naming salmonella (a paccagnella 08 nov 05

8/2/2019 Naming Salmonella (a Paccagnella 08 Nov 05

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Naming the Salmonellae:Naming the Salmonellae:

all you wanted to know aboutall you wanted to know about

Saintpaul, Sandiego, Heidelberg,Saintpaul, Sandiego, Heidelberg,

Muenchen and other destinations.Muenchen and other destinations.Ana M. Paccagnella

SupervisorEnteric Bacteriology Section

BC Centre for Disease Control655 W 12th AveVancouver BC

[email protected]


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SalmonellaSalmonella

2005 Kenneth Todar University of Wisconsin2005 Kenneth Todar University of Wisconsin--Madison Department of BacteriologyMadison Department of Bacteriology


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IdentificationIdentification

The genus Salmonella consists oftwo species: S. entericawhich isdivided into 6 subspecies and S.bongori


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Differential Characteristics ofDifferential Characteristics of SalmonellaSalmonella

Species and SubspeciesSpecies and SubspeciesSPECIES S. bongori

I II IIIa IIIb IV VI V

SUBSPECIES enterica salamae arizonae diarizonae houtenae indica

CHARACTERISTICS

dulcitol + + - - - d +

ONPG(2hr) - - + + - d +

malonate - + + + - - -

gelatinase - + + + + + -

sorbitol + + + + + - +

culture with KCN - - - - + - +

L(+)-tartrate(a) + - - - - - -galacturonate - + - + + + +

(-glutamyltransferase +(*) + - + + + +

$-glucuronidase d d - + - d -

mucate + + + - (70%) - + +

salicin - - - - + - -

lactose - - - (75%) + (75%) - d -

lysis by phage O1 + + - + - + d

USUAL HABITAT

S. enterica

cold blooded animals and environmentwarm blooded

animals

(a) = d - tartrate

(*) = Typhimurium d, Dublin -+ = 90% or more positive reactions

- = 90% or more negative reactions


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NomenclatureNomenclature

WHO Collaborating Centre for Reference and

Research on Salmonelladesignatesserotypes (= serovars)

S. entericasubspecies entericaorsubspecies I are given a name related togeographical place where it was firstisolated


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Full and Family NamesFull and Family Names

SalmonellaTyphimurium

or

Salmonellaser. Typhimurium

or

Salmonella entericasubsp.entericaserotype

Typhimurium


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Full and Family namesFull and Family names

other subspecies are designated by their

antigenic formulae following the subspeciesname,

S.entericasubsp salamaeser. 50:z:e,n,x or

Salmonellaserotype II 50:z:e,n,x


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Subtyping: Serotyping,Subtyping: Serotyping,

Phagetyping and all that jazzPhagetyping and all that jazzIdentification - biochemical

Serotyping - surface antigen detection:somatic = O and flagellar = H

This names the Salmonella and requires threesteps.

Phagetyping - patterns of susceptibility

to various phages

Molecular typing - pick your method: PFGE,

ribotyping, MLST, etc. - DNA /RNA based


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Salmonella SerotypingSalmonella Serotyping

Flagellar = H antigen, from the GermanFlagellar = H antigen, from the German

word Hauch.word Hauch. Phase variation: two types of flagellaPhase variation: two types of flagella

occurring at different times.occurring at different times. Determination of the first phase andDetermination of the first phase and

suppress this with specific antisera andsuppress this with specific antisera anddetermine the second phasedetermine the second phase


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Vi antigen = are actually capsular antigensVi antigen = are actually capsular antigens

and found in 3 serotypes:and found in 3 serotypes: Salmonella TyphiSalmonella Typhi,, Salmonella DublinSalmonella Dublinandand

Salmonella Paratyphi CSalmonella Paratyphi C


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SerotypingSerotypingLook for an agglutination reaction withLook for an agglutination reaction withdefined antisera.defined antisera.

Glass slides are divided into sections withGlass slides are divided into sections withwax pencil.wax pencil.Antisera and the isolate are emulsifiedAntisera and the isolate are emulsifiedand rocked and observed for a minute.and rocked and observed for a minute.

Clumping is looked for with a lens.Clumping is looked for with a lens.

Let us go step by step for SalmonellaLet us go step by step for SalmonellaTyphimurium.Typhimurium.


