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United States Department of Agriculture

Agricultural Research Service

2010 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
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.

1. Determination of E. coli O157:H7 predominant colonization sites in super-shedders. E. coli O157:H7 contamination of beef products has resulted in human disease outbreaks and large financial losses to the beef industry. This pathogen utilizes the intestinal tract of cattle and cattle that shed E. coli O157:H7 in their feces at disproportionately high levels have been 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 super-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.

2. Sequence-based H-typing for the identification of Shiga toxin producing E. coli (STEC) serotypes. An essential part of characterizing STEC isolates is the identification of its serotype. Due to the limited availability of 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-based assay for the fliC gene that can accurately identify 44 of the 53 H-types 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.

3. 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 beef 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 was an unrealistic goal for use as a control because at this concentration half of all samples received no cells when inoculated. Detection of 3 CFU 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.

4. 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) bacteria 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 detection 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 increased fat percentage and that may have elevated background bacteria.

5. Method revalidation is required for test and hold users if changes are made in sample size and 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 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 scientists 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 testing programs.

Review 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.

Last Modified: 5/28/2015
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