MOLECULAR TYPING OF
MICROORGANISMS…..
TAHIRA KHAN
UZAIR HASHMI
FARIA KHAN
INAM KAMAL
BADAR SALAM
TAYYABA KOMAL
INTRODUCTION
 DEFINITION:
Molecular typing procedures can be broadly defined
as methods used to differentiate bacteria and
viruses based on the composition of biological
molecules such as proteins, fatty acids,
carbohydrates, etc., or nucleic acids
 Infection control relies increasingly on the recently
introduced nucleic acid mediated procedures for
addition of microbial pathogens. The newly
designed techniques are for microbial identification
and typing. The presentation in detail covers the
phenotypic, genotypic and other methods of
molecular typing in detail.
NEED FOR MOLECULAR TYPING
 There are a number of reasons why it may be necessary to
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characterize a microbial isolate beyond species level and
determine its sub-species, strain, or even sub-strain.
To relate individual cases to an outbreak of infectious
disease
To establish an association between an outbreak of food
poisoning and a specific food vehicle
To study variations in the pathogenicity, virulence and
antibiotic resistance of individual strains within a species
To trace the source of contaminants within a manufacturing
process
To characterize microorganisms with important industrial
applications
TYPING METHODS
Phenotypic
Sero typing
• Rely on expression of phenotypic
characteristics (genetically
coded)
• Antibiotic resistance, antigens
etc
Phage typing
• Analysis of the genetic material
• DNA, RNA
Antimicrobial
Genotypic
resistance
monitoring
Multilocus enzyme
electrophoresis
(MLEE)
PHENOTYPING METHODS
Sero
typing
• 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
MLEE
MLEE
MLEE Characterizes the
cellular
proteins
by
electrophoretically
.separating them in a gel
matriXExposing the gel to
chromogenic substrates (that
react with the enzymes)
Limitation: Complexity of
interpretation
EMEGRING MOLECULAR
DIAGONOSTIC METHODS
UZAIR HASHMI
EMERGING MOLECULAR METHODS
IN DIAGNOSIS
• Plasmid profiling
• Restriction enzyme analysis (REA)
• Restriction fragment length polymorphism
•
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(RFLP)
Ribotyping
Pulse Field Gel Electrophoresis (PFGE)
Random Amplified Polymorphic DNA
(RAPD)
Nucleic acid sequencing
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
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
Random Amplification of
Polymorphic DNA (RAPD )
 DEFNITION:
• Uses short primers that find a lot
of targetslow-stringency
 Technique that utilizes
• Different size amplicons
• Products separated by
electrophoresis
polymerase
chain reaction PCR amplification
ADVANTAGES
with single primers of arbitrary sequence to
generate strain-specific arrays of
anonymous DNA fragments.
• Identification of suitable primers
Difficult to
interpret
differences
in
the
 RAPD technique •may
be
used
to
determine
intensity of bands
• Inefficient
reactionskinship
LIMTATIONS:
taxonomic
identity,
assess
• Amplification of cryptic genetic
material (prophages,
relationships, analyze
mixedbacteriophages)
genome
samples, and create specific probes.
FARIA KHAN
ANTIBIOTIC SENTISVITY
PULSE FIELD GEL ELECTROPHRESIS
MULTI LOCUS SEQUENCE TYPING
(MLST)
Multi Locus sequence typing
(MLST)
• Targets different DNA pieces and
sequences them
• Compares results with data banks
• Pro: highly comparable
• Con: expensive equipment
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
Examples:
• MRSA methicillin/oxacillinresistant Staphylococcus aureus
• VRE vancomycinresistant enterococci
• Esbls extendedspectrum betaLactamases (which are resistant to
Cephalosporins and monobactams)
• PRSP penicillinresistant streptococcus pneumoniae
Limitations:
 Antibiotic resistance under extraordinary selective pressure
 Multiple mechanisms for a strain to become abruptly resistant
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
 This technique is relatively similar to performing
aADVANTAGES
standard gel electrophoresis except that
instead of constantly running the voltage in one
direction, the voltage is periodically switched
among three directions;
• Time consuming
• Expensivethe central axis of the gel
 one that runs through
LIMITATIONS • Specialized equipment
and two that run at an angle of 60 degrees either
side
 The pulse times are equal for each direction
resulting in a net forward migration of the DNA.
