Lab 8' Strain typing of Bacteria:

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Lab 8. Strain typing of Bacteria Pulse-Net

• Purpose
• Monitor foodborne disease case clusters by
  pulsed-field gel electrophoresis (PFGE).
• Facilitate early epidemiology of common source
  outbreaks,

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PulseNet, began in 1996. The there were 10 labs
typing Escherichia coli O157H7. As of 2001,there
were 46 state and 2 local public health labs and
the food safety labs of the U.S. Food and Drug
Administration and the U.S. Department of
Agriculture all participate
Impetus for a single unified pathogen monitoring
effort to rapidly assess disease
outbreaks. 1993,large outbreak of E. coli
O157H7 in seven western United States. CDC
performed DNA "fingerprinting" by pulsed-field
gel electrophoresis (PFGE) Single strain of E.
coli O157H7 found in patients was same as the
strain found in hamburger patties served at a
large chain of regional fast food
restaurants. Rapid ID estimated to prevent 800
Illnesses
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RFLP, PFGE and INLV DNA macrorestriction
(restriction enzymes that cut genomic DNA
infrequently and thus generates a small number
(usually 10-20) of restriction fragments)
fragments (gt100,000 bp) are resolved by
pulsed-field gel electrophoresis (PFGE) (capable
of separating yeast chromosome-sized DNAs). PFGE
facilitates the differential migration of large
DNA fragments through agarose gels by constantly
changing the direction of the electrical field
during electrophoresis. The contour-clamped
homogeneous electric field (CHEF) gel
electrophoresis method has become the method of
choice for PulseNet PFGE. Other types include
linear pulsed field gel electrophoresis (LPFGE)
and field inversion gel electrophoresis
(FIGE). Lindstedt BA, Heir E, Gjernes E,
Vardund T, Kapperud G. DNA fingerprinting of
Shiga-toxin producing Escherichia coli O157 based
on multiple-locus variable-number tandem-repeats
analysis (MLVA). Ann Clin Microbiol Antimicrob.
20032129. 69 strains by PFGE 44 types
by MLVA 45 types
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VNTR Tandem Repeats
Replication (Striesinger) Slippage Mechanism
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EXAMPLE 2006 Spinach Outbreak of EHEC O157 H7.
Same source? 09/27/06 199 cases 102
hospitalized 31 Hemolytic uremic syndrome (HUS)
Deaths 3 WI, MD ID
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Oregon 6/1-10/9, 2006. Reported O157 EHEC
infections
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Oregon 6/1-10/9, 2006. Reported O157 EHEC
infections, strains ID via PulseNet
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• The CDC now publishes protocols for 6 bacteria
• Campylobacter jejuni
• Escherichia coli O157H7
• Listeria monocytogenes
• Salmonella
• Shigella
• Vibrio cholerae.

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• This Thursday..Dry Lab
• Discussion of two methods
• Pulsed Field Gel Electophoresis (PFGE) Genomic
  Restriction Fragment Length Polymorphism (RFLP)
• Multiple-locus variable-number tandem-repeats
  analysis (MLVA) via Capillary Gel Electrophorsis
  (general concepts only)