OVERVIEW OF THE USDA ARS & FSIS FRANKFURTER STORAGE STUDY

John B. Luchansky, Ph.D.John B. Luchansky, Ph.D.Agricultural Research Service

Eastern Regional Research CenterMicrobial Food Safety Research Unit

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

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 typesWallace et al., 2003

J. Food Prot. 66:584-591.

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 3rd-party contractor

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

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 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

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., 2002J. Food Prot. 65:567-570.

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

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 32800

Contains sodium diacetate** and/or potassium lactate* as an ingredient

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%

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 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?

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

DayPackages

1 6000

10 2400

20 2400

30 2400

45 2400

60 2400

120/150 1100

0.9

3.5

1.5

2.3

0.21

1.5

0.45

0

2.5

5

1 10 20 30 45 60 120/150

Sampling Day

Perc

ent P

ositi

ve

Recovery rate of L. monocytogenes from all packages during storage at10°C

2.11.6

0.92 0.73

3.2 3.3

0

2.5

5

5 10 15 20 25 30

Sampling Day

Per

cent

Pos

itive

DayPackages

5 2400

10 2400

15 2400

20 2200

25 1900

30 2400

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 10C 105 to 107 cfu/package on day 60 following

storage at 4C

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.

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 -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

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

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

Enumeration of L. monocytogenes from Plant 133 Following Storage at 4°C for 150 Days

9

1 12

12

0

2

4

6

8

10

<10 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05

CFU Per Package

Nu

mb

er

of

Pa

ck

ag

es

Rinsates were stored at -20oC 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 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%

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

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!

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

Enhancing the Safety of Frankfurters