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Genetic Subtyping:Food Safety Applications

• A couple of methods• Foodborne disease in the US• Recent outbreaks

– 2009 Salmonella outbreak; peanuts– 2010 Salmonella outbreak; salami

• Genetic subtyping methods– PCR-based– Non-PCR (RFLP) based

• PulseNet• Sequence-based methods

– MLST– MVLST

Methods used for typingE. coli O157:H7 strains

• Toxin gene screening• Plasmid profiling

• Phage typing

• Antibiotic susceptibility testing

• Restriction fragment length polymorphism with bacteriophage λ (λ-RFLP)

• Ribotyping;• Pulsed-field gel electrophoresis• PCR using randomly amplified

polymorphic DNA (RAPD) sequences

• PCR using highly repetitive sequences (rep-PCR)

• Amplified fragment length polymorphism analysis

• Thomas et al., 1996; Ostroff et al., 1989• Ostroff et al., 1989; Paros et al., 1993;

Meng et al., 1995; Radu et al., 2001• Ahmed et al., 1987; Khakhria et al., 1990;

Barrett et al., 1994• Kim et al., 1994; Farina et al., 1996; Radu

et al., 2001; • Paros et al., 1993;Samadpour et al., 1993

• Martin et al., 1996; Roberts et al., 2001• Bohm and Karch, 1992;Barrett et al.,

1994;Meng et al., 1995;Radu et al., 2001• Swaminathan and Barrett,1995; Radu et al.,

2001

• None specific for E. coli-O157:H7; Johnson and O’Bryan, 2000

• Iyoda et al., 1999; Zhao et al., 2000

DNA “Fingerprinting”

• It’s “jargon”

• Fragment based methods (CSI)

• Sequence based methods (MLST, MVLST)

• Phenotypes– Non-weighting of characteristics

• Importance of genetic relationships– Common ancestors

– Changes over time

– Snapshot of current relationships

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Foodborne Diseases Active Surveillance Network (FoodNet)

• Principal foodborne disease component of CDC's Emerging Infections Program

• Active surveillance for foodborne diseases and related epidemiologic studies to better understand the epidemiology of foodborne diseases in the United States.

• “Active” surveillance system, – Public health officials frequently contact laboratories

to find new cases of foodborne diseases

– Cases reported electronically to CDC.

FoodNet Disease Monitoring• Bacteria

– Campylobacter– Escherichia coli O157– Listeria monocytogenes– Salmonella– Shigella– Vibrio– Yersinia enterocolitica

• Parasites– Cryptosporidium– Cyclospora

• Viruses– Hepatitis A– Noroviruses

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FoodNet

• 1995, FoodNet surveillance began in fivelocations: California, Connecticut, Georgia, Minnesota and Oregon

• New York and Maryland in 1998, Tennessee in 2000, Colorado in 2001 and New Mexico in 2004). The total population of the 2004 bacterial catchment is 44.5 million persons, or 15.1% of the United States population.

Mead et al, 1999

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5714a2.htm

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Compare banding patterns with other patterns in the PulseNet system

Role of PulseNet

• Detect foodborne disease case clusters by pulsed-field gel electrophoresis (PFGE) Facilitate early identification of common source outbreaks

• Assist epidemiologists in investigating outbreaks• Separate outbreak-associated cases from other sporadic

cases• Assist in rapidly identifying the source of outbreaks• Act as a rapid and effective means of communication

between public health laboratories

How Does PulseNet Work?

1. PFGE

2. Pattern electronic database at local, state or federal level

3. Uploaded to national database at CDC

4. CDC searches for clusters of patterns

5. Local cluster searches

6. Clusters posted to Listserve.

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Limitations of PFGE and PulseNetPFGE Method and Analysis:

• Time consuming – need pure culture of isolate• Requires a high-level of skill• Does not work for everything (i.e. clonal patterns) • Pattern results vary from person to person, lab to lab even though

standardized protocol used and audited • Can’t optimize separation in every part of the gel at the same time • Bands are bands, not sequences • Don’t really know if bands of same size are same pieces of DNA • Bands are not independent but are treated equally • Change in one restriction site can mean > 1 band change • “Relatedness” should be used as a guide, not true phylogenetic

measure • Some strains are untypeable by PFGE (nucleases)• Indirect method – don’t know significance of bands • Small percentage of genome analyzed: RE sites

What makes interlaboratory comparison of DNA patterns possible?

• Standardized protocols• QA/QC Manual • Same molecular size standards• Standardized software used by all participants • Standardized nomenclature of PulseNet patterns • Training workshops (lab & software): most participating labs

have attended a week of combined laboratory and analysis software training

• Certification: all individuals who submit data must be certified by stringent PulseNet standards

• Proficiency testing: all certified individuals must participate and pass annual proficiency testing in order to maintain certification

• Annual update meetings: provide a forum for the live exchange of information

Changes in Outbreaks

• Food consumption and practices in US have changed during the past 20 years

• Shift from the typical point source, or “church supper” outbreak, which is relatively easy to detect to the more diffuse, widespread outbreaks that occur over many communities with only a few illnesses in each community.

