2010 Annual Report
1a.Objectives (from AD-416)
The objectives include: i) elucidate the ecology (persistence, predominance, behavior, and community analysis) of pathogens in various food matrices; specifically focus on foods considered high risk by the stakeholder regulatory agencies (FSIS and FDA), for example ready-to-eat foods, or foods with a short shelf life. ii) develop and validate intervention strategies used either alone or in combination with other processes for pathogen control. iii) elucidate/define (including at the molecular level) the pathogens physiological responses to various intervention strategies and processes. Examine the influence of the inherent food macro and micro-environments.
1b.Approach (from AD-416)
Microbiological studies will be conducted with commercial and laboratory developed foods to determine how varying food matrices, processing environments, indigenous flora, or conditions associated with food distribution alter the persistence, clonality, or succession of food borne pathogens and threat agents. The predominance, persistence, and succession of pathogens along the food chain and in foods such as ready-to-eat (RTE) meats, dairy products and poultry products will be determined using conventional and molecular methods to detect and track the microorganisms. Studies will identify critical control points for the application of interventions. Isolates that predominate and persist will be used for inoculated package studies and/or will be evaluated for virulence potential. Food borne pathogens or food security threat agents will be purposefully inoculated into high risk foods (e.g. milk, RTE meats, and cheese) and pathogen viability will be monitored throughout food manufacture and projected shelf life to quantify the lethality of select food processes. Product processing conditions will be identified and used to optimize pathogen destruction and food quality. New and existing microbiological and genomic/proteomic technologies will be used to delineate the genes, proteins, and integrated physiological response networks expressed by food with food processing and storage. The genes for the identified traits or networks will be mutated and these strains will be compared to wild types to assess the importance of the genes and related physiological traits for pathogen survival and growth within foods.
Further work was conducted to subtype Escherichia coli, Salmonella spp., and Campylobacter spp., associated with wild birds, nopal, irrigation water, and/or bell peppers and L. monocytogenes at retail and in plants associated with Hispanic-style cheese. The results validated those of our previous studies and confirmed the presence of these pathogens in the targeted foods and established the comparative diversity of the strains recovered. In related studies, we evaluated the fate of L. monocytogenes following inoculation into Queso Fresco (QF) and confirmed that its relatively high pH and moisture content provided an environment quite favorable for growth this pathogen. Studies evaluating food grade chemicals as ingredients and/or surface applied agents to control this pathogen in QF have resulted in some elimination of relatively low levels. With regards to interventions, we expanded our efforts to develop strategies to control L. monocytogenes, Escherichia coli O157:H7, Salmonella, and Trichinella spiralis in specialty/ethnic meats, namely Genoa salami, scrapple, frankfurters, and turkey breast. With the exception of salami, the other products supported growth of listeriae. Those considerable efforts were expended to evaluate food grade chemicals as ingredients or as surface applied agents via the Sprayed Lethality in Container (SLIC®) method to control L. monocytogenes several of these products. Depending on the type of product and the processing and storage conditions, food grade chemicals such as lauric arginate and organic acid blends, were effective at delivering lethality of inhibition of L. monocytogenes. Lastly, micro array and real-time PCR assays were used to monitor the regulation of genes in response to high pressure processing of skim milk containing L. monocytogenes. Our results demonstrated that gene expression levels were appreciably altered by heat and these data may provide some insight into the molecular mechanisms of pathogen survival in milk.
Established the prevalence and sources of pathogens along the food chain. ARS researchers in Wyndmoor, PA have conducted several collaborative studies to recover and characterize pathogens at various points in the continuum from farm to consumption. As an example, a study was designed to recover L. monocytogenes from Queso Fresco (QF) sampled at retail and to identify the source(s) of contaminated products in the corresponding dairy processing plant and farm. From among 75 QF samples tested between January and August of 2007 from retail establishments located in Culiacan, Mexico, 7 tested positive for L. monocytogenes. This study also established that QF from the Culiacan area varies appreciably in composition among processor and may contribute to its ability to support the growth of L. monocytogenes. Studies are ongoing to better track and manage pathogens during the production, further processing, and storage of QF. Related studies are ongoing with dairy products and processing plants in Brazil.
