Transcript NSPCD LABORATORIES - World Health Organization

Typing
Typing methods for bacteria
May 2007
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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Learning objectives
At the end of the presentation, participants should:
• Identify situations when typing is relevant
• Know different methods of typing
• Understand problems that arise when using typing methods
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Isolate versus Strain
Isolate: a pure culture derived
from a single colony that is
presumed to arise from a single
organism/bacterium
Strain: a set of isolates, that
when typed are indistinguishable
from each other and can be
differentiated from other isolates
OUTBREAK
OUTBREAK STRAIN
STRAIN 4
STRAIN 2
STRAIN 5
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STRAIN 6
A simple question ?
Are these isolates the same or different?
Through a typing method we are looking for:
• Epidemiologically linked isolates that represent the
clonal expansion of a single precursor
• Clonal isolates are the same type and unrelated
isolates have a different type
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Typing system evaluation criteria
Typeability
Capacity to produce clearly interpretable results with
most strains of the bacterial species
Reproducibility
Capacity to repeatedly obtain the same typing profile
result with the same bacterial strain
Discriminatory
power
Ability to produce results that clearly allow
differentiation between unrelated strains of the same
bacterial species
Practicality
(ease of
performance &
interpretation)
Method should be versatile, relatively rapid,
inexpensive, technically simple and provide readily
interpretable results
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Phenotype & genotype bacteria
Capsule
Polysaccharides
GENOTYPE
(Chromosomal &
plasmid DNA)
Fimbirae
(M-protein)
Teichoid acid
Lipoteichoic acid
Different
Enzymes
Peptidoglycan
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PHENOTYPE
Typing methods
Phenotypic
• Rely on expression of phenotypic characteristics
(genetically coded)
– Antibiotic resistance, antigens etc.
Genotypic
• Analysis of the genetic material
– DNA, RNA
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Phenotypic techniques
Serotyping
Phage typing
Antimicrobial resistance monitoring
Multilocus enzyme electrophoresis (MLEE)
Other:
•
Protein profiling – SDS PAGE, immunoblotting
•
Based on nutritional requirement e.g. auxotyping
•
Biotyping
•
Bacteriocin typing
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Phenotypic techniques
Serotyping
• Antigenetic determinants expressed on the cell surface
• Still widely used for Salmonella, Shigella, Neiseria, E. coli, V cholerae
• Slide/ tube agglutination
• LIMITATION: Requires extensive stock of absorbed/monoclonal sera
(e.g. >2200 antisera required for definitive Salmonella typing)
Phage typing
• Viruses that infect and destroy bacterial cells –Bacteriophage
• The resistance or susceptibility of strains is used for differentiation
• LIMITATION: Technically demanding, time consuming, typeability is an issue
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Phenotypic techniques
Antibiotic susceptibility testing
• Based on susceptibility of bacterial isolates to a panel of
antimicrobial agents
• Routinely performed on clinical isolates
• A reasonable preliminary indicator to initiate epidemiological
action
LIMITATIONS:
– Antibiotic resistance under extraordinary selective pressure
– Multiple mechanisms for a strain to become abruptly resistant
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Phenotypic Techniques
Phenotypic characteristics can vary in different conditions
• Antibiotic resistance can be expressed under antibiotic
pressure
Methods are not very discriminatory
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MLEE
Characterizes the cellular proteins by electrophoretically
separating them in a gel matrix
Exposing the gel to chromogenic substrates (that react
with the enzymes)
Limitation: Complexity of interpretation
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Phenotypic typing system
characteristics
Typing system
Typeability
Reproducibility
Discrimination
Ease of
interpretation
Ease of
performance
Most
Good
Fair
Good
Fair
Most
Fair
Fair
Fair
Poor
All
Fair
Poor
Excellent
Excellent
All
Excellent
Good
Excellent
Good
Serotyping
Phage typing
Antibiotic
susceptibility
testing
MLEE
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Phenotypic typing during an outbreak
Outbreak of Paratyphi B salmonellosis phage type 1var3
France, 1993
Cases
Phage type "1var3"
30
Other phage types
25
20
15
10
5
0
July
August
September
E P I D E M I C
A L E R T
October
A N D
November
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December
DNA molecule
Source: Wikipedia, created by Michael Ströck
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Genotypic methods
Plasmid profiling
Restriction enzyme analysis (REA)
Restriction fragment length polymorphism (RFLP)
Ribotyping
Pulse Field Gel Electrophoresis (PFGE)
Random Amplified Polymorphic DNA (RAPD)
Nucleic acid sequencing
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The principle
Without amplification
•
Cutting the DNA in pieces
•
Visualizing the pieces
With amplification
•
Amplifying (using PCR) parts of the DNA
•
Visualizing the pieces
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Genotypic typing methods
Methods without prior amplification
•
Isolation of the pathogen
•
Extraction of the DNA
•
Cutting the DNA with Enzymes (restriction endonuclease
enzymatically cuts/ “digests” DNA at a specific/ “restricted”
nucleotide recognition sequence)
•
Separation of the pieces by size using an electric field (GelElectrophoresis)
•
Visualization
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Example of molecular typing

