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United States Department of Agriculture

Agricultural Research Service

2008 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
Studies were conducted to recover L. monocytogenes from dairy farms, cheese processing plants, and retail cheese products located in different regions of Mexico (8% prevalence) and Brazil (10% prevalence) during two different seasons (dry and wet). Genotyping and Molecular subtyping tools were used to assess the relatedness of these isolates and to establish contamination their sources. For example in Brazil, we identified the contamination source of a Minas Frescal cheese product within the processing plant and assisted the producer to implement corrective measures to eliminate the pathogen from the plant. Other experiments were conducted to develop and implement chemical and physical interventions to control E. coli O157:H7, Salmonella spp., and L. monocytogenes in a variety of processed red meat and poultry products. As one example, we further exploited the Sprayed Lethality In Container (SLIC(Registered)) technology to evaluate its effectiveness to deliver antimicrobials into the package to control L. monocytogenes on frankfurters and hams. As another accomplishment, we validated the fate of E. coli O157:H7, Salmonella spp., and L. monocytogenes during storage of specialty/ethnic RTE meats, namely kippered beef and teewurst. These studies demonstrated that such products do not provide a favorable environment for these pathogens during storage at refrigeration and abuse temperatures. In related studies, we evaluated crossflow microfiltration to remove B. anthracis Sterne and Salmonella Enteritidis from liquid egg whites. The results of this study established that crossflow microfiltration was effective in the removal of both pathogens from liquid egg whites and confirms the results from our previous study for removing B. anthracis Sterne from fluid milk. In addition to recovery and control of pathogens, we also conducted experiments to monitor the response of L. monocytogenes to food relevant processing conditions at the molecular level using micro array technology. More specifically, we compared the gene expression profiles of L. monocytogenes in UHT skin milk and in BHI broth during storage at 4°C for 24h. The microarray analyses revealed that there were 26 up-regulated and 14 down-regulated genes in L. monocytogenes exposed to UHT milk compared to those that were exposed to BHI. These results will assist in determining how L. monocytogenes can adapt and survive in a food system. Collectively, the ability to find, characterize, and control pathogens and threat agents in meat and dairy products will further enhance the safety and wholesomeness of our Nation’s food supply. National Program 108, Food Safety, Section 1.2.3; 1.2.5; 1.2.8; 1.2.9. Agency Performance Measure 3.1.2.

1. Interventions are needed to better manage the threat of pathogens in red meat and poultry products. To this end, we evaluated the use of cooking blade tenderized steaks on a commercial gas grill to eliminate E. coli O157:H7. Blade tenderization is a process whereby needles are used to tenderize whole muscle pieces of meat that are then cut into steaks that for our study were 0.75, 1.0, and 1.25 inch thick. The potential problem is that the process of tenderization may force cells of pathogenic bacteria that reside on the outside of the whole muscle piece of meat into the inside of the meat. The question then remains as to whether or not cooking would be adequate to kill cells that are inside rather than on the surface of the steaks. To test this, blade tenderized steaks containing E. coli O157:H7 were cooked on an open-flame gas grill to internal temperatures ranging from 120° to 140°F. Regardless of temperature or thickness, depending on the portion, we were able to kill up to 1000 cells of this pathogen following cooking. These results validate that mechanical blade tenderization transfers E. coli O157:H7 into the interior of steaks, with the majority of the cells remaining in the top 1 cm, and that 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. Program supports NP108, Food Safety, Animal and Plant Products.

2. Outbreaks of salmonellosis, caused by Salmonella enteritidis, still occur with consumption of heat pasteurized liquid egg white (LEW) products because all of the bacteria are not killed by the heat. Heating also damages the functional properties of the egg white proteins. We have developed a filtration process which removes more than 99.9999% of the salmonella in LEW while preserving the functional properties of the LEW proteins, and extends the shelf – life of the LEW compared to unfiltered product. Our data establish that filtration prior to pasteurization of LEW will ensure the safety of LEW while maintaining its nutritional and quality aspects. A US patent protecting this technology has been filed: “Process for Removal of Pathogens From Liquid Eggs; Application Number 12077405; Filing Date 19-MAR-2008. ARS Docket Number 187.07. Program supports NP108, Food Safety, Animal and Plant Products.

