use of enterococcus faecium nrrl b-2354 as a surrogate...e. faecium nrrl b-2354 has been used for...

Use of Enterococcus Faecium NRRL B-2354 as a Surrogate

December 8, 2015

Speakers

Linda Harris, UC Davis (Moderator)

Linda Harris, UC Davis

Maria Marco, UC Davis

Shirin Abd & Carrie Ferstl, The National Food Laboratory

Linda Harris, University of California, Davis

Validating the use of Enterococcus faecium NRRL B-2354 for almond processes

Linda J. Harris, Ph.D. Specialist in Cooperative Extension Food Science and Technology Western Center for Food Safety

Surrogates: when appropriate or necessary?

• In-plant validation studies – usually almost always involve use of surrogates – introduction of the pathogen usually considered an unacceptable risk

• E.g., continuous flat bed dry roaster

Pathogens: when appropriate or necessary?

• When parameters can be mimicked in the laboratory or on pilot scale equipment – E.g., oil roast, water blanch

• In very rare cases pathogens may be used for in-plant validation where: – No surrogate exists – Contamination of equipment/facility is unlikely – Precautions are taken/planned in advance – Experiments are planned/executed with

guidance of experts

What makes an ideal surrogate?

• Non-pathogenic

• In low moisture foods (e.g., almonds) – Similar desiccation tolerance

• Represents pathogen of concern or “target organism” – For almonds and other tree nuts: Salmonella

• Similar inactivation characteristics and kinetics – Predict target organism

• Easy preparation – Stable high-density populations – Easy to enumerate

Salmonella

See: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses/ucm094141.htm

http://www.fda.gov/Food/ScienceResearch/

Almond Board Microbial Surrogate Development 2004-2007

Potential Surrogates for Salmonella Culture Names and Source ID

Salmonella culture Salmonella Enteritidis Phage Type 30 (ATCC BAA-1045) (SE PT 30)

Surrogate cultures

Pantoea agglomerans SPS2F1 (PA) Pediococcus spp. NRRL B-2354

(Reclassified as Enterococcus faecium) (PD) Enterococcus faecalis (ATCC 49452) (EF) Lactobacillus plantarum (ATCC 14917) (LP) Lactobacillus fermentum (ATCC 9338) (LF) Streptococcus faecalis (ATCC 33186) (SF)

Potential Surrogates Screened (Dry Heat)

Salmonella Enteritidis PT30

Pantoea agglomerans

Enterococcus faecium

0.300.400.500.600.700.800.901.001.10

260 270 280 290 300 310

Log

D

Hot Air Temperature (°F)

Pantoea agglomerans and Enterococcus faecium were chosen for further study

E. faecium NRRL B-2354/ATCC 8459

• Originally Micrococcus freudenreichii Guillebeau – Isolated from milk and dairy utensils – Used as a surrogate for thermal processing of dairy products (1947)

• Renamed Pediococcus sp.

• Reclassified as Enterococcus faecium (Ma et al., 2007) – Evaluating use as a surrogate for meat

• Deposited to – American Type Culture Collection ATCC 8459 – Northern Regional Research Laboratory NRRL B-2354

• USDA National Center for Agricultural Utilization Research

http://atcc.org/Products/All/8459.aspx Annous and Kozempel. 1998. J. of Food Protection 61(5):578

Ma et al. 2007. J. of Food Protection 70(4):952 Kornacki, J. L. 2012. Food Safety Magazine.

https://commons.wikimedia.org/wiki/File:Entercoccus_sp2_lores.jpg

PresenterPresentation NotesIsolated from cheese

1970 renamed pedio

2007 renamed faecium (work from the Doyle lab)

Long history of safe use as surrogate organism in almonds, ground beef, and cider, amongst other products.

Reduction of Salmonella Enteritidis PT30 and E. faecium NRRL B-2354 (laboratory ovens, 2007)

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0 1 2 3 4 5 6 7 8

Redu

ctio

n (lo

g CF

U/g

)

265 310 295 280

S E 24

S E 15

S E 10

S E 25

S E 15

S E 45

S E 6

S E 12

Deibel - Salmonella

NFL - Enterococcus

NFL - Salmonella

Deibel - Enterococcus

Time (min) Temperature (°F)

PresenterPresentation NotesWater activity and moisture content not measured post-inoculation

Thermal treatment: 15 min, 280°F; Fisher Ovens

Influence of storage time at 4°C on thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT30

0.0

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Redu

ctio

n (lo

g CF

U/g

)

Week(s) of storage at 4°C/39°F 0 10 8 6 4 2 1 12 0 0

UCD 2012; (625G) NFL

2007; (851F)

Deibel 2007; (851F)

Salmonella significantly greater reduction at all time points No

sig.diff No

sig.diff

0

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4

1 4 8 12

Redu

ctio

n (lo

g CF

U/g

)

Weeks

Gravity Feed Oven Forced Air Oven

15 min, 280°F in gravity feed (Fisher 625 G) or forced air (Fisher 725 F) oven

Ovens are not equal - Enterococcus faecium NRRL B-2354

Hot oil 121°C/250°F

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9

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Log

CFU

/g

Treatment time (min)

Salmonella Enteriditis PT 30

E. faecium NRRL B-2354

Kaur and Harris, unpublished

PresenterPresentation NotesE. faecium NRRL B-2354 has been used for many years as a successful surrogate organism for Salmonella

In this example, work done by the lab of Dr. Linda Harris, *describe graph and medium and oil*, this thermal tolerance curve measures the effect this treatment has on each organism, almost the same, successful surrogate for this medium and this method of pasteurization

E. faecium NRRL B-2354 a useful surrogate in moist air heating

• Conclusions • NRRL B-2354 is a conservative

surrogate for SE PT30 in the targeted reduction range of 4 to 5 log

• a potentially useful surrogate

for steam heating

Jeong et al. 2011. Journal of Food Protection 74(4):603.

Log

redu

ctio

n, E

. fae

cium

NR

RL

2354

Comparisons to other pathogens

0

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3

4

Red

uctio

n (L

og C

FU/g

)

A BC

C C

D

B B B B B B

AB

Reductions during desiccation on almonds

Moussavi and Harris, unpublished

PresenterPresentation NotesMean ± SD reduction of E. faecium, Salmonella Enteritidis PT30 and different �E. coli O157:H7 or L. monocytogenes strains on inoculated almonds before exposure to heat treatments (n = 6). Columns denoted with different letters are significantly different (P < 0.05).