Gel-Electrophoresis


Size of
fragments

Cutting
locations


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Restriction Enzyme Analysis
(REA)
•Extraction
of plasmid or chromosomal
DNA
•Digestion
of the DNA at particular sites
using specific restriction enzymes
•Hundreds
of DNA fragments of various
sizes (0.5-50Kb) separated by gel
electrophoresis
•LIMITATION:
Complex profiles with
hundreds of unresolved or overlapping
bands
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Southern blot analysis of RFLP
& ribotyping
Better analysis of restriction enzyme patterns
•
Specific parts of the pieces are detected by pieces of
DNA as a probe - Southern Blot
•
Variation in number & size of fragments detected by the
markers are referred to as restriction fragment length
polymorphism (RFLP)
•
Ribotyping: when probes mark ribosomal operons
LIMITATIONS: technically complex, organisms with single
copy of ribosomal operons
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Southern/Northern blotting
Separate DNA
fragments on
the agarose gel
Visualization reveals a
band where your
probe bound to the
target sequence
"Blot" DNA to
membran
Membrane
imprinted with
DNA bands
Add a labelled
probe to the
membrane
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Visualization with a marker
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Adapted from: http://lifesciences.asu.edu/resources/mamajis/southern/southern.html
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Pulsed-field gel electrophoresis
(PFGE)
• Rare cutting enzymes
• Alternate current orientations allow separation of large DNA fragments
• Highly discriminatory and reproducible; currently the method of choice for
typing a range of bacteria
LIMITATIONS: time consuming (≥2 days), expensive, specialized equipment
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Pulsed-field gel electrophoresis
(PFGE)
RFLPs of VRE isolates
as determined by
PFGE; all appear
identical
RFLPs of two strains (B & C)
from a patient as determined
by PFGE; both different
implying mixed infection;
lane A is marker
RFLPs of MRSA isolates with
similar ABT ST profile as
determined by PFGE; only
isolates B & C are identical
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Genotypic typing methods
Methods with prior amplification
•
Extraction of the DNA, separation
•
Target with primer
•
Amplification of specific region
•
Separation of amplicons according to size using an
electric field (gel-electrophoresis)
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Molecular typing
Gel-Electrophoresis
Primerlocations
Size of
(amplified)
fragments
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Random Amplification of
Polymorphic DNA (RAPD )
Uses short primers that find a lot of targets
Different size amplicons
Products separated by electrophoresis
LIMTATIONS:
• Identification of suitable primers
• Difficult to interpret differences in the intensity of bands
• Inefficient reactions
• Amplification of cryptic genetic material (prophages,
bacteriophages)
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RAPD-PCR
60
70
80
90
100%
10 Isolates,
two clusters
(3 isolates
each)
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Nucleic acid sequencing
Enumeration of individual nucleotide base pairs
Provides highly reliable and objective data suitable for
subsequent quantitative analysis
Necessary for virus typing
LIMITATIONS:
• Locus with sufficient sequence variability
• Sequencing of a single locus may not be reliable result
• Prohibitively expensive for most settings
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Multi Locus sequence typing
(MLST)
Targets different DNA pieces and sequences them
Compares results with data banks
Pro: highly comparable
Con: expensive equipment
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Genotypic typing system
characteristics
Typeability
Reproducibility
Discrimination
Ease of
interpretation
Ease of
performance
All
Good
Good
Poor
Excellent
All
Excellent
Fair
Good
Good
All
Excellent
Excellent
Excellent
Good
Restriction
digests of
PCR products
All
Excellent
Good
Excellent
Good
PCR based on
repeated
sequences
All
Good
Good
Good
Good
RAPD
All
Fair
Good
Fair
Good
Nucleotide
sequencing
All
Excellent
Excellent
Excellent
Fair
Typing system
REA
Ribotyping
PFGE
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Limitations of typing methods
•
Discriminatory function
