DNABased Methods for Identifying and Subtyping Foodborne Pathogens

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DNA-Based Methods for Identifying and
Subtyping Foodborne Pathogens
Bhushan Jayarao, MVSc, PhD, MPH Extension
Veterinarian Pennsylvania State
University Department of Veterinary Science
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Why do we need to identify bacteria in food ?

• Safeguard human health
• Prevent spoilage of food

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Why do we need to identify foodborne pathogens ?

• Important for
• Diagnosis
• Surveillance
• Epidemiology
• Control of disease

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Conventional Biochemical Tests
Gold Standard

• This classical approach is the basis for
  classification of bacteria into species
• Phenotypic characterization
• Time consuming
• Subject to variability
• Expensive

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Rapid Identification Tests

• First made available in the early 1980s for
  several groups of bacteria
• Alternate approach to conventional tests
• Less time, labor and set up costs
• Cost
• Extensively evaluated
• Now accepted by most microbiologists

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Different Types of Rapid Tests

• Ability to identify bacterial species based on
• Biochemical tests
• Biochemical and enzymatic tests
• Immunoassays

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Biochemical Enzymatic- Based Rapid ID Tests
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VIP for EHEC (from BioControl)
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Agglutination-based ID Tests
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Into the 90s Next Millennium !

• Status on available rapid tests
• 48-72 h variable accuracy (70 - 95)
• Faster the test, more expensive it is !
• Affordable ?
• By whom ?
• International trade
• Less time (8-12 h)
• More accuracy

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Into the 90s Next Millennium !

• DNA-based rapid assays
• More accurate
• Less time
• Cost ?

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• Whats DNA ? Deoxyribonucleic
  acid - a molecule that is
  the primary carrier of genetic information
• Where is DNA located ? DNA is located on the
  chromosome

• Number of chromosomes
• Human 23 pairs
• Cow 30 pairs
• Bacteria 1 circular

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The DNA within a chromosome is double
stranded The two strands pair very specifically
with one another to form a kind of a braid known
as DNA helix
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The Holy Grail ?????
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• Each braid is made-up of individual substances
  called as nucleotides that are held together by a
  sugar and phosphate molecule
• The nucleotides are
• A adenine
• T thymidine
• C cytosine
• G guanine
• The T always links up with an A on the opposite
  braid, while the C always links up with G on the
  opposite braid

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T-T-G-A-C-T-A-T-C-C-A-G-A-T-C I I I
I I I I I I I I
I I I IA-A-C-T-G-A-T-A-G-G-T-C
-T-A-G

• Nucleotides arranged in long line in a particular
  sequence, they carry the information for a
  particular trait or character ----
• the nucleotides that encode the information for
  the trait/character are collectively called as a
  gene.
• On a chromosome
  there may be several hundred genes.

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Why are we excited about using DNA-based tests ?

• BECAUSE !!!!!!
• Techniques are now available that permit analysis
  of a gene to the extent of a finding a
  difference in one nucleotide in the whole gene !

T-T-G-A-C-T-A-A-C-C-A-G-A-T-C I I I I I
I I I I I I I I I
IA-A-C-T-G-A-T-T-G-G-T-C-T-A-G
E. coli isolate A E. coli isolate B
T-T-G-A-C-T-A-C-C-C-A-G-A-T-C I I I I I
I I I I I I I I I
IA-A-C-T-G-A-T-G-G-G-T-C-T-A-G
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Advantages ?

• Accurate
• differences at single base pair level can be
  determined
• independent of culture conditions
• Rapid
• allows identification of species within 12 to 24
  h.
• Test format
• some tests still require some degree of
  culturing
• interference from food matrix
• Cost
• expensive !!!

Dis-advantages ?
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How do DNA-based ID tests work ?

• DNA hybridization
• followed by detection with
• Dot blot assay
• Fluorescence or Chemiluminesence
• Polymerase Chain Reaction (PCR)
• followed by detection with
• Gel electrophoresis
• ELISA
• Fluorescence or Chemiluminesence

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1. Sample Preparation
2. Hybridization 3. Detection
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Gen-Trak Assay
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Qualicon
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DNA-based Assays for Subtyping Foodborne Pathogens
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Why do we need to subtype bacteria ?

• Example
• Between Oct 1 - 30 1997, E. coli O157H7 isolated
  in Kansas State from
• Feces from cull cows
• Beef
• Lettuce
• A 3 year old child with HUS
• CDC P observed that all of the 4 isolates had
  the same biochemical profile
  
  
  
  
  
  Do you conclude that
  all the 4 isolates are same and could be from the
  same source ?

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• Bacteria belonging to the same species could have
  similar biochemical profiles, BUT still be
  genetically very different !
  
  
  
  
  
• DNA-based methods allow discrimination of strains
  that are indistinguishable based on biochemical
  or serological tests
• Several methods have been developed
• Riboprinter
• Pulsed field gel electrophoresis

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Riboprinter (from Qualicon a subsidary of
DUPONT)
Salmonella
Shigella
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Pulsed Field Gel Electrophoresis
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How does PFGE work ?
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PFGE

• PFGE now accepted as a gold standard for
  differentiation of strains
• A technique adopted by CDC-P for subtyping
  foodborne pathogens
• 8 Food net Centers- personnel will be trained in
  PFGE
• Computerized database at CDC-P for cross
  reference of isolates
• Tracking of isolates
• Emergency response
• Assist Epdemiological studies
• Develop Control and Education programs

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Summary

• Rapid tests based on DNA analysis
• More test kits on the market
• Less expensive
• Microbiological laboratories will soon learn and
  accept to use DNA-based tests
• Tests more sensitive
• HOWEVER
• some basic microbiology work will be still
  involved