ROLE OF FOODBORNE PATHOGEN CELL SURFACE MOIETIES AND PLANT DEFENSE SYSTEMS IN COLONIZATION OF CROPS INTENDED FOR HUMAN CONSUMPTION.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0182217
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Food Science

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.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	4099	1040	15%
712	1419	1040	20%
712	1419	1100	20%
723	1419	1040	15%
723	1419	1100	15%
723	4099	1100	15%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
4099 - Microorganisms, general/other; 1419 - Leguminous vegetables, general/other;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

food safety

foodborne pathogens

fresh fruits and vegetables

produce safety

salmonella

escherichia coli o157:h7

Goals / Objectives
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. 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. 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 (β-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. 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. 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. 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. 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. 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. 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. 1. Determine environmental conditions that influence expression of cell surface moieties by Salmonella and E. coli O157:H7. 2. 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. 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.

Project Methods
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 Manevals 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 (β-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.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Research linked to the project was presented at local (New York Institute of Food Technologists) and international meetings (International Association for Food Protection) meetings. Research was also shared with representatives of USDA ARS and private industry. This was in the context of development of collaborative research. The audience for the research included academics, government and industry representatives. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergradaute students completed research associated with the project. Two Ph.D. level graduate students have been involved in the project and their dissertation is based on project goals. Students gained experience in experimental design, problem solving, data analysis, and interpersonal communication skills. How have the results been disseminated to communities of interest?The research has been presented at local, national , and international meetings and to industry representatives. Papers have been published in peer reviewed journals. The information has been conveyed to other interested parties (students, peers) through seminars and invited lectures. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Determine environmental conditions that influence expression of cell surface moieties by Salmonella and E. coli O157:H7. The influence of cell surface structures of E. coli O157:H7, such as flagella, curli fimbriae, lipopolysaccharides, or exopolysaccharides, on plant defense responses and on survival or colonization on the plant was investigated. The population of an E. coli O157:H7 wild-type strain was significantly lower on wild-type Arabidopsis plants compared with that of the flagella-deficient mutant. The population of the E. coli O157:H7 flagella mutant was greater on both wild-type and npr1-1 (nonexpressor of PR genes) plants and resulted in less PR gene induction by the plant defense system. The results suggest that the flagella, among the other pathogen associated molecular patterns (PAMPs), made a substantial contribution to the induction of plant defense response and contributed to the decreased numbers of E. coli O157:H7 wild-type strain on the wild-type Arabidopsis plant. A curli-deficient E. coli O157:H7 strain, survived better on wild-type Arabidopsis plants than the curli-producing wild-type strain did. The curli-deficient E. coli O157:H7 exhibited a GUS signal substantially lower than that of the curli-producing wild-type strain. Curli were recognized by plant defense systems, consequently affecting bacterial survival. The cell surface structures of E. coli O157:H7 have a significant impact on the induction of differential plant defense responses, thereby impacting persistence or survival of the pathogen on plants. 2. Determine change in adherence, colonization and persistence of E. coli O157:H7 and salmonella. Subsequent, study evaluated whether growth medium or exposure conditions influence the production of capsular polysaccharides (CPS) by Escherichia coli O157:H7, and whether changes in CPS impact plant defense responses, consequently affecting survival on plants. Escherichia coli O157:H7 grown in Luria-Bertani (LB) broth supplemented with manure extracts showed an approximately 58% increase in CPS production compared to cells grown in LB medium alone. Levels of CPS were significantly higher for E. coli O157:H7 cells exposed to soil or manure extracts as compared to the non-exposed LB cultured control. Arabidopsis thaliana plants expressing β-glucuronidase (GUS) under the control of the β-1,3-glucanase (BGL2) promoter were used to investigate whether E. coli O157:H7 induces defense-related gene expression. Plants inoculated with E. coli O157:H7 grown in LB containing manure extracts or cells exposed to manure extracts exhibited 3-fold and 2-fold lower GUS activity, respectively, suggesting a limited plant defense response compared to plants inoculated with cells grown in LB. On day 5 post inoculation the population of E. coli O157:H7 grown in LB supplemented with manure on plants was significantly greater than the population of E. coli O157:H7 grown in LB medium alone. E. coli O157:H7 cells exposed to soil or manure exhibited greater survival on plants compared to LB-grown E. coli O157:H7. The results of this phase of the project underscore the need to consider medium composition and cultural conditions when conducting crop challenge studies.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Tana Hintz, Karl K. Matthews, and Rong Di, The Use of Plant Antimicrobial Compounds for Food Preservation, BioMed Research International, vol. 2015, Article ID 246264, 12 pages, 2015. doi:10.1155/2015/246264
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Jung, Y., H. Jang, and K.R. Matthews. 2014. Effect of the food production chain from farm practices to vegetable processing on outbreak incidence. Microbial Biotechnology. 7:517-527.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Seo, S., and K.R. Matthews. 2014. Exposure of Escherichia coli O157:H7 to soil or manure influences capsular polysaccharide production, plant defense response and persistence of that pathogen on plants. Food Microbiology. 38: 87-92

