Bacteriophage typing

This presentation about bacteriophage typing methods.

1. Bacteriophage
typing
Dr. Ravi K Bhatia
Deptt. of Biotechnology HP
University Summer Hill Shimla-05

2. Bacteriophage typing
➢Bacteriophagetypingmeansidentification
ofthevirusesuptostrainlevel.
➢Themainpurposetocontrolinfectionand
alsousedindifferentepidemiological
purpose.
➢Thedifferentstrainsarebasedontheir
phenotypicandgenotypicdifferences
knownas‘typing’.

3. Criteria for Typing
Typeability Capacitytoproduceclearlyinterpretableresults
withmoststrainsofthespecies
Reproducibility Capacitytorepeatedlyobtainthesametyping
profileresultwiththesamestrain
Discriminatory
power
Abilitytoproduceresultsthatclearlyallow
differentiationbetweenunrelatedstrainsofthe
samespecies
Practicality (ease
of performance &
interpretation)
Methodshouldbeversatile,relativelyrapid,
inexpensive,technicallysimpleandprovide
readilyinterpretableresults

4. Phenotypic techniques
Biotyping, Phage typing, Bacteriocine, Serotyping,
Antimicrobial susceptibility typing (Antibiogram),
Protein typing , Multilocusenzymes electrophoresis (MLEE)
Genotypic technique
Plasmid profiling,Ribotyping,
Restriction enzyme analysis (REA) `
Pulse Field Gel Electrophoresis (PFGE)
Random Amplified Polymorphic DNA (RAPD)
Restriction fragment length polymorphism (RFLP)
Nucleotide sequences analysis,
Multi Locus sequence typing (MLST)
Types of typing methods

5. Phenotypic method
•Thosethatdetectcharacteristicsexpressedbythe
organisms.
•Phenotypicpropertiesarelikeshape,size,staining,
biochemicalproperties,thatcanbemeasuredwithout
referenceofgenome.
Limitations:
Alteredphenotypeinresponsetoenvironment
stimuli.
Largefractionsofthestrainsareuntypeable

6. Biotyping
•Itdependsuponthebiochemicalreaction.
•Biotypingmakesuseofpatternofmetabolicactivitiesexpressedbyanisolate,clonical
morphologyandenvironmenttolerance.
•Biotypingmaybeperformedmanuallyorusingautomatedsystem
*Sugarfermentation
*Aminoaciddecarboxylation/deamination
*StandardenzymatictestssuchasIMViC,Citrate,urease
*TolerancetopH,chemicalsanddyes
*Hydrolysisofcompounds
*Haemagglutination
*Hemolysis

7. Advantages:
Easytoperformandmoststrainsaretypeable
Disadvantage:
•Theyhavepoordiscriminatorypower.
•Variationingeneexpressionisthemostcommon
reasonforisolatesthatrepresentsinglestrainto
differinoneormorebiochemicalreactions.
•Pointmutationtoocontributestothisproblem.

8. Phage typing
•Strainscanbecharacterizedbytheirpatternof
resistanceorsusceptibilitytoastandardsetof
bacteriophages.
•Thisreliesonthepresenceorabsenceofparticular
receptorsonthebacterialsurfacethatareusedby
thevirustobindtothebacterialwall.
•Thismethodisusedtotypeisolatesof
StaphylococcusaureusandSalmonellasps.Such
stainsarereferredas'phagetypes'.

9. •Advantages:
•Thistechniquehasfairamountofreproducibility,
discriminatorypowerandeaseofinterpretation.
•Disadvantages:
•Thistechniquerequiresmaintenanceofbiologically
activephagesandhenceisavailableonlyat
referencecenters.
•Evenfortheexperiencedworker,thetechniqueis
demanding.
•Manystrainsarenon-typeable.

10. Bacteriocine typing
•Anisolateisassessedforsusceptibilitytoasetofbacterialpeptides
(bacteriocine)producedbycertainbacteria.
•Bacterocinesproducedbyaparticularstrainareusuallyonlyactive
againstotherstrainsofthesamespecies.
•IthasbeenusedtotypestainsofPseudomonasaeruginosa,E.coli,
Yersiniapestisetc.
•Advantages:
•Thistechniquehasfairamountofreproducibility,discriminatory
powerandeaseofinterpretation.
•Disadvantages:
•Thistechniqueisavailableonlyatreferencecenters.Evenforthe
experiencedworker,thetechniqueisdemanding.
•Manystrainsarenon-typeable.

11. Serotyping
•Serotypingisbasedonfactthatstrainsofsamespecies
candifferintheantigenicdeterminantsexpressedon
thecellsurface.
•Surfacestructuressuchaslipopolysaccharides,
membraneproteins,capsularpolysaccharides,flagella
andfimbriaeexhibitantigenicvariations.Strains
differentiatedbyantigenicdifferencesareknownas
'serotypes'.
•Serotypingisperformedusingseveralserologictests
suchasbacterialagglutination,latexagglutination,co-
agglutination,fluorescentandenzymelabellingassays.

