2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Foodborne bacterial disease associated with the consumption of red meat is a serious problem. The FSIS has estimated that the contamination of meat and poultry products with foodborne pathogenic bacteria results in as many as 5,000,000 illnesses and 4000 deaths per year. In addition to human illness, food safety problems can cause economic losses to producers, processors, and consumers, and jeopardize the international competitiveness of the U.S. agriculture industry. Improved sampling methods and more rapid, sensitive microbial detection tests will allow meat processors to respond more quickly to correct process problems that may result in contamination of meat, and to more effectively detect pathogens in meats. More effective antimicrobial interventions that reduce and control pathogens on carcasses and meat will improve the safety of these products and reduce the risk of foodborne illness. Despite advances in antimicrobial intervention technology and the recent implementation of Hazard Analysis and Critical Control Point (HACCP) plans, the red meat processing industry continues to be challenged by pathogen contamination on animal carcasses and, subsequently, in meat products. Improved sampling procedures and more sensitive, accurate, and rapid pathogen detection tests are being developed for use by the meat industry for detecting microbial contamination that may occur during the meat production process. To improve the microbial safety of meat, more effective intervention procedures to reduce or control pathogens throughout slaughter and processing are being developed. In addition, the conditions and mechanisms that allow bacteria to adapt and develop resistance to antimicrobial treatments are being determined as a basis for the development of methods to interfere with resistance development, thus, improving the effectiveness of antimicrobial interventions. This research is pertinent to National Program Food Safety 108 goals to reduce microbial pathogens in food products, throughout food operations from farm to fork. This work specifically addresses program objectives relating to the development of improved sampling, detection, and enumeration methodology, and the development and evaluation of intervention strategies to reduce and control pathogens in foods. 2.List the milestones (indicators of progress) from your Project Plan. 1) Combination antimicrobial treatment intervention systems for the decontamination of beef and pork trim will be designed and tested. 2) Antimicrobial trim interventions will be developed and tested to reduce gas-forming spoilage bacteria, including Hafnia alvei, that cause ground beef chub blow-ups. 3) The growth and/or survival of E. coli O157 field isolates on beef carcass tissue following antimicrobial treatments and during refrigerated storage will be determined. 4) The growth and/or survival of E. coli O157:H7 field isolates during refrigerated and frozen storage of ground beef will be examined, and factors affecting E. coli O157:H7 recovery will be determined. 5) Carcass microbial load tests utilizing bacterial phosphatase and other potential contamination indicators will be developed for use in HACCP process monitoring. 6) The variation of acid resistance and acid adaptability characteristics among recent bovine isolates of both E. coli O157:H7 and biotype 1 E. coli will be determined. 7) The acid resistance status of E. coli O157:H7 in bovine feces as shed from naturally contaminated cattle will be determined. 8) The potential for the induction of genes involved in the development of acid resistance by E. coli O157:H7 upon organic acid spray washing of beef will be examined. 9) Expression of stx1, stx2, eaeA, ehxA, chuA, and rpoS genes of E. coli O157:H7 exposed to antimicrobial agents will be measured by quantitation of mRNA. 10) Identify E. coli O157:H7 genes involved in the development of resistance to low pH and organic acids. 11) The biochemical and ultrastructural changes of lipopolysaccharide (LPS) and the porin proteins OmpC, OmpF and PhoE of E. coli O157:H7 in response to exposure to organic acids will be determined. 12) Phenotypic and genotypic traits of preharvest and postharvest E. coli O157:H7 isolates will be characterized. 13) Genes unique to E. coli O157:H7 will be identified and their potential for use in rapid, sensitive, and specific detection schemes will be evaluated. 14) Novel methods to concentrate E. coli O157:H7 for improved recovery and detection will be explored. 