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Serotyping step by step:Serotyping step by step:

Somatic Antigen determination:Somatic Antigen determination:

Emulsify a 20 hour growth of Salmonella inEmulsify a 20 hour growth of Salmonella ina series of squares drawn on a glass slide.a series of squares drawn on a glass slide.

In each square mix with specific antisera.In each square mix with specific antisera. Rock for about 1 minute observing forRock for about 1 minute observing for

which square shows agglutination.which square shows agglutination. The squares with clumping are consideredThe squares with clumping are considered

positivepositive


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For this isolate we have clumping occurring inFor this isolate we have clumping occurring in

wells with antisera 4, 5 and 12 andwells with antisera 4, 5 and 12 andnegative for any other.negative for any other.

This means that this organism is in Group B.This means that this organism is in Group B.


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Phase conversion. Salmonella has two setsPhase conversion. Salmonella has two sets

of flagellar antigens. One must suppressof flagellar antigens. One must suppressone to see or be able to agglutinate theone to see or be able to agglutinate the

other.other. This is performed from a 20 hour cultureThis is performed from a 20 hour culture

grown in semisolid medium. This allows thegrown in semisolid medium. This allows the

elaboration of flagella.elaboration of flagella.


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Again emulsify the bacteria with flagellarAgain emulsify the bacteria with flagellar

antisera and observe as before and note theantisera and observe as before and note thereactions.reactions.

In our example we have agglutination inIn our example we have agglutination insera we call flagellar 2, and none in anysera we call flagellar 2, and none in any

other.other.

So far the formula for this isolate is:So far the formula for this isolate is:

4,5,12: ?:4,5,12: ?:1,21,2


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In order to suppress the 1,2 we include aIn order to suppress the 1,2 we include a

drop of antisera 1,2 into molten semisoliddrop of antisera 1,2 into molten semisolidagar and allowed to solidify. This is called aagar and allowed to solidify. This is called aGard plate.Gard plate.

The isolate is inoculated at one point andThe isolate is inoculated at one point andincubated for 20 hours.incubated for 20 hours.

The next day the Salmonella should haveThe next day the Salmonella should haveswarmed all over the plate if it has theswarmed all over the plate if it has thesecond flagellar phase.second flagellar phase.


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The agglutination steps are repeated and inThe agglutination steps are repeated and in

our example find that the isolateour example find that the isolateagglutinates antiserum i.agglutinates antiserum i.

The antigenic formula therefore is now:The antigenic formula therefore is now:4,5,12:i:1,2 or4,5,12:i:1,2 or S. TyphimuriumS. Typhimurium

If it had agglutinated with r, it would be:If it had agglutinated with r, it would be:4,5,12:r:1,2 or4,5,12:r:1,2 or S. HeidelbergS. Heidelberg


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SerotypesSerotypes

SaintpaulSaintpaul 11,4,[5],12:eh:1,2,4,[5],12:eh:1,2

SandiegoSandiego 4,[5],12:eh:e,n,z154,[5],12:eh:e,n,z15

HeidelbergHeidelberg11,4,[5],12:r:1,2,4,[5],12:r:1,2

MuenchenMuenchen 6,8:d:1,26,8:d:1,2


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Are these bugs the same asAre these bugs the same as

those somewhere else?those somewhere else?


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Phagetyping: HistoricalPhagetyping: Historical

backgroundbackground Frederick Twort (1915)Frederick Twort (1915) andand Felix d'Herelle (1917)Felix d'Herelle (1917)

recognized the existence of viruses which infect bacteria,recognized the existence of viruses which infect bacteria,

which d'Herelle called bacteriophages (eaters of bacteria).which d'Herelle called bacteriophages (eaters of bacteria). In the 1930s and subsequent decades, virologists such asIn the 1930s and subsequent decades, virologists such as

Luria, Delbruck and many others utilized them as modelLuria, Delbruck and many others utilized them as model

systems to investigate many aspects of virology, includingsystems to investigate many aspects of virology, includingvirus structure, genetics, replication, etc.virus structure, genetics, replication, etc.

They are still a paradigm for many areas of biology,They are still a paradigm for many areas of biology,especially gene expression.especially gene expression.

They are also used for typing purposes.They are also used for typing purposes.


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PhagetypingPhagetyping

Whats a Phage?Whats a Phage? Bacteriophages, also called phages, areBacteriophages, also called phages, are

viruses that attack bacteria.viruses that attack bacteria. Phages are highly host specific; meaningPhages are highly host specific; meaning

that any given phage will attack a particularthat any given phage will attack a particularspecies or group of species of bacteria.species or group of species of bacteria.