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Aspects of the project were reported at an undergraduate research conference held in Shanghai China. The research was also presented to participants in various continuing education courses which the PI instructs. Changes/Problems: Objective 3 may be modified since construction of lettuce with supressed defense system is proving difficult. Experiments under objective 2 will be broadend and strengthened, providing novel data. What opportunities for training and professional development has the project provided? Four undergraduates have worked on the project during this reporting period. The students gained valuable experience in working with human pathogens, experimental design, data analysis, and problem solving. A Ph.D. candidate served as the lead student investigator. The student gained experience in project mangament, problem solving and logistics/planning. All students involved were required to present research findings; this provided an opportunity to enhance public speaking skills and to learn how to tell a story. How have the results been disseminated to communities of interest? The results have been conveyed verbally to students attending an undergraduate research conference. The students expressed surprise at the basic research nature of the project. Generally, research is directed at application of antimicrobial agents to reduce microbial load on a commodity. The research was also presented to industry representatives who attended one or more continuing education courses offered through Rutgers University. What do you plan to do during the next reporting period to accomplish the goals? Now that appropriate wildtype and mutant isolates have been secure,d studies will focus on interaction of bacteria with growing plants. Having access to the university greenhouses will permit the cultivation of lettuce, spinach and Arabidposis. Experiments will be designed to determine behavior of pathogens of interess on each plant in an effort to determine impact of bacterial cell surface moieties versus the plant in the ability of the pathogen to survive and persist on a plant.

Impacts
What was accomplished under these goals? In reference to objective 2, a series of wildtype and mutant strains lacking specific cell surface moieties has been established. The isolates have been cultured under various conditions to establish phenotypic characteristics. A series of studies have been conducted using lettuce under controlled conditions to establish differences in cell attachment to plant tissue. These studies aid in establishing the direction of future research where lettuce grown under greenhouse conditions will be contaminated with the strains of interest. Comparisons will be made with Arabidopsis.

