodborne pathogen-plant interaction hinders the development of effective strategies to reduce or eliminate foodborne pathogen contamination of leafy greens and other fresh fruits and vegetables. Sanitizers and sanitizing practices presently employed commercially to reduce microbial numbers on fresh fruits and vegetables post-harvest are not adequate. Under the conditions studied, no sanitizer has been able to produce more than a 1-2 log10 CFU reduction in pathogen levels. This was the situation >5 years ago and remains true today. Crops in the field may be exposed to contaminated irrigation water, manure, contact with feral animals, and workers hands during harvest. Individually or collectively such exposure can result in contamination of the commodity with microorganisms that are detrimental to human health. Implementation of Good Agricultural Practices (GAP?s) on the farm and use of Good Management Practices (GMP?S) and Hazard Analysis Critical Control Point (HACCP) programs post-harvest are positive steps in controlling contamination of raw agricultural products, but outbreaks associated with the consumption of fresh and fresh-cut fruits and vegetables continue to occur. The increase in foodborne illness associated with the consumption of fresh fruits and vegetables has been attributed to increased consumption of fresh produce, changes in agronomic practices, and increased importation. Tomatoes, seed sprouts, lettuce, and cantaloupes have repeatedly been implicated as vehicles of human salmonellosis and E. coli O157:H7 infection. There exist a real and immediate need to address foodborne-pathogen plant interaction to move forward in the development and initiation of strategies that will enhance the microbial safety of fresh fruits and vegetables. <P> Approach: Procedure: Objective 1. Determine environmental conditions that influence expression of cell surface moieties by Salmonella and E. coli O157:H7. Bacteria: E. coli O157:H7 strains include 43895OW, noncurliated and 43895OR, a curli producing strain. E. coli O157:H7 strains used are described in detail (39). Salmonella Stanley was isolated from alfalfa sprout linked to an outbreak of foodborne illness. S. Typhimurium (ATCC 14028) flagella positive and the flagella mutant of 14028, (BA3104) are used. Capsule Staining: A negative staining technique employing Maneval's stain is used to test for capsule formation. Flagella staining. The method is a simplified Leifson flagella stain. Curli production. Isolates are streaked onto Congo red plates, curli production is based on morphotype: brown, dry and rough indicates curli production; smooth and white, indicating lack of curli production. Biofilm Assay: Each strain is grown overnight in different media , diluted 1:10 in each fresh medium, and used to inoculate three replicate wells. Biofilms are stained and optical density is measured at 630nm using a plate reader. Objective 1.2. Impact of exposure/growth in manure, water, and soil on exopolysaccharide, flagella, and curli production. Experiments are conducted using methods of exposure designed to be as similar as possible to conditions that may occur on a farm. Two methods of exposure are used - direct inoculation and dialysis tubing. Bacteria are cultured at 37oC overnight in TSB prior to use. Objective 2. Determine change in adherence, colonization and persistence by E. coli O157:H7 and Salmonella exposed to condition demonstrated to influence cell surface moiety expression to plant tissue. Experiments in this proposal are designed to assess bacterial and plant factors that affect foodborne pathogen attachment and colonization. Under this objective Arabidopsis thaliana PR1::GUS and A. thaliana npr1-4 mutant are used. Recall that for A. thaliana PR1::GUS the plant defense response gene, PR1, is fused to the bacterial uidA (?-glucuronidase) reporter gene. Whereas, for the A. thaliana npr1-4 mutant it lacks the ability to produce the NPR1 protein which regulates the DNA binding ability of transcription factors in the plant defense system. An additional advantage of using Arabidopsis thaliana is the plants short (6 wks) life cycle. Objective 3. Development of lettuce with reduced plant defense system; determine change in adherence, colonization and persistence to lettuce of E. coli O157:H7 and Salmonella exposed to conditions demonstrated to influence cell surface moiety expression. Arabidopsis will be used as a model plant to study the interaction of E. coli O157:H7 and Salmonella with plants and plant defense system. However, to understand how these human enteric pathogens colonize and persist on fresh produce, it is undoubtedly important to directly study the interactions of these pathogens with plants such as lettuce and spinach. We choose to use lettuce for this study because it has been associated repeatedly in recent years with outbreaks of foodborne illness linked to enteric pathogen contamination.