RESEARCH PROJECT SUMMARY OUTLINE - FINAL REPORT July 28, addendum September 6, 1995

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1 RESEARCH PROJECT SUMMARY OUTLINE - FINAL REPORT July 28, 1995 addendum September 6, 1995 I. Principal Investigator(s) Principal Investigator: John H. Silliker, Ph.D. Associate Investigator: Ranzell Nickelson, Ph.D. II. Institution Silliker Laboratories Group, Inc. 900 Maple Road Homewood, Illinois (708) III Project Title Methods for Sampling and Compositing Fresh Red Meat Products for Analysis of Pathogenic and Indicator Bacteria IV. Stated Objectives Determine the effect of compositing multiple units compared to analysis of individual units on detection of pathogenic bacteria (Salmonella, Listeria monocytogenes, E. coli O157:H7 and Campylobacter) in beef in order to make statistically based sampling practical and economically feasible. Establish the most efficient and productive methods for sampling fresh red meats for analysis of pathogenic and indicator bacteria. Determine the efficiency of rinsing versus grinding as a method of sample preparation for analysis of pathogenic and indicator bacteria in fresh red meats. V. Background Information About the Need for This Research Currently, it is not economically feasible to test the number of samples of fresh red meat necessary to have statistical confidence in the microbiological condition of the lot. It is common practice to take multiple units of the product which are mixed together to prepare a composite sample for analysis. The rationale for this approach is that the composite sample is more reflective of the environment than would be a single grab sample. Whether the analytical unit tested is a grab sample or composite of multiple units, the same weight of sample is analyzed in both 1

2 cases. Thus, the act of compositing does not increase the statistical validity of the analytical procedure. Within the context of this project, compositing is applied in quite a different manner. Multiple units of product are collected e.g., 25 gram samples, and these are combined (composited) to prepare a large sample which is then examined as a single analytical unit. When tests for pathogens are conducted, the sensitivity of the test is in direct relationship to the amount of sample tested. If large composite samples can be analyzed, as compared to the analysis on multiple smaller samples, the total weight being equal in both cases, the economic savings of compositing is obvious. Indeed, the analysis of sufficient numbers of small units to give statistical validity is economically unfeasible; whereas the analysis of a single composite sample may be a reasonable approach. The National Research Council Committee on Salmonella (1969) recommended sampling plans for various food categories. For example, the plan for foods eaten by high risk populations specified the analysis of sixty 25g samples to clear a lot, the statistical inference being that if each of the 60 samples tested were found negative one could be 95% confident that the level of Salmonella is not greater than one per 500g. These sampling plans were subsequently adopted by the U.S. FDA and were recommended by the International Commission of Microbiological Specifications for Foods (ICMSF), as well as other bodies. Obviously, the cost of analyzing 60 individual 25g samples to clear lots of product would be prohibitive. Accordingly, the ICMSF sponsored studies funded by the USDA to determine the feasibility of compositing multiple 25g samples. These studies showed that such compositing was feasible with no loss of sensitivity as compared to the analysis of multiple 25g samples (Silliker and Gabis, 1973; Gabis and Silliker, 1974). For example, in the sampling of foods in the most sensitive category, the same sensitivity was achieved through the analysis of three 500g composites as with sixty 25g samples (total weight analyzed in each case being 1,500g). These findings made analytical control over the Salmonella defect economically feasible. The compositing approach was accepted by the U.S. FDA (BAM, 1992), and the ICMSF (1974) and other groups. It is widely used in industry quality control programs. More recently, Listeria has been recognized as important food borne pathogen and sampling plans for its detection in foods have been published. The FDA (BAM, 1992) prescribes the analysis of two 25g samples for various foods; whereas the USDA/FSIS Baseline Data Collection Program prescribes the analysis of a 75g composite comprised of three 25g samples (USDA-FSIS). Similarly, the ICMSF plans to recommend the analysis of multiple 25g samples. Studies on compositing larger samples of foods were reported by Silliker Laboratories (Decker et. al., 1992). This work suggested that multiple 25g samples could be used to provide 375g composites without loss of sensitivity, thus providing an economical approach to statistically based sampling and testing. Subsequent to funding this project, FSIS/USDA has taken two actions which further justifies this research. First, E. coli was declared an adulterant in raw ground beef. None of the steps in the 2

3 slaughter or processing of ground beef are lethal to E. coli O157:H7. Thus, at present, many manufacturers of ground beef at the insistence of the major fast food chains have adopted testing programs to detect E. coli O157:H7 in the raw materials prior to grinding and formation of patties. The objective of eliminating contaminated raw materials from ground beef manufacture cannot be accomplished without the use of the statistically valid sampling and testing schemes. The second action taken by FSIS/USDA which increases the importance of this work is the establishment of target levels for Salmonella in meat products in their HACCP proposal. Although FSIS proposed to test only one sample per day of each product type produced by a plant, the reality is that in order to meet a target level of 1 in 82 days, designated for beef carcasses, an average days production would need to meet this target level. The best way for a manufacturer to determine if they are consistently meeting the target is to perform statistically valid sampling and testing on an ongoing basis. For example, a manufacturer might test one sample/day to comply with the USDA regulation, but collect 60 individual samples and test 4 composites of 15 samples each to determine that they are producing product that will meet the target for 82 days. If one or more of the composite samples tests positive, corrective action could be taken prior to failing to meet the target somewhere during the 82nd day window. In this study, we investigated the most efficient way to sample fresh red meat for pathogenic bacteria, but in addition to access the feasibility of analyzing composites of multiple samples in order to facilitate effective surveys at economical costs. VI. Achievement of the Specific Objectives A. Determine the effect of compositing multiple sample units in order to make statistically based sampling and testing of beef for pathogens practical and economically feasible. 1. Salmonella This research has demonstrated that up to fifteen 25g samples of raw beef can be composited and analyzed as 375g samples without a loss of sensitivity i.e., if any one of the 15 samples were positive, even at a low level, the composite will test positive. If a manufacturer tests one 25g sample from a lot, he has little knowledge of the probability of the lot testing positive or negative if analyzed again. However, if 15 random samples are drawn and tested as one 375g composite, and the result is negative, the manufacturer can be 95% confident that there is less than one Salmonella/125g of product. 2. E. coli O157:H7 3

