Foodborne Illnesses

• 76 million illnesses in the US• 325,000 hospitalizations • 5,200 deaths • Known pathogens account for an

estimated 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths annually

Microbes

• Bacteria, fungi, viruses, parasites

Foodborne Pathogens

• Escherichia coli 0157:H7 • Listeria • Salmonella • Shigella • Staphylococcus aureus (toxin) • Clostridium • Bacillus anthracis, B. cereus • Francisella• Enterobacter sakazakii • Fungi • Viruses (norovirus, Hepatitis)• Parasites (Cyclospora, Cryptosporidium)

Emerging pathogens• Campylobacter jejuni• Cryptosporidium parvum• Cyclospora cayetanensis• Escherichia coli O157:H7 and related E. coli (e.g.,

O111:NM, O104:H21 )• Listeria monocytogenes• Norwalk-like viruses • Salmonella Enteritidis• Salmonella Typhimurium DT 104 • Vibrio cholerae 01 • Vibrio vulnificus• Vibrio parahaemolyticus• Yersinia enterocolitica

Clinical Pathogens• Streptococcus• Staphylococcus (MRSA, VRSA)• Bacillus• Neisseria• Pseudomonas• Mycobacterium• Viruses

– HIV, Herpes, HPV, Hepatitis, Pox, Varicella

Infectious Dose

• Escherichia coli (E. coli) 10-100 [0]• Shigella 10-200 [0]• Listeria [0-100]• Salmonella 10 (or less?) [0]• Viruses 1-10 pfu [0]• Toxins pg-ng [0]

New Food Vehicles of Transmission

• Eggs (internal; Salmonella enteritidis)• Apple cider (E. coli O157:H7 )• Raspberries (Cyclospora)• Oysters (Norwalk viruses)• Produce (Salmonella, E. coli O157:H7 )

Indigenous microbial population on produce-

102 to 108 cfu/g

Needle in the Haystack

Environmental factors

Finding that needle

Targets: bacteria, viruses, protozoa, toxins, chemicals

Factors

• Target (pathogen, chemical, protein)• Food matrix• Contamination source(s)• Growth vs survival (cells)• Sampling• Methods: isolation vs detection

Sampling

• Non-homogenous distribution• Non-destructive means• Internalization (e.g. tomato)

Sample preparation

• Sample size (statistically valid)• Processing• Rinse vs soak (cantaloupes)• Stomaching vs blending

Ye Olde Dilemma

• DNA vs Protein– Target– Level (sensitivity)– Presence

Serologically-based

Detection of GMOs in Food

Food matrixExtraction (DNA or Protein)

Analysis (PCR or ELISA)

Confirmation

Our Yellow Corn Inventory at theOur Yellow Corn Inventory at the““CFSAN CommissaryCFSAN Commissary””

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CornCornMealMeal

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Corn FlakesCorn Flakes#1#1 #2#2

ControlControlFlourFlour

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Variability in DNA Template PreparationsVariability in DNA Template PreparationsFrom Finished Corn ProductsFrom Finished Corn Products

Sampling Size: 100 mgSampling Size: 100 mg Sampling Size: 5 gSampling Size: 5 g

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Corn Corn AldolaseAldolase Primers:Primers:

Genomic DNA:Genomic DNA:

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Popular Misconception aboutPopular Misconception aboutFood Sampling and Food Sampling and cry9Ccry9C AnalysisAnalysis

at FDA/CFSANat FDA/CFSAN

Food Matrix

• Inhibition of growth• Difficult to process• Interference in molecular-based assays

Isolation vs Detection

• Regulatory action based on….

FOR IMMEDIATE RELEASE March 1, 2007

FDA Update on Peanut Butter RecallSalmonella found in the ConAgra Plant

As a follow-up to the recent Salmonella outbreak linked to peanut butter, the U.S. Food and Drug Administration (FDA) is conducting an extensive inspection of ConAgra's Sylvester, Georgia processing plant. Samples collected by the FDA revealed the presence of Salmonella. The fact that FDA found Salmonella in the plant environment further suggests that the contamination likely took place prior to the product reaching consumers. Last week, tests by several states identified Salmonella in many open jars of Peter Pan and Great Value peanut butter recovered from consumers. In these instances, the Salmonella found in the plant and in the open jars matched the outbreak strain recovered from consumers who became ill.