12. •Advantages:
•Moststrainsaretypeable.Theyhavegoodreproducibility
andeaseofinterpretationthoughsomehaveeaseof
performance.
•Disadvantages:
•Someautoagglutinable(rough)strainsareuntypeable.
•Somemethodsofserotypingaretechnicallychallenging.
•Thereisdependencyongoodqualityreagentfrom
commercialsources.In-housepreparationofreagentsis
difficultprocess.
•Serotypinghaspoordiscriminatorypowerduetolarge
numberofserotypes,cross-reactionofantigensand
untypeablenatureofsomestrains.

13. •Thistypingtechniqueinvolvescomparisonofdifferentisolatestoasetof
antibiotics.
•Isolatesdifferingintheirsusceptibilitiesareconsideredasdifferentstrains.
•Theidentificationofneworunusualpatternofantibioticresistanceamong
isolatesculturedfrommultiplepatientsisoftenthefirstindicationofan
outbreak..
•Advantages:
•Almostallstrainsaretypeable.Thetechniquehaseaseofperformanceand
interpretationwithfairamountofreproducibility.
•Disadvantages:
•Asaconsequenceofvariousgeneticmechanisms,differentstrainsmay
developsimilarresistancepatternthusreducingthediscriminatingpower.
•Thesusceptibilitypatternofisolatestakenoveraperiod
Antimicrobial susceptibility typing
(Antibiogram)

14. Protein Typing
•Proteintypingreliesonmajororminordifferencesintherangeof
proteinsmadebydifferentstrains.
•Theproteinsandglycoproteinsareextractedfromacultureofthe
strain,separatedbySDS-PAGEandstainedtocomparewiththoseof
otherstrains.
•More-similarorganismsdisplaymore-similarproteinpatterns.
•Immunoblotting,theelectrophoresedproductsaretransferredto
nitrocellulosemembraneandthenexposedtoantiseraraisedagainst
specificstrain.
•Theboundantibodiesarethendetectedbyenzymelabeledanti-
immunoglobulins.
•Thesemethodsarecurrentlyemployedforepidemiologicalstudiesof
StaphylococcusaureusandClostridiumdifficile.

15. •Advantages:
•Almostallstrainsaretypeableandtechniqueshavegood
reproducibilityandeaseofinterpretation.
•Disadvantages:
•Sincethepatternsdetectedareverycomplex,comparisons
amongmultiplestrainsaredifficultandtheinterpretation
becomesdifficult.
•Methodsemployedaretechnicallychallengingand
equipmentsarecostlyandhencearenotavailableinall
laboratories.

16. Multi-locus Enzyme Electrophoresis
(MLEE)
•Here,theisolatesareanalyzedfordifferencesintheeletrophoretic
mobilitiesofasetofmetablolicenzymes.
•Variationsintheelectrophoreticmobilityofanenzyme,referredtoas
'electromorph',typicallyreflectaminoacidsubstitutionthatalterthe
chargeoftheprotein.
•Advantages:
•Almostallstrainscanbetyped.Thetechniquehasexcellent
reproducibilityandeaseofinterpretation.
•Disadvantages:
•Itisonlymoderatelydiscriminatoryfortheepidemiologicalanalysisof
clinicalisolates.Itrequirestechniquesandequipmentsthatarenot
availableinmostlaboratories.

17. Phenotypic typing system
characteristics
Typing systemTypeability Reproducibility Discrimination Ease of
interpretation
Ease of
performance
Serotyping
Most Good Fair Good Fair
Phage typing
Most Fair Fair Fair Poor
Antibiotic
susceptibility
testing
All Fair Poor Excellent Excellent
MLEE
All Excellent Good Excellent Good

18. Genotypic techniques
•Thesemethodsinvolvethestudyofthemicrobial
DNA,thechromosomeandplasmid,their
composition,homologyandpresenceorabsence
ofspecificgenes.
•Originallyperformedonlyinresearch
laboratorieshavenowfoundtheirwayinto
diagnosticlaboratories.
•Duetotheircomplexitiesandcostinvolved,they
arehoweverlimitedtofewlaboratoriesonly.

19. Plasmid analysis
Thenumberandsizesofplasmidiscarriedbyanisolatecanbedeterminedby
preparingaplasmidextractandsubjectingitgelelectrophoresis.
Advantages:verysensitiveveryspecificandgoodeaseofinterpretation
•Disadvantage:Reproducibilityofthismethodsuffersduetotheexistenceof
plasmidindifferentmolecularformssuchassupercoiled,nickedorlinear,eachof
whichmigratedifferentlyonelectrophoresis.
•Sinceplasmidscanbespontaneouslylostorreadilyacquired,relatedstrainscan
exhibitdifferentplasmidprofiles.
•Sincecertaingenesarecontainedintransposonsthatcanbereadilyacquiredor
deleted,thecompositionofplasmidDNAcanchangerapidly.
•Clinicalisolateslackingplasmidsareuntypeable.Thosestrainswithoneortwo
plasmidsprovidepoordiscriminatorypowers.