4 This research demonstrated that it was not possible to composite samples for analysis of E. coli O157:H7 using the current FSIS/USDA recommended procedure, Petrifilm Escherichia coli O157:H7 assay. However, it was also determined that the Petrifilm method could be improved for use on composited samples by increasing the time of enrichment prior to plating and assay. Further, it was determined that use of another rapid assay, the Organon Teknika EHEC-TEK assay, allowed up to fifteen 25g samples to be composited with little loss of sensitivity compared to testing individual 25g samples. This sampling and compositing scheme has already been widely accepted throughout the beef industry for analysis of raw ground beef and raw materials prior to grinding. 3. L. monocytogenes This research has determined that currently available methods for detection and isolation of L. monocytogenes are not selective enough to allow compositing without a loss of sensitivity. Further, this work has demonstrated that the current methods are not selective enough to be reliable for detection of L. monocytogenes even in 25g samples, when L. inocua is also present; and most raw beef samples examined contained L. inocua. Based on these data and related studies the current FSIS/USDA method for L. monocytogenes is not acceptable for detection of this organism in most raw beef samples. 4. Campylobacter Initial investigations determined that the current FSIS/USDA procedure for Campylobacter could not be modified to allow composited samples to be analyzed and was not practical to perform in most meat company laboratories. Preliminary studies suggest that the GENE-TRAK Campylobacter assay is more practical and may allow compositing of samples. Validation studies indicated a sensitivity of 0.42 cells/25g in low count ground beef. However, when the method was applied to compositing trials utilizing larger sample sizes, there was a loss in sensitivity as the sample size increased. Although the identified method shows a loss in sensitivity, this work did demonstrate that a practical method is feasible. Further work in developing the identified method is warranted. B. Establish the most efficient and productive methods for sampling fresh red meat for analysis of pathogenic and indicator bacteria. 1. This research demonstrated that the non-destructive sponge sampling technique was an acceptable alternative to the destructive tissue 4

5 excision method for enumeration of indicator bacteria (e.g., APC, coliform, E. coli) on beef carcasses. Unfortunately, there was an insufficient number of samples positive for pathogenic bacteria to allow a comparison of the efficacy of the methods. However, the favorable results with the sponge sampling technique for indicator bacteria suggest that the method will be suitable for pathogen sampling and analysis. Further research to validate the sponge sampling technique is warranted, especially considering the USDA s proposal to sample carcasses daily for Salmonella. The tissue excision method is labor intensive and mars the carcass. 2. Research on sampling of boxed and combo fresh beef demonstrated that core sampling was an efficient alternative to random selection of surface pieces and that both methods were superior to purge sampling for analysis of indicator bacteria. The number of samples positive for Salmonella and E. coli O157:H7 were not sufficient to allow a comparison of the methods. However, based on the data for the indicator organisms, core sampling is suggested. This method combined with compositing has already been adopted and is widely used in the beef industry to screen products destined for ground beef. C. Determine the efficiency of rinsing versus grinding as a method of sample preparation for analysis of pathogenic and indicator bacteria in fresh red meats. These data suggest that blending is preferable to rinsing for laboratory preparation of beef samples for analysis. However, if situations in which maximum recovery of indicators and pathogens is not necessary; e.g., survey work, rinsing may be a suitable alternative to blending. VII. Summary of Results and Discussion A. Compositing Studies (Objective 4.a. of project) The objective of this research was to determine the effect of compositing multiple units compared to analysis of individual units on detection of pathogenic bacteria (Salmonella, Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter) in beef in order to make statistically based sampling practical and economically feasible. The compositing protocol for each trial can be seen in Table 1. The theoretical cell densities ranged from 1,000 cells/25g to 1 cell/250g. The inoculated meat at these cell densities was tested directly as individual samples or further blended with uninoculated meat. Five replicates were treated for each cell density/blend variable. The effect of the microbial 5

6 flora level on the sensitivity of the analytical method is evaluated by using low and high Aerobic Plate Count (APC) meat. Table 1. Sample Preparation for Compositing Studies. Inoculation Level Individual 25g Samples 375g Samples 350g uninoculated) 250g Samples 225g uninoculated) 125g Samples 100g uninoculated) Total Samples Control /250g /25g /25g /25g ,000/25g Total Salmonella Compositing Studies Data presented in Tables 2 and 3 demonstrate that there was no loss of sensitivity in detection of Salmonella due to compositing up to 375g/sample. In the high APC beef (Table 2) at the levels of 97.5, 9.75 and 0.95 cells/container, there were 5/5, 3/5 and 2/5 positives in the 375g samples compared to 5/5, 3/5 and 0/5 in the 25g samples. Results were similar for the low APC beef samples. There was no loss of sensitivity as the sample size was increased. If one considers the high level of inoculation as a 0 dilution and lower inoculation levels as 1/10, 1/100, 1/1,000, etc. dilutions, then a Most Probable Number (MPN) can be calculated for each size sample. Table 2. Salmonella in Beef with High APC Inoculation Level (cells/container) Individual 25g Samples Samples Positive/Samples Analyzed 375g Samples 250g Samples 350g uninoculated) 225g uninoculated) 125g Samples 100g uninoculated) 6

