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OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY

OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY. John B. Luchansky, Ph.D. Agricultural Research Service Eastern Regional Research Center Microbial Food Safety Research Unit. Examples of Research on L. monocytogenes and Frankfurters.

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OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY

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  1. OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY John B. Luchansky, Ph.D. Agricultural Research Service Eastern Regional Research Center Microbial Food Safety Research Unit

  2. Examples of Research onL. monocytogenes and Frankfurters • Efficacy of potassium lactate as an ingredient in batter • Porto et al., J. Food Prot. 65:308-315, 2002 with HQM • USDA/ARS Package Rinse Method for pathogen recovery • Luchansky et al., J. Food Prot. 65:567-570, 2002 • Effect of re-heating on viability • Porto et al., J. Food Prot. 67:71-76, 2004 • Use of PFGE to determine the persistence of a 5-strain cocktail • Porto et al., J. Food Prot. 69:4177-4182, 2003 • USDA frankfurter storage study • Wallace et al., J. Food Prot. 66:584-591, 2003 with FSIS, AMI, NFPA, NTF • Localization within naturally-contaminated packages • Wallace, Call, Luchansky et al., J. Food Prot., Published • Evaluation of frankfurter casings containing a biopreservative • Call/Luchansky et al., J. Food Prot., Published 2004 with Hatfield, Viskase & Rhodia

  3. USDA Frankfurter Storage Study Sample packages for Listeria monocytogenes during refrigerated storage: Part A = Determine package prevalence Part B = Estimate pathogen levels Part C = Establish pathogen types Wallace et al., 2003 J. Food Prot. 66:584-591.

  4. USDA Frankfurter Storage Study Part A: Package Prevalence!

  5. Distribution of Volunteer Plants • 300 processors contacted • 12 facilities volunteered to participate: • 9 large and 3 small plants as determined by HACCP classification • USDA/FSIS regions 1, 2, 3, and 4 • 8 USDA/FSIS districts in 10 states • ~2700 pounds/packages collected from each facility by a 3rd-party contractor

  6. Sample Size Considerations for an Estimated L. monocytogenes Prevalence of ~3% Dr. John G. Phillips, Statistician, USDA/ARS, NAA

  7. Terms and Conditions - Industry • No identification of plant name or location • No inspection activities • No regulatory actions or recalls • No “fingerprint” data added to PulseNet

  8. Terms and Conditions – USDA • Independent 3rd party interacts with plants • Collects product, shares results with participants • Normal production run, regular HACCP monitoring and GMP • No special sanitation prior to production • Refrigerated transport to ERRC • Temperature recorders placed in select shipping boxes

  9. Sampling Plan • Day 1 = 5 days post-production • 500 packages/pounds sampled • Remainder of packages stored at 4° and 10°C • Storage at 4°C • 200 packages tested on days 10, 20, 30, 45, and 60 • Product tested on days 120 and 150 for some plants • Storage at 10°C • 200 packages tested on days 5, 10, 15, 20, 25, and 30 • Product not tested on days 20 and 25 for some plants

  10. Sampling Strategy:USDA-ARS Package Rinse Method • Add 60 mL peptone water per package and rinse package contents • Analyze 25 mL - enrich, isolate, & confirm • Retain multiple isolates from each positive sample for subtyping • Retain 35 mL at -20°C – enumerate if possible • 3-tube MPN procedure (FDA/CFSAN)

  11. USDA/ARS Package Rinse Method • Six-fold more effective at recovery of L. monocytogenes than the approved USDA/FSIS product composite enrichment method because the package, the purge, and the product are tested • About twice as likely to recover the bacterium from: • rinse > purge > product composite • Less likely to cause product contamination and more likely to decrease the time required to sample the product because it requires less hands-on manipulation of the product Luchansky et al., 2002 J. Food Prot. 65:567-570.

  12. Proximate Composition • Six packages tested from each plant • 2 packages on initial sample day • Day 1 = 5 days after production • 2 packages after 30 days at 10°C • 2 packages after 60 days at 4°C • Portions of each package tested for nitrite, total phenolics, NaCl, pH, protein, moisture, ash, fat, carbohydrates, and lactic acid.

  13. USDA Frankfurter Storage Study Contains sodium diacetate** and/or potassium lactate* as an ingredient

  14. USDA Frankfurter Storage Study Package prevalence = 1.6% (543 of 32,800) Range = 0.07 to 16%

  15. Evidence against laboratory contamination • Non-disposable equipment, supplies, and laboratory surfaces decontaminated frequently • Separation of experiments/incubators to recover the organism from experiments/incubators to type it • Pattern and frequency of positive packages does not support carryover or cross contamination • Negative controls in place • Environmental swabs – all 30 negative • Glove samples - all 147 negative

  16. USDA Frankfurter Storage Study • Package prevalence – all 12 plants • 4°C – 314 of 19,100 (1.64%) • 10°C – 218 of 13,700 (1.59%) Did storage temperature affect recovery rate?

  17. USDA Frankfurter Study • Timeframe • August 17 of 2000 through July 3 of 2002 • Seasonal Distribution • Fall 3 of 12 plants – 2 positive plants • Winter 3 of 12 plants – 1 positive plant • Spring 4 of 12 plants – 3 positive plants • Summer 2 of 12 plants – 1 positive plant Did seasonality affect recovery rate?

