Source: AGRICULTURAL RESEARCH SERVICE submitted to
CONTROL OF PATHOGENIC AND SPOILAGE BACTERIA ON RED MEAT
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0410528
Grant No.
(N/A)
Project No.
5438-42000-013-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 12, 2006
Project End Date
Jan 31, 2011
Grant Year
(N/A)
Project Director
ARTHUR T M
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
CLAY CENTER,NE 68933
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123520110025%
7123620110015%
7123320110060%
Goals / Objectives
1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non-O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing.
Project Methods
Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine-related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS).

Progress 04/12/06 to 01/31/11

Outputs
Progress Report Objectives (from AD-416) 1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non- O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing. Approach (from AD-416) Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine- related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin- producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS). This is the final report for the 5 year project 5438-42000-013-00D, which was terminated in 2011, and replaced with project 5438-42000-014-00D "Pathogen Mitigation in Livestock and Red Meat Production." Over the lifespan of this research project, we have collected a great deal of data regarding foodborne pathogens and their association with beef, pork, and lamb supplies in the United States. Studies were conducted in live animal settings during production and on carcasses and the finished product during processing. Through these efforts, our knowledge of pathogen ecology has grown, giving insights into new intervention development. Many of these experiments could only be realized by utilizing detection methodology also developed in this project. We have compiled evidence that cattle shedding high amounts of E. coli O157:H7 are of particular concern in a feedlot setting, as they serve as the largest source of the bacterial pathogen and lead to high rates of hide contamination among the other animals in the pen. We have determined the contamination present on the carcass during processing is more likely to originate from the processing plant holding pen environment than from the feedlot. A multitude of novel antimicrobial interventions have been evaluated for efficacy including 1, 3-Dibromo-5, 5-Dimethylhydantoin (DBDMH) or commercially called Bovibrome, electrolyzed water, Fresh FX, ozone, and UV light. Prior to initiating much of this work, methodologies for the assessment of microbial pathogen load, at various steps in the beef production process, were lacking. We developed two methods for the direct enumeration of E. coli O157:H7 and Salmonella spp. Also, work was conducted to enhance detection of foodborne pathogens in the finished product. The beef industry has used a process called test-and-hold to sample beef trim and ground beef to test for the presence of E. coli O157:H7 and only release product that tests negative. We determined the optimal medium and growth conditions for improving the speed at which results can be obtained. Further work determined that the volume of media could be reduced by 2/3, thus, creating a significant cost savings for the end user. The cost reduction for these changes has reduced the cost from ~$10 to ~$4 per test. These results demonstrate that beef processors can use a low cost medium and reduce the amount of medium used to enrich for possible contaminating E. coli O157:H7 while maintaining a high level of accuracy in the testing of beef trim and ground beef. Experiments performed under this research project also evaluated the potential contamination risk presented by novel sources. Airborne pathogens in slaughter facilities have the potential to contaminate beef carcasses after interventions have been performed. Our work determined that airborne contamination in areas immediately adjacent to the hide puller equipment could be a source for spreading E. coli O157:H7 and Salmonella. Accomplishments 01 Bacteria colonization sites in cattle excreting disproportionately high levels of E. coli O157:H7. E. coli O157:H7 contamination of beef produc has resulted in human disease outbreaks and large financial losses to th beef industry. This pathogen utilizes the intestinal tract of cattle as primary habitat. Some cattle have been shown to shed E. coli O157:H7 in their feces at disproportionately high levels. It is thought that if th excretion of disproportionately high levels of E. coli O157:H7 by cattle could be prevented, the transmission of E. coli O157:H7 could be controlled. ARS scientists in Clay Center, NE, identified cattle sheddi disproportionately high levels of E. coli O157:H7 and determined that th distal colon was the intestinal tract location most frequently found to harbor E. coli O157:H7. In addition, it was shown that at times of peak shedding E. coli O157:H7 could be isolated from sites encompassing the entire bovine gastrointestinal tract from mouth to rectum. At no time w E. coli O157:H7 ever detected from samples of lymph nodes or gall bladde This study emphasized the contamination potential of cattle that shed disproportionately high levels of E. coli O157:H7 in beef processing. A better understanding of E. coli O157:H7 shedding will aid in reducing th spread of this pathogen among cattle and throughout the beef production chain and thereby prevent economic loss to farmers, ranchers, and United States and foreign consumers of American beef. 02 Method revalidation for test-and-hold programs. Method revalidation is required when changes are made in sample size and/or enrichment media (volume or type). Testing schemes are constantly evolving with improvements to the test protocols. There is a tendency across the beef processing industry to implement changes while not recognizing potential problems that may result later. ARS scientists at Clay Center, NE, demonstrated that changes that reduced the volume of enrichment media in order to concentrate the E. coli, or changes that increased the size of the sample to provide greater sensitivity, must be revalidated by all users in their own systems. In addition, the scientists noted that rapi detection methods can be used on numerous sample types, except those tha might alter the pH of the enrichment media during incubation. These results identified situations when a revalidation is needed and when it not. The results can also be used by in-house laboratories of beef processors, ground beef manufacturers, and others, such as third party laboratories, as supporting documents in their testing programs. 