7 Control 0/5 0/5 0/5 0/ /5 (39) a 5/5 (2.6) 5/5 (3.9) 5/5 (7.8) /5 (3.9) 5/5 (.26) 5/5 (.39) 5/5 (.78) /5 (.39) 3/5 (.026) 4/5 (0.39) 3/5 (.078) /5 (.039) 2/5 (.0026) 0/5 (.0039) 0/5 (.0078) /5 (.0039) 0/5 (.00026) 0/5 (.00039) 1/5 (.00078) a Numbers in parentheses represents number of cells/g of hamburger. Table 3. Salmonella in Beef with Low APC Inoculation Level (cells/container) Individual 25g Samples Samples Positive/Samples Analyzed 375g Samples 250g Samples 350g uninoculated) 225g uninoculated) 125g Samples 100g uninoculated) Control 0/5 0/5 0/5 0/ /5 (370) a 5/5 (25) 5/5 (37) 5/5 (74) 952 5/5 (14) 5/5 (.93) 5/5 (1.4) 5/5 (2.8) /5 (1.4) 2/5 (.09) 3/5 (.14) 2/5 (.28) /5 (.37) 2/5 (.03) 0/5 (.04) 0/5 (.07) /5 (.037) 0/5 (.003) 0/5 (.004) 1/5 (.007) a Numbers in parentheses represents number of cells/g of hamburger. The calculated MPN s and 95% confidence limits for the various size samples, each containing the same number of organisms/container are as follows: MPN/g (95% Confidence Limits) Sample Size High APC Low APC 25g 79/g (30-250) 79/g (30-250) 125g 110/g (40-300) 70/g (30-210) 250g 130/g (50-390) 79/g (30-250) 375g 140/g (60-360) 94/g (40-250) The highest MPN s 140/g, were obtained with the 375g samples, but the 95% confidence limits for all size samples were overlapping. These data clearly indicate the beef samples for Salmonella testing can be composited up to 15 25g = 375g without a loss of sensitivity compared to analyzing 15 individual 25g samples. 2. Escherichia coli O157:H7 Compositing Studies 7

8 Prior to initiating the compositing studies, a preliminary experiment was conducted to validate the sensitivity of the USDA method for detection of E. coli O157:H7 in meats; i.e., the Petrifilm Escherichia coli O157:H7 Test method. Twenty five grams of beef patties or beef from chubs were weighed in duplicate to their respective stomacher bags. One ml of a 24 h Escherichia coli O157:H7 culture adjusted to cell concentrations ranging from 10 5 to 10 0 cells/ml was added to each beef sample. The Petrifilm Hemorrhagic assay enrichment protocol was then followed by adding 225 ml of modified EC with Novobiocin (225 ml) to each sample. The enrichment, selective plating and enzyme immunoassay were performed. The results in Table 4 demonstrate that a two Log 10 decrease in Escherichia coli O157:H7 was experienced upon inoculation and that these organisms would not be recovered by the Petrifilm test method. This data suggests that fewer than 100 cells/25g would not be recovered with this test method. No difference was observed in test method sensitivity when Escherichia coli O157:H7 was inoculated into high APC or low APC beef. Table 4. Petrifilm Escherichia coli O157:H7 Test Method Validation. E. coli O157:H7 cells/ml E. coli O157:H7 cells/g Culture a Low APC Beef A Low APC Beef B High APC Beef A High APC Beef B ,000 Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative Positive Negative Negative Negative Negative 0 0 Negative Negative Negative Negative a The culture used to inoculate the 25g beef patties was tested directly using the Petrifilm method. Results from the compositing experiments conducted with low APC beef are presented in Table 5. Data indicted better recovery, i.e., greater sensitivity, with the individual 25g and 125g samples compared to the 250g and 375g samples. The calculated MPN for 25g samples was 460/g compared to 7.0/g for the 375g sample. One possible explanation for the loss of recovery resulting from compositing is that in the composited samples the target organism would 8

