OVERVIEW OF THE USDA ARS

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