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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.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.

1b.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).

3.Progress Report
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.

1. Increased detection of Listeria species and Listeria monocytogenes in raw beef using a molecular detection system with culture isolation. Testing for Listeria is challenging due to its slow growth rate. Recently, ARS scientists in Clay Center, NE, described a rapid Listeria culture isolation method. This method has been improved by utilizing a rapid molecular detection test. When the molecular test was evaluated as endpoint, it agreed with culture isolation 100% for positives and 84.4% for negatives. Experiments showed that discrepancies were due to increased sensitivity of the molecular test. The results could be known two days earlier when the molecular tests were used instead of plating media, providing processors and laboratories improved turnaround time for results.

2. Using reduced volumes of non-selective enrichment medium for the detection of Escherichia coli O157:H7 from raw beef. Tryptic soy broth (TSB) is a superior enrichment medium for use in test-and-hold methods for E. coli O157:H7 at levels down to a ratio of three volumes of medium to one volume of sample. ARS scientists in Clay Center, NE, examined lower ratios for their effect on culture isolation and the most common commercial detection tests. The volume of TSB used did not affect E. coli O157:H7 detection by culture isolation, but varying results were observed and reported using different commercial test formats. Despite the differences observed, positive detection test results can be considered valid as long as enrichment medium is used, even if it is less than the specified 3 or 10 volumes.

3. Prevalence and characterization of Salmonella in commercial ground beef. ARS scientists in Clay Center, NE, completed a two-year analysis of commercially produced ground beef from multiple grinding establishments across the U.S. Prevalence and level of Salmonella were determined to be very low (4.2%). The serotype and antibiotic susceptibility of the isolated Salmonella were determined, and four serotypes made up one-half of all the isolates. Only 0.6% of samples had Salmonella present that were resistant to antibiotics. Indistinguishable genetic fingerprints could be observed in isolates of the same serotype found in different regions and months of sampling. This demonstrated that attempts to identify sources of Salmonella based on serotype and genetic fingerprints cannot be done without additional documented evidence.

4. Prevalence and characterization of multidrug resistant (MDR) Salmonella Newport associated with cull cattle at harvest. Each year in the United States an estimated 1.4 million people contract foodborne illnesses due to Salmonella. Most cases of salmonellosis are self-limiting, however, some infections result in invasive salmonellosis, a more severe form of illness requiring antibiotic therapy. Recent studies indicate that drug resistant Salmonella may be more invasive than antibiotic-sensitive Salmonella, and the emergence of multidrug resistant (MDR) Salmonella in the past decade is an issue of increasing concern. ARS scientists at Clay Center, NE, examined the prevalence of MDR Newport associated with cull cattle at slaughter in the United States. Drug resistance phenotypes were determined for 513 MDR Newport isolates. The baseline data collected in this study provide key information for HACCP analysis and cattle processing risk assessment modeling. Additionally, the genetic elements mediating these antibiotic resistances are being characterized in order to better understand how Salmonella acquires and disseminates these resistances. The results from these studies will aid cattle producers in implementing farm management practices that will reduce the dissemination of MDR pathogens.

5. Mapping Escherichia coli O157:H7 and Salmonella on cattle hides at a feedlot and a processing plant. Escherichia coli O157:H7 and Salmonella pose not only a significant health risk, but also considerable economic threats to the beef industry. It is clear that cattle hides are the major source of these pathogens which may be transferred to the carcass during harvest. Knowing the distribution of these pathogens on beef cattle during production and harvest is essential to achieve the proper focus of antimicrobial interventions. Additionally, knowing the distribution of these pathogens will aid in the determination of sampling locations that provide optimal detection for measuring pathogen prevalence. ARS scientists in Clay Center, NE, determined the distribution of E. coli O157:H7 and Salmonella from multiple locations on beef cattle hides in a feedlot setting and a commercial processing plant. The results indicated that E. coli O157:H7 and Salmonella are most often found to reside on the belly along the abdominal midline. This site should serve as an essential target for decontamination to prevent hide to carcass contamination during hide removal.

6. Clostridium difficile in commercial beef processing plants. Illnesses from Clostridium difficile infection have increased recently in North America and Europe due to the emergence of a toxigenic strain identified as North America pulsed-field gel electrophoresis type 1 (NAP-1). The cause of illnesses has been speculated as being derived from consumption of contaminated meat products. However, the information on C. difficile contamination of carcasses during processing in the U.S. and its presence in the U.S. commercially produced ground beef was very limited. ARS scientists in Clay Center, NE, collected the necessary data to determine if C. difficile was a relevant threat to food safety in the U.S. beef supply. In this study, C. difficile was found infrequently on cattle hides, but not found on beef carcasses either early or late in processing. No C. difficile was detected in samples of commercial ground beef. None of the isolates from hides was identified as the strain of concern, NAP-1 strain. These results indicate that C. difficile does not pose a relevant threat in the U.S. beef supply.

6.Technology Transfer

Number of the New/Active MTAs (providing only)3

Review Publications
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.

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.

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., 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., 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.

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.

Last Modified: 7/29/2015
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