General Presentation Tips

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General Presentation Tips
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Look at the Guidelines

• Tell a story
• Do not read slides
• Total Points 25
• Introduction/Objectives 5
• Methods 2
• Results Discussion 7
• Conclusion 5
• References 2
• Grammar, neatness professionalism 4

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Detection of Escherichia coli O157H7
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Characteristics of E. coli O157H7

• Non-sporulating, Gram-negative, facultative rod
• Cattle is the main reservoir
• Ground beef, water, milk, apple juice and produce
• Produces Shiga-toxin (Stx)
• Enterohemorrhagic E. coli (EHEC)
• 73,000 cases (HC/HUS) 61 deaths/year (CDC)
• Low infective dose

Jay et al., Modern Food Microbiology, 6th edition
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Controls
x 2
Enrichment Food/mECN
E. coli ATCC 11775
Incubation at37 oC, 24 h
E. coli O157H7
E. faecalis
Rapid Method Multiplex PCR
Isolation Rainbow agar
x3
Only MUG (-)PRMB
Tryptose broth
Confirmation Phenol red mannitol
broth (PRMB)
x3
Confirmation Indole test
x3
Fig. 10.1. Detection of Escherichia coli
O157H7 in food as done in this laboratory
exercise
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Conventional Method - Media

• Modified EC broth with novobiocin (mEC-N)
• Lactose fermented by coliforms to acid and gas
• Bile salts inhibits Gram positive bacteria
• Novobiocin suppresses Gram positive and some
  Gram negative bacteria (most Enterobacteriaceae
  are resistant)
• Rainbow Agar
• Novobiocin inhibits Proteus swarming
• Chromogenic substrates ß-galactosidase
  (blue/black colonies) and ß-glucuronidase (red
  colonies) activity
• E. coli O157H7 produces black or gray colonies
  (lacks ß-glucuronidase)

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Conventional Method - Media

• Phenol Red Sorbitol Broth Phenol red
• pH indicator, detect acidic end products of
  sorbitol fermentation
• Sorbitol sole carbon source
• Durham tubes detect gas production
• E. coli O157H7 does not ferment sorbitol, other
  E. coli ferment sorbitol
• Tryptose Broth
• Non-selective
• Used as a vehicle for indole test
• Indole Test
• EHEC strains
• Decompose tryptophan (from peptone) to indole
• Reaction of indole with Kovacs reagent red
  layer is positive

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Food Sample
Food Sample
Controls(E. coli O157H7, E. coli ATCC 11775,
and E. faecalis)
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Enrichment
Incubation
mEC-N
mEC-N
Transfer
Transfer
Centrifuge tubes
DNA extraction
Centrifuge and Resuspend in Sterile Water
Laboratory Period
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Boil for 10 min
Add DNA sample to PCR tubes.
DNA amplification
PCR Reaction mixture (Water, Reaction Buffer,
primers, dNTPs, Taq)
PCR tube
Run the thermocycler. Cool or refrigerate
Gel electrophoresis
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Gel with DNA bands
Electrophoresis
Identify DNA bands that corresponds to E. coli
O157H7
Fig. 10.2. Multiplex PCR procedure for
identification of the presence of E. coli O157H7.
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Multiplex PCR Result
E. coli O157H7
100 bp DNA ladder
E. coli O157H7
E. coli K12
E. coli K12
P. fluorescens
S. aureus
wec gene involved in synthesis of Enterobacterial
Common Antigen rfb gene encodes enzyme necessary
to synthesize O-antigen (LPS) fliC gene encodes
for flagellar antigen (H)
547 bp (fliC)
259 bp (rfb)
103 bp (wec)