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United States Department of Agriculture

Agricultural Research Service

2007 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
1935-41420-012-01S SCA (Role of Adherence-Associated Surface Antigens in Food Borne Listeriosis.) with University of Wisconsin. (see separate annual report for further details) Genomic analysis of Listeria monocytogenes has revealed a large number of surface proteins, which might participate in adherence or invasion of mammalian cells. We investigated the role of the prominent listerial protein p60, and several serotype 4b-specific surface antigens associated with teichoic acid glycosylation, in the pathogenesis of gastrointestinal listeriosis. At the time we initiated these experiments, there was little or no information as to whether these virulence-related determinants contribute to food contamination and foodborne illness.

1935-41420-012-04G Assistant type of Cooperative (Virulence Mechanisms of Listeria Monocytogenes.) with the University of Wisconsin (see separate annual report for further details) Understanding the risk of foodborne pathogens includes knowing conditions that cause the expression of virulence factors in food. Researchers at the University of Wisconsin in Madison, WI and the ARS Eastern Regional Research Center in Wyndmoor, PA are developing reporter strains of pathogenic E. coli O157:H7 that can be used to measure the expression of virulence factors under real food handling conditions.

1935-41420-012-06S SCA (Enhancing Safety, Shelf Life, and Quality of Ethic Cheeses.) with Drexel University (see separate annual report for further details) Interestingly, the association of listeriosis with the consumption of this cheese in the US has been well documented, but to date there have been no reported cases in Mexico. As such we conducted a survey of farms, cheese processing plants, and retail markets located in various geographical areas of Sonora, Mexico over a one year period to determine the prevalence and types of Listeria monocytogenes.

1935-41420-012-07S - SCA (How do the Serotype 4B Specific Genes of Listeria Monocytogenes Contribute to the Pathogenesis of Listeriosis?) with the University of Wisconsin (see separate annual reprot for further details) Genomic analysis of Listeria monocytogenes has revealed a large number of surface proteins, which might participate in adherence or invasion of mammalian cells. Serotype 4b-specific surface antigens associated with the teichoic acid of the cell wall have been identified by immunological and genetic approaches.

Whole genomic expression profiling of L. moncytogenes. A custom-designed commercial microarray containing genes representative of the whole genome of L. monocytogenes was designed. The microarray procedure was optimized and excellent images were obtained. Nucleic acid was isolated from L. monocytogenes incubated in UHT pasteurized skim milk that was stored at 4 deg C for 24 hours, as well as from otherwise similar cells of the pathogen that were exposed to a laboratory medium rather than milk. Compared to the same strain of L. monocytogenes maintained in the laboratory medium, 27 genes were up-regulated (> 2-fold difference in expression) in UHT pasteurized skim milk while 29 genes were down-regulated. We are currently investigating what genes were up and down regulated to develop strategies such information will be critical to developing strategies to better control pathogens in dairy and meat products.

Proteomics analyses of L. monocytogenes in food. Protein expression experiments were completed and the data analyses are showing which proteins are expressed by L. monocytogenes when the organism is in a model food such as the purge from vacuum-packaged frankfurters stored at refrigeration temperature. These findings project 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 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.

Microfiltration of Bacillus anthracis (BA) spores from milk. 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 to higher risk foods such as milk we conducted studies to develop intervention strategies using microfiltation technology to eliminate BA spores in bulk, fluid milk. In this study we used a 30 gallon capacity pilot plant scale microfiltration unit study to confirm on the pilot plant scale earlier laboratory studies to establish that microfiltration of milk prior to pasteurization has the ability to remove greater than 99.9999% of BA spores while maintaining the quality of milk. The addition of a microfiltration step, in conjunction with an ultrapasteurization step for the retentate, will lessen the likelihood that pathogenic bacteria and/or their spores that are greater than 0.8 µm in size will contaminate the milk supply.

Prevalence and source tracking of Listeria monocytogens in Minas Frescal. A study was designed to recover Listeria 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 of 2005 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 of 2005 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. Our results also established that the 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. Such that all cheese and environmental samples tested negative for L. monocytogenes. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of food borne listeriosis.

