E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists
Typing
May 2007
Typing methods for bacteriaTyping methods for bacteria
E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists
Learning objectivesLearning objectivesAt 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
Isolate versus StrainIsolate versus StrainIsolate: 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 2
STRAIN 3
STRAIN 4
STRAIN 5
STRAIN 6Laboratory Training for Epidemiologists E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists
A simple question ?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 criteriaTyping 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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Teichoid acid
Lipoteichoic acid
Capsule
Polysaccharides
Fimbirae (M-protein)
Different Enzymes
Peptidoglycan
Phenotype & genotype bacteriaPhenotype & genotype bacteria
PHENOTYPEGENOTYPE
(Chromosomal & plasmid DNA)
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Typing methodsTyping methodsPhenotypic
• 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 techniquesPhenotypic techniquesSerotyping
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 techniquesPhenotypic techniquesSerotyping
• 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 techniquesPhenotypic techniquesAntibiotic 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 TechniquesPhenotypic TechniquesPhenotypic characteristics can vary in different conditions
• Antibiotic resistance can be expressed under antibiotic pressure
Methods are not very discriminatory
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E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists
MLEEMLEECharacterizes the cellular proteins by electrophoreticallyseparating them in a gel matrix
Exposing the gel to chromogenic substrates (that reactwith the enzymes)
Limitation: Complexity of interpretation
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Phenotypic typing system Phenotypic typing system characteristicscharacteristics
Typing system
Typeability Reproducibility Discrimination Ease of interpretation
Ease of performance
Serotyping Most Good Fair Good Fair
Phage typingMost Fair Fair Fair Poor
Antibiotic susceptibility
testingAll Fair Poor Excellent Excellent
MLEEAll Excellent Good Excellent Good
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Cases
July August September October November December
Phenotypic typing during an outbreakPhenotypic typing during an outbreakOutbreakOutbreak of Paratyphi B salmonellosis phage type 1var3 of Paratyphi B salmonellosis phage type 1var3
France, 1993France, 1993
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DNA moleculeDNA molecule
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Source: Wikipedia, created by Michael Ströck
Genotypic methodsGenotypic methodsPlasmid profiling
Restriction enzyme analysis (REA)
Restriction fragment length polymorphism (RFLP)
Ribotyping
Pulse Field Gel Electrophoresis (PFGE)
Random Amplified Polymorphic DNA (RAPD)
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The principleThe principleWithout 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 methodsGenotypic typing methodsMethods 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 (Gel-Electrophoresis)
• Visualization
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Example of molecular typingExample of molecular typing
Cutting locations
Gel-Electrophoresis
Size of fragments
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Restriction Enzyme Analysis (REA)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 Southern blot analysis of RFLP & ribotyping& 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 singlecopy of ribosomal operons
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Southern/Northern blottingSouthern/Northern blotting
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Separate DNA fragments on the agarose gel
"Blot" DNA to membran
Membrane imprinted with DNA bands
Add a labelled probe to the membrane
Visualization reveals a band where your probe bound to the target sequence
Visualization with a markerVisualization with a marker
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Adapted from: http://lifesciences.asu.edu/resources/mamajis/southern/southern.html
Pulsed-field gel electrophoresis (PFGE)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 Pulsed-field gel electrophoresis (PFGE)(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 methodsGenotypic typing methodsMethods 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 typingMolecular typing
Primer-locations
Gel-Electrophoresis
Size of (amplified) fragments
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Random Amplification of Polymorphic Random Amplification of Polymorphic DNA (RAPD )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-PCRRAPD-PCR
60 70 80 90 100% 10 Isolates, two clusters
(3 isolates each)
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Nucleic acid sequencingNucleic acid sequencingEnumeration of individual nucleotide base pairs
Provides highly reliable and objective data suitable forsubsequent 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 Multi Locus sequence typing (MLST)(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 Genotypic typing system characteristics characteristics
Typing system Typeability Reproducibility Discrimination Ease of interpretation
Ease of performance
REA All Good Good Poor Excellent
Ribotyping All Excellent Fair Good Good
PFGE 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
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Limitations of typing methodsLimitations 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 1999-2000Llisteriosis outbreaks, France 1999-2000
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Sporadic cases used as controls
Outbreak 2 (32 cases)
Outbreak 1 (11 cases)
October November December January February March 1999 2000
Cases
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Interpretation of strain typing dataInterpretation of strain typing dataSeveral 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 resultsInterpretation of typing resultsGenetic relatedness assessed with clinical andepidemiologic relatedness
Restrict analysis to discrete set of isolates (≤30)
Identify “index isolate” as starting point for analysis that isdefined 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 resultsInterpretation of typing resultsMultiple isolates representing a single type are mostappropriately designated “indistinguishable”
No typing method confirms that entire genomes of twoorganisms 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 interpretationProblems 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 systemsApplication of typing systems
Detection of outbreaks:
Concept of “prior probability”; rigorous epidemiologicalinvestigation and data to avoid misleading results
Epidemiological investigation
Request for Molecular typing
• Increased prevalence
• Same bacteria species from a cluster of cases
• Multiple isolates with distinct biotype/ AST pattern
Typing technique with
good reproducibility & discriminatory
power
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Applications of typing systemsApplications of typing systemsDistinguish 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 Phenotypic vs. genotypic typing of Staphylococcus aureusStaphylococcus aureus
Method Discriminatory index
Percentage Typeability
Ease of performance
Basic set up
Phage typing0.556 30 Reference
laboratory
ARM .880 100 ++++ All laboratories
Coagulase typing .659 100 ++++ All laboratories
Protien profiling – SDS PAGE, Immunoblotting
.978 100 +++Molecular
biology laboratory
Ribotyping .845 100 +
Molecular biology
laboratory
PFGE.986 100 ++
Molecular biology
laboratory
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To summarizeTo summarizeTyping 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
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E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists
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
Typing