15) Bovine feces, feedlot soils, and feedlot runoff will be assayed for the presence and persistence of bacteriophage carrying virulence genes. 4a.What was the single most significant accomplishment this past year? Effects of Low-Dose, Low-Penetration E-Beam Irradiation of Chilled Beef Carcass Surface Cuts on Escherichia coli O157:H7 and Meat Quality Low-dose, low-penetration electron beam (E-beam) irradiation was evaluated by scientists in the MRU, Clay Center, NE, for potential use to kill bacteria on beef carcasses during processing. The objectives of this study were (i) to assess how well E-beam irradiation can reduce levels of Escherichia coli O157:H7 on a large beef surface and (ii) to evaluate the effect of the treatment on the taste, smell, and color of the product. A low dose of E-beam irradiation reduced E. coli O157:H7, inoculated onto sections of beef, 99.99%. In assessing for effects on beef quality, the flank steak was used as the model muscle and was treated with 5, 10, 25, 50, and 75% penetrating irradiation. None of the flank steak sensory attributes were affected by any penetration treatment. Ground beef formulations consisting of 100, 50, 25, 10, 5, and 0% irradiated beef were tested. A trained sensory panel did not detect any difference between the untreated and either the 5 or 10% treatments. These results suggest that if chilled carcasses were subjected to low-dose E-beam irradiation, aroma and flavor of ground beef would not be impacted. The data presented here show that low-dose, low-penetration E-beam irradiation has great potential for use as an antimicrobial intervention on beef carcasses during processing and minimally impacts the quality of the treated beef products. 4b.List other significant accomplishments, if any. Decreased Dosage of Acidified Sodium Chlorite Reduces Microbial Contamination and Maintains Organoleptic Qualities of Ground Beef Products Scientists in the MRU, Clay Center, NE, investigated acidified sodium chlorite (ASC) spray at decreased dosages and application rates to determine its efficacy for reducing bacterial contamination on boneless beef trimmings used for production of raw ground beef products, while maintaining desirable consumer qualities in the finished ground beef products. Although ASC has been shown to be effective at reducing pathogen contamination of beef trim when used at these dosages, the ground beef produced from this treated trim can be discolored and non-typical tasting. Results indicated that decreased dosages of ASC do produce satisfactory microbial reductions in boneless beef trimmings and finished ground beef, extend shelf life of the ground beef products, and maintain desirable qualities of odor, color, and taste. The decreased dosages and application rates have been adopted by a U.S. ground beef supplier to many fast food restaurant chains. Development and Evaluation of an On-Line Hide Decontamination Procedure for Use in a Commercial Beef Processing Plant Scientists in the MRU, Clay Center, NE, identified the most effective reagents to decontaminate beef hides and then evaluated their application in a novel hide washing cabinet system. The hides of cattle are the source of Escherichia coli O157:H7 that contaminates beef carcasses during commercial beef processing. Scientists in the MRU, Clay Center, NE, have previously shown that effective interventions that reduce hide contamination also reduce subsequent carcass contamination. In this current evaluation, sodium hydroxide wash and a chlorinated water rinse were found to provide the best decontamination. When the hide wash cabinet was evaluated using these compounds, hides were cleaner, but more importantly, the contamination of carcasses was significantly reduced. The prevalence of E. coli O157 on pre-evisceration carcasses was reduced from 17% to 2% when the cabinet was in use. As a result of this work, Cargill Meat Solutions has installed hide washing cabinets in all of their processing beef plants. Efficacy of Ozonated and Electrolyzed Oxidative Waters to Decontaminate Hides of Cattle Before Slaughter Scientists in the MRU, Clay Center, NE, determined the efficacy of ozonated and electrolyzed oxidizing (EO) waters to decontaminate beef hides, compared to similar water washing without the active antimicrobial compounds present. Based on previous results reported by scientists in the MRU, Clay Center, NE, a number of alternative interventions that reduce hide contamination and the subsequent carcass contamination are being sought since not all producers can implement hide wash cabinets. Experiments used a model system designed by scientists in the MRU, Clay Center, NE, to simulate hide-on carcasses. Ozonated and EO water both were shown to be effective interventions. The prevalence of E. coli O157 on hides was reduced from 89% to 31% following treatment with ozonated water, and from 82% to 35% following EO water treatment. These results demonstrate that ozonated and EO waters can be used as viable alternatives to hide washing cabinets to decontaminate hides during processing and hold potential as means to reduce pathogen load on beef hides, thereby reducing pathogens on beef carcasses. Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef Scientists in the MRU, Clay Center, NE, evaluated several commercial pathogen detection platforms for use in detecting E. coli O157:H7. Since ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. Also, the test must have a low false positive rate, so product is not needlessly discarded, and false negatives cannot be tolerated. False negative results would allow contaminated product to be released and potentially cause disease. In this study, commercially developed methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. With most commercial beef processor employing systems in which pathogen testing must be completed prior to release of the product, robust detection systems are required. This study has been used by commercial beef processors as a factor in deciding which pathogen detection system to use for detecting E. coli O157:H7. 4c.List any significant activities that support special target populations. None 4d.Progress report. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The research reported is conducted under National Program 108, Food Safety, and contributes directly to research Component I, Microbial Pathogens - Postharvest. Research is further related to ARS Strategic Plan Goal 3, Enhance protection and safety of the nation's agriculture and food supply; Objective 3.1, Provide science-based knowledge on the safe production, storage, processing, and handling of plant and animal products and on the detection and control of toxin-producing and/or pathogenic bacteria and fungi parasites, mycotoxins, chemical residues, and plant toxins so as to assist regulatory agencies and the food industry in reducing the incidence of foodborne illnesses and Objective 3.2, Develop and deliver science-based information and technologies to reduce the number and severity of agricultural pest, insect, weed, and disease outbreaks. Scientists in the MRU, Clay Center, NE, investigated cetylpyridinium chloride (CPC) as a model hide wash antimicrobial compound to reduce carcass microbial load. Previous work had shown significant improvements in carcass cleanliness following chemical dehairing of hides, but that system could not be readily implemented by many producers. The use of CPC was novel, therefore, the parameters of application (such as concentration and dwell time) were determined. The use of CPC also called for the development of special sampling protocols to neutralize residual compound in samples. A field application of CPC hide washing demonstrated that antimicrobials can be an aid in reducing hide bacterial load and result in lower carcass contamination. Scientists in the MRU, Clay Center, NE, are establishing protocols for the implementation of CPC and other antimicrobial treatments with industry officials to further lower beef carcass contamination. (NP 108 Strategic Plan Problem Statement 1.2.4) Scientists in the MRU, Clay Center, NE, studied the regional aspects of E. coli O157:H7 prevalence that previously had been poorly understood. This lack of clarity led to several members of the meat industry and FSIS officials expressing to scientists in the MRU, Clay Center, NE, the belief that E. coli O157:H7 prevalence in western Kansas was consistently lower than other regions of the U.S. To address this question scientifically, the E. coli O157:H7 prevalence rates for fed cattle were determined from nine major beef producing regions. Samples (n=2591) of both hides and carcasses were collected from fed cattle processing plants. Prevalence for E. coli O157:H7 in samples of the animal hides ranged between plants from 9% to 85%. While the differences in prevalence between some plants were statistically significant, we do not believe that these differences are due to regional effects. The reason for this belief comes, in part, from the day-to-day variation observed in sampling. Large fluctuations (79% to 27%) in E. coli O157:H7 prevalence in hide samples occurred