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PFGE as a Component of FDA Compliance Efforts

2009 Annual PulseNet MeetingSeptember 25th 2009

David Melka

Th U S F d & D Ad i i t tiThe U.S. Food & Drug AdministrationCenter for Food Safety and Applied Nutrition

Office of Regulatory Science

FDA PulseNet Certified Field Lab

PRL-NW

FDA CFSAN

FDA CVMDEN-DO

ARLPRL SW

SAN-DO

NRL

ARLPRL-SWSRL

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Where our isolates come from:

• Domestic and Import Surveillance

• Special Assignments– Investigation efforts of food types

– specific firms

Some Specific Concerns to FDA

• Salmonella enterica: • raw seafood, egg products, cake mixes,

unpasturized milk and salad dressings,

• Many types of produce (peppers, leafy greens, sprouts, etc.)

• Listeria monocytogenes: – ready to eat (RTE)

• deli meats, raw milk, soft cheeses

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Peanut Butter – 2006/7(3 concurrent clusters eventually linked)

XbaI – Primary Enzyme BlnI – Secondary Enzyme#1

#2

#3

Patient A

#1 #2

Patient B

#1 #3

Peanut Butter

#1 #3

(With confirmed history of peanut butter consumption)

Changes in 2007

• Increased focus on determining whether or not microorganisms are present in food processing environment

• Field instructed to gather 5 to 10 fold the gnumber of environmental samples previously collected prior to 2007.

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New Strategy Started in 2007• Doubled the inspectional and analytical• Doubled the inspectional and analytical

resources for Environmental sampling

• Implemented new training module in the Certification training regimen given field investigatorsg

• Anticipated that FDA will continue to increase resources for environmental sampling– Importance of analytical results

FDA ORA/CFSAN Entries Uploaded to PulseNet

200

300

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700

Listeria

Salmonella

0

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200

2003

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2009

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CFSANCenter for Food Safety and Applied Nutrition

ORPSSOffice of Regulations,

Policy andSocial Sciences

OCACOffice of Cosmetics

And Colors

ORSOffice of Regulatory

Science

OARSAOffice of Applied

Research and

ONPLDSOffice of Nutrition,

Labeling and

OCOffice of

Research andSafety Assessment

Labeling and Dietary Supplements

Compliance

OFASOffice of FoodAdditive Safety

OFDCEROffice of Food Defense,

Communication, & Emergency Response

OFSOffice of Food

Safety

Compliance Objectives

• To Keep the Public safe from unadulterated products

• To aid industry in having unadulterated product– Ensure that industries system is workingEnsure that industries system is working

• To collect evidence that will hold up in court

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Case Development:Collect Product and/or Environmental Samples

Samples sent to FDA lab for analysis and PFGE.

Review of EIR and/or violative sample findingsPrepares recommendations to CFSAN O.C.

CFSAN Compliance officer evaluates case pCase forwarded to Office of Chief Counsel

FDA/OCC attorney reviews caseReferal package DOJ

Adulteration ChargesFDA’s Legal Authority:FDA s Legal Authority:• Federal Food, Drug and Cosmetic Act

– Section 402 (a)(1)• “A food shall be deemed to be adulterated within the meaning of 402(a)(1) if

it “bears or contains any poisonous or deleterious substance which may render it injurious to health.” For example, this charge is cited when there is FDA laboratory confirmation of a human pathogen in product.”

– Section 402 (a)(4)• “A food shall be deemed to be adulterated within the meaning of

402(a)(4) if it has “been prepared, packed, or held under insanitary conditions whereby it may have become contaminated with filth, or whereby it may have been rendered injurious to health.”

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Interpretations of PFGE

• Environmental samples that match product contamination– Can help show a firm is responsible for

contamination of product

• PFGE is persistent in a processing facility over months or years– Permanent Resident Pathogen

Listeria monocytogenes(in ready to thaw and eat sandwiches)

PFGE-AscI PFGE-ApaI

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Sponge Stick

Sponge Stick

sub sandwich with turkey & cheese

sub sandwich with turkey & cheese

Sponge Stick

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Sponge Stick

Sponge Stick

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Sponge Stick

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2009-01-14

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Sponge Stick

Sponge Stick

Sponge Stick

Sponge Stick

Sponge Stick

Sponge Stick

Sponge Stick

Sponge Stick

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2009-06-09

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Listeria monocytogenes(in ready-to-eat hot & cold smoked fish)

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%]

PFGE-AscI10

0

50

PFGE-AscI PFGE-ApaI

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smoked salmon

smoked salmon

Swab

Env. sponges

Env. sponges

Env. sponges

Env. sponges

Env. sponges

Env. sponges

Env. sponges

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2001-01-23

2001-01-23

2005-09-13

2009-07-08

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2009-07-08

2009 07 08.

