Molecular Techniques for Outbreak Investigations

1
Molecular Techniques for Outbreak Investigations

• Dr. Ross Davidson
• Associate Director, Microbiology
• Queen Elizabeth II Health Sciences Centre

2
Typing Systems

• Hypothesis - series of isolates obtained from an
  epidemiologic cluster are clonally related
• Nosocomial infections VRE, MRSA
• Community E.coli 0157H7, Salmonella
• Nursing Home S.pneumoniae, S.pyogenes

3
Typing Systems

• Premise related isolates will share
  characteristics by which they can be
  differentiated from unrelated isolates
• Characteristic utility for typing is related to
  its stability within a strain and its diversity
  within a species.

4
Typing Systems

• Typeability
• Ease of
  interpretation
• Reproducibility
• Ease of
  performance
• Discriminatory power

5
Typing Systems

• Phenotypic systems Biotyping
• 
  Antibiograms
• Phage
  Typing
• 
  Serotyping
• -based on presence or absence of metabolic /
  biologic activities
• -organisms can alter expression of biologic
  properties
• - mutation, environment

6
Typing Systems

• Genotypic Systems (Molecular)
• substantial genetic diversity within microbial
  species
• evolutionary divergence - accumulation of
  non-lethal mutations, deletion and acquisition of
  DNA

7
Nucleic Acid as Template

• Relatively Stable.
• Ubiquitous in nature.
• Purification processes are virtually identical,
  irrespective of organism.

8
Nucleic Acid Structure
A T C G G C A T C G A T
- - - - - - - - - - - -
- - - - - - - - - - - -

T A G C C G T A G C T A

9
Polymerase Chain Reaction(PCR)

• Target site amplification
• -ability to rapidly and exponentially amplify
  (replicate) a particular DNA sequence
• -Rapid
• -Sensitive
• -Specific

10
Components of the PCR
CGCTTCAAAG
AGATCGCTTA
Primer 2
Primer 1
Template DNA
T
C
DNA polymerase (Thermostable)
T
C
A
A
Taq
G
dNTPs
G
11
Thermocycling Profile
100 80 60 40 20
Denature 94C
Extend 72 C
Temp C
Anneal 55 C
12
PCR
94C
55C
Taq
Taq
72C
13
Analysis of PCR Products
-
M 1 2 3 4

Gel Electrophoresis

14
Pulsed Field Gel Electrophoresis (PFGE)

• Based on digestion of high MW DNA with a rare
  cutting restriction endonuclease
• Electrophoresed using a system of alternating
  current angles

--



- - - - -
- - - - -
- - - - -

15
Pulsed Field Gel Electrophoresis (PFGE)

• Methodology
• Isolates embedded in high-grade agarose
  - inhibits shearing of DNA
• Bacterial cell wall, proteins, etc digested
• DNA cleaved with rare cutting restriction enzyme
  - 10 Kb to 1 Mb

16
Pulsed Field Gel Electrophoresis (PFGE)

• Advantages of PFGE- Highly discriminatory
• - Reproducible (inter and intra lab)
• - Adapted to most organisms
• - outbreak investigation
• - tracking evolution or long term spread of
  pathogens
• Disadvantages
• - Initial capital outlay expensive
• - Requires skilled technologists

17
Pulsed Field Gel Electrophoresis (PFGE)

• Interpretive Criteria
• Indistinguishable banding pattern same number
  and size
• Closely related banding pattern differs due to
  a single genetic event (usually, 2 - 3 band
  difference)
• Possibly related- banding pattern differs due to
  2 independent genetic events (usually, 4 - 6 band
  difference)
• Unrelated banding pattern differs due to
  changes consistent with ? 3 genetic events.

18
PFGEInterpretation
19
Pulsed Field Gel Electrophoresis (PFGE)
20
16s rRNA gene sequencing

• Common to all bacteria
• Essential gene - codes for small ribosomal
  subunit
• 16s rRNA gene evolves at an extremely slow rate.
• Molecular Clock

21
16s rRNA gene sequencing

• Contains both highly conserved and highly
  variable regions
• Conserved regions are very highly conserved among
  all bacterial species
• Variable regions are different between species,
  but highly conserved within a species

22
16s rRNA gene sequencing

• Molecular diagnostics exploits the properties of
  the gene.
• PCR primers designed to anneal to conserved
  regions - Any organism present will be
  amplified
• PCR reaction is designed such that the amplicon
  will contain several variable regions

23
16s rRNA gene sequencing

• The variable regions of the DNA product
  (amplicon) are sequenced
• DNA sequence is compared to a large database of
  known sequences / organisms
• Organism identified according to best match

24
16s rRNA Gene
Variable
Conserved
1.5 Kb
25
16s rRNA gene sequencing

• Advantages
• No need for viable organisms - PCR is
  performed directly on specimens
• Detection / identification of organisms in
  partially treated patients
• Detection / identification of non-growing / slow
  growing organisms

26
16s rRNA gene sequencing

• Disadvantages
• Only useful from normally sterile sites
• Only useful for mono-microbial infections
• Contamination during specimen collection may be a
  significant problem
• May take a week to obtain identification