Reduction of in hot water (176°F/80°C) E. faecium NRRL B-2354, E. coli O157:H7, Listeria monocytogenes, and S. Enteritidis PT30

01234567

0 0.5 1 2 3Treatment time (min)


Reduction in oil (250°F/121°C) Listeria monocytogenes, E. coli O157:H7, E. faecium NRRL B-2354 and S. Enteritidis PT30 on almonds

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0 30 60 90 120Time (s)


E. faecium NRRL B-2354 is an appropriate surrogate for Salmonella in almonds • Jeong et al., 2011 Journal of Food Protection

74(4):603 – Moist air or steam processes (ambient or vacuum)

• NFL and Deibel Laboratories, 2007 – Dry-heat processes:

• dry roast, • brine and pre-wet dry roast, • dry roast flavoring, • dry plasticizing

Jeong et al. 2011.

ABC, 2014

http://www.almondboard.com/Handlers/Documents/Enterococcus-Validation-Guidelines.pdf

Acknowledgements

• Gordon Davidson

• Lillian Khan

• Harbir Kaur

• Mahta Moussavi

• Chris Theofel

Funding Provided By:

Maria Marco, University of California, Davis

Biosafety of Enterococcus faecium NRRL B-2354

Maria L Marco

Associate Professor Department of Food Science & Technology

University of California, Davis

E. faecium: impact on human health

Used in some food fermentations

Sold as a probiotic (humans and animals)

Antibiotic-resistant, opportunistic pathogen

Each bacterial strain is genetically distinct Thermal tolerance

Antibiotic resistance Virulence

Strain specificity of E. faecium

78% genetically related 70% genetically related

Staley 1997 Cur Op Biotechnol

How to evaluate bacterial strains for safety

Phenotype Genotype

Complete genome: Chromosome (2.6 Mbp) One plasmid (214 kbp) Genome of E. faecium ATCC 8459 is over 99% identical

Genome sequence of NRRL B-2354

Kopit et al (2014) Appl Environ Microbiol Kim et al (2013) Appl Environ Microbiol

Clinical Community

Hierarchical clustering based on 128 E. faecium genomes

NRRL B-2354 is a “community” strain

NRRL B-2354 is distinct from other strains

• 45 genes not found in >100 other E. faecium genomes. Contained in loci (SPs)

• Lacks cas –CRISPRs for viral recognition

• Fewer mobile elements, virulence genes, and genes for antibiotic resistance than clinical strains

Cultures grown on tryptic soy agar with 5% defibrinated horse blood

Negative control: E. coli DH5α

Positive Control: E. faecalis ATCC

29212

cyl genes are not present in NRRL B-2354

NRRL B-2354 does not produce hemolysin NRRL B-2354

PresenterPresentation NotesThinking back to the EFSA document, the next step was Antibiotic sensitivity

acm E. faecium NRRL B-2354

acm E. faecium TX0082 – Clinical

E. faecium NRRL B-2354 contains: - scm (collagen I and IV adhesin) - sagA (fibrinogen, fibronectin, collagen I adhesin) - acm (collagen I adhesion)

100% coverage, 99% nucleotide identity

Virulence Factor: adhesion to human proteins

PresenterPresentation Notes1.1 kbp insertion at the 5’ end when compared to clinical strain and …

NRRL B-2354 does not bind collagen

Adherent cells were stained with crystal violet, positive control: clinical strain E. faecium TX0082, Tukey’s HSD, P < 0.05

n=3

Clinical

PresenterPresentation NotesBound to collagen type I, adhered cells stained with crystal violet and measured absorbance at 595nmResults agree with genome, non-functional, adhesion barely above background levelsWe also looked at the production of a hemolysin, a trait commonly seen in clinical strains

NRRL B-2354 does not form biofilms

2000 bp

NRRL B-2354 does not make pili

Transmission electron microscopy B-2354 contains pil operon and efaA but lacks ebp genes

NRRL B-2354 TX0082

Minimum inhibitory concentration, BD Phoenix system, UC Davis Medical Center

NRRL B-2354 is sensitive to antibiotics

PresenterPresentation NotesAs you can see, all were sensitive, but wanted to call attention to

Aminoglycosides Binds to 30S (some to 50S) ribosomal subunit, inhibit protein synthesis Cephalosporins (first generation) Similar to beta-lactams, inhibit peptidoglycan synthesis Cephalosporins (second generation) Similar to beta-lactams, inhibit peptidoglycan synthesis Glycopeptides Inhibit peptidoglycan synthesis Macrolides Binds to 50S ribosomal subunit, inhibit protein synthesis Nitrofurans Damages bacterial DNA (reduced form is very reactive) Penicillins (and combinations) Similar to beta-lactams, inhibit peptidoglycan synthesis Quinolones Inhibit DNA replication/transcription by inhibiting DNA gyrase Tetracyclines Bind to 30S ribosomal subunit and inhibit binding of aminoacyl-tRNA to mRNA-ribosome complex

Incubated in physiological saline, pH 2.4

Clinical

Limit of detection

NRRL B-2354 is acid tolerant

NRRL B-2354 is thermal tolerant

50 °C

60 °C

Incubated in physiological saline, LOD = 20 CFU/mL

Clinical

Limit of detection

Regulatory advice: E. faecium strain biosafety

European Food Safety Authority (2012)* Absence of esp, hyl, IS16 (marker of hospital-

associated strains) Sensitive to ampicillin

* Safety evaluation of E. faecium strains intended as additives in animal feed

Conclusions

E. faecium NRRL B-2354 (ATCC 8459) lacks phenotypic and genotype features of clinical strains According to our assessments E. faecium NRRL B-2354 (ATCC 8459) is safe for use in thermal process validation tests

PresenterPresentation NotesBased on our findings and combined with the long history of safe use of this organism, this strain could continue to be used as a surrogate for the validation of thermal processing.

