Source: TUSKEGEE UNIVERSITY submitted to
FOODBORNE PATHOGENS DETECTION AND EDUCATION TO SUPPORT SUSTAINABLE AGRICULTURE FOR SMALL SCALE PRODUCERS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0227698
Grant No.
2011-51110-31082
Project No.
ALV-YEHUALAESHETET
Proposal No.
2011-04963
Multistate No.
(N/A)
Program Code
111
Project Start Date
Sep 1, 2011
Project End Date
Nov 30, 2013
Grant Year
2011
Project Director
Yehualaeshet, T.
Recipient Organization
TUSKEGEE UNIVERSITY
(N/A)
TUSKEGEE,AL 36088
Performing Department
Microbiology
Non Technical Summary
Foodborne pathogens continue to cause epidemic illnesses and immense economical damage to the US industry and the public. The number of cases of gastroenteritis associated with food is conservatively estimated to be between 68 million and 275 million per year. Lacks of rapid, sensitive and susceptible foodborne pathogen detection assays may exacerbate the issue and also result in under-reporting of foodborne illness outbreaks. The Food Safety and Inspection Service (FSIS) within the USDA inspects and regulates meat, poultry and processed egg products produced in federally inspected plants. Despite the existing efforts to ensure food safety, massive outbreaks continue to happen with enormous health, economical and social consequences. There exists local and national need, among many others to: a. Develop workforce at national level in the areas of genomic data mining and incorporation of microbial genomic data into the pathogen detection tools and platforms. b. Rapidly and reliably detect foodborne pathogens in food before or as soon as outbreaks of infection are reported, c. Develop rapid, sensitive and specific detection strategies to diagnose foodborne illnesses, d. Train students (mainly minority) and laboratory personnel at Tuskegee University on the recent advances in foodborne pathogen detection platforms and molecular technologies, especially those that have field applications. This proposal addresses these issues by attempting to develop hand-held and laboratory-based detection tools, as well as by training minority students in methods in food safety molecular technologies. Traditional methods for foodborne pathogens detection are time consuming and laborious, so there is a need for innovative methods that enable rapid identification of foodborne pathogens. Recent advances in molecular techniques have revolutionized the detection of pathogens in foods. The advent of biotechnology has greatly altered food-testing methods. In this study we will develop PCR-based technique and surface plasmon resonance (SPR) biosensor application for the rapid identification of the foodborne pathogens (Campylobacter, Salmonella, E. coli and Listeria). The overall outcome of this project will be to translate pathogen detection methods developed in our laboratory and validate them at a point of concern.
Animal Health Component
(N/A)
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010110065%
7124010104035%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;
Goals / Objectives
The overall goal of the project is to integrate food pathogen detection laboratory research and education with the ultimate aim to improve food safety. This integrated multi-disciplinary proposal is specifically targeted to translate foodborne pathogen detection methods developed in our laboratory (research) into augmenting the knowledge base about food safety by training minority students in applied food safety (education). The proposed project will have the following specific goals: i) Develop nucleic acid-biosensor -based detection assays which will have simultaneous or multiplexed detection capacity, ii) Train, motivate, mentor and develop skilled manpower in food safety, foodborne pathogen detection technologies among the underrepresented minorities, iii) Enhance the capacity of the faculty in the College of Veterinary Medicine, Nursing and allied Health (CVMNAH) to stay abreast with advances in foodborne pathogen detection technologies and the impact of food safety issues. The research arm of the project we will integrate our specific real time- PCR based foodborne pathogen detection tools and antibodies with impedometric biosensor technologies and hand-held surface plasmon resonance (SPR), respectively. The education component of the research will enable students to be trained in the genomic bioinformatics and the development of the biosensor technologies stated above, and for the faculty to update their knowledge through attendance and presentation at relevant meetings. The measurable outcomes will be a) Graduate two students (MVSc) with theses focused on integrated detection of food borne pathogens, b) Improve the contents of two elective courses offered to graduate students at CVMNAH, by including current advances and tools for the molecular and biosensor-based detection techniques of foodborne pathogens, c) Active participation of project members at national meetings relevant to food safety and food microbial biodetection. Successful development, implementation, testing, and validation of the proposed methodologies will minimize an existing gap in our capacity to rapidly detect multiple pathogenic threats simultaneously in real time. We anticipate that this collaborative research will provide cross-disciplinary experience to the PI's of the project with respect to detection technologies (foodborne pathogen detection, genomic bioinformatics, PCR, nanotechnology, biosensor development). Additionally, it will encourage minority veterinarians to undertake post-DVM education in food safety industry and pursue research-oriented careers. The state-of-the-art research will boost the confidence of CVMNAH graduates and is expected to improve their marketability for jobs in the federal government as well as in industry. The resources developed through funding for this project will enhance the quality of research by graduate students and faculty. Project funds will train minority graduate students who subsequently will contribute to work force diversity in the state, federal agencies and in the industry.