03 Salmonella prevalence is low in commercial ground beef. Salmonella is a major foodborne pathogen in the United States. Data has been lacking regarding the presence of Salmonella in ground beef, especially for thos Salmonella that are resistant to multiple antibiotics. ARS scientists i Clay Center, NE, analyzed several thousand ground beef samples from commercial establishments and determined that the prevalence and level o Salmonella were very low (4.2%). Only 0.6% of samples harbored Salmonel that were resistant to antibiotics. From these results, it can be concluded that Salmonella and specifically antibiotic resistant Salmonel present a very low risk to the safety of the United States beef supply. 04 Prevalence and characterization of multidrug resistant (MDR) Salmonella Newport. Each year in the United States an estimated 1.4 million people contract foodborne illnesses due to Salmonella. Most cases of salmonellosis are not serious; however, some infections result in invasi salmonellosis, a more severe form of illness requiring antibiotic therap The emergence of multidrug resistant Salmonella is concerning. Salmonella Newport is frequently a cause of salmonellosis in both humans and cattle, and also frequently MDR. Cattle have been identified as a potential reservoir for Salmonella and this association presents a food safety risk for Salmonella contamination of ground beef. ARS scientists (Clay Center, NE) examined the prevalence of multidrug resistant Newport associated with cattle at slaughter in the United States. Regional differences in prevalence were observed across the United States. Drug resistance Newport isolates were resistance to ceftriaxone, a key antibiotic for treatment of human illness cases of salmonellosis. The research provided key information for hazard analysis and critical contr point analysis and cattle processing risk assessment modeling. The results from these studies will aid cattle producers in implementing far management practices that will reduce the dissemination of multidrug resistant pathogens and protect American and international consumers of United States beef. 05 Beef carcass contamination linked to cattle transportation and processin plant holding pen environment. Cattle hide has been established as the main source of carcass contamination during beef processing; therefore, is crucial to minimize the amount of Escherichia coli O157:H7 on hides prior to slaughter. There are several potential sources of E. coli O157:H7 encountered in the transportation to and in the holding pen environment at beef processing facilities that could increase the prevalence and levels of E. coli O157:H7 on the hides of cattle. ARS scientists in Clay Center, NE, investigated the extent to which E. coli O157:H7 is transferred to cattle hides during transport and while in holding at beef processing plants and, subsequently, transferred to beef carcasses. They determined that transport to and holding at processing plants leads to increased prevalence and levels of E. coli O157:H7 contamination on hides and carcasses. Thus, intervention efforts by bee producers are negated by pathogen transfer in the beef processing facili 06 Small and inexpensive beef cattle hide wash cabinet to manage bacterial contamination. Harborage of Escherichia coli O157:H7 and Salmonella on animal hides at slaughter is the main source of beef carcass contaminati during processing. Interventions have been designed and implemented to target the hides of cattle following entry into beef processing plants. Previous interventions targeting hides have not been suitable for all be processing plants to implement due to cost and space restrictions. ARS scientists in Clay Center, NE, evaluated a hide wash cabinet design that was smaller and more economical and, therefore, might be more amenable t widespread use in the beef processing industry. They discovered large reductions in E. coli O157:H7 and Salmonella levels after hide washing i the test cabinet. The hide wash cabinet was effective and should provid beef processors, especially small and medium-sized processing plants, wi an affordable hide wash intervention strategy. 07 Microbiological characterization of imported and domestic boneless beef trim used for ground beef. The United States imports lean boneless beef trim from Australia (AUS), New Zealand (NZL), and Uruguay (URY) to meet demand for ground beef production. The reported incidence of, and the bacteria responsible for, foodborne disease differs between these countries and the United States. ARS scientists in Clay Center, NE, investigated if current United States microbiological profiling adequate addresses the potential differences in the foreign and domestic beef tri Additionally, they compared the prevalence of pathogens between importe and domestic samples. Researchers determined that current pathogen monitoring procedures in the United States do not need to be changed for imported beef trim. 08 Determination of E. coli O157:H7 concentration for ground beef and hides carcasses, and feces of cattle. Methodologies for the assessment of microbial pathogen load, at various steps in the beef production process are lacking. In order to quantify the risks associated with the slaught of animals that may harbor or shed E. coli O157:H7 or Salmonella spp., accurate estimates of the prevalence and frequency of distribution of these pathogens and their relative concentration on hides and in feces i needed to assess the food safety risk associated with bacterial pathogen found at various process steps in ground beef production. ARS scientist in Clay Center, NE, developed two methods for the direct enumeration of coli O157:H7 and Salmonella spp. One method enumerated E. coli O157:H7 and Salmonella spp. from samples with high bacterial loads such as cattl hide and fecal samples. The second enumerated E. coli O157:H7 and Salmonella spp. from low bacterial count samples such as beef carcass an ground beef samples. Analysis of pathogen enumeration data, in combination with pathogen prevalence, should provide processors with greater control of potential pathogen contamination and further improve the safety of beef for consumers. In addition, these methods will be ve useful to researchers studying sources of pathogen contamination and its prevention.