Acknowledgements Mary Moore Sandra Appelt Zach Bendiks Ben Golomb Xiaochen Yin Dustin Heeney Annabelle Yu Zhengyao Xue Brendan McCarthy-Sinclair Yanin Srisengfa Irene Yim Zach Quart Elissa Goldberg

Lauren Kopit

Eun Bae Kim

Collaborator: Linda Harris, UC Davis

Shirin Abd & Carrie Ferstl The National Food Laboratory

Validation of Thermal Processes Applied to Almonds Continuing Investigations to Support the Use of E. faecium NRRL B-2354 as a Surrogate

December 8, 2015

Overview

• Review criteria to select a surrogate organism • Review ABC guidelines for preparation and evaluation of surrogate heat

resistance • Summary of ongoing research at The National Food Laboratory to evaluate the

appropriateness of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella Enteritidis PT30 on almonds

Extended storage of surrogate-inoculated almonds Thermal resistance of surrogate on different almond types

PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?

Selection of a Surrogate

• Identify the target organism Based upon food safety plan Outbreaks in similar ingredients/products

• Food matrix composition/parameters Worst case scenario

• Thermal process parameters • Select potential surrogate(s) Non-pathogenic Stable Easy to grow to high levels Similar thermal resistance to the target organism

How is the appropriateness of a surrogate organism determined?

Enterococcus faecium

Pediococcus acidilactici


Selection of a Surrogate

• Understand the thermal resistance of target organisms in the selected food matrix

• Understand the thermal resistance of the surrogate organism to the same conditions

Main goals when evaluating a surrogate:

Surrogate organism should demonstrate similar or greater

resistance to heat


Selection of a Surrogate for Almonds

Consideration Identify the target organism

Food matrix

Process parameters

Selection of a surrogate

Almonds S. Enteritidis PT30

Whole kernel almonds

Dry roasting



ABC Surrogate Evaluation Guidelines

ABC published a protocol, “Guidelines for Using Enterococcus faecium NRRL B-2354 as a Surrogate Microorganism in Almond Process Validation”, to provide guidance to the industry on the following points: Preparation of E. faecium NRRL B-2354 culture for inoculation

Inoculation of almonds with the prepared E. faecium NRRL B-2354 culture

Heat resistance verification for E. faecium NRRL B-2354 on the inoculated almonds

Storage of the inoculated almonds prior to validation testing

Can the protocols be refined further to provide more flexibility in their use for the validation of almond processes?


Reevaluate ABC Surrogate Evaluation Guidelines

Reevaluate the following limitations of the protocol:

The almonds inoculated with the surrogate should be used within two weeks of preparation and stored at 38-40°F (refrigeration temperatures). Can inoculated almonds be stored beyond 14 days without sacrificing heat

resistance? Do inoculated almonds need to be stored at refrigeration temperatures?

The heat resistance of E. faecium NRRL B-2354 and its correlation to S. Enteritidis PT30 was completed using whole intact almonds. Can whole kernel inoculated almonds be used in plant validation studies for other

almond types?

Thermal Resistance of Surrogate on Almonds -Recent Studies at The NFL

Study Design Overview

S. Enteritidis PT30 E. faecium NRRL B-2354

Refrigeration Room temperature

Up to 60 days Forced Air Oven

280°F for 15 minutes

Calculate the log reduction to evaluate the thermal resistance of organisms on almonds

PresenterPresentation NotesTo develop a study protocol to evaluate the following:Stability over the extended shelf life of inoculated almonds (60 days)Storage temperatures: 4°C and 25°CComparative heat resistance of E. faecium and SEPT30Forced Air Oven vs. Gravity Oven

Extended Storage of Surrogate-Inoculated Almonds

The current shelf life of almonds inoculated with E. faecium NRRL B-2354 can be extended from 14 to 60 days when stored at 4°C.


E. faecium NRRL B-2354 is an appropriate surrogate for S. Enteritidis PT30 when the inoculated almonds are stored at 4°C.

PresenterPresentation NotesThe heat resistance of E. faecium NRRL B-2354 on inoculated almonds is similar to heat resistance of S. Enteritidis PT30 when the inoculated almonds are stored at 4°C, but not at 25°C.


The almonds inoculated with the surrogate should be used within two weeks of preparation and stored at 38-40°F (refrigeration temperatures). Can inoculated almonds be stored beyond 14 days without sacrificing heat

resistance? Do inoculated almonds need to be stored at refrigeration temperatures?

Thermal Resistance of Surrogate on Different Almond Types

Natural Dice 12-8 Natural Split

NonPl 27/30 (Nonpareil Whole Kernels without chip of scratch

27/30)

Monterey Whole/Broken

(Monterey natural whole and broken,

36aos)


Almond type has an impact on the heat resistance of E. faecium NRRL B-2354
PresenterPresentation NotesHeat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 at each sampling time was significantly different (p


Heat resistance of S. Enteritidis PT30 is comparable to E. faecium NRRL B-2354 on all four almond types.
PresenterPresentation NotesHeat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 at each sampling time was significantly different (p


The current shelf life of almonds inoculated with the surrogate can be extended beyond 14 days at refrigeration temperature.

0.0

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1.5

2.0

2.5

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3.5

4.0

Log R

educ

tion

E. faecium NRRL B-2354 Day 0Day 14

Day 28

A

AA

AA

AA

AB

B

A A

PresenterPresentation NotesThe heat resistance of E. faecium NRRL B-2354 and S. Enteritidis PT30 on Natural Dice 12-8, Natural Split, and NonPI 27/30 almonds did not change over 28 days of storage at 4°C.

