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Enhancing Microbial Food Safety by Risk Analysis

Thippareddi, Harshavardhan
University of Nebraska - Lincoln
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Risk Assessment: Assess food safety risks in agriculture systems.
Risk Management: Develop science-based interventions to prevent and mitigate food safety threats.
Risk Communication: Communicate food safety messages to stakeholders.
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Non-Technical Summary:
Assuring the microbiological safety of the food products is a primary requirement for the food industry. Foodborne pathogens are reported to cause 9.4 million episodes of foodborne illness, 55,961 hospitalization and 1,351 deaths annually in the U.S. In spite of the extensive efforts of the beef industry, beef products have been implicated in several large recalls although the number of large outbreaks has decreased. Foodborne bacterial pathogens of great concern for the beef industry have been Escherichia coli O157:H7, and more recently, Shiga toxin producing Escherichia coli (STEC). The USDA Food Safety and Inspection Service declared the six serotypes of STEC as adulterants in beef trim, ground beef and non-intact beef. While the vegetative pathogens can be a concern, the spore-forming pathogens present a greater threat to the cooked, ready-to-eat meat products. Major spore forming, anaerobic pathogens of significance to the food industry have been Clostridium botulinum and Clostridium perfringens. Beef and beef products have been implicated in several outbreaks resulting from survival, growth and/or toxin production by these organisms. The spores of these pathogens are widely distributed in nature and have been shown to be present in beef and beef products. The spores can survive typical pasteurization processes used in the meat industry tailored to destroy vegetative pathogens such as E. coli O157:H7 and Salmonella spp. The heat treatment can activate the spores, and provide competitive advantage for their germination and outgrowth due to the destruction of the competitive spoilage flora destroyed during the pasteurization (cooking) step. The food industry has developed methods to address these spore forming pathogens, such as use of nitrite in beef products, rapid cooling to prevent potential germination and outgrowth of the spores, canning process, etc. Rrecent evidence indicates that another organism of the same genus, Clostridium difficile could be emerging as a foodborne pathogen and be a threat to the beef industry. C. difficile (CD) infection (CDI) in humans is increasingly common, severe, and difficult to treat. Most of the infections have been nosocomial in origin, but the incidence of community-associated (CA)-CDI may now account for nearly 50% of cases. Songer et al. (2009) described CDI in food animals, and more recently demonstrated fully-toxigenic CD in > 40% of retail meats. Some food strains are of the same genotype as those from food animals, while others share identifying characteristics with the current epidemic human strain. Thus, foods may be a source of CD for humans. The significance of detecting CD in foods is not precisely known, in large part because (a) the minimum infectious dose for humans is not known and (b) we lack knowledge of the behavior of CD spores in meats during processing and handling. Scientific literature on the viability of CD vegetative cells in foods is non-existent. Thus, there is a need to conduct research and provide answers to the food industry and the regulatory community to mitigate the risk of CD from food. Songer, J. G., H. T. Trinh, G. E. Killgore, A. D. Thompson, L. C. McDonald, and B. M. Limbago. 2009.Clostridium difficilein retail meat products, USA, 2007.Emerg. Infect. Dis.15: 819-821.