Impacts
(N/A)

Publications

  • Harhay, D.M., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2011. Diversity of multidrug- resistant Salmonella enterica strains associated with cattle at harvest in the United States. Applied and Environmental Microbiology. 77(5):1783-1796.
  • Arthur, T.M., Nou, X., Kalchayanand, N., Bosilevac, J.M., Wheeler, T.L., Koohmaraie, M. 2011. Survival of Escherichia coli O157:H7 on cattle hides. Applied and Environmental Microbiology. 77(9):3002-3008.
  • Bosilevac, J.M., Koohmaraie, M. 2011. Prevalence and characterization of non-O157 Shiga toxin producing Escherichia coli isolates from commercial ground beef in the United States. Applied and Environmental Microbiology. 77(6):2103-2112.
  • Bosilevac, J.M., Kalchayanand, N., Schmidt, J.W., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2010. Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods. Journal of Food Protection. 73:2180-2188.


Progress 10/01/09 to 09/30/10

Outputs
Progress Report Objectives (from AD-416) 1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non- O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing. Approach (from AD-416) Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine- related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin- producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS). With the request by the Food Safety Inspection Service that beef processing facilities revalidate their antimicrobial interventions, we have begun to evaluate the use of pH measurements to serve as a monitor for verifying the establishments using lactic acid are achieving adequate antimicrobial coverage of beef carcasses. As it is cost prohibitive to revalidate in-plant interventions for every parameter modification, such as temperature, pressure, volume, etc., it is desirable to develop the ability to validate intervention performance based on a single parameter, such as end-point pH. In cooperation with a commercial beef processor, we are generating the data necessary to determine if end-point pH testing will be an effective method for validating intervention modifications. In light of data indicating that feeding wet distillers grains solubles (WDGS) could increase the level and prevalence of E. coli O157:H7 in feces and on feedlot cattle hides, we have been conducting experiments to determine if feeding cattle with different levels of WDGS will affect hide prevalence of non-O157 STEC strains as well. Although E. coli O157:H7 is currently well recognized, several other serotypes of Shiga toxin-producing E. coli (STEC) have been implicated in human illnesses. It is not clear if these pathogens share the same relationship with cattle as that of E. coli O157:H7, much less if their levels are affected by changes in cattle diets. Another gap in knowledge with respect to STEC is in defining the core virulence factors required to cause human illness. STEC share the common feature of Shiga toxin expression, but it appears that additional virulence factors are required to achieve the full virulence potential of strains such as O157:H7. Gene clusters that have been established as integral in the pathogenesis of STEC were used to design rapid multiplex PCR-based assays that provide a molecular risk assessment of an isolated STEC. Four PCR assays have been designed that identify the presence of 16 different genes. The greater the number of conserved genes possessed by an isolate, the greater the likelihood it is a human pathogen. The results of this work aid in the characterization of STEC as these important organisms are coming under increased regulatory scrutiny. Airborne pathogens in slaughter facilities have the potential to contaminate beef carcasses after interventions have been performed. Previous research has demonstrated that cattle slaughter facilities aerosols contained mesophilic bacteria. However, no published studies have determined if the foodborne pathogens E. coli O157:H7, Salmonella, and Listeria are present in the air of cattle slaughter facilities. In order to address this knowledge gap, we are collecting air samples from three slaughter facilities. Air samples are collected from 5 to 7 locations in each plant. Samples are being cultured to determine if E. coli O157:H7, Salmonella, and Listeria are present. Air samples are being collected during July, August, and September, since previous research has demonstrated that the prevalence of E. coli O157:H7 is highest in these months. Accomplishments 01 Determination of E. coli O157:H7 predominant colonization sites in super shedders. E. coli O157:H7 contamination of beef products has resulted i human disease outbreaks and large financial losses to the beef industry. This pathogen utilizes the intestinal tract of cattle and cattle that sh E. coli O157:H7 in their feces at disproportionately high levels have be labeled as ¿super-shedders.¿ It is believed that if the super-shedder status of cattle could be prevented, the transmission of E. coli O157:H7 could be controlled. ARS scientists at Clay Center, NE, identified supe shedding cattle and identified that the distal colon was the intestinal tract location most frequently found to harbor E. coli O157:H7. In addition, it was shown that at times of peak shedding, E. coli O157:H7 could be isolated from sites encompassing the entire bovine gastrointestinal tract from mouth to rectum but never detected from samples of lymph nodes or gall bladders. A better understanding of E. coli O157:H7 super shedding will aid in reducing the spread of this pathogen among cattle and throughout the beef production chain. 02 Sequence-based H-typing for the identification of Shiga toxin producing coli (STEC) serotypes. An essential part of characterizing STEC isolate is the identification of its serotype. Due to the limited availability o antisera, the identification of the flagellar (H-type) antigen of an isolate has recently become a molecular based test. However the current molecular test relies on interpretation of data that has limited resolution. ARS scientists at Clay Center, NE, designed a sequence-base assay for the fliC gene that can accurately identify 44 of the 53 H-type of E. coli. The assay has been shown to be rapid and robust and use of this system for identifying the H-type of STEC isolates has reduced the expense and time required while increasing the accuracy of H-type identification. 03 Detection of one cell of E. coli O157:H7 in samples of 375 g in size by commercial test and hold systems. Currently used industry testing programs require the ability to detect E. coli O157:H7 in samples of bee trim or ground beef at levels as low as 1 colony forming unit (CFU) per 375 grams. ARS scientists at Clay Center, NE, put forth a reliable protocol for generating a control inoculum for verification testing at this low concentration and evaluated its use. It was shown that 1 CFU w an unrealistic goal for use as a control because at this concentration half of all samples received no cells when inoculated. Detection of 3 C was much more reliable by culture isolation and two commercial assays. These results have immediate impact on testing labs and beef processors monitoring E. coli O157:H7. 04 Identification of factors in ground beef samples that interfere with the rapid detection of E. coli O157:H7. ARS scientists at Clay Center, NE, determined that the level of background aerobic plate count (APC) bacter and fat content in a beef sample can have significant effects on the detection of E. coli O157:H7. Increased background bacteria reduced the ability of rapid methods to detect E. coli O157:H7. Increasing fat content of ground beef samples correlated with decreasing recovery of antibody-linked separation beads that in turn reduce the rate of detecti by culture isolation and certain rapid methods. These results provide caveats for the users of test-and-hold assays and guidance for optimal detection of E. coli O157:H7 in ground beef samples that are of increase fat percentage and that may have elevated background bacteria. 05 Method revalidation is required for test and hold users if changes are made in sample size and enrichment media volume or type. Testing scheme are constantly evolving with improvements to the test protocols. There a tendency across the beef processing industry to implement changes whil not recognizing potential problems that may result later. ARS scientist at Clay Center, NE, demonstrated changes that reduced the volume of enrichment media in order to concentrate the E. coli, or changes that increased the size of the sample to provide greater sensitivity must be revalidated by each user in their own system. In addition, the scientis noted that rapid detection methods can be used on numerous sample types, except those that might alter the pH of the enrichment media during incubation. These results identified situations when a revalidation is needed and when it is not. The results can also be used by in-house laboratories of beef processors, ground beef manufacturers, and others, such as third party laboratories, as supporting documents in their testi programs.