Chart1

Natural Dice 12-8Natural Dice 12-80.3956864160.20161765080.20161765080.22902434340.22902434340.33455496440.3345549644

Natural SplitNatural Split1.27520857870.11526738630.11526738630.34550857540.34550857540.55142445370.5514244537

NonPl 27/30NonPl 27/302.27857056050.59932831210.59932831210.29521903010.29521903011.15645049361.1564504936

Monterey BrokenMonterey Broken2.36511999140.61679869110.61679869110.55410950320.55410950321.08797662561.0879766256

A

A

A

A

A

A

A

A

B

B

A

A

Day 0

Day 14

Day 28

Log Reduction


0.2927502849

0.4537626428

1.0315400027

0.885367817

2.0118809622

2.1486092375

1.4499724326

1.0817828768

Natural Dice 12-8

Natural Dice 12/8

Trial 1Trial 2Trial 3

Day 0Day 14Day 28Day 0Day 14Day 28Day 0Day 14Day 28

UninoculatedSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faecium

AwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoisture

0.46030.44460.43130.45680.47860.47650.4839

0.45580.4241

0.45020.4277

Trial 1

E. faeciumSalmonella

wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevLog reductionwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdevLog reduction

92117104.51045000008.027.894045084564560000008.668.67

516256.5565000007.754405364884880000008.69

Day 0Day 0

Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7

564249490000007.697.720.10505552.55250000008.728.720.01

563143.5435000007.64564550.55050000008.70

696567670000007.83624553.55350000008.73

Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6

212190201201000007.307.250.070.420.4775657070000006.856.850.171.871.86

112181146.5146500007.170.550.07100116108108000007.031.680.17

195184189.5189500007.280.44524548.548500006.692.03

Day 14Day 14


1101021061060000008.038.070.054042414100000008.618.650.03

126105115.51155000008.06464344.54450000008.65

1501201351350000008.135046484800000008.68


492939390000007.597.750.230.480.32292828.5285000007.457.430.021.191.21

100109104.51045000008.020.050.23262525.5255000007.411.240.02

553344440000007.640.43272827.5275000007.441.21

Day 28Day 28


919894.5945000007.987.580.55414241.54150000008.628.600.07

528166.5665000007.82464545.54550000008.66

90909090000006.953234333300000008.52


110809595000006.987.100.140.610.48148128138138000007.147.480.301.461.12

180180180180000007.260.330.14335443.5435000007.640.960.30

130108119119000007.080.51464646460000007.660.94

Trial 2


wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev

685159.5595000007.777.80485149.54950000008.698.64

726066660000007.824236393900000008.59

Day 0Day 0


265138.5385000007.597.560.037351626200000008.798.620.17

403437370000007.573545404000000008.60

373033.5335000007.532929292900000008.46


403035350000007.547.500.110.020.0635454040000006.606.780.172.021.84

247234240.5240500007.380.180.11112668989000006.951.670.17

443238380000007.58-0.0269556262000006.791.83

Day 14Day 14


395547470000007.677.770.08585958.55850000008.778.780.07

508668680000007.837072717100000008.85

685762.5625000007.80436051.55150000008.71


302728.5285000007.457.370.080.310.401011071041040000008.027.870.230.760.91

241203222222000007.350.420.08453439.5395000007.601.180.23

200195197.5197500007.300.471157896.5965000007.980.79

Day 28Day 28


252525250000007.407.430.04144157150.51505000008.188.140.06

183526.5265000007.421481481481480000008.17

352530300000007.48127112119.51195000008.08


354439.5395000007.607.420.24-0.160.01133165149149000007.177.430.390.970.71

133145139139000007.140.290.24846876760000007.880.260.39

254133330000007.52-0.09165182173.5173500007.240.90

Trial 3



9310699.59950000009.009.031664218419241924000000010.2810.15

13210411811800000009.071056105610561056000000010.02

Day 0Day 0


585456560000007.757.720.042737323200000008.518.360.20

555454.5545000007.741351371361360000008.13

613347470000007.672826272700000008.43


189172180.5180500007.267.370.110.460.35475450.5505000007.707.640.090.650.72

259218238.5238500007.380.340.11445047470000007.670.680.09

352429.5295000007.470.25373134340000007.530.83

Day 14Day 14


619276.5765000007.887.880.00523342.54250000008.638.630.01

000.00463741.54150000008.62

000.004145434300000008.63


78144111111000007.057.250.260.840.64694456.5565000007.757.680.100.870.95

151133142142000007.150.730.26564852520000007.720.910.10

432634.5345000007.540.35353937370000007.571.06

Day 28Day 28


708075750000007.887.770.09315844.54450000008.658.730.08

307251510000007.715375646400000008.81

476053.5535000007.73555454.55450000008.74


175122148.5148500007.177.080.080.600.6912099109.51095000008.047.820.270.690.91

96111103.5103500007.010.760.08273933330000007.521.210.27

120103111.5111500007.050.72808281810000007.910.82

Natural Splits

Natural Splits





0.60660.57630.57150.59570.58360.58790.5827

0.57090.5751

0.57360.5733

Trial 1



737674.5745000007.877.963864904384380000008.648.67

116103109.51095000008.04614401507.55075000008.71

Day 0Day 0


796974740000007.877.630.21110109109.51095000008.048.380.30

352731310000007.494426353500000008.54

392833.5335000007.534331373700000008.57


343333.533500006.536.550.031.101.07282727.5275000007.447.420.230.940.97

43333838000006.581.050.03149152150.5150500007.181.210.23

44283636000006.561.07483843430000007.630.75

Day 14Day 14


1137091.5915000007.967.900.08424141.54150000008.628.470.13

1007587.5875000007.943119252500000008.40

705964.5645000007.81242524.52450000008.39


867580.580500006.917.080.391.000.82404040400000007.607.241.000.871.23

422533.5335000007.530.380.39120841021020000008.010.461.00

86446565000006.811.0912014013013000006.112.35

