Presentation on theme: "OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY John B. Luchansky, Ph.D. Agricultural Research Service Eastern Regional Research Center Microbial."— Presentation transcript:
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 Efficacy of potassium lactate as an ingredient in batter Porto et al., J. Food Prot. 65: , 2002 with HQM USDA/ARS Package Rinse Method for pathogen recovery Luchansky et al., J. Food Prot. 65: , 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: , 2003 USDA frankfurter storage study Wallace et al., J. Food Prot. 66: , 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
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:
USDA Frankfurter Storage Study Part A: Package Prevalence!
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 3 rd -party contractor
Sample Size Considerations for an Estimated L. monocytogenes Prevalence of ~3% ConfidenceError in P = 25%Error in P = 10% 80%770 samples4,809 samples 90%1,269 samples7,930 samples 95%1,802 samples11,258 samples 99%3,112 samples19,445 samples Dr. John G. Phillips, Statistician, USDA/ARS, NAA
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
Terms and Conditions – USDA Independent 3 rd 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
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
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)
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:
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.
USDA Frankfurter Storage Study FacilitySizeFormulationSeasonPounds Assayed Plant 42LargePork and BeefSpring2900 Plant 94Large Turkey ** Spring2700 Plant 105LargeBeefFall2800 Plant 133LargeTurkeySpring2800 Plant 172LargeBeefWinter2700 Plant 236SmallPork and BeefWinter2900 Plant 344LargePork, Beef, and ChickenFall2500 Plant 367SmallPorkSummer2900 Plant 385SmallPork and BeefFall2600 Plant 399LargePork and BeefSummer2800 Plant 439LargePork and Beef*Spring2300 Plant 443LargePork and TurkeyWinter2900 Total Packs Tested August 2000 through July Contains sodium diacetate** and/or potassium lactate* as an ingredient
USDA Frankfurter Storage Study FacilityFormulationPackages Assayed/Positive Plant 42Pork and Beef2900 Plant 94 Turkey ** 2 of 2700 = 0.07% Plant 105Beef2800 Plant 133Turkey437 of 2800 = 16% Plant 172Beef3 of 2700 = 0.11% Plant 236Pork and Beef2900 Plant 344Pork, Beef, and Chicken4 of 2500 = 0.16% Plant 367Pork44 of 2900 = 1.5% Plant 385Pork and Beef2 of 2600 = 0.08% Plant 399Pork and Beef2800 Plant 439Pork and Beef*51 of 2300 = 2.2% Plant 443Pork and Turkey2900 Package prevalence = 1.6% (543 of 32,800) Range = 0.07 to 16%
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
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?
USDA Frankfurter Study Timeframe August 17 of 2000 through July 3 of 2002 Seasonal Distribution Fall3 of 12 plants – 2 positive plants Winter3 of 12 plants – 1 positive plant Spring4 of 12 plants – 3 positive plants Summer2 of 12 plants – 1 positive plant Did seasonality affect recovery rate?
Recovery rate of L. monocytogenes from all packages during storage at 4°C DayPackages /
Recovery rate of L. monocytogenes from all packages during storage at 10°C DayPackages
Lactic Acid Bacteria Levels Limited number of packages from each manufacturer evaluated 10 1 to 10 3 cfu/package on day to cfu/package on day 30 following storage at 10 E C 10 5 to 10 7 cfu/package on day 60 following storage at 4 E C
Prevalence of Listeria monocytogenes in Ready-to-eat Foods 1.6% (32,800)FranksUSDA/ARS ( ) 1.8% (2,162)Sm. Diam. SausageUSDA/FSIS (1999) 2.8% (31,009)All meat & poultryUSDA/FSIS ( ) 3.6% (6,820)Sm. Dia. SausageUSDA/FSIS ( ) 7.6% (1,874)Franks – compositeLm Risk Assessment 1.8%(31,700)RTE foodsNFPA ( ) Levine et al., JFP 64: , Wallace et al., JFP 66: , Gombas et al., JFP 66: April, 2003.
USDA Frankfurter Storage Study Part B: Pathogen Levels!
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
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 -20 o C for 1 to 23 months Plate directly onto MOX agar Tested 100 rinsates from plant 133 after 150 days at 4 o C Rinsates held at -20 o C for 7 days
Enumeration using a 3-tube MPN Of the 157 package rinsates analyzed: Most tested negative after storage at -20 o C 50 to 80% reduction within hours/days 4.0 log 10 reduction after weeks/months 3 packages yielded 71, 95, and 191 MPN/package Plant 367 packages held for 30 days at 10 o C, contents rinsed, and rinsates stored at -20 o C for 2 months prior to MPN
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 -20 o C for 7 days and then plated onto MOX agar Levels from < 10 up to 9.6 x 10 4 CFU/package
Enumeration of L. monocytogenes from Plant 133 Following Storage at 4°C for 150 Days Rinsates were stored at -20 o C for 7 days
USDA Frankfurter Storage Study Part C: Pathogen Types!
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
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.
USDA Frankfurter Storage Study Part D: Concluding Remarks!
Risk Management Question Intended 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%
Risk Management Question Intended 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
Risk Management Question Intended 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!
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
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?
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?
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