Impacts
(N/A)

Publications

  • Arthur, T.M., Keen, J.E., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Nou, X., Koohmaraie, M. 2009. Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination. Applied and Environmental Microbiology. 75(20):6515-6523.
  • Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Wells, J., Shackelford, S. D., Wheeler, T.L., Koohmarie, M. 2010. Evaluation of a Direct-Fed Microbial Product Effect on the Prevalence and Load of Escherichia coli O157:H7 in Feedlot Cattle. Journal of Food Protection. 73(2):366-371.


Progress 10/01/08 to 09/30/09

Outputs
Progress Report Objectives (from AD-416) 1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non- O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing. Approach (from AD-416) Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine- related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin- producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS). Significant Activities that Support Special Target Populations We have compiled evidence that cattle shedding high amounts of E. coli O157:H7 are of particular concern in a feedlot setting as they serve as the largest source of the bacterial pathogen and lead to high rates of hide contamination among the other animals in the pen. The details are not known as to the reason an animal sheds the bacterial pathogen at high levels. One factor may be the particular strain type of E. coli O157:H7. Strain differences (i.e., the ability to readily colonize cattle and/or multiply more rapidly in the intestinal tract of cattle) may result in variation in the level of shedding by an animal. Samples have been collected from cattle at slaughter in multiple regions of the U.S. Genetic fingerprints of strains from those animals found to be shedding at high levels are being analyzed to identify commonalities among such strains. Although E. coli O157:H7 is currently well recognized, several other serotypes of Shiga toxin-producing E. coli (STEC) have been implicated in human illnesses. The isolation and detection of these strains are based on labor intensive methods that lack sensitivity. We have developed and validated a suitable discriminatory medium and genetic test for detection and confirmation of these strains of STEC. This medium will increase efficiency of detecting STEC, aid in our understanding of the ecology of STEC organisms in the processing environment, and help in evaluating the effectiveness of current interventions. To properly assess the potential virulence of STEC strains identified in environmental or processing samples, the serotype must be determined. Rapid methods based on the bacterial cell outer membrane have been designed; however, rapid methods utilizing the bacterial flagella are limited and restricted to only a few types. Because traditional flagella typing requires significant labor and resources, a sequence-based typing assay utilizing a flagella gene has been developed. Current validation experiments show that this method can accurately identify known types and accurately predict unknown types. This method is being adopted for use in a number of studies that require the serotype of numerous STEC isolates. Bacteria that enter the beef food chain may gain resistance to antimicrobials due to low-level exposure or by acquiring a resistance gene from another bacteria. Wet distillers grains with solubles (WDGS) are the byproduct of ethanol production and are commonly used as a feed stuff for growing and finishing cattle. Antimicrobials are used to control unwanted bacteria during the fermentation of ethanol and these antimicrobials carry over at very low levels to the WDGS, or WDGS may contain bacteria that possess resistance to these antimicrobials. Examinations of the bacteria carried by cattle on diets containing different levels of WDGS were carried out to determine if diets containing different levels of WDGS alter bacterial antimicrobial resistance. Technology Transfer Number of New/Active MTAs(providing only): 3

Impacts
(N/A)

Publications

  • Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Guerini, M.N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2009. Effectiveness of 1,3-Dibromo-5,5 dimethylhydantoin on reduction of Escherichia coli O157:H7- and Salmonella-inoculated fresh meat. Journal of Food Protection 72(1):151-156.
  • Arthur, T.M., Harhay, D.M., Bosilevac, J.M., Guerini, M.N., Kalchayanand, N., Wells, J., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2008. Prevalence and characterization of Salmonella in bovine lymph nodes potentially destined for use in ground beef. Journal of Food Protection 71(8):1685-1688.
  • Arthur, T.M., Kalchayanand, N., Bosilevac, J.M., Harhay, D.M., Shackelford, S.D., Bono, J.L., Wheeler, T.L., Koohmaraie, M. 2008. Comparison of effects of antimicrobial interventions on multidrug-resistant Salmonella, susceptible Salmonella, and Escherichia coli O157:H7. Journal of Food Protection 71(11):2177-2181.
  • Arthur, T.M., Bosilevac, J.M., Harhay, D.M., Kalchayanand, N., King, D.A., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2008. Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. Journal of Food Protection. 71(9):1752-1760.
  • Bosilevac, J.M., Koohmaraie, M. 2008. Effects of using reduced volumes of nonselective enrichment medium in methods for the detection of Escherichia coli O157:H7 from raw beef. Journal of Food Protection 71(9):1768-1773.
  • Bosilevac, J.M., Guerini, M.N., Kalchayanand, N., Koohmaraie, M. 2009. Prevalence and Characterization of Salmonellae in Commercial Ground Beef in the United States. Applied and Environmental Microbiology. 75(7):1892- 1900.
  • Bosilevac, J.M., Arthur, T.M., Bono, J.L., Brichta-Harhay, D.M., Kalchayanand, N., King, D.A., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2009. Prevalence and Enumeration of Escherichia coli O157:H7 and Salmonella in U.S. Abattoirs That Process Fewer Than 1,000 Head of Cattle Per Day. Journal of Food Protection. 72(6):1272-1278.
  • Bosilevac, J.M., Guerini, M.N., Koohmaraie, M. 2009. Increased Detection of Listeria Species and Listeria Monocytogenes in Raw Beef, Using the Assurance GDS Molecular Detection System with Culture Isolation. Journal of Food Protection. 72(3):674-679.
  • Kalchayanand, N., Brichta-Harhay, D.M., Arthur, T.M., Bosilevac, J.M., Guerini, M.N., Wheeler, T.L., Shackelford, S.D., Koohmaraie, M. 2009. Prevalence Rates of Escherichia coli O157:H7 and Salmonella at Different Sampling Sites on Cattle Hides at a Feedlot and Processing Plant. Journal of Food Protection. 72(6):1267-1271.
  • Harhay, D.M., Guerini, M.N., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. Salmonella and Escherichia coli O157:H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: an evaluation of prevalence and bacterial loads by immunomagnetic separation and direct plating methods. 2008. Applied and Environmental Microbiology 74(20):6289- 6297.