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Study results were reported at an international association of food protection nmeeting in France. The information has also been shared at local meetings. The audience reached ranged from industry representatives to academics. The presentations were directed in anticipation of the expected audience. Therefore, applied or basic aspects of the experimental results were shared making the information relevant to those specific groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Undergraduate students are teamed with MS and Ph.D. students and trained in laboratory skills and experimental protocol. How have the results been disseminated to communities of interest? Information has been shared with members of the produce production and processing community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Experiments have demonstrated that evnironmental growth factors influence expression of bacteria cell surface moeties and that this, in turn, affects plant defense response. The expression of cell surface moieties including curli and capsule were increased when bacteria were cultured in the presence of manure or soil. Those bacteria were then applied to plants and specific plant defense response factors were monitored. Specifically, the PR2 gene was up-regulated, which influenced population of the target microbe on plant tissue. Growth or holding of bacteria in water had little influence on bacterial cell surface moieties. Studies are on-going to address Salmonella and how it behaves. Previous studies were conducted using E. coli O157:H7. Work is also progressing on comparison of bacterial behavior on lettuce and Arabidopsis.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Seo, S., and K.R. Matthews. 2012. Plant defense response to Escherichia coli O157:H7 cell surface structures influences survival of the enteric pathogen on plant surfaces. Appl. Envir. Microbiol. 78:5882-5889.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Matthews, K.R. 2013. Sources of enteric pathogen contamination of fruits and vegetables: future directions of research. Stewarts Post Har. Rev. ISSN: 1945-9656; doi: 10.2212/spr2013.1.2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Seo, S. and K.R. Matthews. 2013. Exposure of Escherichia coli O157:H7 to soil, manure or water influences persistence of that pathogen on plants and initiation of plant defense. IAFP European Symposium on food safety. Marseille France.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The data has been shared at national and international meetings. The interaction with professionals at scientific meetings has resulted in development of new collaborations. The PI has also shared the information at Adult continuing education courses. The courses are intended for individuals industry seeking the latest information. PARTICIPANTS: A Ph.D. student was trained and completed research to fulfill requirements toward the Ph.D. The students developed greater technical competency and ability to plan and analyze data. Two undergraduate students were involved in the project and developed a greater understanding of the plant defense system and behavior of foodborne pathogenic bacteria. TARGET AUDIENCES: The information/results of the project were presented at several scientific meetings and continuing education courses. The others researchers provided information on linkage of the outcomes with studies being conducted in their laboratories. The interaction has resulted in development of collaborative research projects. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The funding provided support for graduate and undergraduate students to conduct research. The activities foster the development of critical thinking skills and the ability to interact with other researchers. The research has provided the base for additional studies investigating foodborne pathogen plant interaction. Additional funding will be sought to pursue this new direction of research.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Significant strides have been made in moving the project to the next level; understanding how plants behave when faced with an enteric pathogen. The progress has resulted in submission of four abstracts so that information may be shared at professional meetings. The data will be used to develop research proposals to secure additional funding to continue the research. PARTICIPANTS: Wen-Hsuan Wu, Graduate student, Rutgers University. Seungwook Seo, Graduate Student, Rutgers University bassam Annous, collaborator, USDA ARS ERRC, Rong Di, Assistant Professor, Plant Pathology, Rutgers University. The research provides training to undergraduate and graduate students and a broad number of areas - from microbiology TARGET AUDIENCES: Researchers (government, academic), regulatory agencies, producers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The research has been shared with industry representatives and recognized for the potential impact it may have on measures designed to ensure the safety of crops. Although we are using a surrogate plant, Arabidopsis, studies will be conducted with lettuce. Such studies should demonstrate that for the purpose of studies being conducted Arabidopsis is a suitable surrogate.

Publications

• Seo, S. and K.R. Matthews. 2012. Influence of Constituents of Water, Soil, or Manure on Colonization of E. coli O157:H7 on Plant by Differential Induction of Plant Defense. In: Program and Abstract book International Association for Food Protection. Annual meeting. Providence, RI.
• Seo, S. and K.R. Matthews. 2012. Plant Defense Response to Escherichia coli O157:H7 Cell Surface Structures Influences Survival of the Enteric Pathogen on Plant Surfaces. In: Program and Abstract book International Association for Food Protection. Annual meeting. Providence, RI.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The project is still in the early phase of execution. Strains of Escherichia coli O157:H7 have been screened for presence of extracellular polysaccharide (EPS) and curli. The isolates were grown in manure slurry, water, and soil slurry and impact on EPS and curli production determined. Results suggest that various environments impact expression of EPS and curli expression. The isolates were also cultured at various temperatures to determine influence on curli production. Temperature did have a decided influence of curli expression, which was variable by strain. We are now in the process of growing several variants of Arabidopsis. By June we anticipate completion of several plant experiments to determine role of bacterial cell surface moieties in the interaction of E. coli with plants. PARTICIPANTS: Wen-Hsuan Wu, Graduate student, Rutgers University. Seungwook Seo, Graduate Student, Rutgers University bassam Annous, collaborator, USDA ARS ERRC, Rong Di, Assistant Professor, Plant Pathology, Rutgers University. The research provides training to undergraduate and graduate students and a broad number of areas - from microbiology to plant genetics. TARGET AUDIENCES: The expected target audience is other researchers. A portion of the information will likely be of interest to government agencies, extension specialists and farmers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
At this stage of the study no substantive outcomes have been realized. The first phase of research was presented at an International Meeting. The study demonstrated that culturing of E. coli O157:H7 in the presence of water, soil, or manure had a direct impact on expression of cell surface moieties. However, change in cell surface moiety expression did not have a significant influence on initial adherence of the pathogen to lettuce tissue.

