OVERVIEW%20OF%20THE%20USDA%20ARS%20

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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

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Examples of Research onL. monocytogenes and
Frankfurters

• Efficacy of potassium lactate as an ingredient in
  batter
• Porto et al., J. Food Prot. 65308-315, 2002 with
  HQM
• USDA/ARS Package Rinse Method for pathogen
  recovery
• Luchansky et al., J. Food Prot. 65567-570, 2002
• Effect of re-heating on viability
• Porto et al., J. Food Prot. 6771-76, 2004
• Use of PFGE to determine the persistence of a
  5-strain cocktail
• Porto et al., J. Food Prot. 694177-4182, 2003
• USDA frankfurter storage study
• Wallace et al., J. Food Prot. 66584-591, 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

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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. 66584-591.

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USDA Frankfurter Storage Study

• Part A Package Prevalence!

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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

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Sample Size Considerations for an Estimated L.
monocytogenes Prevalence of 3
Confidence Error in P 25 Error in P 10
80 770 samples 4,809 samples
90 1,269 samples 7,930 samples
95 1,802 samples 11,258 samples
99 3,112 samples 19,445 samples
Dr. John G. Phillips, Statistician, USDA/ARS, NAA
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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

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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

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Sampling Plan

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

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Sampling StrategyUSDA-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 -20C enumerate if possible
• 3-tube MPN procedure (FDA/CFSAN)

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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 gt purge gt 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. 65567-570.
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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 10C
• 2 packages after 60 days at 4C
• Portions of each package tested for nitrite,
  total phenolics, NaCl, pH, protein, moisture,
  ash, fat, carbohydrates, and lactic acid. 

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USDA Frankfurter Storage Study
Facility Size Formulation Season Pounds Assayed
Plant 42 Large Pork and Beef Spring 2900
Plant 94 Large Turkey Spring 2700
Plant 105 Large Beef Fall 2800
Plant 133 Large Turkey Spring 2800
Plant 172 Large Beef Winter 2700
Plant 236 Small Pork and Beef Winter 2900
Plant 344 Large Pork, Beef, and Chicken Fall 2500
Plant 367 Small Pork Summer 2900
Plant 385 Small Pork and Beef Fall 2600
Plant 399 Large Pork and Beef Summer 2800
Plant 439 Large Pork and Beef Spring 2300
Plant 443 Large Pork and Turkey Winter 2900
Total Packs Tested August 2000 through July 2002 August 2000 through July 2002 August 2000 through July 2002 32800
Contains sodium diacetate and/or potassium
lactate as an ingredient
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USDA Frankfurter Storage Study
Facility Formulation Packages Assayed/Positive
Plant 42 Pork and Beef 2900
Plant 94 Turkey 2 of 2700 0.07
Plant 105 Beef 2800
Plant 133 Turkey 437 of 2800 16
Plant 172 Beef 3 of 2700 0.11
Plant 236 Pork and Beef 2900
Plant 344 Pork, Beef, and Chicken 4 of 2500 0.16
Plant 367 Pork 44 of 2900 1.5
Plant 385 Pork and Beef 2 of 2600 0.08
Plant 399 Pork and Beef 2800
Plant 439 Pork and Beef 51 of 2300 2.2
Plant 443 Pork and Turkey 2900
Package prevalence 1.6 (543 of 32,800) Range
0.07 to 16
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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

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USDA Frankfurter Storage Study
Did storage temperature affect recovery rate?

• Package prevalence all 12 plants
• 4C 314 of 19,100 (1.64)
• 10C 218 of 13,700 (1.59)

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USDA Frankfurter Study
Did seasonality affect recovery rate?

• 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

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Recovery rate of L. monocytogenes from all
packages during storage at 4C

• Day Packages
• 1 6000
• 10 2400
• 20 2400
• 30 2400
• 45 2400
• 60 2400
• 120/150 1100

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Recovery rate of L. monocytogenes from all
packages during storage at10C

• Day Packages
• 5 2400
• 10 2400
• 15 2400
• 20 2200
• 25 1900
• 30 2400

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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

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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 641188-1193, 2001.
• www.foodsafety.gov/dms/lmrisk.html
• Wallace et al., JFP 66584-591, 2003.
• Gombas et al., JFP 66 April, 2003.

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USDA Frankfurter Storage Study

• Part B Pathogen Levels!

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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 -20C enumerate if possible

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Sampling StrategyUSDA-ARS Package Rinse Method

• Retain 35 mL at -20C 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

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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

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Enumeration for L. monocytogenes by direct plating

• 100 packages from plant 133 were tested following
  storage at 4C 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 lt 10 up to 9.6 x 104 CFU/package

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

• Part C Pathogen Types!

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USDA Frankfurter Storage StudyPathogen Types

• How many different types of
• L. monocytogenes were recovered?
• Among 1102 isolates typed
• gt90 displayed ribotype A"
• all of these isolates were serotype 1/2a

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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.

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USDA Frankfurter Storage Study

• Part D Concluding Remarks!

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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
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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
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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!
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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

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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?

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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?

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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

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Enhancing the Safety of Frankfurters