Progress 10/01/07 to 09/30/08

Outputs
Progress Report Objectives (from AD-416) 1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non- O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing. Approach (from AD-416) Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine- related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin- producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS). Significant Activities that Support Special Target Populations We evaluated the efficacy of a novel antimicrobial agent in decontamination of beef tissue. 1, 3-Dibromo-5, 5-Dimethylhydantoin (DBDMH), commercially called Bromitize, and hot water spray treatments were evaluated for efficacy in decontamination of pathogenic bacteria attached to beef carcass surfaces represented by flank tissue sections and beef hearts. Following Bromitize or hot water spray treatments, bacterial populations were counted immediately and after storage for 48 h at 4°C. This study demonstrated Bromitize spray washing could be effective as an antimicrobial intervention for red meat carcasses and variety meats. Much of the research to date on reducing E. coli O157:H7 from the food supply has been focused on the post-harvest side of the production chain. Pre-harvest interventions do not share the same wealth of techniques that have been proven effective against these bacterial pathogens. Direct Fed Microbials (DFM) have been identified as potential pre-harvest interventions for the reduction of foodborne bacterial pathogens. We determined the effect on E. coli O157:H7 and Salmonella prevalence and load from feeding a DFM to feedlot cattle. Hide and fecal samples obtained from 526 heifers were followed over an 84-day finishing period. The data from this experiment leads to the conclusion the DFM tested would not be an effective pre-harvest intervention against E. coli O157:H7. There were not sufficient observations of Salmonella to enable substantive conclusions to be made. Salmonella, and Salmonella resistant to multiple antibiotics (MDR-Salmonella), have been observed in the meat production environments, but solid data identifying their source is lacking. We conducted research projects to 1) determine if lymph nodes are a source of Salmonella that could potentially contaminate ground beef products; 2) determine if MDR Salmonella can be attributed to cattle from a particular production environment such as cull beef cows, dairies, or feed lots; and 3) enumerate Salmonella throughout the commercial pork harvesting process. These projects will aid our understanding of the ecology of Salmonella in processing environments. There is ample data on the prevalence and levels of E. coli O157:H7 and Salmonella found during the different steps of harvest at large U.S. beef processing plants. However, a significant portion of the U.S. beef supply passes through small processing plants, for which little data is available. For this study "small processing plants" was defined as processors who harvest less than 1,000 cattle a day. More data on contamination rates was needed to assist in developing technology to reduce the risk of pathogen contamination of carcasses and help determine the best processes to put into place to reduce hide levels of pathogens. It is well known higher levels of pathogens on hides positively correlate to greater risk of pathogen on carcasses. USMARC scientists determined the prevalence and enumerated the levels of E. coli O157:H7 and Salmonella on hides and pre- evisceration carcasses of cattle processed through smaller plants. The processors were able to rapidly improve pathogen control by changing hide removal steps.

Impacts
(N/A)

Publications

  • Harhay, D.M., Arthur, T.M., Koohmaraie, M. 2008. Enumeration of Salmonella from poultry carcass rinses via direct plating methods. Letters in Applied Microbiology 46(2):186-191.
  • Harhay, D.M., Arthur, T.M., Bosilevac, J.M., Guerini, M.N., Kalchayanand, N., Koohmaraie, M. 2007. Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. Journal of Applied Microbiology 103(5):1657-1668.
  • Guerini, M.N., Harhay, D.M., Shackelford, S.D., Arthur, T.M., Bosilevac, J. M., Kalchayanand, N., Wheeler, T.L., Koohmaraie, M. 2007. Listeria prevalence and Listeria monocytogenes serovar diversity at cull cow and bull processing plants in the United States. Journal of Food Protection. 70(11):2578-2582.
  • Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Harhay, D.M., Guerini, M. N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2007. Microbiological characterization of lamb carcasses at commercial processing plants in the United States. Journal of Food Protection 70(8):1811-1819.
  • Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Guerini, M.N., Wheeler, T.L., Koohmaraie, M. 2008. Evaluation of various antimicrobial interventions for the reduction of Escherichia coli O157:H7 on bovine heads during processing. Journal of Food Protection 71(3):621- 624.


Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) 1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non- O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing. Approach (from AD-416) Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine- related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin- producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS). Accomplishments Transportation and Lairage Environment Effects on Prevalence and Levels of Escherichia coli O157:H7 on Hides and Carcasses of Beef Cattle at Processing - Hide has been established as the main source of carcass contamination during processing, therefore, it is crucial to minimize the amount of Escherichia coli O157:H7 on cattle hides prior to slaughter. There are several potential sources of E. coli O157:H7 encountered in the transportation to and lairage environment (holding pens, alley ways, etc.) at beef processing facilities that could increase the prevalence and levels of E. coli O157:H7 on the hides of cattle. Scientists in Clay Center, NE, conducted a study to determine the extent to which E. coli O157:H7 is transferred to cattle hides and, subsequently, to carcasses during transport to and while in holding at beef processing plants. The results of this study demonstrate that transport to and lairage at processing plants leads to increased prevalence and levels of E. coli O157:H7 contamination on hides and carcasses. A major implication of this finding is that pre-harvest intervention effects would be negated by pathogen transfer in the lairage environment. This accomplishment addresses National Program 108 Problem Statement 1.2.3: Production and Processing Ecology. Effects of a Minimal Hide Wash Cabinet on the Levels and Prevalence of E. coli O157:H7 and Salmonella on the Hides of Beef Cattle at Slaughter - Harborage of Escherichia coli O157:H7 and Salmonella on animal hides at slaughter is the main source of beef carcass contamination during processing. Given this finding, interventions have been designed and implemented to target the hides of cattle following entry into beef processing plants. Previous interventions targeting hides have not been suitable for all beef processing plants to implement due to cost and space restrictions. USMARC scientists evaluated a hide wash cabinet design that was smaller and more economical and, therefore, might be more amenable to widespread use in the beef processing industry. This study showed large reductions in E. coli O157:H7 and Salmonella levels after hide washing in the test cabinet. Based on these results, the hide wash cabinet described in this study was effective and should provide beef processors, especially small and medium-sized processing plants, with an affordable hide wash intervention strategy. This accomplishment addresses National Program 108 Problem Statement 1.2.4: Processing Intervention Strategies. Microbiological Characterization of Imported and Domestic Boneless Beef Trim Used for Ground Beef - The United States imports lean boneless beef trim from Australia (AUS), New Zealand (NZL), and Uruguay (URY) to meet demand for ground beef production. The reported incidence of, and the bacteria responsible for, foodborne disease differ between these countries and the United States. USMARC scientists determined if current United States microbiological profiling adequately addresses the potential differences in the foreign and domestic beef trim, by comparing the hygienic status of imported and domestic (USA) beef trim by enumeration of aerobic bacteria, Enterobacteriaceae, coliforms, Escherichia coli and Staphylococcus aureus. We also compared the prevalence of pathogens between imported and domestic samples by screening for the presence of Salmonella, Campylobacter spp., Listeria spp., and non-O157 Shiga toxin-producing Escherichia coli (STEC). The results of this work showed that the current pathogen monitoring procedures in the United States do not need to be changed for imported beef trim. This accomplishment addresses National Program 108 Problem Statement 1.2.7: Risk Assessment. Rapid Enrichment Strategy for Isolation of Listeria from Bovine Hide, Carcass, and Meat Samples - In keeping with the need to continually improve the food safety of beef products, USMARC scientists developed and validated a novel rapid method for detection of Listeria monocytogenes. An enrichment medium, Tryptic soy broth (TSB), was tested in place of the traditional enrichment media University of Vermont modification medium (UVM) to allow for resuscitation of injured bacterial cells. No difference between TSB and UVM was observed during primary enrichment for the isolation of Listeria spp. The significance of this work is that using TSB as a primary enrichment medium for the isolation of Listeria results in a time savings and a cost savings, and the number of manipulations required for each sample is decreased. Additionally, this work provides a method development that may enable the end user to screen food samples for Listeria spp. using a high-throughput method. This accomplishment addresses National Program 108 Problem Statement 1.2.1: Detection and Validation. Microbiological Characterization of Lamb Carcasses at Commercial Processing Plants in the United States - Although the United States (U.S.) produces 203 million pounds of domestic lamb and mutton each year, thorough studies of the microbiological safety during lamb processing are lacking. To address this missing information, USMARC scientists collected samples from multiple large commercial lamb processing plants to determine levels of generic bacteria and the prevalences of Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), and Salmonella. The results of this study establish a baseline for microbiological quality and prevalences of Salmonella, E. coli O157:H7, and STEC in U.S. lamb processing plants as well as provide information as to the efficacy of various antimicrobial interventions in reducing bacterial pathogens on lamb carcasses. This accomplishment addresses National Program 108 Problem Statement 1.2.3: Production and Processing Ecology and Problem Statement 1.2.4: Processing Intervention Strategies. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 2 Number of Newspaper Articles,Presentations for NonScience Audiences: 3

Impacts
(N/A)

Publications

  • Guerini, M.N., Bosilevac, J.M., Koohmaraie, M. 2007. Rapid enrichment strategy for isolation of Listeria from bovine hide, carcass, and meat samples. Journal of Food Protection 70(1):53-57.
  • Bosilevac, J.M., Guerini, M.N., Harhay, D.M., Arthur, T.M., Koohmaraie, M. 2007. Microbiological characterization of imported and domestic boneless beef trim used for ground beef. Journal of Food Protection 70(2):440-449.
  • Arthur, T.M., Bosilevac, J.M., Harhay, D.M., Guerini, M.N., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2007. Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. Journal of Food Protection 70(2):280-286.
  • Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2007. Effects of a Minimal Hide Wash Cabinet on the Levels and Prevalence of Escherichia coli O157:H7 and Salmonella on the Hides of Beef Cattle at Slaughter. Journal of Food Protection 70:1076-1079.
  • Arthur, T.M., Bosilevac, J.M., Nou, X., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2007. Comparison of the molecular genotypes of Escherichia coli O157:H7 from the hides of beef cattle in different regions of North America. Journal of Food Protection. 70(7):1622-1626.
  • Nou, X., Arthur, T.M., Bosilevac, J.M., Harhay, D.M., Guerini, M.N., Kalchayanand, N., Koohmaraie, M. 2006. Improvement of immunomagnetic separation for Escherichia coli O157:H7 detection by the pickpen magnetic particle separation device. Journal of Food Protection 69:2870-2874.