Publications

• Seo, S., and K.R. Matthews. 2010. Influence of constituents of water, soil, or manure on adherence of Escherichia coli O157:H7 to plant tissue. Poster P. In: Program and Abstract book International Association for Food Protection. Annual meeting. Anaheim, CA.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: E. coli O157:H7 persistence studies with spinach have been conducted twice. Plants were harvested at 1, 5, and 10 days post-exposure to E. coli O157:H7 (Spray or surface irrigation). Following harvest spinach leaves were surface sterilized or processed directly for presence of E. coli O157:H7. A greater number of samples of spinach leaves from plants spray irrigated were positive at each sample day compared to leaves from soil irrigated plants. A total of 8 of 36 surface sterile samples from surface irrigated plants were positive for E. coli O157:H7 based on enrichment. Samples (including surface sterilized) from spray irrigated plants were positive based on plate count and enrichment. A similar set of experiments have been completed for green onion. No surface irrigated surface sterilized samples were positive for E. coli O157:H7 following direct plating of serial dilutions or enrichment. Spray irrigated samples harvested one-day post-challenge had 2 to 3-log E. coli O157:H7. At day 10 post-challenge all spray irrigated plants were negative for E. coli O157:H7. Presently, microscopy studies are being conducted to determine sites of localization of E. coli O157:H7 on spinach and green onions. Studies are being conducted using only spray irrigated plants. Outreach/Education: Providing educational opportunities and training for undergraduate and graduate students is an important component of the grant proposal. A first year Master's candidate has taken on the responsibility of cultivating green onions and spinach in the Rutgers greenhouse facilities. She has shared the experience with other students in the laboratory. During this past summer two SEED students sponsored through the American Chemical Society worked on the project. The students are gifted students from inner city high schools. The students had an opportunity to work in the laboratory processing samples for microbial analysis and learned about production of spinach and green onions. PARTICIPANTS: Lauren Landgrebe, Undergraduate student, Rutgers University. Wen-Hsuan Wu, Graduate student, Rutgers University. Seungwook Seo, Graduate Student, Rutgers University bassam Annous, collaborator, USDA ARS ERRC TARGET AUDIENCES: This has not changed from previous project report. The information is important to producers and packers of fresh and fresh-cut lettuce, green onions, spinach and tomatoes. Information may be helpful to regulatory agencies that provide recommendations to the produce industry. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Similar to previous studies with lettuce, results of the present study indicate that surface irrigation minimizes the likelihood that the edible portion of a plant will become contaminated following irrigation with contaminated water. In addition, under conditions of the present study, exposure of green onions to contaminated irrigation water greater than 10 days prior to harvest represented no risk of harvesting contaminated onions. Outreach/Education: Providing educational opportunities and training for undergraduate and graduate students is an important component of the grant proposal. An senior undergraduate student spent the summer working with Dr. Annous at the USDA ARS ERRC facility in Wyndmoor PA. The student was eager to continue research on the project, but also wanted to expand the research she was conducting. Based on what she had learned through working with Dr. Annous and discussion with me she prepared a grant proposal designed to support undergraduate research. The proposal was funded (see attached). The education and training she received the result of working on the USDA NIFSI proposal significantly contributed toward her success in receiving an Aresty Grant for undergraduate research at Rutgers University.

Publications

• Matthews, K.R. 2009. Foodborne and commensal bacteria associated with produce: Illness, antibiotic resistance, mitigation strategies. Session S1606. P. 108. In: Book of Abstracts 11th ASEAN Food conference. Bandar Seri Begawan, Brunei.
• Mootian G., W-H. Wu, and K.R. Matthews. 2009. Transfer of Escherichia coli O157:H7 from Soil, Water, and Manure Contaminated with Low Numbers of the Pathogen to Lettuce Plants of Varying Age J. Food Prot. 72:2308-2312.
• Hirneisen, K.A., H. Chen, R.W. Worobo, K.R. Matthews, and K.E. Kniel. 2009. Internalization of enteric viruses in spinach and green onion. Poster P3-17. In: Program and Abstract book International Association for Food Protection (IAFP). Annual meeting. Grapevine, TX.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: A large study was conducting addressing the contamination of lettuce following exposure to low numbers of E. coli O157:H7 associated with soil, manure amended soil, or irrigation water. The results of the study were presented at several professional meetings and shared with industry representatives. An oral presentation highlighting the research was given at the annual meeting of the International Association of Food Protection. The research was also used as preliminary data associated with two USDA NIFSI grant proposals. Both proposals were funded. A manuscript based on the reseach has been submitted for publication. PARTICIPANTS: Graduate students Shanta Adeeb, Seungwook Seo, Gabriel Mootian, and Wen-Hsuan Wu conducted experiments related to the project. Two high school students participating in the American Chemical Society SEED program worked for 10 weeks duirng the summer to complete carious aspects of the project. The students gained hands on experience working with plants, microbes, and data analysis programs. The PI was provided the opportunity to teach students laboratory techniques and how to develop critical thinking skills. The PI was responsible for coordinating research activities to ensure that all students had an enriching learning experience. TARGET AUDIENCES: The produce industry and particularly the fresh and fresh-cut leafy greens sector have been faced with major challenges following several large outbreaks of foodborne illness linked to the consumption of contaminated product. The industry is thirsty for knowledge on risk associated with production practices. The research conducted under the present project suggest that plants are at greatest risk of becoming contaminated after exposure to irrigation water compared to growth in contaminated soil or manure amended soil. Future studies will seek to determine changes that may occur in the bacteria while in water that may influence the interaction and persistence of the bacteria on leafy plant tissue. PROJECT MODIFICATIONS: None