Day 28Day 28


703653530000007.727.800.14202924.52450000008.398.420.05

969093930000007.972525252500000008.40

663751.5515000007.71253630.53050000008.48


19020019519500006.296.610.391.511.19303733.5335000007.537.360.240.901.07

114109111.5111500007.050.750.391212120000007.081.340.24

342730.530500006.481.32293029.5295000007.470.95

Trial 2



674656.5565000007.757.784244434300000008.638.66

706065650000007.81504949.54950000008.69

Day 0Day 0


413739390000007.597.650.095545505000000008.708.700.02

473641.5415000007.62535252.55250000008.72

674757570000007.764850494900000008.69


57595858000006.766.660.140.890.99303130.5305000007.487.630.181.221.07

283531.531500006.501.160.14607567.5675000007.830.870.18

525553.553500006.730.93483039390000007.591.11

Day 14Day 14


333735350000007.547.600.09180199189.51895000008.288.150.14

416151510000007.711601341471470000008.17

432936360000007.5610499101.51015000008.01


1126588.588500006.956.980.160.660.62574852.5525000007.727.810.110.430.34

128158143143000007.160.450.16744459590000007.770.380.11

716869.569500006.840.76948087870000007.940.21

Day 28Day 28


145159152152000007.187.330.14113134123.51235000008.098.160.07

212250231231000007.361332031681680000008.23

292728280000007.45153138145.51455000008.16


18019818918900006.286.580.271.050.7592115103.5103500007.017.110.091.151.05

46444545000006.650.680.27190130160160000007.200.960.09

52766464000006.810.52130121125.5125500007.101.06

Trial 3



10666868600000008.938.96960206415121512000000010.1810.21

9793959500000008.981888163217601760000000010.25

Day 0Day 0


537765650000007.817.800.111351311331330000008.128.080.07

798079.5795000007.90119134126.51265000008.10

435348480000007.68971031001000000008.00


73837878000006.896.770.160.911.03178170174174000007.247.160.110.830.91

56806868000006.830.970.1612694110110000007.041.030.11

374038.538500006.591.21175144159.5159500007.200.87

Day 14Day 14


706567.5675000007.837.630.20363736.53650000008.568.600.04

292627.5275000007.444337404000000008.60

592542420000007.62474043.54350000008.64


303934.534500006.546.420.181.091.2197899393000006.977.130.291.631.48

173154163.516350006.211.420.18681088888000006.941.660.29

38263232000006.511.13322629290000007.461.14

Day 28Day 28


781401091090000008.048.000.04303532.53250000008.518.600.08

5712591910000007.963260464600000008.66

9310699.5995000008.004242424200000008.62


16818617717700006.256.120.141.751.88424543.5435000007.647.630.080.960.97

7511092.59250005.972.030.14425850500000007.700.900.08

12015813913900006.141.86343635350000007.541.06

NonP XI 2730 FPO

NonP XI 27/30 FPO





0.53690.55430.53930.54270.54370.53040.5468

0.55820.5534

0.556250.5464

Trial 1



314437.5375000007.577.652192312252250000008.358.40

466053530000007.722533012772770000008.44

Day 0Day 0


73636868000006.836.860.02444142.5425000007.637.730.11

88627575000006.88766570.5705000007.85

76747575000006.88674053.5535000007.73


7212096960004.984.910.361.881.955969646400005.815.920.271.931.82

180155167.51675005.221.640.36174159166.516650006.221.510.27

372832.5325004.512.35624553.55350005.732.01

Day 14Day 14


93758484000006.927.050.13617668.5685000007.847.740.09

101130115.5115500007.06454746460000007.66

160140150150000007.18406954.5545000007.74


160181170.51705005.235.060.281.821.995242474700005.675.780.322.071.96

2101241671670005.221.830.2810617013813800006.141.610.32

496054.5545004.742.324424343400005.532.21

Day 28Day 28


1049599.599500007.006.970.12847680800000007.907.740.25

90466868000006.83263128.5285000007.45

130108119119000007.08865671710000007.85


9411210310300006.015.880.180.961.091845812112100006.085.810.241.651.92

4254484800005.681.290.18505351.55150005.712.020.24

809989.58950005.951.024840444400005.642.09

Trial 2



432835.5355000007.557.6423695165.51655000008.228.11

505954.5545000007.74901101001000000008.00

Day 0Day 0


64686666000006.826.720.15292024.5245000007.397.450.09

30403535000006.54173425.5255000007.41

84426363000006.80333835.5355000007.55


183210196.51965005.295.270.351.431.45374440.54050005.615.410.371.842.04

5262764014010005.601.120.354739434300005.631.820.37

719181810004.911.818910597970004.992.46

Day 14Day 14


14197119119000007.087.030.04534951510000007.717.720.05

81134107.5107500007.03563847470000007.67

1099099.599500007.00675058.5585000007.77


2221782002000005.305.030.241.732.018561737300005.865.920.181.851.79

1175385850004.932.110.245765616100005.791.930.18

865872720004.862.1814811613213200006.121.60

Day 28Day 28


253.535000006.546.690.2111293102.5102500007.017.080.08

71440000006.60138155146.5146500007.17

988.585000006.9397128112.5112500007.05


799084.5845004.934.640.261.762.05433740400004.604.690.093.113.03

143725.5255004.412.290.26455148480004.683.030.09

542539.5395004.602.10566560.5605004.782.93

Trial 3



647569.56950000008.848.99380423401.540150000009.609.51

8019413713700000009.14241276258.525850000009.41

Day 0Day 0


353635.535500006.556.650.19583044440000007.647.710.06

254233.533500006.53575154540000007.73

271197373000006.86536458.5585000007.77


193276234.5234504.374.020.382.282.6316314115215200006.186.050.121.531.66

38464242003.623.020.388811210010000006.001.710.12

114116115115004.062.5911670939300005.971.75

Day 14Day 14


25889173.5173500007.247.010.21392833.5335000007.537.520.01

65656565000006.81293632.5325000007.51

118749696000006.98392733330000007.52


283933.5335004.534.560.092.492.456094777700005.896.120.271.631.39

514146460004.662.350.0910013211611600006.061.450.27

253831.5315004.502.51282526.526500006.421.10