9 need to go through more multiplications in the composited enrichments to reach detectable numbers. For example, if one cell were present in a 250 ml enrichment (25g of sample plus 225 ml of enrichment), it would need to go through 10 multiplications (generations) to reach 1,000/ml. In a 3,750 ml enrichment (375g of sample in 3,375 ml of enrichment) one E. coli O157:H7 cell would need to go through 14 generations to reach 1,000/ml. Unlike the Salmonella method which employs a 24 h enrichment, the Petrifilm Escherichia coli O157:H7 test method employs only a 6-8 h incubation. This reduced incubation time may not allow adequate time for the E. coli O157:H7 to reach detectable levels in the larger enrichments used for composited samples. Table 5. Escherichia coli O157:H7 in Beef with Low APC Inoculation Level (cells/container) Individual 25g Samples Samples Positive/Samples Analyzed 375g Samples 250g Samples 350g uninoculated) 225g uninoculated) 125g Samples 100g uninoculated) 3000/g 5/5 (120) a 5/5 (8) 5/5 (12) 5/5 (24) 300/g 5/5 (12) 2/5 (.8) 3/5 (1.2) 5/5 (2.4) 30/g 5/5 (1.2) 1/5 (.08) 1/5 (.12) 0/5 (.24) 3/g 1/5 (.12) 0/5 (.008) 1/5 (.012) 2/5 (.024) 0.3/g 1/5 (.012) 0/5 (.0008) 1/5 (.0012) 0/5 (.0024) a Numbers in parentheses represents number of cells/g of hamburger. To test this hypothesis, the modified EC with novobiocin enrichments were incubated a full 24 h at 35 C, plated, and tested via the Petrifilm Hemorrhagic test method. Table 6 shows the results for this trial when the 24 h results are substituted for the 6-8 h test results for those groups. The groups in Table 6 affected by the additional positive results are noted in brackets. Four additional positive test results were generated with the increased incubation time. Of these four additional positive test results, three positives were in the 375g composite group and one positive was in the 250g composite group. These results confirmed the hypothesis that additional incubation time may be required to maximize recovery by the Petrifilm method applied to composited sample. Table 6. Escherichia coli O157:H7 in Beef with Low APC 24 Hour Enrichment Data Compared to 6 Hour Enrichment Samples Positive/Samples Analyzed 9

10 Inoculation Level (cells/container) Individual 25g Samples 375g Samples 350g uninoculated) 250g Samples 225g uninoculated) 125g Samples 100g uninoculated) Control 0/5 0/5 0/5 0/ /5 (120) c 5/5 (8) 5/5 (12) 5/5 (24) 300 5/5 (12) [5/5] a (.8) [4/5] b (1.2) 5/5 (2.4) 30 5/5 (1.2) 1/5 (.08) 1/5 (.12) 0/5 (.24) 3 1/5 (.12) 0/5 (.008) 1/5 (.012) 2/5 (.024) 0.3 1/5 (.012) 0/5 (.0008) 1/5 (.0012) 0/5 (.00024) a Three additional positive test results were generated in this group following Petrifilm Hemorrhagic Escherichia coli O157:H7 analysis of enrichments incubated 24 hours at 35 C. b One additional positive test results were generated in this group following Petrifilm Hemorrhagic Escherichia coli O157:H7 analysis of enrichments incubated 24 hours at 35 C. c Numbers in parentheses represents number of cells/g of hamburger. Table 7 presents results of the compositing experiments using high APC beef, and incubating the enrichments for 24 h before plating to Petrifilm. Table 7. Escherichia coli O157:H7 in Beef with High APC Inoculation Level (cells/container) Individual 25g Samples Samples Positive/Samples Analyzed 375g Samples 250g Samples 350g uninoculated) 225g uninoculated) 125g Samples 100g uninoculated) Control 0/5 0/5 0/5 0/5 24,000 5/5 (960) a 5/5 (64) 5/5 (96) 5/5 (190) /5 (96) 5/5 (6.4) 5/5 (9.6) 5/5 (19) 240 3/5 (9.6) 0/5 (.64) 1/5 (.96) 4/5 (1.9) 24 1/5 (.96) 0/5 (.064) 0/5 (.096) 0/5 (.19) 2.4 0/5 (.096) 0/5 (.0064) 0/5 (.0096) 1/5 (.019) a Numbers in parentheses represents number of cells/g of hamburger. Although extending the incubation period from 6-8 h to 24 h appeared to improve recovery of E. coli O157:H7 from the composited samples (Table 6), recovery was still lower than from individual 25g samples (Table 6 and 7). Calculated MPN s for E. coli O157:H7 in high APC and low APC beef using the modified (24 h enrichment) Petrifilm method were as follows: MPN/g (95% Confidence Limits) Sample Size High APC Low APC 10

11 25g 460 (200-1,500) 110 (40-300) 125g ) 170 (70-480) 250g 63 (22-180) 33 (14-120) 375g 33 (14-120) 31 (13-110) The MPN s and 95% limits for the 250g and 375g samples were considerably lower than those associated with the 25g samples. The 125g data was similar to the 25g data for the high APC beef, but was demonstrated lower recovery for the low APC beef (49 vs. 460). Based upon these results, an experiment was conducted to compare the Organon-Teknika (O/T) EIA method to the Petrifilm method for recovery of E. coli O157:H7 in 25g and 375g samples. These data are presented in Table 8. The data for the 3M method, as expected, indicated better recovery with 25g samples than with 375g samples. With the O/T assay similar recovery was observed with 25g and 375g. Table 8. Effect of Compositing on Detection of Escherichia coli O157:H7 in Ground Beef Samples Positive/Samples Analyzed 3M Petrifilm Method O/T EIA Method Cells/Container 25g 375g 25g 375g Control 0/10 0/10 0/10 0/10 <3 1/10 0/10 2/5 1/ /10 1/10 10/10 10/ /10 2/10 ND* ND /10 10/10 ND ND *Not Determined Additional compositing experiments were conducted with the O/T method to determine if it could be employed with composited samples (Tables 9 and 10). In low APC product all inoculated samples were positive, regardless of sample size. In the high APC meat, recovery of E. coli O157:H7 from 375g samples was lower than that observed with 25g and 125g samples. The calculated MPN for recovery from 375g samples was 220/g with 95% confidence limits of ( ), compared to an estimated level of inoculation of 1,000/container. Table 9. Escherichia coli O157:H7 in Beef with High APC Samples Positive/Samples Analyzed 11

