Foodborne Disease Role of Pulsedfield Gel Electrophoresis and PulseNet

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Foodborne DiseaseRole of Pulsed-field Gel
Electrophoresis and PulseNet

• Dave Warshauer, Ph.D D(ABMM)
• Deputy Director, Communicable Diseases
• Wisconsin State Laboratory of Hygiene

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The Public Health Burden of Foodborne Disease in
the US

• Mead et al 1999 - Emerg Infect Dis Vol. 5
• 76 million cases per year
• 325,000 hospitalizations
• 5000 deaths
• Cost 8-10 billion/year

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Other Factors Influencing Emergence of Foodborne
Disease

• Inexperienced food workers
• Average turnover rate about 350 / year
• International transport of contaminated foods
• Introduction of foodborne pathogens to new
  geographic areas
  

From M. T. Osterholm, 2002 ICAAC Presentation
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Trends in Foodborne DiseasePercent of Food
Spending by Site
Food Review 1623-7, 1993
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Emerging Foodborne Pathogens
Parasites Cryptosporidium parvum Cyclospora
cayetanensis Viruses Noroviruses Prions Spongifo
rm encephalopathies

• Bacteria
• Shiga toxin-producing E. coli (STEC)
• Salmonella Typhimurium DT104
• Salmonella Enteritidis
• Listeria monocytogenes
• Campylobacter jejuni
• Campylobacter fetus
• Yersinia enterocolitica
• Vibrio cholerae O139
• Vibrio vulnificus
• Vibrio parahemolyticus

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WSLH WEPS Program
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WEPS Program

• Wisconsin Enteric Pathogen Surveillance Program
• Request submission of all bacterial enteric
  pathogens to WSLH as soon as possible for PFGE
  analysis and antimicrobial resistance monitoring
  (for select organisms)
• Vital to sustain our foodborne disease
  surveillance

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What is PFGE?

• Molecular subtyping method used to generate the
  DNA fingerprint of a bacteria
• Other subtyping methods
• Phage typing
• Serotyping
• Biotyping
• Various molecular methods

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PFGE Method
Step

• Liquid suspension of bacteria
• Encase in agarose plug
• Lyse with detergent and protease
• Wash away detergent and protease
• Digest with restriction enzyme
• Separate restriction fragments of genome DNA on
  PFGE apparatus

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Specimen Slice
Comb
Gel
Casting Stand
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Pump
CHEF Mapper
Cooler
Electrophoresis Chamber
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Gel Doc Imaging System
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PFGE Analysis of E.coli O157H7 isolates
M Milwaukee outbreak O Other outbreak ?
marker/internal reference
M
M
M
M
O
O
O
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What is PulseNet?

• A national network of public health laboratories
• State health departments
• Local health departments
• Federal agencies (CDC, USDA, FDA)
• Perform standardized molecular typing of
  foodborne disease-causing bacteria by PFGE
• Sharing DNA fingerprints electronically
• Enhancing foodborne disease surveillance

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PulseNet

• Inception in 1996
• CDC-based APHL administration
• Molecular surveillance network for foodborne
  bacterial pathogens
• Primary subtyping method is PFGE
• Organisms subtyped include E. coli O157 and
  non-O157 STEC, Salmonella, Shigella, Listeria and
  Campylobacter

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PulseNet- Contd

• PulseNet International
• - Canada - Middle East
• - Europe - Asia Pacific
• - Latin America

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How Does PulseNet Enhances Surveillance?

• Detects outbreaks by identifying cases with
  indistinguishable PFGE patterns
• Links apparently unrelated outbreaks
• Speeds outbreak investigations through focusing
  efforts on cases with indistinguishable PFGE
  patterns
• Monitors for illnesses associated with a product
  retail samples ? recalls

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single food supplier
outbreak in New York
outbreak in California
outbreak in Wisconsin
same PFGE pattern
-Hypothesis for disease transmission -Test food
samples with PFGE
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Reporting Timeline
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2006 Multi-State Spinach E. coli O157 H7
Outbreak
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Spinach Outbreak Timeline

• Sept. 5----Cluster of 5 E.coli O157H7 cases
• Livestock at Manitowoc county fair suspected
• Sept. 7----Report to State Medical Officer of 5
  adults with HUS who had undergone plasma exchange
• E. coli O157H7 confirmed in 3 patients
• Sept. 8----WSLH posts PFGE pattern of 8 isolates
  on PulseNet
• 7 had indistinguishable patterns, 1 a small
  variation

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PFGE Gel - E. coli O157H7
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Spinach Outbreak (2)

• Sept. 7-9-----12 more E.coli O157H7 received at
  WSLH
• Sept 11----DPH questionnaires indicate high
  percentage of females and link with spinach
  consumption
• Sept 12----Oregon investigation suggests 6
  patients with E. coli O157H7 linked to spinach
• Sept 14----CDC confirms 50 cases in 8 states
• Wisconsin, New Mexico, Utah, Oregon
• One death----77 yr old Wisconsin woman
• Sept 14----Conference call with CDC, state HDs,
  and FDA
• FDA issues advisory for consumers not to eat
  bagged spinach

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Spinach Outbreak (3)

• Sept. 15----National Selection Foods of San Juan
  Bautista, CA recalls all products containing
  spinach (use-by dates from Aug 17 Oct 1)
• Sept. 19----New Mexico confirms E. coli from
  patients open bag of spinach
• Identical PFGE pattern to outbreak strain
• Other states later identify outbreak strain in
  bags of spinach
• 13 bags from 8 states including 2 from Wisconsin
• Non-O157 isolate from New Mexico spinach
• Sept. 20----2 yr old Utah boy dies
• Oct. 5----Elderly Nebraska woman dies

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National Cases
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Outbreak Statistics

• 204 cases
• 26 states
• 1 Canadian province
• Approximately 50 hospitalized (104)
• Hemolytic uremic syndrome (HUS) (31)
• Higher than usual
• 39 of children
• 10 of adults
• 3 Deaths

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Spinach Outbreak Strain

• E. coli O157H7 identified as EXHX01.0124 strain
• Accounts for lt1 of strains reported each year
• Produces only Type 2 shiga toxin
• Usually more virulent than strains that produce
  only Type 1 or both toxins
• Characterized as Stx2d-activatable----activated
  by human and mouse intestinal mucus

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What was the Source?

• Extensive environmental sampling
• Water
• Processing plant
• Cattle feces
• Wild pig feces
• Investigation by FDA and CDC suggests wild pigs
  or contaminated irrigation water as culprits

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How do we Prevent Future Outbreaks

• Preventive measures
• At the field
• Irrigation water
• Proximity to cattle, pig, and other animal
  ranches
• At the processing plant
• Decontamination steps
• Distribution
• Maintaining appropriate temperatures
• Consumer education

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Conclusions I

• PFGE is extremely valuable as part of
  epidemiologic and environmental investigations
• Helps separate outbreak from sporadic cases
• Identifies cases that are likely to be linked
• Helps trace source of contamination
• Most of the time, lab and epidemiology correlate
• PFGE is only part of the picture results must be
  correlated with epidemiology

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Conclusions II

• PulseNet
• Provides a means for laboratorians to rapidly
  exchange cata
• Allows active participation of laboratorians in
  evolving outbreak investigations
• Provides effective two-way communication among
  laboratories and CDC

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The Latest Victim