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Env. sponges

Env. sponges

Cold Smoked White Fish

Cold Smoked White Fish

Env. sponges

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2009-07-08

2009-07-08

2009-07-08

2009-07-08

2009-07-08

Sporadic L. mono contamination and lapses in sanitation?

Voluntary Recall was announced for products sold Dec. 7, 2008 through June 18th 2009

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%]

PFGE-AscI PFGE-AscI PFGE-ApaI

herring in oil

Environmental Swabs

Swab

Environmental Swabs

Environmental Swabs

Environmental Swabs

Swab

swabs

Environmental Swabs

Cold Smoked Salmon

2003-07-23

2008-09-11

2006-01-11

2008-09-11

2008-09-11

2008-09-11

2009-05-27

2003-07-22

2008-09-11

2009-05-28

Environmental Swabs

Swab

Environmental Swabs

Environmental Swabs

Environmental Swabs

Swab

2008-09-11

2006-01-11

2008-09-11

2008-09-11

2008-09-11

2009-05-27

Listeria monocytogenes in a Seafood Processing Firm

Cold Smoked Salmon

Cold Smoked Salmon

Smoked Salmon

Smoked Salmon

Frozen Smoked Sal.

Frozen Smoked Sal.

swabs

swabs

swabs

Vaccum packed pick.

swabs

herring in oil

smoked salmon

swabs

swabs

Swab

Environmental Swabs

2009 05 28

2009-05-28

2009-06-02

2009-06-02

2009-06-02

2009-06-02

2003-05-09

2003-07-22

2003-05-09

2008-09-11

2003-07-22

2003-07-22

2003-07-23

2003-07-22

2003-07-22

2006-01-11

2008-09-11

S ab 009 05

swabs

Environmental Swabs

Cold Smoked Salmon

Cold Smoked Salmon

Smoked Salmon

Smoked Salmon

Frozen Smoked Sal.

Frozen Smoked Sal.

b

2003-07-22

2008-09-11

2009-05-28

2009-05-28

2009-06-02

2009-06-02

2009-06-02

2009-06-02

2003 05 09

Environmental Swabs

swabs

swabs

salmon trimmings

salmon trimmings

herring in corn oil

swabs

2008-09-11

2003-05-09

2003-05-09

2003-05-09

2003-05-09

2003-06-19

2003-07-22

Environmental Swabs

Environmental Swabs

Environmental Swabs

swabs

swabs

salmon trimmings

salmon trimmings

herring in corn oil

swabs

smoked salmon

Environmental Swabs

Vaccum packed pick.

Vaccum packed pick.

Swab

Smoked Salmon

2008-09-11

2008-09-11

2008-09-11

2003-05-09

2003-05-09

2003-05-09

2003-05-09

2003-06-19

2003-07-22

2003-07-22

2008-09-11

2008-09-11

2008-09-11

2009-05-27

2009-06-02

smoked salmon

Environmental Swabs

Vaccum packed pick.

Vaccum packed pick.

Swab

Smoked Salmon

2003-07-22

2008-09-11

2008-09-11

2008-09-11

2009-05-27

2009-06-02

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Salmonella Agona(in cereal)

JABX01 0001

PFGE-XbaI

Cereal Toasted Oats 1998 06 26

Interesting…

PFGE-BlnI

JABX01.0001

0.67% of all S. Agona

.....Cereal - Toasted Oats . 1998-06-26

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Swab

Swab

Swab

Swab

Swab

Swab

Swab

Cereal

Swab

Puffed Rice Cereal

Puffed Rice Cereal

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2008-04-16

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Puffed Rice Cereal

Swab

Swab

Swab

Swab

Unsweeten Puffed Rice Cereal

Unsweeten Puffed Rice Cereal

Puffed Wheat Cereal

Puffed Wheat Cereal

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COLLABORATIVE SCIENCE WITH CDC

►FDA-PULSENET ●in step with CDC innovations and new platforms (i.e., MLVA)●continued populating of Salmonella national database with food and veterinary associated PFGE patterns

