Novel Methods for Tracking and Controlling Foodborne Pathogens

NON-TECHNICAL SUMMARY: Zoonotic pathogens, such as Salmonella spp., E. coli O157:H7, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus, are increasingly the cause of foodborne disease outbreaks, which are due to specific epidemic clones and outbreak clones of these pathogens. Preventing outbreaks due to these pathogens rests on two very important critical control points: 1) Preventing their introduction into both raw and pasteurized foods and 2) Ensuring their destruction if and when foods are contaminated. In order to prevent contamination of foods we must first understand the pathways or routes by which these pathogens are transmitted to foods. Once these routes of transmission have been identified we can then implement targeted intervention strategies to more effectively prevent transmission. In order to track the dangerous strains of these zoonotic pathogens we propose to develop novel DNA-sequence-based approaches that can rapidly and accurately identify and differentiate epidemic clones and outbreak clones of these pathogens. We will then use these novel methods to subtype isolates from various environments, foods and infected humans. This data will be analyzed using modern epidemiological tools to identify routes of transmission and the critical points where intervention strategies can be applied to prevent transmission. The second critical control point for ensuring food safety is destruction of pathogens after foods are contaminated. In order to ensure destruction we need to know more about the factors that influence resistance and inactivation of pathogens during destruction by heat and high pressure processing. Understanding more about the mechanisms of resistance and inactivation will allow us to optimize these processes to ensure complete destruction of all vegetative (non-spore-forming) pathogens in foods.

<P>APPROACH: 1. Novel multiplex PCR methods will be developed and used to screen Listeria monocytogenes isolates from various sources. Those isolates that are selected for further analysis will be subjected to MVLST and prophage sequencing and the results shared with government agencies and the food industry. The success of this research will be evaluated by determining how many intervention strategies have been implemented by the food industry and how much reduction in L. monocytogenes contamination and listeriosis has occurred as a result. <P>2. Whole genome comparisons between different E. coli strains will be conducted to identify potential targets that can differentiate epidemic clones and outbreak clones of E. coli O157:H7. Special attention will be given to identifying hypervariable loci that are present in all strains of E. coli O157:H7. Results of this research will be shared with government agencies and the food industry to see if this method can be used to track the transmission of epidemic clones and outbreak clones of E. coli O157:H7 to raw beef products and high-risk RTE foods, such as lettuce, spinach, sprouts, dry-fermented meats, etc. The research will be evaluated by determining how many intervention strategies have been implemented by the food industry and how much reduction in E. coli O157:H7 contamination and disease has occurred as a result. <P>3. Multiplex PCR methods will be developed to screen the large number of potential methicillin-resistant Staphylococcus aureus (MRSA) isolates to answer two questions: 1) Are the isolates community-associated (CA) or healthcare associated (HA)? and 2) Are the isolates epidemic clones or outbreak clones? To answer the first question a MP-PCR will be set up to determine SSCmec Type and the presence or absence of the arginine catabolic mobile element (ACME) and Panton-Valentine leukocidin (PVL). To answer the second question, a multi-virulence-locus sequence typing method targeting core virulence genes will be developed. The above methods will be applied to a set of well-characterized MRSA isolates to optimize their ability to identify and differentiate epidemic clones and outbreak clones of MRSA. The research will be evaluated by determining how many intervention strategies have been identified and implemented, and how much reduction in MRSA infection has occurred as a result. <P>4. Cells of L. monocytogenes will be subjected to various stresses and then subjected to high pressure processing to determine if they become significantly more pressure and heat resistant. Resistant cells will be subjected to 400 and 600 MPa for 150 s and then analyzed using differential scanning calorimetry (DSC). Cells that have achieved maximum pressure and heat resistance will also be analyzed using expression microarrays to determine which genes are up-regulated during the stress condition. Site-directed knock-out mutagenesis will be used to determine which gene(s) are responsible for pressure resistance. Success will be evaluated by the creation of novel processes that ensure complete inactivation of this pathogen.