MOLECULAR TYPING OF
MICROORGANISMSBACTERIA
BY: TAYYABA KOMAL.
Phenotyping
 Biotyping. ( based on detailed
biochemical structure)
 Bacteriocin typing.
 Protein typing.
 Extracted cell proteins can be
typed using MALDI-TOF.
 Analysis of cellular fatty acid
methyl esters (FAMEs) by gas
chromatography.
 Profile databases and Softwares.
Genotyping
1. Multilocus sequence typing.
2. Pulsed- field gel electrophoresis.
3. Ribotyping.
4. Repititive sequence based PCR.
TECHNIQUE
ADVANTAGES
DISADVANTAGES
1.
Multilocus sequence Highly discriminatory if the genes
typing.
are correctly chosen.
Quite time
consuming and
costly.
2.
Pulsed- field gel
electrophoresis.
Method of choice in the typing of
human bacterial pathogens and
the investigation of disease
outbreaks.
Costly and requires
at least three days
to obtain a result.
3.
Ribotyping.
It is rapid (<24 hours to result),
reproducible and works for a wide
range of bacterial species.
Costly in terms of
equipment.
4.
Repititive sequence
based PCR.
Widely used for typing human
pathogens.
Variation in
analysis and
interpretation of
data.
HYBRIDIZATION AND NUCLEAR PROBING
SOUTHERN BLOT
RESTRICTION LENGTH POLYMOPRPHISM(RFLP)
INAM KAMAL
Hybridization
 Nucleic acid hybridization is the formation of a
duplex between two complementary sequences
 Intermolecular hybridization: between two
polynucleotide chains which have complementary
bases
 DNA-DNA
 DNA-RNA
 RNA-RNA
 Annealing is another term used to describe the
hybridization of two complementary molecules
Denaturation - Renaturation
Renaturation
Denaturation
Doublestranded
DNA
Singlestranded
DNA
Initial
Base
pairing
Renatured DNA
Southern Blots
 Southern blotting is a procedure for transferring
denatured DNA from an agarose gel to a solid
support filter where it can be hybridized with a
complementary nucleic acid probe
 The DNA is separated by size so that specific
fragments can be identified
 Procedure:
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Restriction digest to make different sized fragments
Agarose gel electrophoresis to separate by size
Since only single strands bind to the filter, the DNA
must be denatured.
Denaturation to permit binding to the filter (NaOH)
Transfer to filter paper (capillary flow)
Hybridization to probe
Visualization of probe
Southern Blot
Restriction enzyme
DNA of
various sizes
Electrophorese on agarose gel
gel
Denature - transfer to
filter paper.
blot
Restriction fragment length polymorphism
 RFLP is a polymorphic allele identified by the
presence or absence of a specific restriction
endonuclease recognition site:
 GAATTC versus GATTTC
 RFLP is usually identified by digestion of genomic
DNA with specific restriction enzymes followed by
Southern blotting
 Regions of DNA with polymorphisms:
Introns
 Flanking sequences
 Exons
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Applications, Advantages and
limitations
 Badar Slam
Applications
 Nonculturable agents
 Fastidious, slow-growing agents
 Highly infectious agents that are dangerou
culture
 In situ detection of infectious agents
 Agents present in low numbers
 Culture confirmation
 Locating specific genes
 Organisms present in small volume
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specimens
Differentiation of antigenically similar agents
Antiviral drug susceptibility testing
Non-viable organisms
Molecular epidemiology
Advantages
 High sensitivity
 Can theoretically detect the presence of a single
organism
 High specificity
 Can detect specific genotypes
 Can determine drug resistance
 Can predict virulence
 Speed
 Quicker than traditional culturing for certain organisms
 Simplicity
 Some assays are now automated
Disadvantages
 Expensive
 So specific that must have good clinical data to
support infection by that organism before
testing is initiated.
 Due to high sensitivity and specificity, proper
quality control is critical for molecular testing.
 May be a problem with mixed cultures – would
have to assay for all organisms causing the
infection.