20. Restriction Endo-nuclease Analysis
(REA) of Chromosomal DNA
•ArestrictionendonucleaseenzymaticallycutsDNAataspecific
nucleotiderecognitionsequence.
•Thenumberandsizesofrestrictionfragmentsareinfluencedbythe
recognitionsequenceofenzymeandcompositionofDNA.
•DNAisdigestedwithendo-nucleasesthathaverelativelyfrequent
restrictionsites,therebygeneratinghundredsoffragmentsranging
from~0.5to50kbinlength.
•Suchfragmentscanbeseparatedbysizeusingagarosegel
electrophoresis.Thepatternstainedbyethidiumbromideand
examinedunderUVlight.
•DifferentstrainsofthesamespecieshavedifferentREAprofiles
becauseofvariationsintheirDNAsequences.

21. •Advantages:
•Allthestrainscanbytypedwithgoodreproducibility.
•Disadvantages:
•Thecomplexprofileconsistsofhundredsofbandsthat
maybeunresolvedoroverlappingthusmaking
comparisondifficult.
•Thepatternmayconsistofbandsgeneratedfrom
digestionofplasmidstoo.Thesereducetheeaseof
interpretationanddiscriminatorypower.

22. Southern Blot Analysis
•IncontrasttoREAofDNA,southernblotdetectonlythe
particularrestrictionfragment.
•TheDNAisdigestedbyendo-nuclease,thefragmentsare
separatedbygelelectrophoresisandthefragments
transferredtonitrocellulosemembranes.
•Thefragmentscontainingspecificsequencesarethen
detectedbylabeledDNAprobes.
•Variationsinthenumberandsizesofthefragmentsdetected
arereferredtoasrestrictionfragmentlengthpolymorphism
(RFLP).

23. Southern/Northern blottingE P I D E M I C A L E R T A N D R E S P O N S E
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

24. •Advantages:
•Allstrainscarryinglocihomologoustoprobeare
typeable.Theyarereproducible,havegoodease
ofinterpretation.
•Disadvantages:
•Thediscriminatorypowerdependsonthechoice
ofprobes.Theprocessrequirescostlyreagents
andequipmentbesidesbeinglabourintensive.

25. Ribotyping
•Itistheblottingofrestrictionenzymedigestionof16s
rRNA,25srRNAandoneormoretRNAs.
•Asthe16srRNAissohighlyconserveditisaveryuseful
moleculeforcomparingrelatednessoforganismsoverthe
courseofevolution.
•Organismsareclassifiedasseparatespeciesiftheir
sequencesshowlessthan98%homologyandareclassified
asdifferentgeneraiftheirsequencesshowlessthan93%
identity.

26. PFGE of Chromosomal DNA
•ThistechniqueovercomesthelimitationsofREA.Itisa
variationofagarosegelelectrophoresisinwhichthe
orientationoftheelectricfieldacrossthegelischanged
periodically.
•Thismodificationenableslargefragmentstobeeffectively
separatedbysize.
•Advantages:
•Allthestrainscanbytypedwithgoodreproducibility.
Restrictionprofilesareeasilyreadandinterpreted.

27. 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 equipmentE P I D E M I C A L E R T A N D R E S P O N S E

28. 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)E P I D E M I C A L E R T A N D R E S P O N S E

29. Nucleotide Sequence Analysis (NSA)
•Genotypeinformationathighestprecisionmaybedeterminedas
DNA(orRNA)nucleotide-basesequences.
•RNA'sareoftensequencedeitherbyconvertingtheRNAsintoDNA
orbysequencingtheDNAgenethatgivesrisetotheRNA.
•ByusingPolymeraseChainReaction(PCR)toamplifyaknownDNA
segmentandautomatedtechniquestosequencetheamplified
product,itispossibletocomparemultipleisolates.
•Advantages:
•Thistechniquecanapplyonallstrains;resultsarereproduciblewith
easeininterpretation.
•Disadvantages:
•Theprocessrequirescostlyreagentsandequipmentbesidesbeing
labourintensive.

30. Multi Locus sequence typing
(MLST)
•Used to type multiple loci of housekeeping genes
•Targets different DNA pieces and sequences
Compares results with data banks
Pro: Highly comparable
Con: Expensive equipment

31. Genotypic typing system 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

32. 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

33. Application of typing systems
Detection of outbreaks:
Concept of “prior probability”; rigorous epidemiological
investigation 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

34. Applications of typing systems
•Distinguish relapse from re-infection
•Identify types associated with increased transmission &
virulence
•Used to identify a number of pathogens
•Help to find out the serotypes easily
•Help to find out any epidemiology or any outbreak
•Emergence of new types ; implications on control measures