Pathogen Reduction DialoguePanel 4

May 7, 2002

Characterization and Control of Food Borne Pathogens

John B. Luchansky, Ph.D.

Agricultural Research Service

Eastern Regional Research Center

Microbial Food Safety Research Unit

Characterization and Control of Food Borne Pathogens

John B. Luchansky, Ph.D.

Agricultural Research Service

Eastern Regional Research Center

Microbial Food Safety Research Unit

Input helpful for developing interventions

What is the targeted pathogen or indicator microbe? Where does it reside and how long does it persist or

predominate? How many types are present and at what levels? How does it respond to environmental cues? Where did it come from and where might it end up? What levels and types of the targeted microbe are

tolerable and under what situations?

Research Strategy

Raw Material

Slaughter Fabricate Process

Finished Product

• Chemical (Na Lactate)

• Mechanical (Design change)

Interventions• Physical (Heat)

• Biological (LAB)

Recovery/Characterization• Culture & Antibody (IMS)

• Nucleic Acid (PCR, PFGE)

• Genomics & Proteomics

Modeling• Predictive Microbiology

(PMP, ComBase, CEMMI)

Download atwww.arserrc.gov/mfs/pathogen.htm

Pathogen Modeling Program (PMP)

The PMP is a group of models that estimate the behavior of bacterial pathogens in specific environments

Through a user-friendly interface, information is provided about the effects of environmental factors on growth, toxin production, and inactivation (thermal and non-thermal)

Pathogen Modeling Program (PMP)

• ~5000 downloads per year• Used by ~30% of food industry to design

HACCP systems• PMP-6.1 (release – May ’02) contains:

• growth, survival, inactivation models• dynamic temperature and thermal

inactivation models• reference database• enhanced help functions

ComBase - a relational database ofpredictive microbiology information

GOALS:

• to organize the storage of raw microbial data sets• to assist in the development of microbial models• to provide an efficient user interface for data

acquisition

BENEFITS:

• Enhances development of models and risk assessments

• Reduces user resources necessary to locate and analyze pathogen-food specific data

Center of Excellence in Microbial Modeling &

Informatics

Microbial Detection

ARS/NAFS Downer Dairy Cattle Survey NAHMS 2000 Swine Survey HIMP Microbial Surveillance Project

States Participating in the National Animal Health Monitoring System (NAHMS) Swine 2000 Study

- 17 states, 160 farms, 60 samples/farm

- 93% of U.S. hogs represented

- 92% of producers with 100 hogs represented

National Animal Health Monitoring System (NAHMS) Swine 2000 Study

Microbial Food Safety Research Unit: Examined feces for: E. coli O157:H7, STEC,

Y. enterocolitica, and L. monocytogenes. Determined the clonality of isolates using phenotypic

and genotypic methods.

Recovery of E. coli O157 from Swine Feces(NAHMS 2000 Swine Surveillance Study)

Samples tested 2,526

Serotype O157-positive samples 102 (4%)

Serotype O157:H7-positive samples None

Ingrid Feder, Jeffrey Gray, Rachel Pearce, Eric Bush, Dave Dargatz, Pina Fratamico, F. Morgan Wallace, Anna Porto, Paula Fedorka-Cray, Robert L. Dudley, Richard Perrine, Jeffrey E. Call, and John B. Luchansky. Annual Meeting of the International Association for Food Protection (2002)

Recovery of E. coli O157 from Intact Colons of Swine at a Slaughter Facility

(HIMP Microbial Surveillance Project)

Samples tested 305

Serotype O157-positive samples 12 (4%)

Serotype O157:H7-positive samples 6 (2%)

Ingrid Feder,, F. Morgan Wallace, Jeffrey E. Call, Pina Fratamico, Rachel Pearce, Paula Fedorka-Cray,Richard Perrine, and John B. Luchansky. Annual Meeting of the American Society for Microbiology (2002)

Conclusions– E. coli O157:H7 and Swine

Within the timeframe and geographic scope: Prevalence of serotype O157 isolates was similar in

colon samples obtained at slaughter and fecal samples obtained on farms

Serotype O157:H7 isolates were recovered from intact colons, but not from feces

Talking Points – E. coli O157:H7 and Swine

What is the impact of collection, storage, shipment, and/or methodology on recovery?

Relative to finding E. coli O157:H7 in colon samples at slaughter but not in fecal samples from farms, what is the impact of transport and holding on shedding and/or viability?

Should studies be initiated to determine the prevalence of the pathogen in matched animal/fecal samples on the farm and at slaughter?

Characterization of E. coli O157:H7 from Downer and Healthy Dairy Cattle in the Upper Midwest

Caitriona Byrne, Irfan Erol, Jeffrey E. Call, Dennis Buege, Charles W. Kaspar, Clayton Hiemke, Paula Fedorka-Cray, Jovita Hermosillo, Takiya Ball, Andrew K. Benson, Morgan Wallace, Marcus Handy, and John B. Luchansky.

USDA/ARS and National Alliance of Food Safety Funding. Annual Meeting of the International Association of Food Protection (2002).