Project Methods
Foodborne pathogens included in the research for single and multiplex PCR will be Salmonella, E. coli, Campylobacter and Listeria. As this will be the first study of its kind, we will establish the Surface Plasmon Resonance (SPR) technology for the detection of only two foodborne pathogens: E. coli and Salmonella species. Commercially available anti-Salmonella (Abcam #31555) and anti-E. coli (Abcam #25823) antibodies will be used. As these antibodies have already been characterized and well validated for ELISA assays, we can directly immobilize them on the protein-G chip surface. Once the system is established, the SPR device could also be further modified to detect other pathogens or agents. Reference strains will incorporated in each validation. Approach: Nucleic acid-based detection assay: In our previous work, we validated the strain specific primers and probes for most of the selected foodborne pathogens. We will identify appropriate amplification parameters for the Salmonella subspecies that we had not addressed in our previous work. These will be combined with the probes we already identified, to establish the PCR microarray. The probes will be initially validated in-silico, and then in vitro using isolated bacterial genomic DNA. In our previous work, a number of published sequences showed cross-reactions with related or distantly related bacteria when tested against a larger pool of genomic data (in silico) and bacterial genomic DNA (in vitro). Therefore, every probe we will synthesize will be vigorously tested against about 30 genomes of species and strains of pathogens for cross reactivity b. Label free SPR biosensors: On-Site Monitoring of Foodborne Pathogens Using Hand-Held Surface Plasmon Resonance (SPR): SPR-based technologies have the advantage of simplicity and adaptation for field use. In this experiment, main focus will be on the development of SPR-based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. Moreover, SPR-based pathogen detection devices are now available in hand-held formats, which we can modify by functionalizing the surfaces with our own probes and use for the detection of the pathogens listed above. In this proposal, for orientation-controlled immobilization of antibody onto a SPR surface, protein G modified with cysteine residues will be used as antibody-capturing molecule, since protein G selectively binds to the heavy chain constant Fc region of antibodies. Publication and presentation and practical application of the product to the user will be used as output evaluation. Arrangements will be done once in 6 months to meet all the co-investigators and collaborators to evaluate the progress and future directions of the project. The experimental design and data analysis will be supported by the Center for Computational Epidemiology, Bioinformatics & Risk Analysis (CCEBRA) and Biomedical Information Management Systems (BIMS) located in the College of Veterinary Medicine, Nursing and Allied Health.