12 Inoculation Level (cells/container) Individual 25g Samples 375g Samples 350g uninoculated) 125g Samples 100g uninoculated) Control (0) 0/5 0/5 0/ /5 5/5 5/ /5 4/5 5/5 10 5/5 2/5 5/5 Table 10. Escherichia coli O157:H7 in Beef with Low APC Inoculation Level (cells/container) Individual 25g Samples Samples Positive/Samples Analyzed 375g Samples 350g uninoculated) 125g Samples 100g uninoculated) Control (0) 0/5 0/5 0/ /5 5/5 5/ /5 5/5 5/5 10 5/5 5/5 5/5 These data indicated that samples of low APC beef can be composited up to 15 25g = 375g with similar sensitivity to running individual 25g samples, and that in high count beef samples can be composited up to 125g (5 25g) with no loss of sensitivity, or up to 375g (15 25g) with only a marginal reduction of recovery seen with high APC beef. 3. Listeria monocytogenes Compositing Studies Initial studies on detection of Listeria monocytogenes in fresh meats were to validate the sensitivity of the current USDA method. Necessary to such work was meat that was free of Listeria monocytogenes. In screening many supplies of ground meat there was no problem meeting this requirement. However, virtually every sample of fresh ground meat tested contained Listeria inocua, an organism not considered a human pathogen, but one that has similar growth characteristics to L. monocytogenes. We were concerned that the presence of L. inocua would interfere with detection of L. monocytogenes by the USDA method which does not select for L. monocytogenes over L. inocua, but relies on identification of isolates to differentiate these organisms. Therefore, the sensitivity of the GENE-TRAK Listeria monocytogenes Assay for detection of L. monocytogenes was examined in addition to the USDA method in an effort to overcome this problem. 12

13 In a multi-laboratory study conducted by Curiale and Lewus of Silliker Laboratories, meats and environmental samples were inoculated with both L. monocytogenes and L. inocua. Consistently, L. inocua out-grew L. monocytogenes, and samples inoculated with L. monocytogenes were found negative when analyzed. This work, Detection of L. monocytogenes in Samples Containing Listeria inocua is in press (Journal of Food Protection). Therefore, in addition to the USDA method, a DNA hybridization method reported to be specific for L. monocytogenes was investigated. Meat patties were inoculated at decreasing levels of L. monocytogenes and tested by both the USDA method and the GENE- TRAK Listeria monocytogenes Assay (Table 11). Two trials of this experiment were performed. In both trials, only at the highest levels of inoculation was L. monocytogenes reliably detected by either method. These data were expected for the USDA method based on prior studies (Journal of Food Protection, in press), but unexpected for the GENE- TRAK specific probe method. Apparently, L. inocua naturally contaminating the meat out-grew the inoculated L. monocytogenes and suppressed its growth such that it was not able to multiply in the enrichment to levels detectable by the probe based assay (ca /ml). Table 11. Comparison for the USDA/FSIS Method for Listeria monocytogenes and the GENE-TRAK Listeria monocytogenes Assay. Confirmed USDA and confirmed GENE-TRAK Listeria monocytogenes Assay results for meat patties inoculated with Listeria monocytogenes. TRIAL 1 TRIAL 2 USDA a GENE-TRAK b USDA GENE-TRAK Inoculum Sample A c Sample A Sample B Sample B 10 5 L. mono L. innocua L. mono L. innocua 10 4 L. innocua L. innocua L. innocua L. innocua 13

14 10 3 L. innocua L. innocua 10 2 L. innocua L. mono L. innocua L. innocua 10 1 L. innocua L. innocua L. innocua L. innocua 10 0 L. mono L. innocua L. innocua L. innocua L. innocua 10-1 L. innocua L. mono L. innocua L. innocua L. innocua 10-2 L. mono L. innocua L. mono L. innocua L. innocua Uninoculated L. mono L. innocua Uninoculated L. innocua L. innocua Uninoculated L. mono L. innocua Uninoculated L. innocua L. innocua a Meat patties (25g) inoculated with 1 ml of Listeria monocytogenes culture and tested according to USDA protocol. Five CFU picked from MOX agar for confirmation. b Meat patties (25g) inoculated with 1 ml of Listeria monocytogenes culture and tested according GENE- TRAK protocol. Two CFU picked from MOX agar for confirmation. c Inoculated meat patties were enriched 24 hours at 35 C. Following incubation, enrichment was transferred to Fraser Broth for the USDA protocol and to MOX agar for the GENE-TRAK assay. Thus with fresh meat containing L. inocua there is currently no reliable method of detecting L. monocytogenes. We considered working with meat treated to kill naturally occurring L. inocua (as with irradiation), but eliminated this idea because this would not represent a normal real world situation, namely that fresh red meats, based on our studies, are commonly contaminated with L. inocua. In view of this work one should question survey data on the occurrence of L. monocytogenes in fresh meats. Negative findings frequently may reflect the fact the presence of L. inocua prevents detection of L. monocytogenes. Further studies on the effect of compositing on detection of L. monocytogenes were not conducted. 4. Campylobacter Compositing Studies The current USDA/FSIS method for Campylobacter (Laboratory Communication No. 69. USDA/FSIS, 1992) requires that the enrichments be constantly gassed during incubation. Headspace to volume ratios and agitation affect the efficacy of gas uptake by the medium. The glassware and apparatus employed in the FSIS Campylobacter method are designed for 25g samples in a specific size enrichment flask. Because of these constraints, this method does not lend itself to running larger sample sizes. Furthermore, the requirement for gasses and constant agitation makes the method impractical for performance in most food company laboratories. 14

