Transcript Identification of Genes Essential for L. monocytogenes

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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Efficacy of potassium lactate as an ingredient in batter
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USDA/ARS Package Rinse Method for pathogen recovery
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Wallace et al., J. Food Prot. 66:584-591, 2003 with FSIS, AMI, NFPA, NTF
Localization within naturally-contaminated packages
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Porto et al., J. Food Prot. 69:4177-4182, 2003
USDA frankfurter storage study
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Porto et al., J. Food Prot. 67:71-76, 2004
Use of PFGE to determine the persistence of a 5-strain cocktail
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Luchansky et al., J. Food Prot. 65:567-570, 2002
Effect of re-heating on viability
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Porto et al., J. Food Prot. 65:308-315, 2002 with HQM
Wallace, Call, Luchansky et al., J. Food Prot., Published
Evaluation of frankfurter casings containing a biopreservative
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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. 66:584-591.
USDA Frankfurter Storage Study
Part A: Package Prevalence!
Distribution of Volunteer Plants
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300 processors contacted
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12 facilities volunteered to participate:
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USDA/FSIS regions 1, 2, 3, and 4
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9 large and 3 small plants as determined by HACCP
classification
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
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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
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Independent 3rd party interacts with plants
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Collects product, shares results with participants
Normal production run, regular HACCP
monitoring and GMP
No special sanitation prior to production
Refrigerated transport to ERRC
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Temperature recorders placed in select shipping boxes
Sampling Plan
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Day 1 = 5 days post-production
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500 packages/pounds sampled
Remainder of packages stored at 4° and 10°C
Storage at 4°C
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200 packages tested on days 10, 20, 30, 45, and 60
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Product tested on days 120 and 150 for some plants
Storage at 10°C
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200 packages tested on days 5, 10, 15, 20, 25, and 30
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Product not tested on days 20 and 25 for some plants
Sampling Strategy:
USDA-ARS Package Rinse Method
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Add 60 mL peptone water per package and
rinse package contents
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Analyze 25 mL - enrich, isolate, & confirm
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Retain multiple isolates from each positive sample for
subtyping
Retain 35 mL at -20°C – enumerate if possible
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3-tube MPN procedure (FDA/CFSAN)
USDA/ARS Package Rinse Method
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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.
Proximate Composition
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Six packages tested from each plant
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2 packages on initial sample day
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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
Contains sodium diacetate** and/or potassium lactate* as an ingredient
32800
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
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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
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Environmental swabs – all 30 negative
Glove samples - all 147 negative
USDA Frankfurter Storage Study
Did storage temperature affect recovery rate?
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Package prevalence – all 12 plants
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4°C – 314 of 19,100 (1.64%)
10°C – 218 of 13,700 (1.59%)
USDA Frankfurter Study
Did seasonality affect recovery rate?
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Timeframe
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August 17 of 2000 through July 3 of 2002
Seasonal Distribution
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Fall
Winter
Spring
Summer
3 of 12 plants – 2 positive plants
3 of 12 plants – 1 positive plant
4 of 12 plants – 3 positive plants
2 of 12 plants – 1 positive plant
Recovery rate of L. monocytogenes from all
packages during storage at 4°C
Day
Packages
1
10
20
30
45
60
120/150
6000
2400
2400
2400
2400
2400
1100
Percent Positive
5
3.5
2.3
2.5
1.5
1.5
0.9
0.45
0.21
0
1
10
20
30
45
Sampling Day
60
120/150
Recovery rate of L. monocytogenes from
all packages during storage at10°C
Day
Packages
5
10
15
20
25
30
2400
2400
2400
2200
1900
2400
Percent Positive
5
2.5
3.2
3.3
25
30
2.1
1.6
0.92
0.73
15
20
0
5
10
Sampling Day
Lactic Acid Bacteria Levels
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Limited number of packages from each
manufacturer evaluated
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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
Prevalence of Listeria monocytogenes
in Ready-to-eat Foods
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1.6% (32,800)
1.8% (2,162)
2.8% (31,009)
3.6% (6,820)
7.6% (1,874)
1.8%(31,700)
Franks
Sm. Diam. Sausage
All meat & poultry
Sm. Dia. Sausage
Franks – composite
RTE foods
USDA/ARS (2000-2002)
USDA/FSIS (1999)
USDA/FSIS (1990-1999)
USDA/FSIS (1990-1999)
Lm Risk Assessment
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
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Rinse package contents with 60 mL of peptone
water to recover L. monocytogenes
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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
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Perform 3-tube MPN test (FDA/CFSAN)
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Tested 157 rinsates representing all plants
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Rinsates held at -20oC for 1 to 23 months
Plate directly onto MOX agar
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Tested 100 rinsates from plant 133 after 150 days at 4oC
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Rinsates held at -20oC for 7 days
Enumeration using a 3-tube MPN
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Of the 157 package rinsates analyzed:
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Most tested negative after storage at -20oC
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50 to 80% reduction within hours/days
4.0 log10 reduction after weeks/months
3 packages yielded 71, 95, and 191 MPN/package
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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
o
 Rinsates were stored at -20 C for 7 days and
then plated onto MOX agar
4
 Levels from < 10 up to 9.6 x 10 CFU/package
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Number of Packages
Enumeration of L. monocytogenes from
Plant 133 Following Storage at 4°C for 150 Days
10
9
8
6
4
2
2
1
1
1.E+01
1.E+02
2
1
0
<10
1.E+03
1.E+04
1.E+05
CFU Per Package
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
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Molecular Subtyping Results
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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!
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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!
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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!
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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!
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Partners:
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National Food Processors Association
American Meat Institute
National Turkey Federation
USDA/FSIS
ERRC Special Projects Team
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Morgan Wallace and Jeff Call
Anna Porto and Laura Wonderling
Gaylen Uhlich and Darrell Bayles
Enhancing the Safety of Frankfurters