Progress 09/01/11 to 11/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two underrepresented students were supported from this grant. The graduate students experienced the related research activities in the laboratory and completed their theses. Additionally, faculty involved in the project updated their knowledge, share experience across disciplines, and prepared for educating the students, including those that enrolled in the new advanced degree programs in the College of Veterinary Medicine, Nursing and Allied Health (CVMNAH). The graduates were trained and experienced in modern pathogen detection technologies, hands-on experience at food chain network (through their laboratory experiences), food safety issues and the principles and applications of the Hazard Analysis and Critical Control Points (HACCP) system adopted by the Codex Alimentarius Commission. Additionally, three work-study students participate and give support for the graduate student’s research activities. They work-study students are Akeya Cole, Zena Wilson and Mulugeta Gebremedehin. The main laboratory training includes specifically but not limited to: culture, isolation and identification of bacteria from food samples, DNA extraction Polymerase chain reaction, antibiotics resistance test, phage isolation and handling, molecular and phage data mining, use and application of resonant frequency of magnetoelastic (ME) sensors and scanning electron microscopy (SEM), and also data analysis, interpretation and presentation. This grant is completed with the expected goals. Adequate preliminary data are harvested during the grant period. The main plan in the future is to prepare and submit bigger grant that will foster interdisciplinary research, education and training, which will enrich the student learning experiences. Strategic partnerships are already in place for the future plan. How have the results been disseminated to communities of interest? Results of research were disseminated through local and national symposium and meetings. The graduate students participated and presented their results in Annual Meeting of the Southeastern Branch of the American Society for Microbiology (ASM) and Annual Biomedical Research Symposium at Tuskegee University. Additionally research results were presented at the College’s Seminar Series. The interaction during the survey work and the results will be vital to communicate with the consumers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The main mission of the project was to train and educate graduate students in food safety and advanced molecular detection technologies and contributes diversity in increased cadres working in food safety. In addition to classroom and laboratory exposures during their graduate studies, two students were supported by this project to conduct and complete their Masters’ research and write their theses. Accomplishments of the funded project was: a) Identify and characterize the most important foodborne pathogens from restaurant foods and its facilities b) Develop phage-based biosensor detection for Salmonella Typhimurium from romaine lettuce c) Train, motivate, mentor and develop skilled manpower in pathogen detection technologies; d) Enhance the capacity of the College to stay abreast with advances in pathogen detection technologies and be able to disseminate the knowledge to the college community and the surrounding environs; d) Advance the capacity and capability of Tuskegee University College of Veterinary Medicine, Nursing and allied Health (CVMNAH) to provide the students with cutting edge knowledge on pathogen detection and food safety. Additionally, this project strengthened the current graduate programs of the College (Masters in Veterinary Medicine, Masters of Science in Public Health, Masters in Public Health and other graduate students from agriculture and basic science). The resources developed through funding for this project enhanced the quality of research by graduate students and faculty. The funded Project was instrumental to train minority graduate students who subsequently will contribute to work force diversity in the state, federal agencies and in the food industry. Major accomplishments Short summary of the accomplished tasks of the graduate students including their background is listed below. Summary, data collected, results and discussion Phage-Based Biosensor Used on Romaine Lettuce as Real-Time Detection Method for Salmonella Typhimurium The primary approach of this study was to apply phage-based biosensors to romaine lettuce as a rapid detection method for Salmonella Typhimurium. Attachment of Salmonella Typhimurium to lettuce leaves has been established in recent studies, however, limited biosensor detection methods have been explored on romaine lettuce. Romaine lettuce hearts (Lactuca sativa L. var. longifolia) were obtained from a local grocery store and inoculated with Salmonella Typhimurium. The resonant frequency of magnetoelastic (ME) sensors was measured before and after contact with Salmonella to distinguish if binding took place on the sensor surface. Analysis of three blocking agents, bovine albumin serum (BSA), Casein and polyethylene glycol (PEG), were compared for optimization in attachment of Salmonella Typhimurium on the