Bacteria

Fujino, et al., 1953

Microbes

• Found in most foods• Most are not a health issue for man• Many benefits• However, can be a problem

– Illnesses– Intoxications (toxins)– Food spoilage

Spoiled Food• A good rule is, “When in doubt, throw

it out!”• Do not taste food that smells bad or

looks moldy or slimy. • Avoid cracked jars, or leaking, bulging,

badly dented or rusted cans.

Numbers game

• Infectious dose-number of pathogen (bacteria, viruses) that make people ill

• Range-very low (1-10) to very high (10,000)

• Ill people can shed millions to billions per day

• Pathogen can easily spread

Transmission

• Person to person• Food, water, air• Inanimate object to people

Methods

BacteriologicalConventionalModificationsChromogenic agarsEnrichment

Molecular-basedPolymerase chain reaction (PCR)

conventionalreal-time

DNA probesMicroarrays

Molecular Methods

• PCR– Conventional– Real time

• Microarrays• Biosensors

Conundrum• Live vs dead

– Demonstrate growth• Isolate colony• Broth (enrichment)/plate• Tissue culture• Phage

• Infectious vs non-infections– Viruses– Parasites

PCR

• In vitro DNA amplification system• Rapid (5 minutes to 2 hours)• Specific• Sensitive• Components: buffer, Mg, dNTP,

primers, template, enzyme

Potential PitfallsFalse negative reactions

Bacteria present but negative result- missing reactant- inhibitors present

False positive resultBacteria not present, PCR product

- contamination of reactants or pipettes

Live cells vs dead cells

PCR Steps1. Denaturation

heat (94-95 C)strand separation

2. Annealingtemperature variableprimers hybridize to template

3. Extensiontemperature (72 C)heat stable polymerase, dNTPs

DENATURATION• Strand separation• 94-96° C

ANNEALING• Oligo ‘attach’ to specific sites• Temperature critical

EXTENSION• Heat stable DNA polymerase

ANOTHER ROUND OF PCR

• Defined PCR products

PCR CYCLE• 25-30 cycles• Plateau affect

Agarose Gel Electrophoresis

Successful PCR

1. REACTION CONDITIONS- primer specificity- Mg concentration- temperature- no carryover- sensitivity

2. QUALITY OF TEMPLATE- no inhibitors- intact vs sheared

3. THERMOCYCLERS

Molecular Detection Methods

REAL-TIME PCR Detection

simultaneously quantifies and amplifies a strain-specific part of a given DNAmolecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction.

An Example of a Real-Time PCR detection probe

http://www.genetics.ucla.edu/sequencing/graphics/seminar_diagram.gif

Considerations for the Development of a

Universal PCR Detection Method• Independent of sample

matrix• food/water• environmental• clinical isolates

• Large spectrum of pathogens

• bacterial, parasitic protozoa, viral (?)

• Limited sample handling• extraction-less (?)• little or no template preparation

M l i l C bili

Food- and Water-borne Viral Diseases

• Viruses grow only inside host cells and are very host specific

• Much more difficult to culture than bacteria

• All foodborne viral pathogens are naked viruses– Very resistant to the environment

Viral Illnesses in the U.S. Estimated Cases

Norovirus- 23 million total cases annually9.2 million foodborne cases310 total deaths (6.9% fb)

Hepatitis A- 31 million total cases annually9.3 million foodborne cases 83 total deaths (0.2% fb)

Norovirus• Genus Norovirus, family Caliciviridae - a group of

related, single-stranded RNA, naked viruses that cause acute gastroenteritis in humans.

• Norovirus was recently approved as the official genus name for the group of viruses provisionally described as “Norwalk-like viruses” (NLV).

• Noroviruses are named after the original strain “Norwalk virus,” which caused an outbreak of gastroenteritis in a school in Norwalk, Ohio, in 1968.

• Currently, there are at least four norovirusgenogroups (GI, GII, GIII and GIV), which in turn are divided into at least 20 genetic clusters.