15 Our work with Campylobacter has been to identify and validate a method that can be run without agitation and constant gassing, and that will accommodate larger sample sizes. These studies were not a part of this contract and thus will not be reported on. However, a method, GENE- TRAK Campylobacter Assay has been identified and preliminary trials have been performed to validate the method. Composition experiments with Campylobacter in low and high APC were conducted. The results of the validation trial are shown in Table 12. Campylobacter was detected 3 of 3 times at a level of greater than 0.42 cells/g. The sensitivity of the method for 25g sample sizes was 0.42 cells/g. This trial demonstrated that the identified method was capable of detecting Campylobacter at a sensitivity level of at least 10 cells/g. Table 12. Validation Trial using Individual 25 gram Samples Inoculation Levels Target/25g Actual/25g Actual/g Samples Positive/Samples Analyzed / / / /3 The results for the low and high count ground beef composite study can be found in Tables 13 and 14. The data for both low and high count beef indicate the 25g sample sizes are more sensitive for detecting Campylobacter. For example, there are a total of 15 of 15 detections for the 25g composite versus 6 of 15 for the 375g composite in low count beef and 15 of 15 detections for the 25g composite versus 5 of 15 for the 375g composite in high count beef. The inoculum level for the trials were higher than expected. However, when compared to the validation run the data suggests that Campylobacter would not have been detected at the lower levels in the larger composite sizes. In general, there was a loss in sensitivity as the sample size increased. Table 13. Campylobacter in Low Count Beef Samples Positives/Samples Analyzed Cells/Container 25g 125g 250g 375g 0 0/5 0/5 0/5 0/ /5 (5.2) a 2/5 (1.0) 1/5 (.52) 1/5 (.34) 15

16 1,300 5/5 (52) 5/5 (10) 4/5 (5.2) 0/5 (3.4) 13,000 5/5 (520) 5/5 (100) 5/5 (52) 5/5 (34) a Numbers in parentheses represents number of cells/g of hamburger. Table 14. Campylobacter in High Count Beef Samples Positives/Samples Analyzed Cells/Container 25g 125g 250g 375g 0 0/5 0/5 0/5 0/5 40 3/5 (1.6) a 0/5 (.32) 1/5 (.16) 0/5 (.11) 400 5/5 (16) 2/5 (3.2) 4/5 (1.6) 0/5 (1.1) 4,000 5/5 (160) 5/5 (32) 4/5 (16) 5/5 (11) a Numbers in parentheses represents number of cells/g of hamburger. A most probable number (MPN) can be calculated for each sample size (Table 15). For this, consider the high level of inoculation as a zero dilution and lower inoculation levels as 1:10, 1:100, 1:1,00 etc. dilutions. The highest MPN's were obtained with the 25g samples, while the 95% confidence limits were overlapping. These data indicate the beef samples for Campylobacter testing were more sensitive with the 25g samples. Table 15. Most Probable Number (MPN) Comparison MPN/g (95% Confidence Limits) Low Count High Count Composite Size Expected Observed Expected Observed 25g 520 >1, (300-2,900) 125g (220-2,000) (21-170) 250g (70-480) (15-77) 375g (13-110) (9-68) The results from the trials indicate that 25g samples provided more positive test results than the 375g sample sizes. There was only a marginal decrease in the sensitivity in high count beef. 16

17 B. Establish Most Efficient and Productive Methods for Sampling Combo Bins and Boxed Beef (Objective 4.b.i. and iii. of project proposal) A core sampling device (Appendix I) was identified that allowed relatively aseptic samples to be drawn from boxed beef and combo samples. The diameter and length of the core sampling tubes can be modified to accommodate combos and boxes. Sample tubes are easily removed for cleaning and sanitation. An air filter can be inserted into the compressed airline to allow product to be expelled from the sample core tube without contamination from the compressed air source. Samples of fresh beef combo bins and boxes were collected by three methods: purge, individual pieces from the top of each container, and cores collected with the core sampling device described in Appendix I. Two sets of individual piece samples were collected. One set for blending and one for rinsing as methods of preparation for analysis. A total of 200 samples of each type from four plants were collected. Each sample was analyzed to determine Aerobic Plate Count, coliform count, E. coli count, Salmonella and E. coli O157:H7. Summary data are presented in Tables 16 through 18 and in Appendix II. There was some variation from plant to plant in the level of the APC (Table 16A) ranging from <10/sample to 85,000,000/sample (Log ). However, the relationship between the sampling and preparation methods was similar for all four plants. The individual piece samples prepared for analysis by blending generally had higher APC s than those prepared by rinsing. The Log 10 average APC for all samples was 4.86/g (72,000/g) for blended samples compared to 4.69/g (49,000/g) for rinsed samples. Table 16A. Combo and Boxed Beef Evaluation: Summary of Aerobic Plate Count Data Log 10 Average/Sample (Range) Plant Samples Blend Rinse Core Purge ( ) 3.69 ( ) 3.35 ( ) 3.23 ( ) ( ) 3.37 ( ) 3.56 ( ) 3.58 ( ) ( ) 6.20 ( ) 6.15 ( ) 4.70 ( ) 17