from one day to the next. Every plant except one had at least one sampling day where the hide prevalence of E. coli O157:H7 was over 60%. The one exception was a plant that was sampled for a previous study and found to have hide prevalence for E. coli O157:H7 over 80%. Another point of interest was the transfer rate of E. coli O157:H7 from hide to the carcass surface. The pre-evisceration carcass prevalence for E. coli O157:H7 ranged from 8% to 55% but did not mirror the hide prevalence fluctuations. Some plants did better at not transferring the pathogen from the hide to the carcass surface. This is likely to be a reflection of the carcass dressing practices employed at the individual plants. (NP 108 Strategic Plan Problem Statement 1.2.3) Scientists in the MRU, Clay Center, NE, sampled, over a five-month period, cattle hides, carcasses, and 15 environmental sites in two processing plants and determined the prevalence of E. coli O157:H7 during processing. The results indicate that there is a seasonal and geographical difference in the prevalence of this pathogen between the plants tested. These findings will provide regulatory-action agencies and industry with information to be used to reevaluate its HACCP plans to minimize the likelihood of in-plant cross-contamination of carcasses. (NP 108 Strategic Plan Problem Statement 1.2.4) Scientists in the MRU, Clay Center, NE, established sampling guidelines to measure the levels and prevalence of E. coli O157:H7, aerobic bacteria, and Enterobacteriaceae at various steps in processing. The guidelines were presented at the National E. coli Summit and adopted and implemented by a large segment of the beef industry. Using these sampling procedures, beef processors now have established benchmarks for monitoring and verifying their processes. (NP 108 Strategic Plan Problem Statement 1.2.1) In order to increase the safety of the final product, a majority of beef processors are implementing "test and hold" programs, where no product is released unless it has been tested and found to be negative for E. coli O157:H7. Since ground beef is a perishable product, minimizing the time of testing is critical. Scientists in the MRU, Clay Center, NE, evaluated several bacterial media and incubation conditions for E. coli O157:H7 growth characteristics. These results will be used to increase the speed at which test results are achieved. (NP 108 Strategic Plan Problem Statement 1.2.1) Scientists in the MRU, Clay Center, NE, performed the first comprehensive study of non-O157 STEC carcass contamination in the U.S. and provided FSIS and producers with information to be used in establishing baseline prevalence levels. (NP 108 Strategic Plan Problem Statement 1.2.3) Scientists in the MRU, Clay Center, NE, obtained a marinade utilized by a major producer of enhanced beef products. Using this marinade, a procedure was developed to assess the antibacterial properties of beef marinades in order to address the emerging concern of pathogen contamination of interior tissue surfaces of non-intact beef products such as those injected with marinades. (NP 108 Strategic Plan Problem Statement 1.2.4) Scientists in the MRU, Clay Center, NE, worked with a commercial processor employing chemical dehairing and demonstrated that beef cattle hides are the source of E. coli O157-carcass contamination during processing. This hide antimicrobial intervention procedure (chemical dehairing) was proven to prevent carcass contamination with E coli O157. Industry safety officials and FSIS are using the results of this work to implement hide decontamination protocols at beef processing plants. (NP 108 Strategic Plan Problem Statement 1.2.4) Scientists in the MRU, Clay Center, NE, developed a needed method to ship samples from beef processing plants to a distant laboratory for analysis of E. coli O157:H7, Listeria monocytogenes, Salmonella spp., and other food pathogens of concern without loss of organisms. Now commercial beef processing plants from many regions can be studied concurrently to compare regional differences and seasonality of pathogen contamination. (NP 108 Strategic Plan Problem Statement 1.2.3) 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Scientists in the MRU, Clay Center, NE, have been invited to make presentations, give lectures, or host with the following companies and organizations: Food Safety and Inspection Service, American Meat Science Association, National Cattlemen's Association, National Meat Association, Smithfield Beef Group, American Food Group, Tyson Fresh Meats, Tyson Food