Progress 10/01/05 to 09/30/06

Outputs
Progress 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? Why does it matter? This project is aligned with the National Program 108 for Food Safety. FSIS has estimated that contamination of meat and poultry products with pathogenic bacteria results in as many as 5,000,000 illnesses and 4,000 deaths per year in the U.S. In addition to causing human illness, food safety problems can cause economic losses (estimated at $10 billion per year in the beef industry alone) to producers, processors, and consumers, and jeopardize the international competitiveness of the U.S. agricultural industry. Research to develop better sampling methods and rapid tests for hazard analysis and critical control point (HACCP) monitoring, and to discover more effective pathogen intervention techniques, is needed to reduce the risk of foodborne illness. Despite effective HACCP systems, emerging bacterial pathogens sometimes evade intervention strategies and detection technologies, and are, thus, a continual threat. Proactive research on the mechanisms of emerging pathogen development is needed to develop strategies aimed at preventing or limiting this occurrence. Knowledge of virulence acquisition mechanisms will afford the opportunity to eliminate situations conducive to pathogen development, and to further ensure that intervention strategies do not enhance the potential for pathogen development. To better understand pathogens and the means by which we can reduce the risk of foodborne illness, we have initiated a project plan containing four objectives. Objective 1 is to develop phylogenetic and phenotypic markers for E. coli O157:H7, non-O157 Shiga toxin-producing E.NBcoli (STEC) /enterohemorrhagic E. coli (EHEC), and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. The second objective is to determine prevalence of unrecognized foodborne pathogens such as STEC on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. A third objective entails work aimed at identifying sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, and feces) and develop novel antimicrobial intervention strategies. To further contribute to food safety, we have proposed to determine the microbiological safety of lamb processed in the U.S. and determine the efficacy of currently used intervention technologies during various stages of lamb processing. The meat processing industry will gain additional tools in the fight against pathogen contamination of carcasses and, ultimately, retail meat products from information learned by pursuing these four stated objectives. FSIS will receive pertinent information on which to base food safety policies and regulations. Successful completion of the objectives will benefit processors by increasing their ability to monitor production and develop improved interventions to minimize carcass contamination and product loss, and will benefit consumers by decreasing the risk of foodborne illness. 2. List by year the currently approved milestones (indicators of research progress) FY 2006 1. MALDI-TOF identification of E. coli O157:H7 factors specifically expressed when grown in various culture media. 2. Receive and process samples for Aerobic Plate Counts, Enterobacteria- ceae E. coli, coliforms, and Staph. aureus. 3. Culture and isolate Salmonella strains from samples. Archive for further characterization. 4. Isolate and confirm Campylobacter from samples as received. 5. Isolate and archive Listeria from samples. 6. Screen enrichments for presence of Shiga toxin genes and prepare freezer stocks of each enrichment. 7. Investigate contamination of hide through feedlot housing, lairage, and transport; determine the number cattle shedding E. coli O157:H7 that are necessary to contaminate the hides of over 50% of the animals in the pen. 8. Develop methodology for accurate airborne and ventilation system sampling in beef processing plants. 9. Compare sampling methodologies for fleece and carcass. FY 2007 1. Compare classification by PFGE with that of intact cell MALDI-TOF. 2. 2-D PAGE analysis of E. coli O157:H7 strains. 3. Array 1000 non-O157 strains. 4. Develop a multiplex PCR protocol for the rapid and specific identification of S. Typhimurium and S. Newport. 5. Identify isolated Salmonella sub-species and serotype. Determine antibiotic sensitivity. 6. Determine Listeria species and Listeria monocytogenes serovars. 7. Use positive enrichments for colony lifts and hybridize with stx1/stx2 to isolate STEC colonies. 8. Determine if E. coli O157:H7 supershedders exist and if there is a causal relationship with increased carcass contamination; molecular fingerprinting of E. coli O157:H7 to determine the source of E. coli O157:H7 hide isolates; tracking E. coli O157:H7 isolates via PFGE and/or MALDI-TOF. 9. Screen and sort feedlot cattle based on colonization by E. coli O157:H7. 10. Evaluate traditional MPN methods verses spiral plating, hydrophobic grid analysis, and immunomagnetic recovery. 11. Sample processing plants and identify on-line sources of airborne contamination. Evaluate efficacy of novel interventions aimed at decreasing hide contamination in husbandry and processing environments. 12. Collect hide and carcass samples from sheep processing plants for detection and isolation of E. coli O157:H7 and Salmonella. FY 2008 1. Screen strains with whole cell MALDI-TOF. 2. Comparison of expressed protein profiles and correlation with virulence. 3. Construction of scFv library. 4. Detection of virulence factors by colony hybridization. 5. Determine the antibiotic resistance profiles of S. Typhimurium and S. Newport isolates. 6. Examine isolates for putative virulence factors. 7. O and H Serotype and characterize virulence factors. 8. Sample the hides and feces of sorted cattle periodically to determine the presence of E. coli O157:H7. 9. Apply best method identified previously to feedlot and processing plant samples to evaluate real world applicability. 10. Process hide and carcass sample for non-O157 STEC. FY 2009 1. Identification of virulence markers, development of detection assay, and validation. 2. Selection and expression of scFv. 3. Determine correlation between marker presence and virulence outcome. 