  18. Recovery rate of L. monocytogenes from all packages during storage at 4°C Day Packages 1 6000 10 2400 20 2400 30 2400 45 2400 60 2400 120/150 1100

  19. Recovery rate of L. monocytogenes from all packages during storage at10°C Day Packages 5 2400 10 2400 15 2400 20 2200 25 1900 30 2400

  20. Lactic Acid Bacteria Levels • Limited number of packages from each manufacturer evaluated • 101 to 103 cfu/package on day 1 • 108 to 1010 cfu/package on day 30 following storage at 10EC • 105 to 107 cfu/package on day 60 following storage at 4EC

  21. Prevalence of Listeria monocytogenesin Ready-to-eat Foods • 1.6% (32,800) Franks USDA/ARS (2000-2002) • 1.8% (2,162) Sm. Diam. Sausage USDA/FSIS (1999) • 2.8% (31,009) All meat & poultry USDA/FSIS (1990-1999) • 3.6% (6,820) Sm. Dia. Sausage USDA/FSIS (1990-1999) • 7.6% (1,874) Franks – composite Lm Risk Assessment • 1.8%(31,700) RTE foods NFPA (2000-2002) • Levine et al., JFP 64:1188-1193, 2001. • www.foodsafety.gov/~dms/lmrisk.html • Wallace et al., JFP 66:584-591, 2003. • Gombas et al., JFP 66: April, 2003.

  22. USDA Frankfurter Storage Study Part B: Pathogen Levels!

  23. USDA-ARS Package Rinse Method:Sampling Strategy • Rinse package contents with 60 mL of peptone water to recover L. monocytogenes • Analyze 25 mL – determine presence and types • Retain 35 mL at -20°C – enumerate if possible

  24. Sampling Strategy:USDA-ARS Package Rinse Method • Retain 35 mL at -20°C – enumerate if possible • Perform 3-tube MPN test (FDA/CFSAN) • Tested 157 rinsates representing all plants • Rinsates held at -20oC for 1 to 23 months • Plate directly onto MOX agar • Tested 100 rinsates from plant 133 after 150 days at 4oC • Rinsates held at -20oC for 7 days

  25. Enumeration using a 3-tube MPN • Of the 157 package rinsates analyzed: • Most tested negative after storage at -20oC • 50 to 80% reduction within hours/days • 4.0 log10 reduction after weeks/months • 3 packages yielded 71, 95, and 191 MPN/package • Plant 367 packages held for 30 days at 10oC, contents rinsed, and rinsates stored at -20oC for 2 months prior to MPN

  26. Enumeration for L. monocytogenes by direct plating • 100 packages from plant 133 were tested following storage at 4°C for 150 days • 16 of 100 packages tested positive • Rinsates were stored at -20oC for 7 days and then plated onto MOX agar • Levels from < 10 up to 9.6 x 104 CFU/package

  27. Enumeration of L. monocytogenes from Plant 133 Following Storage at 4°C for 150 Days Rinsates were stored at -20oC for 7 days

  28. USDA Frankfurter Storage Study Part C: Pathogen Types!

  29. USDA Frankfurter Storage Study:Pathogen Types How many different “types” of L. monocytogenes were recovered? Among 1102 isolates typed: • >90% displayed ribotype “A" • all of these isolates were serotype 1/2a

  30. Molecular Subtyping Results • In most instances, multiple isolates from a single package and/or from a single producer displayed the same ribotype/serotype. • In some instances, it was possible to recover isolates displaying more than one ribotype/serotype from a given producer. • In rare instances, multiple isolates from a single package displayed a different ribotype/serotype.

  31. USDA Frankfurter Storage Study Part D: Concluding Remarks!

  32. Risk Management QuestionIntended to Answer What is the “true prevalence” of L. monocytogenes in a high-volume, higher risk, RTE meat? Package prevalence = 1.6% (543 of 32,800) Range = 0.07 to 16%

  33. Risk Management QuestionIntended to Answer How many L. monocytogenes are likely to be recovered from naturally-contaminated RTE meat? Levels ranged from 1 to 100,000 cfu/package

  34. Risk Management QuestionIntended to Answer What types of L. monocytogenes are likely to be recovered from naturally-contaminated RTE meat? Some strains predominate/persist within vacuum-sealed packages - most isolates were ribotype “A” and serotype 1/2a!

  35. Caveats! • Manufacturers were not selected at random and only a single lot from each was tested • Manufacturers were not reflective of all producers in USA • A more effective method (ARS package rinse) was used to sample product/packages • Numerous packages were sampled on several sampling days over extended storage of the product. • Information was not available about the order in which the packages were produced during a given production run • Enumeration was problematic because pathogen numbers decreased appreciably in rinsates during frozen storage

  36. Lessons Learned/Improvements! • What types are tolerable and under what situations? • Are there differences among strains in viability or virulence - how much insight can be provided by genomics/proteomics? • How often would a given plant be positive on consecutive and/or multiple visits? • What is the frequency and distribution of contamination across a positive lot? • Should more emphasis be placed on collecting data on pathogen levels in positive samples?

  37. Lessons Learned/Improvements! • Where does it reside and how long does it persist or predominate? • How many types are present and at what levels? • Where did it come from and where might it end up? • What is the ecology of the bacterium in the environment and on the product – how well does it respond to stress/cues? • Should more emphasis be placed on environmental sampling to compliment targeted testing of finished products?

  38. THANK YOU! • Partners: • National Food Processors Association • American Meat Institute • National Turkey Federation • USDA/FSIS • ERRC Special Projects Team • Morgan Wallace and Jeff Call • Anna Porto and Laura Wonderling • Gaylen Uhlich and Darrell Bayles

  39. Enhancing the Safety of Frankfurters

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