18 ( ) 4.46 ( ) 4.23 ( ) 3.85 ( ) TOTAL ( ) 4.69 ( ) 4.61 ( ) 3.97 ( ) Core samples were prepared for analysis by blending. The average Log 10 APC data for core samples were slightly lower but within a similar range to data for the individual piece samples (blend and rinse). The Log 10 average APC for all core samples was 4.61/g (41,000/g). The Log 10 average APC for the purge samples were typically lower than the other three methods, with a Log 10 average APC of 3.97 (9,300). Statistical comparison of the data for the four methods (Table 16B) indicated that the APC s for blended samples were significantly different from the rinse and the core samples. However, there was no significant difference in the APC s for rinsed and cored samples. All three of the methods (pieces blended and rinsed, and cored) had significantly higher APC than the purge samples. Although the blended individual piece samples provided significantly higher Log 10 APC s than the core samples, the core samples are likely more representative of the product in the combo or box. Individual piece samples consisted of only a few pieces of trim or meat pieces, whereas the core samples contained portions of pieces of meat or trim per core from throughout the container. Table 16B. Combo and Boxed Beef Evaluation: Comparison of Aerobic Plate Counts Between Methods Methods Compared t-value Significantly Different a Blend vs. Rinse Yes Blend vs. Core Yes Blend vs. Purge Yes Rinse vs. Core No Rinse vs. Purge Yes Core vs. Purge Yes a When t> or t< , then the methods are significantly different at the 5% level. 18

19 A synopsis of the coliform data is presented in Table 17 (A&B). Data for coliforms are similar for the individual piece samples (blend and rinse) and for the core samples; Log 10 Avg/g for all samples of 2.14 (140), 1.96 (91), and 2.02 (100), respectively. There was no significant differences in the coliform data for these sample. However, coliform counts for the purge samples were generally lower, Total Log 10 Avg/Sample = 1.63 (43) and was significantly lower than all three of the other methods. Table 17A. Combo and Boxed Beef Evaluation: Summary of Coliform Count Data Log 10 Average/Sample (Range) Plant Samples Blend Rinse Core Purge ( ) 1.57 ( ) 1.51 ( ) 1.72 ( ) ( ) 1.30 ( ) 1.59 ( ) 1.15 ( ) ( ) 2.64 ( ) 2.69 ( ) 1.93 ( ) ( ) 1.88 ( ) 1.64 ( ) 1.37 ( ) TOTAL ( ) 1.96 ( ) 2.02 ( ) 1.63 ( ) 19

20 Table 17B. Combo and Boxed Beef Evaluation: Comparison of Coliform Counts Between Methods Methods Compared t-value Significantly Different a Blend vs. Rinse Yes Blend vs. Core No Blend vs. Purge Yes Rinse vs. Core No Rinse vs. Purge Yes Core vs. Purge Yes a When t> or t< , then the methods are significantly different at the 5% level. A summary of the E. coli data are presented in Table 18A. Levels of E. coli were generally very low. The Log 10 average APC for all samples ranged from 1.36 (23) to 1.28 (19). Table 18A. Combo and Boxed Beef Evaluation: Summary of E. coli Count Data Log 10 Average/Sample (Range) Plant Samples Blend Rinse Core Purge ( ) 1.35 ( ) 1.27 ( ) 1.36 ( ) ( ) 1.02 ( ) 1.19 ( ) 1.02 ( ) ( ) 1.49 ( ) 1.38 ( ) 1.42 ( ) ( ) 1.04 ( ) 1.13 ( ) 1.10 ( ) TOTAL ( ) 1.28 ( ) 1.28 ( ) 1.28 ( ) 20

21 There was no significant difference between the methods for E. coli enumeration (Table 18B). Table 18B. Combo and Boxed Beef Evaluation: Comparison of E. coli Counts Between Methods Methods Compared t-value Significantly Different a Blend vs. Rinse No Blend vs. Core No Blend vs. Purge No Rinse vs. Core No Rinse vs. Purge No Core vs. Purge No a When t> or t< , then the methods are significantly different at the 5% level. Combo and boxed beef samples also were tested for Salmonella and Escherichia coli O157:H7 shown below: Number of Positive Samples Sample Method Salmonella E. coli O157:H7 Individual pieces-blended 7 1 Individual pieces-rinsed 8 0 Core 4 0 Purge 5 0 Because of the low number of positive samples, it is impossible to discern significant differences in pathogen recovery by the methods. However, it appears that the purge sample is not a reliable method for pathogen recovery. Although there were three more samples positive for Salmonella by the individual piece blended method than by the core method, the latter is recommended because the samples should be more representative of the product being tested. C. Establish most Efficient and Productive Methods for Sampling Cacasses/Sides/Subprimals. Three methods were employed for sampling beef carcasses/ sides/subprimals: Tissue Excision (TE), Cellulose Sponge (Sponge-S), 21

22 and Cellulose Sponge with an Abrasive pad (Sponge-A). Duplicate TE samples were collected for each carcass; one was prepared for analysis by Blending (TE-Blend) and the other by Rinsing (TE-Rinse). A total of 190 samples of each type were collected from four plants and analyzed to determine APC, coliform count, Escherichia coli count, Salmonella, and E. coli O157:H7. A summary of the APC data are presented in Table 19 (A&B). A complete set of data is presented in Appendix III. The APC s ranged from less than 100/sample to 790,000/sample (Log ). Data for the TE-Blend and Sponge-S methods were similar; Log 10 average 2.96 and 2.83, respectively. There was no significant difference between these methods at the 5% probability level. APC s for TE-Blend and Sponge-S sample units were significantly higher than APC s for the TE-Rinse and Sponge-A methods. Interestingly, the Sponge-S provided higher APC s than the Sponge-A. At present, there is no clear explanation for this observation. However, one possibility is that the abrasive action entrapped bacteria within fat particles which were not soluble in the diluent used for sample preparation. It was observed that the abrasive pads were coated with fat before and after sample preparation. Use of an emulsifying diluent could overcome this problem. Table 19A. Beef Carcass Evaluation: Summary of Aerobic Plate Count Data Log 10 Average/Sample (Range) Plant Samples TE-Blend a TE-Rinse b Sponge-S c Sponge-A d ( ) 2.38 ( ) 2.47 ( ) 2.07 ( ) ( ) 2.30 ( ) 2.70 ( ) 2.31 ( ) ( ) 2.40 ( ) 2.51 ( ) 2.33 ( ) ( ) 2.81 ( ) 3.62 ( ) 3.01 ( ) TOTAL ( ) 2.47 ( ) 2.83 ( ) 2.43 ( ) a Tissue Excision Blend b Tissue Excision Rinse c Sponge, Standard d Sponge, Abrasive 22