►FACILE SEROTYPING METHODS●CDC-developed ‘BIOPLEX’ assay for O-group antigen assignment●FDA scientists are training at CDC to adopt technology●validation and evaluation efforts underway

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ONGOING PROJECTSTe

sts

per

Sam

ple

multiplex RT-PCR

microarray

Mass Spec xMAP TechnologyIBIS

Sample throughput

T p

Real-Time PCRnorthern blot

Mass Spec gyIBIS

ONGOING PROJECTS

Genome scanning strategies:454 Technologies whole-genome sequencing systems:

► allows for multiple coverage of acomplete bacterial pathogen’s genomein 3 days

► provides raw data for SNP discovery

► allows for identification of rapidlychanging genetic markers

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ONGOING PROJECTS

►SPROUT SAFETY●strategies for reducing Salmonella growth under commercialsprouting conditions●chemical and biological interventions being evaluated●part of FDA ‘produce assignment’

►SALMONELLA SURVIVAL STUDIES►SALMONELLA SURVIVAL STUDIES●studies underway to assess survivability of Salmonella on fruit surfaces and in the food processing environment●studying resistances to desiccation stress and storage

►TOMATO SAFETY…

TOMATO OUTBREAKS

►associated with specific geographical locations and growing seasons where there have been multiple outbreaks:

Eastern United StatesLate Spring – FloridaSummer and Fall - VirginiaWinter – Florida

►widely dispersed, individual patient-cases in many states

►trace backs are difficult due to complexity of the supply chain

►implicated produce is rarely still available, the crop is no longer in the field

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TOMATO OUTBREAKS

• 1998 S. Baildon 86 cases• 2000 S. Thompson 29 casesp• 2002 S. Newport 512 cases

S. Newport 12 casesS. Javiana 90 cases

• 2004 S. Javiana 471 casesS. Braenderup 123 cases

• 2005 S. Newport 71 casesS. Enteriditis 77 casesS. Braenderup 76 cases

• 2006 S. Newport 107 casesS. Typhimurium 186 cases

= single PFGE type

TOMATO OUTBREAKS

• Four Sources of Potential Contamination – Soil

– Domestic and Wild Animals

– Water

– Farm Workers

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PRELIMINARY TOMATO RESEARCH

CURRENT SUBTYPING/MOLECULAR EPIDEMIOLOGY SUPPORT

partners: FDA-CDC-USDA/ARS-NCSU-UMD-VTECH

CURRENT SUBTYPING/MOLECULAR EPIDEMIOLOGY SUPPORT►Genetic and molecular epidemiological analysis of outbreak and environmental strains of S. Newport from tomato farms located on the Virginia eastern shore -

●PFGE pattern comparisons with FDA foods and veterinary databases across several enzymes●antibiotic resistance profiling●MLST analysis

ECOLOGY STUDIES►growth and survival characteristics on tomato fruit, pond water, other niches►sampling of potential reservoirs associated with tomato farms (i.e., ponds, native flora, migratory fowl)►planned competition studies of S. Newport and other serovars on tomatoes

INTERVENTION►in vitro and in vivo screening of tomato farm epiphytic microflora for antagonistic activity against S. Newport ►potential field application for biological control of S. Newport

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CURRENT METHOD DEVELOPMENTS IN STRAIN DIFFERENTIATION

PFGE-BASED

►exploration of novel restriction enzyme combinations and cluster methodsp y►enhanced PFGE schemes with up to six enzymes for retrospective analysis of closely related Salmonella strains

DNA SEQUENCE-BASED

►application of conventional multi-locus sequence typing (MLST) schemes to various homogeneous serovars►currently exploring other ‘hypervariable’ gene targets for increased discriminatory power of highly homogeneous serovars (i.e., S. Enteritidis)y p g y g ( , )►application of several novel SNP detection platforms for rapid subtyping of Salmonella strains

OTHER SUBTYPING METHODS BEING DEVELOPED

►LC-MS protein profiling►Phenotype microarray

Acknowledgements:Office of Compliance: Division of Microbiology:Office of Compliance:

– Priya Rathnam

– Glenn Bass

– Bill Correll

– Ronald Pace

Division of Microbiology:

– Christine Keys

– Eric Brown

– Marc Allard

– Marianna Naum

– Rebecca Bell

– Jie ZhengEric Brown, Ph.D.(301) 436-2020Eric.Brown@fda.hhs.gov

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The Risk