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 typingPhage typing Antimicrobial resistance monitoring Multilocus enzyme electrophoresis (MLEE) Rely on expression of phenotypic characteristics (genetically coded) Antibiotic resistance, antigens etc Phenotypic Analysis of the genetic material DNA, RNA Genotypic

PHENOTYPING METHODS 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) Sero 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 Phage typing

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 (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:  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. Uses short primers that find a lot of targets Different size amplicons Products separated by electrophoresis ADVANTAGES Identification of suitable primers Difficult to interpret differences in the intensity of bands Inefficient reactions Amplification of cryptic genetic material (prophages, bacteriophages) LIMTATIONS:

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)  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. 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 ADVANTAGES Time consuming Expensive Specialized equipment LIMITATIONS

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.

TECHNIQUEADVANTAGESDISADVANTAGES 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.

HYBRIDIZATION AND NUCLEAR PROBING SOUTHERN BLOT RESTRICTION 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 DNA Initial Base 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.