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United States Department of Agriculture

Agricultural Research Service

2009 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.

3.Progress Report
Further work was conducted to subtype isolates of Listeria monocytogenes recovered from dairy farms, cheese processing plants, and retail establishments in northwestern Mexico. The results validated those of our previous studies and confirmed the prevalence of the pathogen in Hispanic-style soft cheese at about 10% and established the comparative diversity of strains recovered. In related studies, we evaluated the fate of this pathogen following inoculation into Queso Fresco and confirmed that its relatively high pH and moisture content provided an environment quite favorable for growth of Listeria monocytogenes. Studies are ongoing to include food grade chemicals as ingredients and/or surface applied agents to control pathogen viability during shelf life. With regards to interventions, we expanded our efforts to develop strategies to control L. monocytogenes, Escherichia coli O157:H7, and Salmonella in specialty/ethnic meats, namely beef and turkey jerky, pork scrapple, and turkey bacon. Although some of these products, such as scrapple, supported growth of select pathogens, proper storage/handling, cooking, and/or inclusion of food grade chemicals eliminated the pathogen and reduced the likelihood of outgrowth during extended refrigerated storage. As another approach to control pathogens on the surface of RTE meat and poultry products, experiments were conducted to implement and optimize the Sprayed Lethality in Container (SLIC®) method to deliver lauric arginate along with derivatives of liquid smoke to control L. monocytogenes on frankfurters and hams comprised of different species of meat. Depending on the type of product and the processing and storage conditions, the SLIC® technology was effective at delivering a 0.5- to 6.5-log reduction of L. monocytogenes. In related studies, to address potential breaches to the security of our Nation’s food supply that may arise due to a terroristic addition of threat agents such as Bacillus anthracis (BA) to higher risk foods, such as milk, in collaboration with the Dairy Processing and Products Research Unit at ERRC we also conducted studies to evaluate pasteurization in combination with microfiltration to eliminate 99.9999% of BA spores while maintaining the quality of milk and liquid eggs. Lastly, RNA was isolated from BA spores after heating/pasteurization, microfiltration, and pasteurization plus microfiltration and subjected to real-time PCR assays. Our results demonstrated that gene expression levels were appreciably altered by heat and microfiltration, whereas pasteurization and/or pasteurization plus microfiltration resulted in less alteration of gene expression. Our results may provide some insight into the molecular mechanisms of spore survival in milk.

1. Use of Micro-array and PCR technologies for analyses of genes expressed under food relevant conditions. The bacterium Listeria monocytogenes is an important food-borne pathogen that causes disease in humans and animals. This bacterium was able to grow and survive at food storage conditions such as refrigeration temperatures, low pH and high salt. However, the factors contributing to the survival and growth of this bacterium in food remain unclear. Molecular technologies are needed to better monitor the fate of pathogens in our food supply. Microarray, a new cutting-edge technology that can be used to study the bacterium at the genome level, was used to study the behavior of L. monocytogenes in skim milk and a synthetic medium. 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 assist us to develop management strategies and validate interventions to better control pathogens in dairy and meat products.

2. Better intervention technique developed from proteomics analyses of Listeria monocytogenes in food. Protein expression experiments were completed and the data analyses revealed proteins expressed by L. monocytogenes in a model food system, namely the purge from vacuum-packaged frankfurters stored at refrigeration temperature. These findings will lead to the identification of the genes and proteins that are responsible for conferring growth or survival advantages to pathogens in foods, food environments, and/or under conditions associated with food processing or storage. Ultimately the information gained on these survival processes will be used to develop targeted interventions that inhibit, control, or otherwise destroy pathogens associated with foods, which will better protect consumers against food borne disease.

3. Evaluation of microfiltration to remove Bacillus anthracis (BA) spores from liquid egg white. Pasteurization, a food processing operation, which is used to reduce or eliminate the natural microflora in foods such as fluid milk or liquid egg whites (LEW), is ineffective against threat agents such as spores of BA if intentionally added to these foods through a terroristic act. In this study, the efficacy of cross-flow microfiltration (CFMF), a membrane process, was investigated as an intervention strategy for removal of BA from LEW. A 30 gal capacity pilot scale microfiltration unit was used to filter LEW inoculated with the surrogate strain of BA (Sterne) spores. Prior to CFMF, the viscosity of the LEW was adjusted to facilitate its transport through the membrane. Under the optimum operating conditions that maintained the functional properties of LEW and ensured that all the constituent egg white proteins permeated through the membrane, greater than 99.9999% of BA spores were intercepted. These studies demonstrate that the addition of a microfiltration step followed by pasteurization will ensure the safety of LEW while preserving its nutrients and quality.