Impacts
The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and wildlife likely contribute. A major emphasis of this study has been to determine transfer of low numbers of E. coli O157:H7 from soil, manure amended soil, and water to growing lettuce plants. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant or within internal locations. Young plants (12 d) were harvested at 1, 10, 20, and 30 d post exposure. No samples were positive for E. coli O157:H7 following direct plating of serial dilutions. Enrichment of all samples (young plants) demonstrated approximately 21% (113 out of 552) of plants (exposed to contaminated soil, manure amended soil, irrigation water) were positive for E. coli O157:H7. Approximately 30% (36 out of 120) of the mature plants initially irrigated with or grown in contaminated soil (includes manure amended soil) for 15 d were positive for E. coli O157:H7. Based on sterilization of surface tissue E. coli O157:H7 was internalized or in protected locations of lettuce tissue. The route of contamination was not determined. The results suggest that lettuce exposed to and grown in the presence of low numbers of E. coli O157:H7 may become contaminated and present a human health risk. These studies provide the foundation for research investigating how consumer handling practices influence behavior of E. coli O157:H7 associated with lettuce or other leafy greens. Future studies will also focus on strategies to reduce populations of foodborne pathogens through modification of packaging systems.

Publications

• Mootian, G., W-H. Wu, H-J. Pang, and K. R. Matthews. 2008. Transfer prevalence of Escherichia coli O157:H7 from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants of varying age. p. 67. In: Program and Abstract book IAFP Annual meeting. Columbus, OH
• Matthews, K.R. 2008. Internalization: Whats the magic number. p. 34. In: Program book IAFP Annual meeting. Columbus, OH

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: During the past year the outcome of experiments included under the project have been reported/presented at several professional scientific meetings and to grower/producer groups. A presentation on E. coli O157:H7 association with lettuce was given to predominantly industry representatives and growers. The meeting was by design small (~100 participants) to facilitate the interaction of speakers with attendees. The outcome of experiments comparing the efficacy of 20,000 ppm sodium hypochlorite to Tsunami 100 for inactivation of Salmonella associated with alfalfa seed was reported at the annual meeting of the International Association of Food Protection. The poster received a great deal of attention form sanitizer manufacturers and sprout growers. As part of a "hot topics" session at the China Food Safety and Quality Conference, the impact of low numbers of enteric pathogens in soil and subsequent contamination of edible crop was presented. Information from all studies related to the project has been shared with producer groups various company meetings. PARTICIPANTS: Graduate students, Gabriel Mootain, Wen-Hsuan Wu, and Hoan-jen Pang conducted experiments related to the project. The research was a unique experience for each student to conduct research outside of the scope of their respective thesis or dissertation projects. Two high school students participating in the American Chemical Society SEED program worked for 10 weeks during the summer to complete various aspects of the project. The students gained hands on experience working with plants, microbes, and data analysis programs. TARGET AUDIENCES: The fresh and fresh cut produce industry has been the subject of great scrutiny following a number of large outbreaks of foodborne illness linked to the consumption of contaminated product. The research will be of interest to growers, producers, commodity groups, regulatory agencies, and researchers. Knowledge of how enteric microbes interact with crops and whether they localize within tissue will have a dramatic impact on development of methods to inactivate the pathogens. Continuation of the present research will focus on control strategies that ensure the safe of the product.

