TAHIRA KHAN BADAR SALAMUZAIR HASHMI TAYYABA KOMALFARIA KHANINAM KAMAL

MOLECULAR TYPING OF MICROORGANISMS…..

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 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

Sero typing

Phage typing

Antimicrobial resistance monitoring

Multilocus enzyme

electrophoresis (MLEE)

Phenotypic

•Rely on expression of phenotypic characteristics (genetically coded)•Antibiotic resistance, antigens etc

Genotypic

•Analysis of the genetic material •DNA, RNA

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

MLEEMLEE 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

(RFLP)• Ribotyping• Pulse Field Gel Electrophoresis (PFGE) • Random Amplified Polymorphic DNA (RAPD)• Nucleic acid sequencing

)

RESTRICTION ENZYME ANALYSIS (REA)Extraction of plasmid or chromosomal DNADigestion of the DNA at particular sites using specific restriction enzymesHundreds of DNA fragments of various sizes (0.5-50Kb) separated by gel electrophoresisLIMITATION: 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 analysisNecessary for virus typingLIMITATIONS:

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: Technique that utilizes low-stringency

polymerase chain reaction PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous  DNA fragments.

RAPD technique may be used to determine taxonomic identity, assess kinship relationships, analyze mixed genome samples, and create specific probes.

ADVANTAGES

• 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)

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/oxacillin resistant Staphylococcus aureus• VRE   vancomycin resistant enterococci• Esbls   extended spectrum beta Lactamases (which are resistant to 

Cephalosporins and monobactams)• PRSP   penicillin resistant streptococcus pneumoniae

Limitations: Antibiotic resistance under extraordinary selective pressure Multiple mechanisms for a strain to become abruptly resistant

PULSED-FIELD GEL ELECTROPHORESIS (PFGE)

This technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions;

one that runs through the central axis of the gel 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.

ADVANTAGES

• 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• Expensive• Specialized equipment

MOLECULAR TYPING OF MICROORGANISMS- BACTERIA

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 typing.

Highly discriminatory if the genes 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.

INAM KAMAL

HYBRIDIZATION AND NUCLEAR PROBINGSOUTHERN BLOTRESTRICTION LENGTH POLYMOPRPHISM(RFLP)

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

Double-stranded

DNA

Denaturation

Single-stranded

DNAInitialBase

pairing

Denaturation - Renaturation

Renatured DNA

Renaturation

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: 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

Applications, Advantages and limitations

Badar Slam

Applications

Nonculturable agents Fastidious, slow-growing agents Highly infectious agents that are

dangerous to culture In situ detection of infectious agents Agents present in low numbers Culture confirmation

Locating specific genes Organisms present in small volume

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.