Norovirus infection

• The incubation period: 24 and 48 h• Symptoms usually last 24 to 60 h• Norovirus infection usually presents as

– Acute-onset vomiting– Watery non-bloody diarrhea with abdominal

cramps, and nausea– Low-grade fever also occasionally occurs– Dehydration is the most common complication,

especially among the young and elderly, and may require medical attention.

Norovirus Transmission• Highly contagious, and infectious dose as

few as 10 viral particles • Noroviruses are transmitted primarily

through the fecal-oral route, either by consumption of fecally contaminated food or water or by direct person-to-person spread. – Several modes of transmission

initial foodborne transmission in a restaurant, followed by secondary person-to-person transmission to household contacts.

Reverse Transcription• Necessary for conversion of ssRNA to

cDNA which serves as the template for the Polymerase Chain Reaction.

Norovirus GI- FAM labeled

Noro- Enterovirus 4 plex

Lane Descriptions (4% Agarose Gel)Lane 1- (0) dilution; Lane 2 (-1) dilution; Lane 3 (-2) dilutionLane 4- (3) dilution; Lane 5 (-4) dilution; Lane 6 (-5) dilutionLane 7- (6) dilution; Lane 8 (-7) dilution; Lane 9 (-8) dilution

100b

pla

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25 b

pla

dder

25 b

pla

dder

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G1GII

1 2 3 4 5 6 7 8 9

IRC

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BSE (Mad Cow Disease) Humans

Pure Protein ReplicativeParticles

• Originally believed to be unusual viruses• 1980, Stanley Prusiner of UCSF proposed

that the disease agent was a pure protein which could direct the formation of more protein like itself by serving as a template for folding.

• Term “prion” for proteinaceous infectious particle, with the o and the I reversed to make a nicer-sounding word.

Prions

• Single molecules containing about 250 amino acids. They are abnormal variants of proteins which normally occur in cells. Prions have the ability to convert the normal forms into abnormal forms.

• Cause transmissible spongiform encephalopathy (TSE)

• Fatal neurodegenerative diseases in humans and animals

Prions

• small proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids

• Prion diseases are often called spongiform encephalopathies– post mortem appearance of the brain with

large vacuoles in the cortex and cerebellum

Prions and Transmissible Spongiform Encephalopathy

• Prion hypothesis explains the transmission of several similar diseases of brain– Some appear to be genetic– Other appear to be transmissible– Disease is progressive and fatal,

beginning with one or two symptoms of brain dysfunction, such as confusion; ending with coma followed by death

Prions in animals• Scrapie: sheep • TME (transmissible mink encephalopathy):

mink • CWD (chronic wasting disease): muledeer,

elk • BSE (bovine spongiform encephalopathy):

cows • Feline spongiform encephalopathy (FSE):

Cat

Probably most mammalian species develop these diseases

Human prion diseases• CJD: Creutzfeld-Jacob Disease • GSS: Gerstmann-Straussler-Scheinker

syndrome • FFI: Fatal familial Insomnia • Kuru• Alpers Syndrome• Fatal Familial Insomnia (FFI)

The incidence of sporadic CJD is about 1 per million per year.GSS occurs at about 2% of the rate of CJD.

Toxins• Endotoxins• Exotoxins

– Cytotoxins: kill host cells in general or affect their function (anthrax toxin)

– Neurotoxins: specifically interfere with nerve cells function (bot toxin)

– Enterotoxins: affect cells lining the gastrointestinal tract (cholera, shiga and shiga-like toxins)

Endotoxins,

Part of the cell wall of Gram-negative bacteria. Only small amounts may escape into surrounding fluids from living bacteria. Greater amounts are released when the bacteria die and their cell walls disintegrate. Endotoxins are less potent, and larger amounts are needed to induce disease symptoms. Also, they are heat resistant and cannot be converted into toxoids.

ExotoxinsMainly proteins that are secreted by a bacterial cell into surrounding fluids, and are produced by both Gram-negative and Gram-positive bacteria. Most are readily destroyed by heat, but they can be converted into toxoids that are used as vaccines. Exotoxinsare extrememly powerful biological poisons as in the case of tetanus, where it has been estimated that 1 mg of purified tetanus toxin could kill millions of mice.