Interventions to better manage the threat of pathogens in raw, red meat and poultry products. Blade tenderization is a process whereby needles are used to tenderize whole muscle pieces of meat; however there is relatively little information available concerning safe cooking guidelines that would eliminate internalized E. coli O157:H7 in such non-intact products. To validate whether or not commonly used cooking temperatures would be adequate to kill cells of E. coli O157:H7 that are into the interior of the meat, tenderized steaks were cooked on an open-flame gas grill to internal temperatures ranging from 120° to 160°F. In general, regardless of temperature or thickness, cooking tenderized steaks at commonly used cooking temperatures resulted in a substantial reduction of this pathogen. ARS researchers in Wyndmoor PA results validated that mechanical blade tenderization transfers E. coli O157:H7 into the interior of subprimals, with the majority of the cells remaining in the top 1 cm. In addition, cooking on a commercial-style gas grill is effective at eliminating cells of the pathogen that may be distributed throughout a steak that was blade-tenderized.
Control of Listeria monocytogenes on ready-to-eat meats. ARS researchers in Wyndmoor, PA have collaborated with several stakeholders to evaluate the potential for delivering both lethality and inhibition towards L. monocytogenes on the surface of a variety of RTE red meat and poultry products including frankfurters, turkey breast logs, and scrapple. Each meat product was formulated and/or surface treated via Sprayed Lethality in Container (SLIC) method with different levels of select food grade antimicrobials and then stored at 4C for up to 120 days. As an example, the viability of L. monocytogenes was monitored on frankfurters that were formulated with or without blends of organic acids and then surface treated with lauric arginate (LAE) via SLIC. Results showed that inclusion of the blends of organic acids as ingredients and the application of LAE on the surface of frankfurters as a post lethality treatment would effectively control L. monocytogenes on this product in the event of post process contamination. These findings should allow meat processors to meet existing regulatory policies while producing a safer product.
Determined genes expressed under food intervention conditions (high hydrostatic pressure). The bacterium Listeria monocytogenes is an important food-borne pathogen that causes disease in humans and animals. High hydrostatic pressure (HPP) has been used by ARS researchers in Wyndmoor, PA to control L. monocytogenes in food. However, the factors contributing to the survival and growth of this bacterium under HPP treatment remain unclear. Microarray technology, a new technology that can be used to study the bacterium at the genome level, was used to study the behavior of a pressure-tolerant mutant in UHT skim milk following treatment for 3 minutes at a pressure of 450 MPa. Genes that were identified by the microarray assay were verified by real-time reverse transcriptase- polymerase chain reaction (RT-PCR) assays. Information from this study will enhance our understanding of how L. monocytogenes survives under HHP and may contribute to the design of safe, accurate and economically feasible HHP treatment in food processing.
Adekunle, A.O., Porto Fett, A.C., Call, J.E., Shoyer, B.A., Gartner, K., Tuft, L., Luchansky, J.B. 2009. Effect of storage and subsequent re-heating on viability of Listeria monocytogenes on pork scrapple. Journal of Food Protection. 72(12):2530-2537.
Porto Fett, A.C., Campano, S., Oser, A., Smith, J., Call, J.E., Luchansky, J.B. 2010. Control of Listeria monocytogenes on commercially-produced frankfurters prepared with and without potassium lactate and sodium diacetate and surface .....using the Sprayed Lethality in Container (SLIC®) delivery method. Meat Science. 85:312-318.
Porto Fett, A.C., Call, J.E., Shoyer, B.A., Hill, D.E., Pshebniski, C., Cocoma, G., Luchansky, J.B. 2010. Evaluation of fermentation, drying, and high pressure processing on viability of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in raw pork and/or Genoa salami. International Journal of Food Microbiology. 140:61-75.
Liu, Y., Ream, A.R. 2009. Sporulation and germination gene expression analysis of Bacillus anthracis Sterne spores in skim milk under heat and different intervention techniques. Journal of Food Science and Technology. 74(3):120-124.
Porto Fett, A.C., Call, J.E., Muriana, P., Freier, T., Luchansky, J.B. 2010. Listeria monocytogenes. In: Juneja, V.K., Sofos, J.N., editors. Pathogens and Toxins in Foods: Challenges and Interventions. Washington, DC: Amreican Society for Microbiology Press. 6:95-107.
Liu, Y., Ream, A.R. 2008. Gene Expression Profiling of Listeria Monocytogenes Strain F365 during Growth in Ultrahigh-Temperature-Processed Skim Milk. Applied and Environmental Microbiology. 74(22):6859-6866.