Day 28Day 28


126148137137000007.147.160.077210086860000007.937.940.02

175166170.5170500007.23699582820000007.91

107148127.5127500007.11839991910000007.96


20282424003.383.310.083.633.701109610310300006.016.100.091.921.83

10322121003.323.690.08144171157.515750006.201.740.09

15191717003.233.7812512912712700006.101.83

Monterey whole broken

Monterey natural whole and broken, 36aos





0.53830.57180.56600.54290.56710.54360.5605

0.56690.5571

0.569350.56155

Trial 1



206205205.5205500007.317.522482782632630000008.428.50

574852.5525000007.72372383377.53775000008.58

Day 0Day 0


233179206206000007.317.060.28635760600000007.787.890.09

58565757000006.76879289.5895000007.95

144120132132000007.12927885850000007.93


11396104.510450006.026.010.181.041.058250666600005.825.880.302.072.01

150161155.515550006.190.870.1817015216116100006.211.680.30

7165686800005.831.234042414100005.612.27

Day 14Day 14


523342.542500006.636.620.01999999990000008.007.970.02

57254141000006.611018894.5945000007.98

562540.540500006.611007989.5895000007.95


1607611811800006.076.050.430.540.5710989999900006.006.520.831.981.46

433840.54050005.611.010.43121120120.512050006.081.890.83

362329.529500006.470.15273330300000007.480.50

Day 28Day 28


452836.5365000007.567.600.07966480800000007.908.000.08

574149490000007.69110115112.51125000008.05

324136.5365000007.561101101101100000008.04


43293636000006.566.480.071.051.1312110111111100006.056.160.211.951.83

25312828000006.451.160.071309011011000006.041.960.21

242926.526500006.421.18262525.525500006.411.59

Trial 2



555856.5565000007.757.8111899108.51085000008.048.04

856173730000007.861111151131130000008.05

Day 0Day 0


74204139139000007.147.170.03469369.5695000007.847.780.05

134169151.5151500007.18506457570000007.76

137175156156000007.19436855.5555000007.74


99132115.511550006.066.080.311.111.099710098.59850005.995.920.131.791.86

497059.55950005.771.400.315565606000005.782.000.13

162341251.525150006.400.771079299.59950006.001.78

Day 14Day 14


190263226.5226500007.367.370.061442001721720000008.248.030.19

169245207207000007.32130721011010000008.00

253027.5275000007.44796371710000007.85


13812413113100006.126.370.231.251.00817678.578500006.896.620.241.141.41

322528.528500006.450.920.2336263131000006.491.540.24

254635.535500006.550.8225353030000006.481.55

Day 28Day 28


124127125.5125500007.107.200.10292125250000007.407.460.05

91300195.5195500007.29273430.5305000007.48

170150160160000007.20293331310000007.49


898.585003.934.560.573.272.631468611611600006.066.320.271.391.14

123100111.51115005.052.150.5743374040000006.600.860.27

356952520004.722.4811760020202020000006.301.16

Trial 3



56116868600000008.938.8027325726526500000009.429.47

474445.54550000008.66317334325.532550000009.51

Day 0Day 0


121143132132000007.127.160.04845971.5715000007.857.920.09

160136148148000007.17100109104.51045000008.02

159159159159000007.20797878.5785000007.89


107132119.51195005.084.960.312.092.2122021021521500006.336.390.421.591.53

354540400004.602.560.3166706868000006.831.090.42

189120154.51545005.191.981139110210200006.011.91

Day 14Day 14


163162162.5162500007.217.420.21324136.53650000008.568.590.04

252726260000007.414838434300000008.63

513342420000007.62334036.53650000008.56


3789636300005.805.730.201.621.68282928.528500006.456.620.272.131.96

787777.57750005.891.530.20362530.530500006.482.100.27

353032.53250005.511.9096748585000006.931.66

Day 28Day 28


18083131.51315000008.127.920.18167166166.51665000008.228.220.08

556158580000007.761981901941940000008.29

807577.5775000007.891381361371370000008.14


40625151003.714.590.774.223.3355816868000006.836.590.391.381.63

828684840004.923.000.7755696262000006.791.420.39

1181601391390005.142.78165110137.513750006.142.08

Summary

Enterococcus faecium NRRL B-2354Salmonella Enteritidis PT30

Natural Dice 12-8Natural SplitNonPl 27/30Monterey Whole BrokenNatural Dice 12-8Natural SplitNonPl 27/30Monterey Whole BrokenEntrococcusSalmonella PT30

Day 0Trial 1Rep 10.421.101.881.04Day 0Trial 1Rep 11.870.941.932.07Day 0Day 14Day 28Day 0Day 14Day 28EntrococcusSalmonella PT30

Rep 20.551.051.640.87Rep 21.681.211.511.68Natural Dice 12-80.290.450.401.471.020.91Day 0Day 14Day 0Day 14

Rep 30.441.072.351.23Rep 32.030.752.012.27Natural Split1.030.891.280.981.011.03Natural Dice 12-80.470.321.861.21

Avg0.471.071.951.05Avg1.860.971.822.01NonPl 27/302.012.152.281.841.722.26Natural Split1.070.820.971.23

Stdev0.070.030.360.18stdev0.170.230.270.30Monterey Broken1.451.082.371.801.611.53NonPl 27/301.951.991.821.96

Trial 2Rep 10.020.891.431.11Trial 2Rep 12.021.221.841.79Monterey1.050.572.011.46

Rep 20.181.161.121.40Rep 21.670.871.822.00Stdev0.200.230.330.580.190.33

Rep 3-0.020.931.810.77Rep 31.831.112.461.780.120.350.550.170.740.140.070.230.170.02

Avg0.060.991.451.09Avg1.841.072.041.860.600.301.160.280.340.590.030.390.231

Stdev0.110.140.350.31Stdev0.170.180.370.130.620.551.090.340.520.400.360.280.270.32

Trial 3Rep 10.460.912.282.09Trial 3Rep 10.650.831.531.590.180.430.30.83

Rep 20.340.973.022.56Rep 20.681.031.711.09

Rep 30.251.212.591.98Rep 30.830.871.751.91

Avg0.351.032.632.21Avg0.720.911.661.53E. faecium NRRL B-2354S. Enteritidis PT30EntrococcusSalmonella PT30

0.110.160.380.31stdev0.090.110.120.42Day 0Day 0Day 14Day 14

Avg of 9 samples0.291.032.011.45Avg of 9 samples1.470.981.841.80

Stdev of 9 samples0.200.120.600.62Stdev of 9 samples0.580.170.280.34

Day 14Trial 1Rep 10.481.001.820.54Day 14Trial 1Rep 11.190.872.071.98Natural Dice 12-80.471.860.321.21