•
Type of material and pathogen
•
Reproducibility , cost, technique, etc.
•
No “gold standard”
RESULTS OF A TYPING SYSTEM SHOULD BE
CONSIDERED RELATIVE TO THE AVAILABLE
EPIDEMIOLOGICAL DATA OR TO THE RESULTS OF
OTHER SYSTEMS
•
The technique used needs to be adapted to the question
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Llisteriosis outbreaks, France 1999-2000
14
12
10
Other sporadic cases
Sporadic cases used as controls
Outbreak 2 (32 cases)
Outbreak 1 (11 cases)
8
6
4
2
0
Laboratory Training for Epidemiologists
8
6
4
January February
2000
2
52
50
48
46
November December
1999
44
42
October
40
Cases
March
Interpretation of strain typing data
Several factors affect interpretation:
Natural biologic variation
• Epidemiologically related isolates of the same strain
demonstrate minor typing differences due to phenotypic
variations or actual genotypic alterations, when
collected and examined over an extended interval
Technical variations
• Limited reproducibility and discriminatory power
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Interpretation of typing results
Genetic relatedness assessed with clinical and
epidemiologic relatedness
Restrict analysis to discrete set of isolates (≤30)
Identify “index isolate” as starting point for analysis that is
defined on the basis of:
• Epidemiological data (first case in an outbreak)
• Clinical data (initial isolate from patient with multiple infections)
• Strain typing data (most common strain type in the set)
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Interpretation of typing results
Multiple isolates representing a single type are most
appropriately designated “indistinguishable”
No typing method confirms that entire genomes of two
organisms are identical
Indistinguishable vs. closely related vs. possibly related
→Final assessment lies with integration of molecular and
epidemiological analyses
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Problems with result interpretation
Types versus subtypes
Isolates assigned as different types if differ in some specified manner (2
or more band shift in S blot)
Isolates that differ but not sufficiently to be designated as distinct types
are designated as subtypes of similar types
Restriction fragments of different sizes may represent same
chromosomal DNA
Insertion or deletion of extra-chromosomal DNA such as
bacteriophage DNA
DNA fragment data are not suitable for quantitating genetic
relatedness among different isolates
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Application of typing systems
Detection of outbreaks:
Concept of “prior probability”; rigorous epidemiological
investigation and data to avoid misleading results
• Increased prevalence
Epidemiological
investigation
• Same bacteria species
from a cluster of cases
• Multiple isolates with
distinct biotype/ AST
pattern
Request for Molecular
typing
Laboratory Training for Epidemiologists
Typing
technique with
good
reproducibility &
discriminatory
power
Applications of typing systems
Distinguish relapse from re-infection
Identify types associated with increased transmission & virulence
Emergence of new types ; implications on control measures
Clonality of acute infection:
•
Infection vs. colonization vs. sample contamination
•
Pseudo-outbreaks related to:
– Clinician or clinical entity
– Laboratory
– Case finding
– Chance clustering
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Phenotypic vs. genotypic typing of
Staphylococcus aureus
Method
Discriminatory
index
Percentage
Typeability
0.556
30
ARM
.880
100
++++
All laboratories
Coagulase typing
.659
100
++++
All laboratories
+++
Molecular
biology
laboratory
+
Molecular
biology
laboratory
++
Molecular
biology
laboratory
Phage typing
Protien profiling –
SDS PAGE,
Immunoblotting
.978
100
Ease of
Basic set up
performance
Reference
laboratory
Ribotyping
.845
100
PFGE
.986
100
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To summarize
Typing data are most appropriately evaluated in the
context of a hypothesis and questions thoughtfully
developed by the clinician or the epidemiologist. They
should augment rather that replace those analyses
Typing is performed independently by the laboratory to
avoid any bias but the results are considered
collaboratively
Laboratory Training for Epidemiologists
Typing
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
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