<OL> <LI> Optimize recovery/enrichment systems for the detection of foodborne pathogens that have been injured by processes other than heat. <LI> Compare the degree of injury at key sites within foodborne pathogens subjected to various food processes (high temperature, high pressure processing [HPP], antimicrobials, etc.) <LI> Determine how injury in those sites is repaired and the kinetics of that repair in different food systems under various storage conditions. <LI> Develop novel molecular methods for tracking Listeria monocytogenes during the manufacture of minimally processed foods. <LI> Use the information collected from objectives 1 - 4 to develop new strategies for detecting, tracking and controlling pathogens in various food systems.
NON-TECHNICAL SUMMARY: Control of foodborne pathogens relies on both destroying them in foods by various processes and preventing these Ready-To-Eat (RTE) foods from being recontaminated after processing. Food processing often produces injured foodborne pathogens that can recover and grow in various foods. These injured cells often are not detected. The purpose of this study is to develop methods that detect injured pathogens in foods and methods that can track pathogens in food processing plants. This will allow the development intervention strategies for their control. <P>
APPROACH: Various selective agents will be screened to determine if they inhibit the growth of background flora without affecting the recovery of the injured target pathogen. For each pathogen and process, those selective agents that fit the above requirements will be combined in a multi-factorial experimental design (various selective agents x different levels of each agent) using EChip. The output from Echip will be analyzed to determine the optimum combination and levels of selective agents that yield the maximum % detection. Those selective agents/food additives that inhibit the recovery of the injured pathogens will be screened for use as antimicrobials in foods. Foodborne pathogens (i.e., Listeria monocytogenes, E. coli O157:H7, etc) will be added to various food systems (dairy, meat, poultry, fruits and vegetables) and subjected to different food processes (high temperature, HPP, antimicrobials, etc). Key potential injury sites (membranes, enzymes and ribosomes) will be analyzed by various methods to determine the degree of damage caused by each process. Membrane damage will be assessed by leakage of intracellular contents (260 nm absorbing material) and flow cytometry. Catalase and superoxide dismutase activity will be analyzed using standard enzymatic methods for these enzymes. Ribosome damage will be assessed using differential scanning calorimetry. Results will be compared to determine if the different processes produce similar degrees of injury at each key injury site within the various pathogens. The various food systems containing the injured cells will be stored at different temperatures and time intervals, and the above assays repeated to determine if repair at the key injury sites is occurring. If so, experiments will be performed to assess how the injury is being repaired and the kinetics of that repair. For example, following HPP, cells L. monocytogenes will be analyzed to determine if their membranes have been repaired and what effect this has on their ability to grow in selective media and in the food itself. Cells will be enumerated on selective and non-selective media at various time intervals to assess repair kinetics in various food systems. An optimized Multilocus Sequence Typing (MLST) scheme for Listeria monocytogenes will be developed by identifying the genes with the highest sequence-information content. These genes will be amplified using PCR and sequenced to produce MLSTs that will allow tracking of these pathogens in meat and dairy processing plants. The optimized MLST method will be compared with PFGE (Pulsed Field Gel Electrophoresis) to determine which is more discriminatory. Information from all of the above experiments will be assessed to develop novel strategies for the detection, tracking and control of foodborne pathogens. For example, understanding how HPP injures pathogens might allow us to combine HPP with other hurdles that subsequently kill these cells in minimally processed foods. Also understanding how they are transmitted in processing plants may allow us to develop strategies to prevent recontamination from occurring.
PROGRESS: 2003/04 TO 2008/03<BR>
OUTPUTS: Results of research on high pressure processing and molecular subtyping were presented at the Annual Meetings of the Institute of Food Technologists and the American Society for Microbiology. Results of multi-virulence-locus sequence typing were shared with scientists from USDA FSIS and ARS. As a result, we are now collaborating with FSIS scientists to analyze suspect L. monocytogenes isolates in our laboratory using multiplex PCR, MVLST and prophage sequencing to determine if the isolates are epidemic clones and outbreak clones of this pathogen. A lecture on MVLST was presented by one of my Ph.D. students in my Microbial Diversity course at Penn State. A seminar on the mechanism of inactivation by high pressure processing was presented by another of my Ph.D. students at the Microbiologists at Penn State Meeting. Both of these Ph.D. students graduated from Penn State in 2007. The results of our molecular subtyping project were disseminated to the Eastern Meat Packer Association. The attendees were cautioned about the potential hazard of Epidemic Clone II Listeria monocytogenes in meat plants in the Northeastern U.S. <BR> PARTICIPANTS: Partner Organization: Scientists at the USDA Food Safety Inspection Service and Agricultural Research Service are currently collaborating with me on the analysis of Listeria monocytogenes isolates that have been collected from Northeast U.S. meat plants during normal surveillance testing. Collaborators and contacts: My collaborators and contacts (above) are Dr. Peter Evans (FSIS) and Dr. Todd Ward (ARS). Dr. Edward Dudley, a new Assistant Professor in the Food Science Department, is now collaborating with me on the new Milk Safety Project to develop a similar DNA-based subtyping scheme for tracking E. coli O157:H7. We are currently co-advising a graduate student on this project. Dr. Cynthia Whitener, Co-Chair of Clinical Affairs at Hershey Medical Center, is currently collaborating with me to develop a similar subtyping scheme for Methicillin-Resistant Staphylococcus aureus. I am currently co-advising (with Dr. Phillip Mohr in the Biochemistry and Molecular Biology Department) an honors student on this project. Dr. Peter Hudson, Director of the Huck Institute is currently seeking funds for Dr. Whitener and I to support a postdoctoral scholar on this project. Training or professional development: A visiting scientist from the University of Turin, Italy is currently working in my laboratory to learn about our new DNA-sequence-based subtyping methods. <BR> TARGET AUDIENCES: This project supported 4 graduate students during the period, two of which were females. The project also supported a technician in my laboratory who is an African-American. The project also supported an honors student in my laboratory who was from an economically disadvantaged family in the coal-mining region of Pennsylvania. Efforts. The results of our HPP research were presented to members of the Center for Food Manufacturing and to the Microbiologists at Penn State. The results of our molecular subtyping research were presented to the Eastern Meat Packers Association and to my Microbial Diversity class.
IMPACT: 2003/04 TO 2008/03<BR>
Multi-virulence-locus sequence typing and sequencing of prophage genes has revolutionized molecular subtyping of Listeria monocytogenes. These DNA-sequence-based methods yield almost perfect discriminatory power and epidemiological concordance, the two most important criteria in the field of molecular epidemiology. FSIS scientists have acknowledged the superiority of these novel methods, which is why they are now sending us isolates to analyze to see if they are epidemic clones and outbreak clones of this important pathogen. These new techniques will allow food industries and governmental agencies to track L. monocytogenes both between and within food processing plants. This will clarify the routes by which these dangerous clones are transmitted to foods, which will then allow application of more effective intervention strategies to prevent contamination of foods and thus prevent foodborne disease. MVLST data were utilized in a major court case to demonstrate that epidemic clone II L. monocytogenes was present in two different processing plants associated with a major outbreak of listeriosis and thus both plants were held responsible. Research demonstrated that growth temperature, growth phase, heat shock and water activity all significantly affect the destruction of L. monocytogenes during high pressure processing. Food industries that utilize this new non-thermal technology need to take these factors into account to ensure destruction of this pathogen during high pressure processing.