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Changes in OutbreaksContinued• Large food producing facilities that disseminate

productss throughout the country• Some few outbreaks that some low level

contamination of food productss• Productss are distributed among many states• Only a few illnesses occur in each community, • New laboratory and statistical tools, such as

PulseNet and the surveillance outbreak detection algorithm (SODA), impact ability to identify and investigate these new types of outbreaks

Some Recent PulseNet Articles• Hyytia-Trees E, Smole SC, Fields PA, Swaminathan B, Ribot EM. Second generation

subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157). Foodborne Pathog Dis. 2006. 3:118-31.

•Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ.Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis. 2006. 3:59-67.

• Cooper KL, Luey CK, Bird M, Terajima J, Nair GB, Kam KM, Arakawa E, Safa A, Cheung DT, Law CP, Watanabe H, Kubota K, Swaminathan B, Ribot EM. Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae. Foodborne Pathog Dis. 2006. 3:51-8.

•Swaminathan B, Gerner-Smidt P, Ng LK, Lukinmaa S, Kam KM, Rolando S, Gutierrez EP, Binsztein N. Building PulseNet International: an interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases. Foodborne Pathog Dis. 2006. 3:36-50.

•Barrett TJ, Gerner-Smidt P, Swaminathan B. Interpretation of pulsed-field gel electrophoresis patterns in foodborne disease investigations and surveillance.Foodborne Pathog Dis. 2006. 3:20-31.

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Meat recall

1993 Western States E. coli O157 Outbreak

732 cases4 deaths

39 d

2002 Colorado E. coli O157 Outbreak

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http://www.who.int/foodsafety/fs_management/No_04_PulseNet_Nov09_en.pdf

Since 2000, PulseNet networks have been established in six regions with more than 80 PulseNet country members worldwide.

Peanut Outbreak – 2008-09

FDA Press ReleaseJanuary 12, 2009

• Salmonella recovered from King Nut peanut butter• Minnesota Dept. Agriculture lab results• Genetic match to national cases• Strain associated with 30 illnesses in MN

– About 400 cases nationwide• Distributed to 7 states• Distributed in MN to:

– Long-term care facilities– Hospitals– Schools– Universities– Restaurants– Delicatessens– Cafeteria– Bakeries

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Salmonella OB: Peanuts

• 550 Illnesses: 2/2/09; 9 PM; 8 deaths may be associated

• >162 Manufacturers

• >133 Separate recalls

• 1085 Products: 2/4/09; 12 PM

http://www.accessdata.fda.gov/scripts/peanutbutterrecall/index.cfm

Some Product TypesBrownie Products - 10Cake and Pie Products - 11Candy Products - 9Cookie Products - 39Cracker Products - 7Donut Products - 1Fruit and Vegetable Products - 4Ice Cream Products - 86Peanut Butter Products - 5Peanut Paste Products - 1Peanuts Products - 3Pet Food Products - 8Pre-Packaged Meals Products - 2Snack Bar Products - 42Snack Mix Products - 14Topping Products - 3

http://www.accessdata.fda.gov/scripts/peanutbutterrecall/index.cfm

Some Companies• Aspen Hills• Best Brands• Lovin Oven• Landies Candies• Weis Markets• Blanton’s Candies• Dinners Ready Meridian• Boca Grande Foods• Premier Nutrition• NutriSystem• Ready Pac Foods• PetSmart• Nature’s Path• Country Maid

• Evening Rise Bread• Clif Bar• Kroger• Abbott Nutrition• Meijer• Peanut Corp. Amer.• Ralcorp• South Bend Chocolate• McKee• Perry’s Ice Cream• Hy-Vee• Kellogg• King Nut• Wegmans• Little Debbie

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CDC Outbreak UpdateJanuary 28, 2009

• 529 cases in 43 states; 8 related deaths

• Latest illness onset January 8, 2009

• 15 clusters of infections in five states reported in schools and other institutions

• Among 14 clusters with detailed information, King Nut was the only brand of peanut butter used in those facilities.

imported jalapeño and serrano peppers

peanut butter

http://www.cdc.gov/salmonella/typhimurium/map.html

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The Epidemic Curve

• Progression of an outbreak over time.• Epi curve is complex and incomplete. • Delay between onset and reported to

public health authorities.– Typically takes 2-3 weeks for Salmonella

• Some background cases likely to occur without an outbreak.– This makes it difficult to say exactly which

case is the first in an outbreak

Delay between illness onset and confirmation that a patient is part of an outbreak.

• 1. Incubation time– Salmonella: typically 1 to 3 days, sometimes longer.

• 2. Time to contact with health care provider/doctor:– Time from the first symptom until diarrhea sample is collected for testing– 1 to 5 days, sometimes longer.

• 3. Time to diagnosis: – Time from sample to Salmonella recovered– 1 to 3 days from the time the sample is received in the laboratory.