3. The bacterium Listeria monocytogenes is an important food-borne pathogen that causes disease in humans and animals. This bacterium is 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 a bacterium at the genome level, was used to study the behavior of L. monocytogenes in skim milk and in a synthetic medium. Genes that were identified by the microarray assay were verified by another molecular-based technology, the real-time reverse transcriptase- polymerase chain reaction (RT-PCR) assay. Information from this study will help further our understanding of how L. monocytogenes survives in skim milk, and eventually aid in developing new intervention strategies to control this bacterium in food. Program supports NP108, Food Safety, Animal and Plant Products.

4. We evaluated the effectiveness of the spray lethality in container (SLIC) method in combination with the antimicrobial blend of lauric arginate (LAE) with smoke extract to control Listeria monocytogenes on the surface of frankfurters. Packages of frankfurters were inoculated with L. monocytogenes (ca. 10 million cells per package) and then treated with various volumes and concentrations of LAE containing smoke extract. The packages were treated and stored refrigerated for up to 120 days. The application of LAE containing smoke extract by the SLIC method was effective at killing about 100 to 1000 cells of L. monocytogenes per package within 2 h, whereas a reduction of ca. 10,000 cells per package was observed after 120 days of storage. A similar study was conducted using hams. After 24 h the application of LAE on hams with a fibrous casing by SLIC method killed about 10 cells of L. monocytogenes per package, whereas on hams without the fibrous casing, the numbers of L. monocytogenes per package was reduced by up to 10,000 cells. These data confirm that the application of LAE using the SLIC method is effective to reduce the levels of L. monocytogenes on the surface of frankfurters and hams within 24 h at refrigeration temperatures. Program supports NP108, Food Safety, Animal and Plant Products. Program supports NP108, Food Safety, Animal And Plant Products.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of Active CRADAs2
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1

Review Publications
Moreno-Enriquez, R.I., Garcia-Galaz, A., Accedo-Felix, E., Gonzalez-Rios, H., Call, J.E., Luchansky, J.B., Diaz-Cinco, M. 2007. Prevalence, types, and geographical distribution of Listeria monocytogenes from a survey of retail Queso Fresco and associated cheese processing plants and dairy farms in Sonora, Mexico. Journal of Food Protection. 70(11):2596-2601.

Orozco, L., Iturriaga, M., Tamplin, M., Fratamico, P.M., Call, J.E., Luchansky, J.B., Escartin, E. 2008. Animal and Environmental Impact on the Presence and Distribution of Salmonella spp. in Hydroponic Tomato Greenhouses. Journal of Food Protection. 71(4):676-683.

Luchansky, J.B., Call, J.E., Cocoma, G. 2007. Hot water post-process pasteurization of cook-in-bag turkey breast treated with and without potassium lactate and sodium diacetate and acidified sodium chlorite for control of listeria monocytogenes. Journal of Food Protection. 69(1):39-46.

Tamplin, M.L., Stewart, T.E., Phillips, R., Luchansky, J.B., Kelley, L. 2008. The behavior of bacillus anthracis strains sterne (avirulent) and ames k01610 (virulent) in sterile raw gound beef. Applied and Environmental Microbiology. 74:1111-1116.

Brito, J.R., Santos, E.M., Oliveira, M.M., Arcuri, E.F., Lange, C.C., Brito, M.A., Souza, G.N., Beltran, J.M., Call, J.E., Liu, Y., Porto Fett, A.C., Luchansky, J.B. 2008. A retail survey of brazilian milk and minas frescal cheese, and a contaminated dairy plant, to establish the prevalence, relatedness, and sources of listeria monocytogenes. Applied and Environmental Microbiology. Vol.74, No.15. pg 4954-4961.

Last Modified: 11/27/2015
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