4. Application of molecular subytping methods to establish the prevalence and sources of Listeria monocytogenes along the food chain. We have conducted several collaborative studies to recover and characterize pathogens at various points in the continuum from farm to consumption. Of note, we have tracked and subtyped Listeria monocytogenes, Salmonella spp., and Campylobacter spp., in beef and pork processing plants and L. monocytogenes at retail and in plants associated with Hispanic-style cheese. As an example of the latter, a study was designed to recover L. monocytogenes from pasteurized milk and Minas Frescal cheese (MFC) sampled at retail and to identify the source(s) of contaminated products in the corresponding dairy processing plant and farm. Fifty pasteurized milk samples and 55 MFC samples were tested between June and October from retail establishments located in 8 areas of Juiz de Fora, Minas Gerais, Brazil. Only “Brand F” of MFC tested positive for L. monocytogenes. Thus, in October the farm/dairy that produced Brand F MFC was sampled; several sites/samples from the processing plant environment and several MFC samples obtained directly at Plant F tested positive for the pathogen. Coolers/refrigeration units served as the point source within Plant F that contaminated the MFC. Failures in the hygienic process and plant design were identified and subsequently corrected, thus allowing the producer to remain in business while lessening the likelihood of listeriosis. Studies are ongoing to better track and manage pathogens during the production, further processing, and storage of these foods. Related studies are ongoing with dairy and produce products and processing plants in Mexico.

5. Validation of food processing parameters for lethality towards pathogens in specialty/ethnic meats. Over the past 5 years we have made significant contributions to small/very small producers of specialty/ethnic meats and dairy products. We have collaborated with several stakeholders to optimize and validate processes to control Listeria monocytogenes, Salmonella, and/or Shiga-toxin producing Escherichia coli in soudjouk, beef and turkey jerky, kippered beef, Queso Fresco, Minas Frescal, and Queso Blanco, deli-style/prepared (chicken) salad, teewurst, and scrapple. We assisted the manufacturer in enhancing the safety of their respective products/processes, and in many instances were essential for validating their HACCP plans and/or allowing them to address potential concerns raised by USDA/FSIS inspectors. It is also relied upon by USDA/FSIS to make science-based policy decisions. The results confirm that proper procesing, storage, and/or cooking can reduce the likelihood of food borne illness.

6. Use of “Sprayed Lethality In Container” method to control L. monocytogenes in RTE meats. Studies were conducted to evaluate the effectiveness of the Sprayed Lethality in Container (SLIC®) method in combination with the antimicrobial lauric arginate (LAE) to control L. monocytogenes on RTE red meat and poultry products. Each meat product was inoculated with L. monocytogenes and then added to packages that were subsequently dosed with various volumes and concentrations of lauric arginate. Pathogen levels were substantially decreased on samples treated with 5% or 10% concentrations of lauric arginate. The results of this study show that applying lauric arginate using SLIC® reduced L. monocytogenes levels on the surfaces of vacuum-packaged roast beef, turkey breast, and frankfurters.

6.Technology Transfer

Number of Active CRADAs2

Review Publications
Jacob, R., Porto Fett, A.C., Call, J.E., Luchansky, J.B. 2009. Fate of surface inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium on kippered beef during extended storage at refrigeration and abusive temperatures. Journal of Food Protection. 72:403-407.

Dourou, D., Porto Fett, A.C., Shoyer, B.A., Call, J.E., Nychas, G.E., Illg, E.K., Luchansky, J.B. 2009. BEHAVIOR OF ESCHERICHIA COLI O157:H7, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMURIUM IN TEEWURST, A RAW SPREADABLE SAUSAGE. International Journal of Food Microbiology. 130 245-250.

Porto Fett, A.C., Hwang, C., Call, J.E., Juneja, V.K., Ingham, S.C., Ingham, B.H., Luchansky, J.B. 2008. Viability of a multi-strain mixture of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage. Food Microbiology. 25:793-801.

Luchansky, J.B., Porto Fett, A.C., Shoyer, B.A., Phebus, R.K., Thippareddi, H., Call, J.E. 2009. THERMAL INACTIVATION OF ESCHERICHIA COLI O157:H7 IN BLADE TENDERIZED BEEF STEAKS COOKED ON A COMMERCIAL OPEN-FLAME GAS GRILL. Journal of Food Protection. 72(7):1404-1411.

Luchansky, J.B., Phebus, R.K., Harshavardan, T., Call, J.E. 2009. Translocation of surface inoculated Escherichia coli O157:h7 into beef subrimals flollowing blade tenderization. Journal of Food Protection. 71:2190-2197.



Porto Fett, A.C., Juneja, V.K., Luchansky, J.B., Tamplin, M. 2009. VALIDATION OF COOKING TIMES AND TEMPERATURES FOR THERMAL INACTIVATION OF YERSINIA PESTIS STRAINS KIM5 AND CDC-A1112 IN GROUND BEEF. Journal of Food Protection. 72(3): 564-571.

Last Modified: 12/1/2015
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