Impacts
The number of outbreaks of human illness associated with the consumption of leafy greens has increased in recent years. The Escherichia coli O157:H7 outbreak approximately one-year ago linked to the consumption of contaminated spinach underscores the need to readdress and evaluate present production and processing practices and the research that is being conducted in the area of produce safety. The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, flooding of fields, and wildlife likely contribute. Results of the on-going studies suggests that young (12 day old ) and mature (30 Day old) plants initially exposed to low levels of E. coli O157:H7 in soil contaminated directed or through incorporation/application of contaminated manure or irrigation water may become contaminated. However, the numbers of the pathogen associated with the lettuce was extremely low since enrichment of samples was required for detection. Only a limited number of samples that had been surface sterilized were culture positive for the target bacteria. This suggests that the microbe remains predominantly on the exposed surface of the plant, although in some cases it may be at subsurface or internal locations. In the present study, whether the microbe was truly internalized in the lettuce plant was not determined. Funding of the project permitted additional variables to be included in the project design. The dose range of E. coli O157:H7 was expanded to include levels that more likely reflect those found under natural conditions. This type of information is particularly useful to commodity groups, growers, and those developing guidelines for growing and processing of fruits and vegetables.

Publications

• Abstracts Matthews, K.R. 2007. Survival of E. coli O157:H7 associated with leafy greens its all about location, location, location. E. coli 2007: Understanding, detecting, preventing. Jamesburg, NJ.
• Buchholz, A. and K.R. Matthews. 2007. Comparison of the efficacy of 1% and 3% Tsunami 100 to 20,000 ppm calcium hypochlorite treatment for the reduction of Salmonella on alfalfa seed. p. 130. In: Program and Abstract book IAFP Annual meeting. Lake Buena Vista, FL.
• Matthews, K.R. 2007. Microbial Safety of Fresh Produce. p. 22. In: China International Food Safety & Quality Conference. Beijing China.

Progress 01/01/06 to 12/31/06

Outputs
The impact of low numbers of E. coli O157:H7 in soil, irrigation water or manure on contamination of growing leafy greens is being investigated. Previous studies have utilized high numbers of the pathogen in order to follow the route of contamination, surface or internalization. The concern is whether low numbers of the pathogen present a risk to crop contamination and subsequently human health. In conjunction with those studies the persistence of E. coli O157:H7 on young and mature plants is under investigation. Previous studies have not conclusively demonstrated that contamination of growing young and old plants occurs at a similar rate or that the pathogen persists on both young and old plants.

Impacts
Given the number of recent foodborne outbreaks linked to the consumption of fresh vegetables there is great interest by government, industry, and consumer groups in developing strategies to control the contamination of fresh vegetables. The research being conducted has direct application to the development of strategies that can be implemented pre-harvest to minimize the contamination of crops with human pathogens. Research demonstrates that strategies to eliminate pathogens during processing are limited at best.

Publications

• Solomon, E.B., and K.R. Matthews. 2006. Interaction of live and dead Escherichia coli O157:H7 and fluorescent microspheres with lettuce tissue suggests bacterial processes do not mediate adherence. Lett. Appl. Microbiol. 42:88-93.
• Solomon, E.B., and K.R. Matthews. 2005. Use of fluorescent microspheres as a tool to investigate bacterial interactions with growing plants. J. Food Protect. 68:870-873.

Progress 01/01/05 to 12/31/05

Outputs
Research is now focused on addressing plant factors that influence association/adherence of foodborne pathogens to plant tissue. Studies are focused on the role that commensal bacteria may play in facilitating adherence of foodborne bacteria to plant tissue. Specifically, whether biofilms formed by the commensal bacteria facilitate adherence. Studies are being conducted to investigate association of fluorescent beads, and live and killed bacteria with plant tissue supporting commensal bacteria biofilm and tissues void of biofilm. A separate set of experiments is addressing inactivation of bacteria associated with these biofilms. Several types of sanitizing agents are being used. To date, foodborne pathogens appear to preferentially localize in areas that commensal bacteria biofilms are present. However, studies are not complete and no conclusive determination can be made. A series of experiments are being conducted using lettuce and tomato explants

Impacts
Linking plant factors and commensal bacteria to the process of interaction of foodborne pathogens with plant tissue will provide the foundation for developing new strategies to remove or kill foodborne pathogens on fruits and vegetables. Present sanitizing strategies are not effective in killing bacteria associated with human illness (Escherichia coli O157:H7, Salmonella) that may be on fruits or vegetables. The strategies that we are developing will have application in industry. Developing effective strategy(ies) to kill bacteria asaocited with produce would reduce incidence of foodborne illness associated with consumption of fresh fruist and vegetables and extend shelf-life.