ME biosensor surface. The resonant frequency for ME sensors decreased as mass (bacteria) is attached. In this study, resonant frequency change results obtained from both measurement and control sensors showed significant change in relation to various concentrations used. Visual conformation of bacterial attachment to the phage-based sensor surfaces was obtained using scanning electron microscopy (SEM). This examination provided the distribution of the Salmonella Typhimurium attachment to the phage in contrast to the concentration of cells used. As a result, this research on the optimization of application methods yielded a permissible real time detection method that can be used in the field. Conclusion: In this study, romaine lettuce was examined for structural difference, attachment and distribution of S. Typhimurium on the surface. ME sensor platforms were used for direct detection of S. Typhimurium on the romaine lettuce surface. Several measurement and control ME sensor platforms were positioned on the romaine lettuce surface for 30 min in humidity controlled chamber. The resonant frequency shifts of the measurement and control ME sensor platforms were measured and compared with the increase of S. Typhimurium concentration and were in proposition. SEM imagery confirmed the resonant frequency shift of both the measurement and control ME sensor platform with attachment of S. Typhimurium and minimal non-specific binding. This study exhibits ME sensor platform as a prospective rapid method for direct detection of food-borne pathogen on fresh-cut produce. Collaboration: This research activity was supports in collaboration with Dr. Brian Chin, Director of The Department of Materials Engineering, Material Research & Education Center, Auburn University, Auburn, AL 36832, USA. Auburn University is working on hand held ME-Biosensor, therefore, this detection technology can easily be translated and used at the point of interest. ii) Summary, data collected, results and discussion A. Bacteria Isolated from Restaurants, Fast Food Restaurants and Food Facility Environments and further Characterization of Staphylococcus species in Tuskegee, Alabama A total of 160 swabs were collected from the representative sites of the groceries and restaurants; swabs from dining tables, food drawers, wash rooms, floors, cashier desks, cash register, main door holder, washrooms door holders, soda refills machines, and different food-contact points to the customers and the workers. Additionally 16 meals were taken (chicken and beef sandwich) from each fast food restaurants. The predominant isolates from the food facility environment were Staphylococcus spp (46.6 %), Bacilli spp (11 %), and Klebsiella spp (9 %). The Staphylococcus species isolated were S. aureus ss aureus (36.8 %), S. haemolyticus (21.1 %), S. sciuri ss sciuri (21.1 %), S. hominis ss hominis (10.5 %) and S. epidermis (10.5 %). All the establishments had Staphylococcus spp. except establishment 1 which had a higher number of Bacilli as compared to establishment 3. Establishment 2 had the highest number of Staphylococcus spp. Klebsiella was found in establishment 1, 3 and 4 but none was found in Establishment 2. From the Staphylococcus spp isolates, S. aureus was the most predominant spp (46.7 %) and out of the isolate, which was tested for drug resistance, it was found that all S. aureus were resistant to oxacillin, penicillin and vancomycin. However, 50% of the S. aureus isolates were resistant to beta-lactam, gentamicin, ciprofloxacin and clindamycin. From the identified Staphylococcus spp, 100 % of the samples were resistant to penicillin, 93 % to vancomycin, 64.3 % to oxacillin 36 % to ciprofloxacin, 36 % to gentamicin, 50 % to clindamycin and 14.2 % were resistant to beta lactam. Results of Real Time PCR for the Detection of Staphylococcus Toxin Genes: In total 20 Staphylococcus isolates were tested for the presence of entrotoxogenic genes. The isolates were tested for SEA, SEB, NUC, SEQ and SEK genes by real time PCR. According to PCR analysis results 19 isolates were found to harbored one or more enterotoxigenic genes. B. Survey on Food Safety Awareness : A total of 140 random surveys were collected from Tuskegee (Macon County) area. The main objective of the survey was to determine the Food Safety Awareness of the community. The survey includes questions, which include the diversity issues related to education, food habits, awareness level on food safety and understanding of how to prevent the food contamination events. The survey results are in arrangement to analysis and interpretation.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Preliminary Survey on the Importance of Cross-contamination as a Source of Food Contamination
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Comparison and Optimization of Blocking Agents for Phage-Based Biosensor Used as Real-Time Detection Method for Salmonella Typhimurium
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Identification and Characterization of Staphylococcus species Isolated from Fast Food Restaurants and its Environments in Tuskegee, Alabama
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Phage-Based Biosensor Used on Romaine Lettuce as Real-Time Detection Method for Salmonella Typhimurium