4. Examine the potential for the transfer of antibiotic resistant plasmids among various species of the bovine or ovine enteric microbial flora. 5. Track E. coli O157:H7 isolates using PFGE and/or MALDI-TOF. 6. Work with processing facilities to implement strategies to use for enumeration. 7. Evaluate efficacy of novel interventions aimed at decreasing hide contamination in husbandry and processing environments. FY 2010 1. Concentration of E. coli O157:H7 using scFv approach. 2. Develop assay for virulence marker detection. 3. E. coli O157:H7 isolates will be genotyped using PFGE and/or MALDI- TOF. 4. Evaluate existing interventions in the context of efficacy in removing emerging pathogenic organisms. 4a List the single most significant research accomplishment during FY 2006. Method Development for Enumeration of E. coli O157:H7 for Ground Beef and Hides, Carcasses, and Feces of Cattle. Methodologies for the assessment of microbial pathogen load, at various steps in the beef production process, are lacking. In order to quantify the risks associated with the slaughter of animals that may harbor or shed E. coli O157:H7 or Salmonella spp., accurate estimates of the prevalence and frequency of distribution of these pathogens and their relative concentration on hides and in feces is needed. ARS scientists at the USMARC developed two methods (NP 108 Strategic Plan Problem Statement 1.2.1) for the direct enumeration of E. coli O157:H7 and Salmonella spp. The first method involves the use of the spiral plate count method (SPCM) for the enumeration of E. coli O157:H7 and Salmonella spp. from cattle hide and fecal samples. The second method involves the use of hydrophobic grid membrane filtration (HGMF) for the enumeration of E. coli O157:H7 and Salmonella spp. from carcass and ground beef samples. Analysis of pathogen enumeration data, in combination with pathogen prevalence, should provide processors with greater control of potential pathogen contamination and further improve the safety of beef for consumers. In addition, these methods will be very useful to researchers studying sources of pathogen contamination and its prevention. 4b List other significant research accomplishment(s), if any. Evaluation of Escherichia coli O157:H7 Growth Media for Use in Test-and- Hold Procedures in Ground Beef Processing The beef industry has used a process called test-and-hold to sample beef trim and ground beef to test for the presence of E. coli O157:H7 and release any product that tests negative. Scientists at the USMARC determined the optimal medium and growth conditions for improving the speed at which results can be obtained (NP 108 Strategic Plan Problem Statement 1.2.1). Further work determined that the volume of media could be reduced by 2/3, thus, creating a significant cost savings for the end user. The cost reduction for these changes has reduced the cost from ~$10 to ~$4 per test. These results demonstrate that beef processors can use a low cost medium and reduce the amount of medium used to enrich for possible contaminating E. coli O157:H7 while maintaining a high level of accuracy in the testing of beef trim and ground beef. E. coli O157:H7 Survival on Cattle Hides Reducing bacterial contamination to the hide has been shown to improve the microbiological quality of the meat products. The survival of E. coli O157:H7 on hides of living cattle is not well understood. From work conducted by USMARC scientists, the survival time for E. coli O157:H7 on the hides of cattle was determined to be less than nine days (NP 108 Strategic Plan Problem Statement 1.1.2). Study of E. coli O157:H7 survival on the hide will be of great importance in designing pre-harvest intervention strategies so they will be effective. 5. Describe the major accomplishments to date and their predicted or actual impact. This new project plan was initiated on April 12, 2006. 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? USMARC scientists developed methods for the enumeration of E. coli O157:H7 and Salmonella from bovine fecal, hide, carcass, and ground beef samples. Cattle hide and fecal samples are enumerated using the spiral plate count method, while carcass and ground beef/trim samples are enumerated using hydrophobic grid membrane technology. Using these methods, the efficacy of intervention strategies can be evaluated by estimating the load of these pathogens at different steps in the slaughter process. Technology transfer has already been initiated, as USMARC scientists have held training sessions with representatives of three large commercial U.S. beef processing companies, and a standard enumeration protocol detailing the methods was released in May 2006. Two constraints of the enumeration methods have been identified. The first involves issues with competing bacterial flora, which, if present in high numbers, can interfere with pathogen detection. The second concerns the limits of pathogen detection. While these methods mark an improvement over existing technologies, continued efforts are underway to improve them and address their current limitations. USMARC scientists met with the microbiologist-in-charge at the USDA Food Safety and Inspection Service (FSIS) to present data and review technology improvements for detection of E. coli O157:H7 in ground beef and for a review of method improvements for the isolation of Listeria monocytogenes from beef products. 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). April 2006 - "Recent Advances in Post-harvest Meat Safety"NBpresented at USMARC to members of the Jordanian Ministry of Agriculture. April 2006 - A Summary of the Results of Food Safety Projects Funded by the National Cattleman's Beef Association. Jacksonville, FL. May 2006 "Post Harvest Food Safety" presented at USMARC to Oklahoma State University graduate students. May 2006 "MDR Salmonella" presented at USMARC to Oklahoma State University graduate students. June 2006 - "Results Obtained from USMARC Studies on Escherichia coli O157:H7" National Cattlemen's Beef Association Quality Assurance state coordinators. Kansas City, MO.

Impacts
(N/A)

Publications

  • Guerini, M.N., Arthur, T.M., Shackelford, S.D., Koohmaraie, M. 2006. Evaluation of Escherichia coli O157:H7 growth media for use in test-and- hold procedures for ground beef processing. Journal of Food Protection. 69(5):1007-1011.