• 4. Sample shipping time– Shipping isolate from lab to state lab performing serotyping and PFGE– Usually 0 to7 days

• 5. Time to serotyping and “DNA fingerprinting”: – Time serotype and PFGE– Compare with outbreak pattern. Serotyping may take up to 3 days.– 1-10 days depending on lab resources

• Thus, onset to part of OB 2-3 weeks• Case counts during outbreak investigation preliminary

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King Nut Peanut Butter

• Produced by Peanut Corporation of America (PCA), a small, family-owned and operated business. HQ in Lynchberg, VA.

• Plant positive for Salmonella in Blakely, GA. <50 employees• No longer producing any products• Expanded recall to include all peanut butter and peanut

paste produced at this plant since July 1, 2008. (1/1/07)• Peanut butter and peanut butter paste was not sold directly

to consumers– Peanut Butter: Containers 5 to 1,700 pounds– Peanut Paste: Containers 35 pounds to tank car – Distributed to institutions, food service providers, food manufacturers

and distributors in many states, Canada, Korea, and Haiti. – Peanut butter and peanut paste is commonly used in many products,

including cookies, crackers, cereal, candy, ice cream and others.

FDA Inspectional Observations• Peanut Paste manufactured on January 25. 2008 under

lot # 8028 tested positive for Salmonella by a private laboratory. After the firm retested the product and received a negative status. the product was shipped in interstate commerce.

• Medium Chopped Granules manufactured on January 24, 2008 under lot # 8024 tested positive for Salmonella by a private laboratory. After the firm retested the product and received a negative status, the product was shipped in interstate commerce.

• Failure to maintain equipment, containers and utensils used to convey. hold, and store food in a manner that protects against contamination.

• Failure to perform mechanical manufacturing steps so as to protect food against contamination.

• Failure to store finished food under conditions that would protect against microbial contamination.

FDA Inspectional Observations (con’t)

• The plant is not constructed in such a manner as to allow ceilings to be kept in good repair

• The design of equipment and utensils fails to preclude the adulteration of food with contaminants

• Proper precautions to protect food and food-contact surfaces from contamination with microorganisms cannot be taken because of deficiencies in plant construction and design

• Devices and fixtures are not designed and constructed to protect against recontamination of clean, sanitized hands

• Failure to conduct cleaning and sanitizing operations for utensils and equipment in a manner that protects against contamination of food

• Effective measures are not being taken to protect against the contamination of food on the premises by pests

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http://www.cdc.gov/salmonella/montevideo/map.html

http://www.cdc.gov/salmonella/montevideo/epi_curve.html

•During 1/16-21/2010, epidemiologic study compared foods eaten by 41 ill and 41 well persons.

•Suggested peppered salami as a possible source of illness •Percent eating salami--Ill persons (58%); well persons (16%)

•Thirteen ill persons purchased the same type of sliced salami atdifferent grocery store locations.

•Suggested product may be the source of some illnesses. •Salami recalled 1/23/2010 (1,240,000 pounds).

•Products can have an extended shelf life of up to one year.•Manufacturer halted production of salami products.

•Using DNA analysis, Iowa's public health laboratory confirmed Salmonella isolated from leftover salami wasindistinguishable from the outbreak strain of Salmonella Montevideo

•Subsequent investigation points to pepper as Salmonella source

Salmonella Montevideo Outbreak - 2010

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MVLST

Multi-

Virulence-

Locus

Sequence

Typing

PCR DNA sequencing

Which has greater epidemiological relevance and information?

• PFGE (fragments)• MVLST (sequences)• Differences and consequences for

data interpretation and analysis– Direct vs. Indirect– Analog vs. Digital– Continuous vs. Discrete.

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MVLST

• MLST targets slowly diversified housekeeping gene sequences

• Virulence important in outbreaks• Virulence genes can evolve more rapidly

due to changes in environment– More sequence diversity– Greater discriminatory power

• Selected 3 virulence genes (prfA, inlB, and inlC) and 3 virulence-associated genes (dal, lisR, and clpP) of L. monocytogenes

28 SNPs in 6 MVLST genes were capable of differentiating all unrelated isolates

16 SNPs in 4 genes (prfA, inlB, inlC and lisR) could differentiate all 4 epidemic clones of Listeria monocytogenes and outbreak strains

Chen et al.

Neighbor-joining tree of 86 L. monocytogenes isolates based on the number of nucleotide differences in the 6 MVLST virulence gene fragments analyzed

Additional virulence genes (inlA and actA) had identical sequences within all 4 epidemic clonesand outbreak strains

Chen et al.

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Some conclusions

• Sequence-based vs RFLP approaches offer numerous advantages– Provide data that are not ambiguous

• “digital” not “analog”

– Provide much more information

– Take advantage of recent advances in:• Molecular Biology

• Genomics

• Bioinformatics

– Are highly portable via the web

Continued• MVLST provides data with high

epidemiological relevance– Targets virulence genes that are required

to cause an epidemic – Nucleotide sequences in virulence genes

are often highly variable in unrelated strains and high conserved in epidemic clones

• Identification of epidemic clones and epidemics

• Separation of outbreak and non-outbreak strains

Continued

• MVLST may be an excellent tool for molecular epidemiology:

– High discriminatory power

– High epidemiological relevance

– High reproducibility

– High typeability

– High portability

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Questions?