Rep 20.050.381.831.01Rep 21.240.461.611.89Natural Split1.070.970.821.23

Rep 30.431.092.320.15Rep 31.212.352.210.50NonPl 27/301.951.821.991.96

Avg0.320.821.990.57Avg1.211.231.961.46Monterey1.052.010.571.46

Stdev0.230.390.280.43stdev0.021.000.320.83

Trial 2Rep 10.310.661.731.25Trial 2Rep 10.760.431.851.140.070.170.230.02

Rep 20.420.452.110.92Rep 21.180.381.931.540.030.230.391

Rep 30.470.762.180.82Rep 30.790.211.601.550.360.270.280.32

Avg0.400.622.011.00Avg0.910.341.791.410.180.30.430.83

0.080.160.240.230.230.110.180.24

Trial 3Rep 10.841.092.491.62Trial 3Rep 10.871.631.632.13

Rep 20.731.422.351.53Rep 20.911.661.452.10

Rep 30.351.132.511.90Rep 31.061.141.101.66

Avg0.641.212.451.68Avg0.951.481.391.96

0.260.180.090.200.100.290.270.27



Day 28Trial 1Rep 10.6061.510.961.05Day 28Trial 1Rep 11.460.901.651.95

Rep 20.3290.751.291.16Rep 20.961.342.021.96

Rep 30.5091.321.021.18Rep 30.940.952.091.59E. faecium NRRL B-2354S. Enteritidis PT30EntrococcusSalmonella PT30EntrococcusSalmonella PT30

Avg0.481.191.091.13Avg1.121.071.921.83Day 0Day 0Day 14Day 14Day 28Day 2800.291.032.011.45

0.140.390.180.070.300.240.240.21140.450.892.151.08

Trial 2Rep 1-0.161.051.763.27Trial 2Rep 10.971.153.111.39280.401.282.282.37

Rep 20.290.682.292.15Rep 20.260.963.030.86Natural Dice 12-80.291.470.451.020.400.91

Rep 3-0.090.522.102.48Rep 30.901.062.931.16Natural Split1.030.980.891.011.281.03

Avg0.010.752.052.63Avg0.711.053.031.14NonPl 27/302.011.842.151.722.282.26

0.240.270.260.570.390.090.090.27Monterey Broken1.451.801.081.612.371.53

Trial 3Rep 10.601.753.634.22Trial 3Rep 10.690.961.921.38

Rep 20.762.033.693.00Rep 21.210.901.741.42Stdev0.200.580.230.190.330.33

Rep 30.721.863.782.78Rep 30.821.061.832.080.120.170.350.740.550.14

Avg0.691.883.703.33Avg0.910.971.831.630.600.280.300.341.160.59

0.080.140.080.770.270.080.090.390.620.340.550.521.090.40




Natural Dice 12-8Natural SplitNonPl 27/30Monterey Broken

Day 00.291.032.011.45

Day 140.450.892.151.08

Day 280.401.282.282.37

Stdev0.200.120.600.62

0.230.350.300.55E. faecium NRRL B-2354

Natural Dice 12-8Natural SplitNonPl 27/30Monterey Broken

0.330.551.161.09Day 00.471.071.951.05

Day 140.320.821.990.57

S. Enteritidis PT30

Natural Dice 12-8Natural SplitNonPl 27/30Monterey0.070.030.360.18

Day 01.470.981.841.80

0.230.390.280.43

Day 141.021.011.721.61

S. Enteritidis PT30

Day 280.911.032.261.53Natural Dice 12-8Natural SplitNonPl 27/30Monterey

Day 01.860.971.822.01

Stdev0.580.170.280.34Day 141.211.231.961.46

0.190.740.340.520.170.230.270.3

0.330.140.590.400.021.00.320.83

Summary

0.07304669040.07304669040.23348658220.2334865822

0.02746130720.02746130720.38713210470.3871321047

0.36213163130.36213163130.28341311550.2834131155

0.17965615960.17965615960.4316104540.431610454

Day 0

Day 14

Log Reduction


Summay-Analysis Results

0.170.170.020.02

0.230.2311

0.270.270.320.32

0.30.30.830.83

Day 0

Day 14

Log Reduction

S. Enteritidis PT30

0.07304669040.07304669040.170.17

0.02746130720.02746130720.230.23

0.36213163130.36213163130.270.27

0.17965615960.17965615960.30.3


S. Enteritidis PT30

Log Reduction

Day 0

0.23348658220.23348658220.020.02

0.38713210470.387132104711

0.28341311550.28341311550.320.32

0.4316104540.4316104540.830.83


S. Enteritidis PT30

Log Reduction

Day 14

00.07304669040.07304669040.02746130720.02746130720.36213163130.36213163130.17965615960.1796561596

��

0.23348658220.23348658220.38713210470.38713210470.28341311550.28341311550.4316104540.431610454

Natural Dice 12-8

Natural Split

NonPl 27/30

Monterey Broken

Log Reduction


0.170.170.230.230.270.270.30.3

��

0.020.02110.320.320.830.83

Natural Dice 12-8

Natural Split

NonPl 27/30

Monterey Broken

Log Reduction

S. Enteritidis PT30

0.20161765080.20161765080.22902434340.22902434340.33455496440.3345549644

0.11526738630.11526738630.34550857540.34550857540.55142445370.5514244537

0.59932831210.59932831210.29521903010.29521903011.15645049361.1564504936

0.61679869110.61679869110.55410950320.55410950321.08797662561.0879766256

A

A

A

A

A

A

A

A

A

A

A

B

Day 0

Day 14

Day 28

Log Reduction


0.57967493250.57967493250.19160793590.19160793590.33079175920.3307917592

0.1682274030.1682274030.73523481560.73523481560.14314909540.1431490954

0.28477431260.28477431260.34045558490.34045558490.59182500940.5918250094

0.33769661260.33769661260.52482660330.52482660330.40468609290.4046860929

A

A

A

A

A

A

A

A

A

A

A

A

Day 0

Day 14

Day 28

Log Reduction

S. Enteritidis PT30

0.20161765080.20161765080.57967493250.5796749325

0.11526738630.11526738630.1682274030.168227403

0.59932831210.59932831210.28477431260.2847743126

0.61679869110.61679869110.33769661260.3376966126

A

A

A

A

A

A

A

A


S. Enteritidis PT30

Log Reduction

Day 0

0.22902434340.22902434340.19160793590.1916079359

0.34550857540.34550857540.73523481560.7352348156

0.29521903010.29521903010.34045558490.3404555849

0.55410950320.55410950320.52482660330.5248266033

A

A

A

A

A

A

A

A


S. Enteritidis PT30

Log Reduction

Day 14

0.33455496440.33455496440.33079175920.3307917592

0.55142445370.55142445370.14314909540.1431490954

1.15645049361.15645049360.59182500940.5918250094

1.08797662561.08797662560.40468609290.4046860929

A

A

A

A

A

A

A

A


S. Enteritidis PT30

Log Reduction

Day 28

Natural Dice 12-8

Natural Split

NonPl 27/30

Monterey Broken

00.20161765080.20161765080.11526738630.11526738630.59932831210.59932831210.61679869110.6167986911