Salmonella is a major pathogen and poultry is a primary vehicle. Mapping the distribution of Salmonella on the carcass of Cornish game hens. To accomplish this objective, Cornish game hens purchased from local retail outlets were divided into 12 parts before isolation of Salmonella. A one-year survey was completed that resulted in 148 isolates of Salmonella from 47 Cornish game hens and a preliminary qualitative (presence or absence) map was developed. The map indicated that all parts of the carcass were contaminated with Salmonella but that some parts (e.g. sacral back) had higher rates of contamination. To complete the map, the isolates will be further characterized by DNA fingerprinting. A quantitative approach for mapping the numbers and distribution of Salmonella on the chicken carcass was successfully developed and submitted for publication. The qualitative and quantitative maps developed in these studies will allow the poultry industry and regulatory agencies to better target processes and interventions to reduce Salmonella contamination on poultry and improve food safety.

Process lethality towards pathogens in jerky. In this study, we evaluated the effectiveness of a commercial cooking process to destroy pathogens on the surface of jerky made with turkey. Mixtures of Salmonella typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes were applied to the surface of marinated and non-marinated strips of turkey. In general, In general, heating/drying marinated turkey jerky at 165°F or 180°F resulted in a substantial reduction of all three pathogens. These data confirm that processing marinated turkey jerky at 3.5 h at 165°F or for 2.5 h at 180°F is adequate to meet the performance standard of 7.0-log10 lethality established for Salmonella spp. in ready-to-eat poultry products.

Use of sprayed lethality in container method the control L. monocytogenes in Ready-to-Eat meats. Listeriosis, a serious disease that can cause death in susceptible populations is commonly associated with ready-to-eat (RTE) food products which support the growth of Listeria monocytogenes and are consumed without additional cooking. As such, the presence of this pathogen in these food products is strictly regulated by Federal agencies. Studies were conducted to evaluate the effectiveness of the Sprayed Lethality in Container method in combination with the antimicrobial lauric arginate (LAE) to lower the levels of L. monocytogenes on the surface of frankfurters, roast beef and turkey breast logs. Each meat product was inoculated with L. monocytogenes and then added to packages containing various volumes and concentrations of lauric arginate. Each package was sealed, and stored at 4°C for 24 h. Pathogen levels were substantially decreased 2.0 to 5.0 log10 CFU/package on samples treated with 5% or 10% concentrations of lauric arginate. The results of this study show that applying lauric arginate using the Sprayed Lethality in Containter method reduced L. monocytogenes levels on the surfaces of vacuum-packaged roast beef, turkey breast, and frankfurters within 24 h at 4°C. The application of LAE as a post-process intervention should assist manufacturers in achieving the USDA/FSIS alternative 2 status. The proposed research specifically addresses the goals of National Program 108, Food Safety. More specifically, this project focuses on genomic/proteomic analyses of pathogens at points throughout food processing to provide the data necessary for characterizing, monitoring, and controlling undesirable bacteria under sections 1.2.3 Production and Processing Ecology; 1.2.5 Omics; 1.2.8 Pathogenicity: and 1.2.9 Food Security. The research addresses Agency Performance Measure 3.1.2:

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of new CRADAs and MTAs1
Number of active CRADAs and MTAs2
Number of patent applications filed1
Number of non-peer reviewed presentations and proceedings31

Review Publications
Schultze, K.K., Linton, R.H., Cousin, M.A., Luchansky, J.B., Tamplin, M.L. 2007. Effect of pre-inoculation growth conditions and fat levels on thermal inactivation of a serotype-4b strain of listeria monocytogenes in frankfurter slurries. Food Microbiology. 24(4):352-361.

Smith, J.L., Bayles, D.O. 2007. Post-infectious irritable bowel syndrome: a long term consequence of bacterial gastroenteritis. Journal of Food Protection. Vol.70(7):1762-1769.

Last Modified: 11/28/2015
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