Design – E. coli O157:H7 and dairy cattle

Two cattle types Healthy and downer ~200 samples from each cattle type

Two slaughter facilities 4 (healthy) and 7 (downer) plant visits between

April and October of 2001 404 total fecal samples from intact colons

Prevalence – E. coli O157:H7 and dairy cattle

Of 404 total fecal samples: 6% (12/203) downer cattle

47 isolates retained 2% (4/201) healthy cattle

20 isolates retained

DownerHealthyNegative

Conclusions– E. coli O157:H7 and dairy cattle

Within the timeframe and geographic scope: 3-fold higher prevalence of serotype O157:H7 isolates in

downer cattle than healthy cattle. 1.7-fold higher prevalence of antibiotic-resistant isolates in

healthy cattle. High degree of heterogeneity among all isolates as

demonstrated by PFGE.

Talking Points – E. coli O157:H7 and dairy cattle

Exclude downer/suspect animals and those receiving antimicrobials from meat supply???

Channel downer/suspect and/or animals receiving antimicrobials into cooking operations???

Conduct additional sampling to address the impact of methodology, geography, and/or seasonality etc. on prevalence.

Develop interventions to further reduce levels of undesirable microbes associated with the carcass, primal cuts and trim, and/or finished products.

Practice more prudent use of antimicrobials???

Hot Dogs are . . .

“… the quintessential American food …” “… the ultimate handheld food …” “… always rated in the top ten of

America’s favorite foods …”

James Ratchford (Natl. Hot Dog & Sausage Council)

Prepared Foods, August 1999

Some Facts About Hot Dogs

850 million pounds sold at retail in 1997 100 million pounds were poultry hot dogs 160 million pounds were fat-free and light hot

dogs 20 billion hot dogs consumed annually

7 billion hot dogs consumed between Memorial and Labor Days

National Hot Dog and Sausage CouncilPrepared Foods, August 1999

Research on L. monocytogenes in commercially-prepared frankfurters

Evaluation of recovery methods Determination of levels & types Optimization of formulation

ARS & FSIS L. monocytogenes Frankfurter Shelf Life Study

ARS & FSIS L. monocytogenes Frankfurter Shelf Life Study

Determine the prevalence, levels, and types Sample 3000 packages/pounds of frankfurters

from each of 12 commercial manufacturers Sample packages within 5 days of manufacture

and at regular intervals over a 60-day storage period at 4 and 10°C

Complete prevalence component by July of 2002

USDA- ARS Package Rinse Method

Recovery of Listeria monocytogenes from vacuum-sealed packages of frankfurters: comparison of the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service Product Composite Enrichment Method, the USDA Agricultural Service Product Composite Rinse Method, and the USDA-ARS Package Rinse Method

J. B. Luchansky, A. C. S. Porto, F. M. Wallace, and J. E. Call

Journal of Food Protection 65:567-570, 2002.

Recovery of L. monocytogenes from frankfurters

Conclusions: USDA/ARS package rinse method is about 6-fold more

sensitive than the approved USDA/FSIS product composite enrichment method because the package, the purge, and the product are tested

USDA/ARS package rinse method requires less hands-on manipulation of the product, which minimizes the likelihood of product contamination and decreases the time required to sample the product

Fate of L. monocytogenes on Frankfurters Containing Potassium Lactate (Klac) at 4°C

0

1

2

3

4

5

6

0 7 15 21 28 60 90

Time (days)

Log1

0C

FU

/pac

kage

0.0% Klac

2.0% Klac

2.8% Klac

Fate of L. monocytogenes on Frankfurters Containing Potassium Lactate

Bacteriostatic during product storage at 4° and 10°C

Results validate the Pathogen Modeling Program

Future studies will optimize levels to achieve a desirable flavor and an appreciable antilisterial effect

Listeria monocytogenes genomics:from sequence to function

Collaborators/Team Members: USDA/ARS

NPS - Leland Ellis, Steve Kappes, James Lindsay, & Caird Rexroad

ERRC - Gaylen Uhlich, John Luchansky, Darrell Bayles, Laura Wonderling, Connie Briggs, John Cherry, & Wilda Martinez

NADC - Irene Wesley, Alissa Jourdan, & Keith Murray NCAUR - Peter Johnsen, Cletus Kurtzman, & Todd Ward WRRC - Robert Mandrell

TIGR - Claire Fraser, Karen Nelson, & William Nierman

Listeria monocytogenes Genome Sequencing Project - Status

Closure date of 4/16/02 Total assembly length = 2,874,238 bp

Edit and confirm single coverage areas Annotation in progress Comparative genomics initiated Manuscript in preparation

What can be done with the completed sequence of Listeria monocytogenes

Study gene regulation and phenotypes of interest to enhance the safety/quality of foods, such as:

Tolerance to high salt, low pH, increased water activity, cold and warm temperatures, and modified atmospheres.

Persistence in foods and/or food processing plants. Survival in animal and human hosts. Resistance to biological and chemical antimicrobials

and sanitizers

“The goal of every bacterium is to become bacteria.”

Stanley Falkow