��

0.22902434340.22902434340.34550857540.34550857540.29521903010.29521903010.55410950320.5541095032

��

0.33455496440.33455496440.55142445370.55142445371.15645049361.15645049361.08797662561.0879766256

A

B

C

D

A

B

B

C

A

A

B

C

Natural Dice 12-8

Natural Split

NonPl 27/30

Monterey Broken

Log Reduction


00.57967493250.57967493250.1682274030.1682274030.28477431260.28477431260.33769661260.3376966126

��

0.19160793590.19160793590.73523481560.73523481560.34045558490.34045558490.52482660330.5248266033

��

0.33079175920.33079175920.14314909540.14314909540.59182500940.59182500940.40468609290.4046860929

A

A

B

C

A

A

A

A

A

B

C

C

Natural Dice 12-8

Natural Split

NonPl 27/30

Monterey Broken

Log Reduction

S. Enteritidis PT30

EntrococcusDay 0Day 14Day 28Conf. Level

Natural Dice 12-80.290.450.40NSD

Natural Split1.030.891.28NSD

NonPl 27/302.012.152.28NSD

bba

Monterey Broken1.451.082.3795%

Salmonella PT30

Natural Dice 12-81.471.020.91NSD

Natural Split0.981.011.03NSD

NonPl 27/301.841.722.26NSD

Monterey Broken1.801.611.53NSD

E. faecium NRRL B-2354S. Enteritidis PT30

Day 0Day 0Conf. Level

Natural Dice 12-80.291.47NSD

Natural Split1.030.98NSD

NonPl 27/302.011.84NSD

Monterey Broken1.451.80NSD





NonPl 27/302.151.72NSD






NonPl 27/302.282.26NSD


E. faecium NRRL B-2354Natural Dice 12-8Natural SplitNonPl 27/30Monterey BrokenConf. Level

dcab

Day 00.291.032.011.4595%

cbab

Day 140.450.892.151.0895%

cbaa

Day 280.401.282.282.3795%

S. Enteritidis PT30

bcaa

Day 01.470.981.841.8095%

Day 141.021.011.721.61NSD

ccab

Day 280.911.032.261.5395%

Analysis Method: Analysis of Variance

Software used: SPSS

NSD: Not significantly different at the confidence level of 95%.

Mean ratings with different superscripts differ significantly at the 95% confidence level (Duncan's Multiple Comparison Test).


The heat resistance of E. faecium NRRL B-2354 and its correlation to S. Enteritidis PT30 was completed using whole intact almonds. Can whole kernel inoculated almonds be used in plant validation studies for other

almond types?

Conclusions

• Shelf life of almonds (whole kernel) inoculated with E. faecium NRRL B-2354 can be extended from 14 to 60 days when stored at 4°C.

• Almonds inoculated with E. faecium NRRL B-2354 should not be stored at 25°C, or heat resistance will change.

• Shelf life of the Natural Dice 12-8 and Natural Split almonds inoculated with E. faecium NRRL B-2354 can be extended to 28 days at 4°C.

• Heat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 is significantly different depending on almond type.

PresenterPresentation NotesThe inoculated almonds with E. faecium NRRL B-2354 should not be stored at 25°C as the heat resistance of the surrogate will change, and will no longer have comparable resistance to S. Enteritidis PT30.

Contributors

Shirin J. Abd, M.S.

Research Microbiologist, Food Safety [email protected]

925-556-4812

Carrie Ferstl, Ph.D. Director, Food Safety

[email protected] 925-551-4293

Questions?

Use of Enterococcus Faecium NRRL �B-2354 as a SurrogateSpeakersLinda Harris, �University of California, DavisValidating the use of �Enterococcus faecium NRRL B-2354 �for almond processesSurrogates: when appropriate or necessary?Pathogens: when appropriate or necessary?What makes an ideal surrogate?Almond Board Microbial Surrogate Development 2004-2007Potential Surrogates Screened (Dry Heat)E. faecium NRRL B-2354/ATCC 8459Reduction of Salmonella Enteritidis PT30 and E. faecium NRRL B-2354 �(laboratory ovens, 2007)Influence of storage time at 4°C on thermal tolerance of �E. faecium NRRL B-2354 and Salmonella Enteritidis PT30Influence of storage time at 4°C on thermal tolerance of �E. faecium NRRL B-2354 and Salmonella Enteritidis PT30Ovens are not equal - Enterococcus faecium NRRL B-2354 Hot oil 121°C/250°FE. faecium NRRL B-2354 a useful surrogate in moist air heatingComparisons to other pathogensReduction of in hot water (176°F/80°C) E. faecium NRRL B-2354, �E. coli O157:H7, Listeria monocytogenes, and S. Enteritidis PT30Reduction in oil (250°F/121°C) Listeria monocytogenes, E. coli O157:H7, �E. faecium NRRL B-2354 and S. Enteritidis PT30 on almondsE. faecium NRRL B-2354 is an �appropriate surrogate for Salmonella �in almondsAcknowledgementsMaria Marco, �University of California, DavisSlide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Shirin Abd & Carrie Ferstl�The National Food LaboratoryValidation of Thermal Processes Applied to AlmondsOverviewSelection of a SurrogateSelection of a SurrogateSelection of a Surrogate for AlmondsABC Surrogate Evaluation GuidelinesReevaluate ABC Surrogate Evaluation GuidelinesThermal Resistance of Surrogate on Almonds�-Recent Studies at The NFL�Study Design OverviewExtended Storage of Surrogate-Inoculated AlmondsExtended Storage of Surrogate-Inoculated AlmondsExtended Storage of Surrogate-Inoculated AlmondsThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesConclusionsContributorsQuestions?