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United States Department of Agriculture

Agricultural Research Service

2011 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
Research was conducted to recover and/or characterize Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Campylobacter spp. associated with raw, further processed, and/or ready-to-eat foods from across the food production continuum. Isolates were recovered from Hispanic-type cheese, reduced-sodium Cheddar cheese, red meat and poultry products, and produce, as well as from water and environmental samples. In addition to determining the prevalence and comparative levels of these pathogens in higher volume, higher risk foods, molecular subtyping was performed to establish the relatedness among isolates, including multiple isolates from positive samples, and to determine their source and niche. Strains that predominated or persisted in food-relevant environments were retained for further characterization and/or were used in pilot-scale, inoculated packaged, challenge studies. In related experiments, several biological, chemical, and physical interventions were developed and validated to control L. monocytogenes, STEC, Salmonella spp., and Trichina spiralis in specialty/ethnic meats, such as Genoa salami and scrapple, as well as in more traditional red meat and poultry products, such as ham, frankfurters and turkey breast. Interventions validated included high pressure processing, fermentation/drying, food grade chemicals, heat/cooking, and microfiltration. Such interventions were used alone or in combination and were applied as ingredients or were surface-applied, pre- and post-processing, via the Sprayed Lethality in Container (SLIC) method. Depending on the type of antimicrobial used and its concentration and amount applied, it was possible to achieve reductions of between 2 and 7 log and/or prevent pathogen outgrowth during product shelf life. Lastly, microarray and real-time PCR assays were used to monitor the regulation of genes in L. monocytogenes in response to high pressure processing and nisin treatments in a model liquid system. Our results demonstrated that gene expression levels were appreciably altered by both pressure and nisin treatments. These data may provide some insight into the molecular mechanisms of pathogen survival in food matrices. Collectively, these findings assisted manufacturers in meeting current regulatory guidelines and assisted regulators in making science-based policy decisions and resulted in a more safe and wholesome food supply.

1. Listeria monocytogenes on ready-to-eat (RTE) meats can be controlled. ARS researchers at Wyndmoor, PA, collaborated with several industry and government stakeholders to evaluate the efficacy of food grade antimicrobial blends for suppressing outgrowth of L. monocytogenes on the surface of a variety of RTE red meat and poultry products including scrapple, frankfurters, and turkey breast logs during shelf life. Each meat product was formulated and/or surface treated via the Sprayed Lethality in Container (SLIC®) method with different levels of select food grade antimicrobials and then stored at 4 deg C for up to 90 days. Results established that lauric arginate, levulinic acid, or a blend of lactate-diacetate-propionate provided an initial lethality towards L. monocytogenes and/or when used in combination as an ingredient provided inhibition of the pathogen throughout shelf life. These findings will allow meat processors to meet existing regulatory policies while producing a safer product.

2. Interventions to better manage the threat of pathogens in raw, red meat and poultry products. Mechanical and chemical tenderization of meat is performed by ARS researchers at Wyndmoor, PA, using solid or hollow needles, respectively, to tenderize/enhance whole muscle pieces of meat. Despite being practiced by the red meat and poultry industry for several years, there have been relatively few, if any, publications on the comparative translocation of Escherichia coli O157:H7 (ECOH) and other serotypes of Shiga toxin-producing E. coli (STEC) into mechanically or chemically tenderized steaks and/or their fate following proper cooking. Thus, we quantified the translocation and distribution of ECOH and STEC in beef subprimals caused by mechanical and chemical methods of meat tenderization and validated whether or not commonly used cooking temperatures ranging from 120 deg to 160 deg F would be adequate to kill cells of ECOH and STEC that are into the interior of the tenderized steaks. Our data validated that ECOH and STEC behave similarly with respect to translocation and thermal stability within non-intact subprimals and steaks. Our findings also established for the first time that mechanical and chemical tenderization transfers ECOH and STEC into the interior of subprimals, with the majority (ca. 40 to 60%) of the cells remaining in the top 1 cm. In addition, proper cooking on a commercial open-flame gas grill appreciably reduced the levels of both ECOH and STEC in mechanically and chemically tenderized meat, but does not completely eliminate the pathogen due to non-uniform heating of portions of the meat. Related studies are ongoing to quantify the effect of both fat and grill type on the fate of these same pathogen types in refrigerated and frozen ground beef patties during cooking.

3. Application of molecular subtyping methods to establish the prevalence and sources of pathogens along the food chain. ARS researchers at Wyndmoor, PA, conducted several collaborative studies to recover and characterize pathogens at various points in the continuum from farm to consumption. Examples include the subtyping of Escherichia coli, Salmonella spp., and Campylobacter spp., associated with wild birds, nopal, irrigation water, and/or bell peppers and subtyping of L. monocytogenes associated with Hispanic-style cheese purchased at retail establishments. The results established the prevalence and persistence of these pathogens in the environment, food, and food processing plants and established their relatedness. In collaboration with food safety professionals in academia and government, studies were initiated to determine the “true” prevalence, levels, and types of L. monocytogenes associated with pre-packaged versus deli-packaged ready-to-eat (RTE) foods purchased at retail. Over the ensuing 12 months ca. 14,500 samples, representing some 10 categories of foods, will be collected at retail establishments from four states and analyzed for the pathogen. These data will be useful to support ongoing/planned risk assessment related to L. monocytogenes in RTE retail foods.

4. Gene expression profiling of a nisin-sensitive Listeria monocytogenes Scott A CtsR Deletion Mutant. The bacteriocin nisin has been used for decades to control Listeria monocytogenes in foods; however, the modus operandi of nisin towards L. monocytogenes has not been fully elucidated. A spontaneous pressure-tolerant ctsR deletion mutant of L. monocytogenes that showed increased sensitivity to nisin was identified by ARS researchers at Wyndmoor, PA. Microarray technology was used to monitor the gene expression profiles of the ctsR mutant under nisin treatments. Total RNA was isolated from the nisin-treated (20ug/ml) ctsR mutant and L. monocytogenes Scott A wild type, labeled with fluorescent dyes, and hybridized to commercial oligonucleotide (35-mers) microarray chips representing the whole genome of L. monocytogenes. Compared to the nisin-treated wild type, 158 genes were up-regulated (> 2-fold increase) in the ctsR deletion mutant whereas 7 genes were down-regulated (< -2-fold decrease). The up-regulated genes included genes encoding for ribosomal proteins, membrane proteins, cold-shock domain proteins, translation initiation and elongation factors, cell division, a ATP-dependent ClpC protease, a putative accessory gene regulator protein D, transport and binding proteins, a phosphotransferase system (PTS) beta-glucoside-specific; IIABC component and hypothetical proteins. The down-regulated genes included genes that encode for virulence, a transcriptional regulator, stress proteins and hypothetical proteins. The gene expression changes determined by microarray assays were confirmed by real-time RT-PCR analyses. This study enhances our understanding of how nisin interacts with ctsR gene in L. monocytogenes and may contribute to the understanding of the antimicrobial mechanisms of nisin.

Review Publications
Porto-Fett, A., Campano, S., Call, J.E., Shoyer, B.A., Yoder, L., Gartner, K., Tufft, L., Oser, A., Lee, J., Luchansky, J.B. 2011. Validation of food grade salts of organic acids as ingredients to control Listeria monocytogenes on pork scrapple during extended refrigerated storage. Journal of Food Protection. 74:394-402.

Luchansky, J.B., Porto Fett, A.C., Shoyer, B.A., Call, J.E., Schlosser, W., Shaw, W., Bauer, N., Latimer, H. 2011. Inactivation of Shiga toxin-producing O157:H7 and non-O157:H7 Escherichia coli in brine-injected, gas-grilled steaks. Journal of Food Protection. 74:1054-1064.

Mukhopadhyay, S., Tomasula, P.M., Luchansky, J.B., Porto Fett, A.C., Call, J.E. 2011. Removal of Bacillus anthracis sterne spore from commercial unpasteurized liquid egg white using crossflow microfiltration. Journal of Food Processing and Preservation. 35:550-562.

Liu, Y., Ream, A.R., Joerger, R.D., Liu, J., Wang, Y. 2011. Gene expression profiling of a pressure-tolerant Listeria monocytogenes Scott A CtsR deletion mutant. Journal of Industrial Microbiology and Biotechnology. DOI 10.1007/s10295-011-0940-9.

Mukhopadhyay, S., Tomasula, P.M., Luchansky, J.B., Porto Fett, A.C., Call, J.E. 2010. Removal of Salmonella Enteritidis from commercial† unpasteurized liquid egg white using pilot scale crossflow tangential microfiltration. Internationl Journal of Microbiology. DOI: 10.1016/j.ijfoodmicro.2010.07.009.

Last Modified: 11/30/2015
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