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The overall goal of this project is to integrate research, teaching, and outreach efforts to facilitate broad use of molecular subtyping by all sectors of the food system. To achieve this goal, we will perform molecular subtyping of Listeria monocytogenes, Salmonella, and Shiga Toxin producing Escherichia coli (STEC), focusing on isolates from non-food associated environments, to provide a diverse set of foodborne pathogen subtypes, which is critical for interpreting subtype data, including during outbreak investigations. Use of these data in outreach and teaching efforts will educate current and future food safety professionals on molecular subtyping.We will complete the following objectives: </p>
<p> Objective 1. Generate molecular subtyping data and populate the publicly available PathogenTracker database with pulsed field gel electrophoresis (PFGE) patterns for > 600 L. monocytogenes, Salmonella, E. coli O157:H7 and non-O157 STEC isolates, focusing on environmental isolates. </p>
<p> Objective 2. Develop sustainable systems for addition of human clinical, animal clinical, and food isolate PFGE data for L. monocytogenes, Salmonella, E. coli O157:H7, and non-O157 STEC to PathogenTracker. </p>
<p>Objective 3. Develop and conduct workshops on molecular subtyping for food industry professionals. </p>
<p>Objective 4. Develop and conduct teaching modules on molecular subtyping for undergraduate and graduate students. </p>
<p>Expected Outputs: Upon completion of Objective 1, we will have analyzed > 5,000 environmental samples from agricultural and natural environments for L. monocytogenes, Salmonella, E. coli O157:H7 and non-O157 STEC. Isolates representing these foodborne pathogens will have been characterized by PFGE typing. After completion of Objectives 1 and 2, we will have compiled PFGE patterns for > 1,200 isolates for our four target pathogens (i.e., > 300 PFGE patterns for each pathogen). All PFGE patterns and appropriate isolate information will be available through a publicly WWW-accessible database (PathogenTracker). Upon completion of Objective 3, we will have developed and conducted three workshops on molecular subtyping and molecular epidemiological of foodborne pathogens. All workshop materials, including lectures and laboratory exercise manuals will be posted on the workshop WWW page and thus made publicly available. We also plan to publish an article (e.g., in the Journal of Food Science Education) on these workshops, including evaluation results. After completion of Objective 4, we will have instructed a four-week undergraduate module in project years 1, 2, and 3, while the seven-week graduate module will be taught in years 2 and 3. Outcomes of this objective include (i) course outlines and laboratory manuals for both modules (to be posted on the project WWW page), (ii) a cadre of well-trained TAs with experience in teaching and troubleshooting molecular techniques in food microbiology, and (iii) a cadre of undergraduate and graduate students familiar with fundamental concepts in bacterial genetics, molecular subtyping, and technical expertise to implement, develop, and interpret molecular subtyping methods for foodborne pathogens.
NON-TECHNICAL SUMMARY: Most recent estimates project that each year in the U.S. foodborne pathogens caue in 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths. Furthermore, 1,100 of 1,800 deaths due to known foodborne pathogens can be attributed to a few key pathogens, including shiga toxin producing Escherichia coli, Listeria monocytogenes, and Salmonella. "DNA fingerprinting" or molecular subtyping techniques can differentiate isolates belonging to a given foodborne pathogen beyond the species level and molecular subtyping is routinely in outbreak investigations to identify a food source responsible cases of foodborne illness. While regulatory agencies routinely subtype foodborne pathogen isolates from clinical cases of human foodborne illness, these molecular subtyping data are not publicly accessible and subtype data for isolates obtained from environmental sources are critical to determine the significance of a shared human clinical and food subtype. The overall goal of this project is to expand research, teaching, and outreach efforts to facilitate the wide-spread implementation of molecular subtyping to track and control foodborne pathogens. In order to accomplish this goal, we will complete the following objectives: <P>Objective 1. Generate molecular subtyping data and populate the publicly available PathogenTracker database with subtype data for key foodborne pathogens isolated from non-food associated environmental sources.<P> Objective 2. Develop sustainable systems for adding human clinical, animal clinical, and food isolate molecular subtyping data for key foodborne pathogens to PathogenTracker. <P>Objective 3. Develop and conduct workshops on molecular subtyping for food industry professionals. <P>Objective 4. Develop and conduct teaching modules on molecular subtyping for undergraduate and graduate students.<P> APPROACH: We will collect environmental samples from agricultural and natural environments to isolate L. monocytogenes, Salmonella, E. coli O157:H7, and non-O157 STEC. Equal numbers of sampling sites will be selected in Colorado and New York and sample collections will be performed at each sample site once per season (i.e., fall, winter, spring, and summer) for a total of four sample collections at each site during a given calendar year. Each sample collected will be analyzed to detect and isolate all four pathogens targeted here. Isolates will be characterized by conventional serotyping and pulsed-field gel electrophoresis (PFGE; following PulseNet protocols for each pathogen) and subtype data will be deposited into PathogenTracker. As part of this project, we will expand our collaborations to receive isolates and PFGE patterns for the four target pathogens from other sources. PI Nightingale will work with the CO Department of Public Health and Environment and the Animal Diagnostic Laboratory at CSU to obtain human and animal clinical isolates for target pathogens. In addition, we will also work with USDA-FSIS (through their isolate sharing program) to obtain selected food isolates (for the four pathogens targeted here) for PFGE typing and inclusion in our database. Both PIs will work with commercial testing laboratories (e.g., Food Safety Net Services), food industry partners, trade organizations, and academic researcher to facilitate transfer of isolates for the four target pathogens and their PFGE patterns as available. We will develop and instruct a workshop on molecular subtyping of foodborne pathogen, which will be conducted as a 1-week program that includes a (i) one and a half-day lecture series and symposium and (ii) three-day hands-on workshop. During the lecture series and symposium portion of the workshop, participants will become familiarized with the core concepts of bacterial genetics and molecular subtyping approaches. During the hands-on workshop participants will learn to perform molecular subtyping protocols commonly used to characterize foodborne pathogens. All attendees will be asked to complete an evaluation after completion of the lecture series to assess quality of the speakers, relevance of each topic covered, overall logistics, technology used, and facilities. Finally, all attendees will be asked to complete a follow-up on-line questionnaire 4 to 6 months after completion of the workshop; this evaluation will assess attendee use of the knowledge and technical expertise gained through the workshop. In order to improve undergraduate and graduate student training in molecular subtyping, we will develop and integrate a basic four-week module (1/4 semester) and an advanced seven-week (1/2 semester) module on molecular subtyping of foodborne pathogens (which includes instructor guided independent projects) into the current curricula at CSU and Cornell. At the beginning of the basic and advanced modules, students will complete pre-course questionnaires to assess their prior knowledge. Students will also complete evaluations at the middle and end of the module.