Some phages are specific enough to attackSome phages are specific enough to attack

only some members of a serotype.only some members of a serotype. For Salmonella this is done by the NationalFor Salmonella this is done by the National

Microbiology Laboratory in Winnipeg.Microbiology Laboratory in Winnipeg.


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PhagetypingPhagetyping

Plaque formation:Plaque formation:

A plaque is aA plaque is a(small) clearing in a(small) clearing in a

lawn of bacterialawn of bacteria

grown on a plate.grown on a plate.Patterns of plaquePatterns of plaque

formation can beformation can be

used to group orused to group ortype bacteria.type bacteria.


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Plaques:Plaques:


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Molecular subtypingMolecular subtyping

PFGE = Pulsed Field Gel ElectrophoresisPFGE = Pulsed Field Gel Electrophoresis

RibotypingRibotyping

MLST = Multilocus Sequence TypingMLST = Multilocus Sequence Typing

Plasmid AnalysisPlasmid Analysis


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PFGE subtypingPFGE subtyping

PFGE stands for Pulsed Field GelPFGE stands for Pulsed Field Gel

Electrophoresis. The procedure takes 24Electrophoresis. The procedure takes 24hours from extracting the DNA to looking athours from extracting the DNA to looking at

the picture.the picture.


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The procedure:The procedure:

Extract the organisms genetic material and cutExtract the organisms genetic material and cutthis at specific sites with an enzyme which resultsthis at specific sites with an enzyme which resultsin pieces of DNA of differing lengths. Place this onin pieces of DNA of differing lengths. Place this on

a gel at one end and under the influence of aa gel at one end and under the influence of apulsed electric current the pieces will migrate andpulsed electric current the pieces will migrate anddeposit themselves according to size. The largestdeposit themselves according to size. The largest

ones will not migrate as far as the smaller ones.ones will not migrate as far as the smaller ones.This results in a series of bars. This characteristicThis results in a series of bars. This characteristicis utilized to subtype identical serotypes of anis utilized to subtype identical serotypes of anorganism.organism.


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We assume that organisms that are the same orWe assume that organisms that are the same orfrom the same source will have their DNA cut infrom the same source will have their DNA cut inthe same places and the resultant pieces of DNAthe same places and the resultant pieces of DNAwill be of the same size.will be of the same size.

Their pattern will be indistinguishable from eachTheir pattern will be indistinguishable from each

other. This helps to determine whether isolatesother. This helps to determine whether isolatesfrom different sources are the same or different.from different sources are the same or different.

This has been used to detect, monitor and helpThis has been used to detect, monitor and help

stop outbreaks since 1999 in BC. This procedurestop outbreaks since 1999 in BC. This procedureis used for Salmonella, Shigella and Listeria asis used for Salmonella, Shigella and Listeria aswell as E. coli O157:H7.well as E. coli O157:H7.


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PFGE resultsPFGE results

This is an example of a gel. TheThis is an example of a gel. Thenumbers across the top are thenumbers across the top are the

lanes in which different isolateslanes in which different isolatesare placed. Lanes 1, 5 and 9are placed. Lanes 1, 5 and 9are standards which help usare standards which help usmeasure. In this particular gelmeasure. In this particular gellanes 2, 4, 6 and 7 arelanes 2, 4, 6 and 7 areindistinguishable and areindistinguishable and areassumed to be from the sameassumed to be from the samesource. At least the patientssource. At least the patientsfrom whom these E. coli Ofrom whom these E. coli O

157:H7s were isolated had the157:H7s were isolated had thesame exposure.same exposure.


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SalmonellaSalmonellaParatyphi var. JavaParatyphi var. Java

Lane 4 is a human isolate Lane 2 is the tank water isolate


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Salmonella StanleySalmonella Stanley


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We are not aloneWe are not alone

PulseNet USA

PulseNet Canada PulseNet Europe

PulseNet Asia


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Basis Of Sharing InformationBasis Of Sharing Information

Strict adherence to protocol

Strict adherence to interpretation guidelines Using secure reporting structures

Dissemination of information is open yetrespects confidentiality


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Thank you,Thank you,

naming salmonella (a paccagnella 08 nov 05

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