Safety and Laboratory Services, Standard Meat Company, Outback Steakhouse, National Beef, Bristol University (UK), Lincoln University and Ag Research (New Zealand), and Foundacion (Chile). Based on the evaluation of pathogen detection platforms for use with ground beef performed by scientists in the MRU, Clay Center, NE, several beef processing companies determined which detection technology to utilize. In some cases, company-wide conversions to a new pathogen detection platform were made. Based on data collected by scientists in the MRU, Clay Center, NE, Cargill Meat Solutions has installed hide washing cabinets in all of their beef processing plants. The reductions in pathogen loads on hides due to the hide washing cabinets have led to cleaner carcasses and less product loss. This technology has led to an annual savings for this company of 18 million dollars. As a result of work completed by scientists in the MRU, Clay Center, NE, a U.S. producer of ground beef is using acidified sodium chlorite, at the levels investigated by MRU scientists, in their production of ground beef distributed to lengthen shelf life and increase safety of their product. Scientists in the MRU, Clay Center, NE, participated in the Beef Safety Research Planning Meeting for the National Cattlemen’s Beef Association. Scientists in the MRU, Clay Center, NE, consulted with microbiologists from a large commercial beef processor regarding the involvement of Clostridium estertheticum in excess gas-blown beef chubs. Scientists in the MRU, Clay Center, NE, consulted with leaders in the sheep industry regarding antimicrobial interventions and bacterial sampling in lamb processing. Scientists in the MRU, Clay Center, NE, hosted university and industry scientists for training on microbiological sampling for antimicrobial intervention evaluation. Scientists in the MRU, Clay Center, NE, met with the Senior Vice President of Food Safety, Quality, and Innovation, Director of Microbiology, and Director of Technical Services of a large commercial beef processing company to present data regarding the persistence of E. coli O157:H7 in a feedlot setting. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Scientists in the MRU, Clay Center, NE, presented seminars "Non-O157 Shiga Toxin-Producing E. coli: Status and Relevance to Food Safety" and "Relative Significance of Feces and Hide as Sources of E.coli O157:H7 on Beef and Current Interventions" at the Southwest Meat Science Conference held at West Texas A&M University. M. Koohmaraie, T. M. Arthur, J. M. Bosilevac, M. Guerini, S. D. Shackelford, and T. L. Wheeler. 2005. Meat Sci. 71:79-91. (51st International Congress of Meat Science and Technology, Presentation in the Food Safety Session: Post-Harvest Intervention to Reduce/Eliminate Pathogens in Meat at the 51st ICoMST Aug. 7-12, 2005, in Baltimore, MD; for publication in the Special Issue of Meat Sci.). Scientists in the MRU, Clay Center, NE, were invited to attend the National Meat Association and participate in a number of food safety related round tables, British Columbia, Canada, August 2005. Scientists in the MRU, Clay Center, NE, were invited to attend the 59th Annual Convention of the National Meat Association and participate in the roundtable discussion entitled "Microbiological Testing," Las Vegas, NV, February 2005. A scientist in the MRU, Clay Center, NE, was invited to be one of 20 selected individuals to organize and speak at the by invitation only 2004 "Beef Safety Summit" sponsored by the National Cattlemen's Beef Association, April 2005. Scientists in the MRU, Clay Center, NE, were invited to attend the National Cattlemen's 2005 summer convention and present a talk on "Recent Advances in Combating E. coli O157:H7 in Beef," Denver, CO, July 2005. Review Publications Bosilevac, J.M., Shackelford, S.D., Fahle, R., Biela, T., Koohmaraie, M. 2004. Decreased dosage of acidified sodium chlorite reduces microbial contamination and maintains organoleptic qualities of ground beef products. Journal of Food Protection 67:2248-2254. Bosilevac, J.M., Nou, X., Osborn, M.S., Allen, D.M., Koohmaraie, M. 2005. Development and evaluation of an on-line hide decontamination procedure for use in a commercial beef processing plant. Journal of Food Protection. 68(2):265-272. Arthur, T.M., Wheeler, T.L., Shackelford, S.D., Bosilevac, J.M., Nou, X., Koohmaraie, M. 2005. Effects of low-dose, low-penetration electron beam irradiation of chilled beef carcass surface cuts on Escherichia coli O157:H7 and meat quality. Journal of Food Protection. 68(4):666-672.