23 Table 19B. Beef Carcass Evaluation: Comparison of Aerobic Plate Count Between Methods Methods Compared t-value Significantly Different e TE-Blend a vs. TE-Rinse b Yes TE-Blend a vs. Sponge-S c No TE-Blend a vs. Sponge-A d Yes TE-Rinse b vs. Sponge-S c Yes TE-Rinse b vs. Sponge A d No Sponge-S c vs. Sponge-A d Yes a Tissue Excision Blend b Tissue Excision Rinse c Sponge, Standard d Sponge, Abrasive e When t> or t< , then the methods are significantly different at the 5% level Summary coliform count data are presented in Table 20. Complete data are presented in Appendix III. Coliform levels ranged from <10/sample to 42,000 (Log )/sample. There were not significant differences (p<0.05) in the coliform counts obtained by the sample methods. (Statistical data not presented.) Data for E. coli were similar to the coliform data (Table 21) counts for E. coli ranged from <10/sample to 39,000/sample (Log ), and there were no significant difference between the methods (statistical data not presented). Table 20. Beef Carcass Evaluation: Summary of Coliform Count Data Log 10 Average/Sample (Range) Plant Samples TE-Blend a TE-Rinse b Sponge-S c Sponge-A d ( ) 1.32 ( ) 1.10 ( ) ( ) 1.02 ( ) 1.01 ( ) 1.11 ( ) ( ) 1.07 ( ) 1.18 ( ) 1.05 ( ) TOTAL ( ) 1.11 ( ) 1.07 ( ) 1.04 ( ) 23

24 a Tissue Excision Blend b Tissue Excision Rinse c Sponge, Standard d Sponge, Abrasive Seven TE-Blend samples and two TE-Rinse samples were positive for Salmonella. None of the sponge samples were positive for Salmonella. None of the carcass samples, regardless of sample method, were positive for E. coli O157:H7. Because of the low number of samples positive for Salmonella and E. coli O157:H7 it is not possible to determine if one of the methods provides superior recovery of these organisms. However, it is concerning that there were no sponge samples positive for Salmonella compared to seven positive samples for TE-Blend. A further comparison of sponge versus TE-Blend for Salmonella and E. coli O157:H7 is recommended. Table 21. Beef Carcass Evaluation: Summary of E. coli Count Data Log 10 Average/Sample (Range) Plant Samples TE-Blend a TE-Rinse b Sponge-S c Sponge-A d ( ) ( ) 1.02 ( ) ( ) ( ) 1.06 ( ) 1.14 ( ) 1.04 ( ) TOTAL ( ) 1.02 ( ) 1.03 ( ) 1.01 ( ) a Tissue Excision Blend b Tissue Excision Rinse c Sponge, Standard d Sponge, Abrasive 24

25 VIII. Publications, Abstracts, Manuscripts in Progress, These or Presentations That Resulted From This Research Portions of data from this study have been presented at the AMI Foundation s Briefing on USDA s Monitoring Program for E. coli in Raw Ground Beef held in Chicago, Illinois on November 2-3, Some of the data has been subsequently shared with several members of the beef industry. Two manuscripts are being prepared for submission to the Journal of Food Protection; one addressing the compositing studies, and another presenting the data on carcass and combo/boxed beef sampling. IX. Additional Funding Secured as a Result of Support from the Beef Industry Council of the National Live Stock and Meat Board At present, no additional funding has been requested from other funding sources to support related research. However, it is anticipated that one or more industry members will support a follow-up study to compare the sponge and the tissue excision methods for detection of Salmonella and E. coli O157:H7, using a larger number of samples to obtain a sufficient number of positive results to allow for statistical comparison of the data. X. Brief Lay Interpretation of Results Suitable for Public Release This research evaluated methods for sampling fresh beef and for preparation of samples for microbiological analysis. The primary objective of the research was to evaluate sampling and sample preparation methods that allow fresh beef to be efficiently sampled and analyzed to provide statistically valid data. These studies demonstrated that samples could be collected and up to 15 sample units composited to provide large (375g) samples representative of the product. These samples can be analyzed with no loss of detection sensitivity compared to testing 15 individual 25g samples for Salmonella and E. coli O157:H7. Use of this sampling and testing scheme makes statistically based sampling of fresh red meats for pathogen analysis practical and economically feasible. Further, a sponge sampling method was compared to the tissue excision method for sampling carcasses for microbiological analysis. The results indicate that the nondestructive and more efficient sponge sampling method is practical alternative to the tissue excision method. Use of the methods developed and validated by this research will greatly improve industry s ability to perform microbiological testing and to evaluate the efficacy of intervention procedures designed to improve the microbiological safety and quality of fresh beef products. 25