Publications

• No publications reported this period

Progress 01/01/04 to 12/31/04

Outputs
New dimensions continue to be added to the project to expand the information on association of foodborne pathogens with plant tissue. Numerous avenues of entry exist for phytopathogenic bacteria including stomata, lenticels, broken trichomes and areas of emergence of lateral roots. Bacteria may not require any specific structures or gene products for attachment or entry. Ralstonia solanacearum, the causative agent of wilt in tomato plants enters through wounds in the root system, colonizes the cortex, and finally causes clogging of the xylem vessels by producing copious amounts of exopolysaccharide. Previous research has demonstrated the presence of live cells of E. coli O157:H7 in the root system as well as in the aerial tissue, whether this interaction was host or microbe specific was not determined. In the present study, we investigated whether the uptake of E. coli O157:H7 was mediated by the microbe. Fluospheres were used to demonstrate that a particle similar in size to a bacterial cell, yet devoid of bacterial surface moieties (e.g. capsule, enzymes), appendages (e.g. fimbriae), and adaptive response are taken in through the root system and carried to the edible plant tissue. Microscopic examination of root, stem, and leaf tissue sections demonstrated that Fluospheres were internalized into growing plants. Laser scanning confocal microscopy revealed that Fluospheres were present within the root tissue. The presence of Fluospheres in internal portions of stem and leaf tissue suggests transport of the spheres from the root upward into the edible tissue. The level of uptake of Fluospheres and E. coli O157:H7 was quantitatied using filtration. Numbers of Fluospheres and E. coli O157:H7 in plant tissue were similar. Results suggest that the entry of E. coli O157:H7 into lettuce plants may be a passive event since the level of Fluospheres was similar to that of the foodborne pathogen. Research is now being focused on the role of contaminated equipment in contamination of product during processing. This is of particular concern to the fresh cut produce industry. The role of contaminated product and contaminated wash water in product contamination is also being investigated.

Impacts
These studies will aid in development of strategies to prevent the contamination of produce in the field and post-harvest. If bacteria are not involved in adherence process than can focus research on plant factors.

Publications

• Solomon, E.B., and K.R. Matthews. 2004. Use of fluorescent microspheres as a tool to investigate bacterial interactions with growing plants. J. Food Protect. Accepted.
• Solomon, E.B., Y. Shafaie, and K.R. Matthews. 2004. Attachment of Escherichia coli O157:H7 to lettuce tissue is not mediated by specific bacterial processes. Submitted. IJFM.

Progress 01/01/03 to 12/31/03

Outputs
We have completed experiments using live and killed E. coli O157:H7 and fluorescent beads. These experiments were initiated to determine whether specific bacterial mediators are required for the interaction of the bacterium with plant tissue. The results of these studies suggest that bacterial mediators are not required. Studies are now in progress to determine the impact of single versus multiple exposure to a pathogen on microbial populations of the plant. Experiments have been designed and are under way to test this effect under laboratory conditions. We plan to conduct field experiments using fluorescent beads.

Impacts
These studies will aid in development of strategies to prevent the contamination of produce in the field and post-harvest. If bacteria are not involved in adherence process than can focus research on plant factors.

Publications

• Solomon, H-J. Pang, and K.R. Matthews. 2003. Persistence of Escherichia coli O157:H7 on lettuce plants following spray irrigation with contaminated water J. Food Protect. 66:2198-2202.