Exotoxins

• Important bacterial virulence factor• Small amounts exhibit toxic affect

– 1 mg of bot tox could kill 1 million guinea pigs

• Categorized by– Structure– Enzymatic mechanism of action– Subcellular target, e.g. host cell surface,

cytosol, nucleus

Subtyping methodsPulsed-field gel electrophoresis (PFGE) is a

standardized molecular subtyping (or “fingerprinting”) method for characterizing foodborne disease-causing bacteria. PFGE can be used to distinguish strains of organisms such as Escherichia coli O157:H7, Salmonella, Shigella, Listeria, or Campylobacter at the DNA level. DNA “fingerprints,” or patterns, are submitted electronically to a dynamic database at the CDC.

PFGE XbaI Pattern

CA Clinical

WI Clinical

PA Clinical

CA Cow

CA Spinach

CA Environment

CA Environment

Subtyping methods

• Multi-Locus VNTR Analysis (MLVA)• Single Nucleotide Polymorphisms

(SNPs)• Multi Locus Sequence Typing analysis

(MSLT)• 16S rDNA sequence analysis

Exploits the specificity of host-virus relationship and use the viruses as "tags" to identify microorganisms in the environment at the sub-species level.

SerotypingAntibodies are reacted against various surfaceantigens on the bacterium allowing for a diagnostic identification of the organism

Phage typing

Biochemical typingSome bacteria contain the enzyme urease, which catalyzes the hydrolysis of urea, yielding alkaline ammonia products which turn the pH indicator in the urease agar red.

Current Phenotypic Typing Strategies(non-automated)

(1) API (Biomerieux) (e.g., 20E)

Species/subspecies identification of Enterobacteriacae and other bacteria

(2) Enterotube (BBL)

Enterotube™ II Prepared multimedia tube for rapid identification of Enterobacteriaceae

Current Phenotypic Typing Strategies(automated)

(1) Dade-Microscan Walk-Away Systems

(2) Biomerieux Vitek

(3) Biolog Omnilog ID

Pulsed Field Gel Electrophoresis

XbaI-digested gel of Salmonella enterica subspecies I

PCR-Based Strain Fingerprinting Methods

Ribotyping REP-PCR RAPD

Size differences among amplicons distinguish closely-related strains

Single Nucleotide Polymorphism S

NucleotideSequence

GeneralPopulationAllele

DiscriminatingAllele

SNPs can be detected by:

Microarray

Pyrosequencing

Conventional methods

Pseudomonas Enterobacter

DNA Microarray Technology

Pyrosequencing Technology

http://www.nature.com/nmeth/journal/v2/n10/images/nmeth800-F1.gif

Multi Locus Sequence Typing analysis

• Based on nucleotide sequences of housekeeping loci• Uses conserved HK genes with minimal nucleotide changes and

important protein function• Multiple loci are targeted in this subtyping method• Simultaneous analysis of multiple genes scattered around the chromosome should yield a picture of the general taxonomic (evolutionary) relationships of bacteria

• Amenable to cladistic analysis (A classification method in which the members of a

taxa have been grouped together on the basis of a more recent common ancestor with

each other than with the other members of any other group)

MLST analysis of Escherichia coli

4 HK Genes

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“uropathogenic”

“diarrheagenic”

“commensal”

Spoilage• Food spoilage is caused by the growth of

microorganisms, primarily bacteria, molds and fungi, that convert nutrients into energy which they use for their own growth.

• Depletion of the nutrient content of foods as well as the secretion of byproducts from this biochemical process are two things which contribute to the spoilage of foods rendering it inedible (degradation of protein, carbohydrates, and fats by the microorganisms or their enzymes).

• Control of microbiological spoilage

Conventional Methods Are Often Time-Consuming

• Enumeration 2 days (molds longer)• Enrichment 1- 7 days• Purification and partial ID of isolates 1-2

days• Conventional ID of isolates 3 + days

• Total time for negative result 9 days• Total time for positive result 4 – 12 days

Molecular Detection Methods

Conventional and Real-Time PCR allows usto target specific bacterial pathogensdespite the background microbial load

So, what to eat…

• Handwashing- #1 means to prevent illness

• Pay attention• Anything-bon apetite