Methodology for each of the objectives will be different based on the specific aspects of the research. Prevalence in raw beef and beef products: The microbiological profile of at least ten beef processing operations for vegetative and spore populations will be evaluated to establish the microbiological baseline. Ten sample sets will be collected from ten beef slaughter operations at four processing stages (locations) of the processing operation (during processing), on 3 trips. Sample locations, size, and other parameters will be selected based on USDA-FSIS sampling protocols and in consultation with the industry collaborators (PI has extensive contact network in the beef industry in NE). Improvements to microbial populations similar to those described by Arthur et al. (2004), such as increased sample area, adequate samples, and so forth, will be made to collect meaningful data, which will allow statistical analysis and conclusions on processing systems to be made. Sponges will be transferred to a Stomacher bag with 225 mL of pre-reduced brain heart infusion (BHI-S) with 0.5% yeast extract, 0.05% DL-cysteine and 0.1% taurocholate. Bags will be vacuum-sealed to preclude oxygen entrainment. Bags will be stomached for 2 min and transferred to the anaerobic chamber. Serial 10-fold dilutions will be prepared. Aliquots (100 ?L) will be plated directly onto TCCFA and incubated anaerobically, as above. Five ml amounts of each dilution will be subjected to enrichment (as above), and after enrichment and EtOH shock, a 100 ?L aliquot will be plated on TCCFA. Suspect colonies will be sub-cultured onto anaerobic blood agar and confirmed as C. difficile by p-cresol odor, yellow-green fluorescence under UV illumination, a positive L-proline aminopeptidase reaction, and negative indole reaction. The results will be expressed as log CFU/cm2 or the % prevalence of C. difficile on the beef carcasses. Survival of C. difficile vegetative cells in ground beef and beef products (sausages) Ground beef (90/10; 70/30 lean:fat ratio) and similarly, three varieties of beef sausages (selected in consultation with the beef industry contacts) will be obtained from NE beef processors. In case NE beef processors do not produce the sausages, product produced from surrounding states will be collected and used in the study. The products will be inoculated with C. difficile vegetative cells at low and high levels (2.0 and 5.0 log CFU/g) and mixed. The product will be packaged following industry protocols (tray packaging and case ready, high oxygen and low oxygen packaging; 3 types). The survival or destruction of the C. difficile organisms will be evaluated throughout the shelf life of the product following methods described above and expressed as log CFU/g. Thermal destruction kinetics of the vegetative C. difficile and its spores At least five C. difficile strains will be inoculated individually onto BHI agar and plates incubated at 37?C for 48 h. Single colonies will be streaked for isolation on multiple antibiotic free BHI agar plates and incubated. Cultures will be harvested with sterile cotton swabs and used to prepare lawns on ~ 300 BHI plates, followed by incubation. Spores will be collected into 5 ml phosphate-buffered saline (PBS; pH 7.4, 0.01 M) per plate, pooled, harvested by centrifugation (10,000x g, 20 min), washed with 180 ml of 1M KCl:0.5 M NaCl, and re-suspended in 100 ml 50 mM Tris-HCl, pH 7.2 with 10 mg lysozyme per ml. After incubation at 37?C for 1 h, spores will be washed 3X with 100 ml amounts of HPLC-grade water and 5 ml aliquots (9 log CFU/ml) stored at 4?C. The ground beef (80:20 lean:fat ratio) will be inoculated with the spores and the thermal destruction of C. difficile spores will be determined using standard thermal death tube (TDT) method. Ten different temperatures will be selected from preliminary experiments and ten samples will be collected during the heating process. Three independent replications will be conducted, as defined by new spore crop. A five strain cocktail of C. difficile will be used. The D values for spores will be determined at ten selected temperatures, and ten sampling points for each temperature. Three independent replications for each temperature will be performed. The z values for CD will be calculated from the determined D values following standard methods. Germination, outgrowth and toxin production of C. difficile spores in beef and beef products At least five different beef products (bologna, roast beef, corned beef, naturally cured roast beef and soup containing beef chunks) will be used. The beef components will be inoculated with C. difficile spores to obtain initial concentrations of ca. 3.0 log spores/g. The products will be prepared according to industry formulations and protocols, 5-25 g aliquots will be vacuum packaged and used for the cooling studies. The vacuum bags containing the inoculated product will be heat treated (80?C for 10 min) to simulate cooking as well as to heat activate the spores. The product will be transferred to a water bath with chilling capabilities and cooled exponentially from 54.4?C to 7.2?C within 6.5, 9, 12, 15, 18, 21 and 24 h. The PI has conducted and published extensively on potential germination and outgrowth of C. perfringens in meat and poultry products during exponential cooling and will adopt similar methodology.

This research will identify safe cooling protocols to control C. difficile spore germination and outgrowth and evaluate the adequacy of the current cooling performance standards (USDA-FSIS and FDA) to control the spore germination and outgrowth. In addition, at least five antimicrobial agents commonly used in meat processing (lactate salts, citrate salts, lemon juice/vinegar blends, and buffered vinegar) will be evaluated for inhibition of C. difficile spore germination and outgrowth during exponential cooling. This research will provide the industry with practical means to control C. difficile risk during cooling of cooked beef products. Predictive models for germination and outgrowth of C. difficile spores in beef products Two beef products will be selected for development of growth models for C. difficile. Briefly, the products will be prepared according to meat industry formulations, with lower concentration of NaCl (ca. 1%) to represent a worst case scenario and to evaluate growth of C. difficile under those conditions. The products will be inoculated with C. difficile five strain spore cocktail, and aliquots (10 g) will be transferred to vacuum bags and vacuum packaged. The inoculated product will be heat treated and transferred to water baths set at specific temperatures that allow the growth (bio-kinetic range) of the organism. Growth of C. difficile will be determined to include lag, log and stationary phase. The lag phase duration, specific growth rate, and maximum populations (parameters) attained will be determined for each of the temperatures. At least three independent replications will be performed for each treatment combination. A modified Ratkowsky's square root model will be used as the secondary model to describe the effect of temperature on growth rate. The model parameters will be determined and used to predict the potential growth of C. difficile during dynamic (time-varying) temperature conditions during processing of meat products. The PI has published extensively on development of predictive models for a variety of foodborne pathogens in a variety of matrices and will use similar modeling techniques for developing dynamic model for C. difficile in beef products.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Risk Assessment, Management, and Communication
Bacterial Pathogens
Sanitation and Quality Standards
Natural Toxins
Meat, Poultry, Game