Progress 01/01/02 to 12/31/02

Outputs
Research has been completed demonstrating the uptake of foodborne pathogens through the root systems of plants. Experiments are on-going addressing whether adherence and uptake are active or passive processes. A microscopy continum, fluorescence microscopy to transmission electron microscopy, is used to investigate the nature of the interaction. Fluorecence microspheres are used in place of bacteria to assist in answering the objective. Studies are also being conducted to determine the impact of irrigation water on microbial load of produce. In the present study, we test samples of irrigation water and crops in the field for Enterobacteriaceae and Pseudomonas. High numbers of these bacteria can significantly reduce the shelf-life of a product and presence of certain Enterobacteriaceae may result in foodborne illness when ingested in sufficient quantity. Rather than determining the presence of coliforms or E. coli, enterobacteriaceae are screened for since they include intestinal pathogens, indicators of hygiene, sanitation, food safety, and food spoilage microorganisms. The presence of enterobacteriaceae may suggest that animal manure (domestic or feral) was recently introduced into the irrigation waters. Laboratory based studies have been completed uisng irrigation water collected on the farm and spiked with foodborne pathogens. Studies are now designed and set to begin that will address transfer of microbes from irrigation water to crops in the field.

Impacts
Regardless, of the outcome of studies investigating adherence the information is valuable with repect to developing novel methods for cleaning and sanitizing fruits and vegetables. Indeed, if the process is passive then greater attention can be directed at mechanical methods of removal rather than focusing on methods that may affect a given bacterial cell surface moiety.

Publications

• No publications reported this period

Progress 01/01/01 to 12/31/01

Outputs
The microbial quality of irrigation water applied to crops may significantly impact the microbial load of crops. Similarly, manure applied to a field may serve to contaminate crops grown in the field. The bacteria may be localized on the surface of the edible portion of the plant or found at subsurface locations. Research demonstrates that sanitizing fresh produce with a solution of chlorine only marginally reduces the level of bacteria associated with the crop. The poor efficacy of such treatment may be related to a number of factors including subsurface location of bacteria. We have demonstrated that bacteria localize in internal structure of a plant and research suggest that bacteria may enter the plant through the root system. Lettuce plants in which bacteria were found in subsurface locations were surface treated with a 100 ppm solution of chlorine. After treatment approximately 90% of lettuce samples tested positive for the target organims. Studies are in progress to evaluate various sanitizers and treament times to determine if internalized bacteria can be killed using surface sanitizing practices.

Impacts
Results reported in the journal Applied and Environmental Microbiology have received worldwide attention. Indeed, our research suggest that Good Agricultural Practices may need to be rethought and that packers of fresh produce may need to consider alternatives to current practices for sanitation of produce

Publications

• Solomon, E. B., S. Yaron, and K.R. Matthews. 2002. Transmission and internalization of Escherichia coli O157:H7 from contaminated manure and irrigation water into lettuce plant tissue. Appl. Environ. Microbiol. 68:397-400.
• Solomon, E.B., C.J. Potenski, and K.R. Matthews. 2001. Effect of irrigation method on transmission to and persistence of Escherichia coli O157:H7 on lettuce. J. Food Prot.65, 4, Accepted.

Progress 01/01/00 to 12/31/00

Outputs
Each year cases of foodborne illness linked to the consumption of contaminated produce increase. Produce may become contaminated at any stage during production and processing. We have constructed GFP expressing Escherichia coli and Salmonella Enteritidis inorder to follow these pathogens in food systems. We have demonstrated that S. Enteritidis is found at subsurface location in alfalfa sprouts and by virtue of the location are protected from the action of antimicrobial agents. Other studies demonstrate the uptake of E. coli O157:H7 by plant roots and subsequent migration of the pathogen to edible portion of the plant.

Impacts
Results of these studies suggest that contamination of produce in-the-field or during early growth can result in contamination of the edible tissue. Therefore, new strategies must be developed to control these pathogens before they can contaminate the plant.

Publications

• Yaron, S., and K.R. Matthews. 2001. A reverse transcriptase-PCR assay for detection of viable Escherichia coli O157:H7. Appl. Environ. Microbiol.
• Gandhi, M., S. Golding, S. Yaron, and K.R. Matthews. 2001. Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts. J. Food Prot. Accepted.

Progress 01/01/99 to 12/31/99

Outputs
Using GFP-expressing marker bacteria we demonstrate bacteria are able to penetrate to subsurface locations in the sprout. Marker bacteria are able to survive for prolonged periods on stored seed. Studies are in progress to determine the efficacy of chloride treatment to kill marker bacteria during sprout growth.

Impacts
Results from these studies may precipitate in a rethinking of how sprouts are grown and the use of sanitizer to kill pathogenic bacteria associated with sprouts prior to the sprout reaching maturity.

Publications

• No publications reported this period