Salmonella Serotyping

Patricia Fields National Salmonella Reference LabCDC

10th Annual PulseNet Update MeetingApril 5, 2006

What is Salmonella serotyping?The “first-generation” subtyping method

Established in 1929Now the “first tier” subtyping for PulseNet

Phenotypic characterization of strains based on the immunologic reactivity of two surface structures:

Lipopolysaccharide (O antigen)Flagellin protein (H antigen)

In Salmonella, includes species and subspecies identification

Isolates of different subspecies can have the same O and H antigens

Schematic Representation of Salmonella Serotype Antigens

H antigen

i

i

45 12

ii

i

Salmonella Typhimurium4,5,12:i:1,2

Salmonella Typhimurium4,5,12:i:1,2

FlagellaFlagellaFlagellaiLPSLPS

4

12

5

O antigen

Basis for the US National Salmonella Surveillance System

About 35,000 isolates serotyped per year by SHDs50 years worth of data based on serotype

Useful for epidemiologic classification of strains and for outbreak investigations

Strains of the same serotype, especially a rarer one, may be relatedSerotype can correlates with disease or epidemiology

S. Typhi, other invasive serotypesSubspecies IIIb serotypes common in reptiles

An international “language”

Why is serotyping important?

Salmonella taxonomyTwo species of Salmonella

Salmonella entericaIts official! Judicial Commission, Opinion 80. IJSEM 2005

Salmonella bongori (formerly subspecies V)S. enterica further divided into 7 subspecies

Approximately 99% of human isolates are subspecies ISubspecies IV, IIIb, II, IIIa, VI (order of frequency in human isolates)Subspecies VII recognized but not used for the purpose of serotype designation

Species/subspecies typically determined by biochemical testing

I S. enterica subspecies entericaII S. enterica subspecies salmonae

IIIa S. enterica subspecies arizonaeIIIb S. enterica subspecies diarizonaeIV S. enterica subspecies houtenaeVI S. enterica subspecies indica

Salmonella enterica subspeciesSubspecies designated by taxonomic name or, more commonly for serotype designation, Roman numeral

Subspecies IIIa and IIIb originally described as the genus Arizonae

Still identified as “Arizona” by some automated ID systems

Differentiating Salmonella subspecies

*Whittam and Bumbaugh, Curr Opin Gen Dev 12:719-725 (2002)

Ssp I

Salmonella O antigenOutermost portion of lipopolysaccharide (LPS)

Carbohydrate antigenDifferent sugars and different linkages between sugars produce the different antigens

O antigen

Salmonella O AntigensTwo types

O Group antigens“Ancillary” O antigens

O Group antigensMost important for determining serotyperfb region contains genes responsible for O groupFound in all Enterobacteriacae

Ancillary O antigensTypically encoded by extra-chromosomal elements (bacteriophages, plasmids)Found in specific O groupsMost can vary within a given serotype, so are less important for serotype determination

Salmonella O Serogroups46 O serogroupsO groups initially designated by capital letters

Ran out of letters … started using numbersNow, all O Groups are designated by numbersLetter designations still commonly used

These O groups represent about 97%

of human isolates

O Group (number

designation)

O Group (letter

designation)Typical

O antigens2 A 2,124 B 4,127 C1 6,78 C2 6,89 D1 9,12

3,10 E1 3,1013 G 13

Distribution of Salmonella O Groups

* Dendrogram taken from Whittam and Bumbaugh, Curr Opin Gen Dev 12:719-725 (2002)

O:7 O:11 O:38 O:44 O:48

Subspecies determination is critical for serotype identification, particularly for “higher” O groups

Salmonella H antigenFlagellin, the flagellar filament

A protein antigenVariation in the middle surface-exposed portion of the protein

Salmonella is unique in having2 different H antigens:

Phase 1/Phase 2Phase 1 has a homolog in other entericsPhase 2 gene is in a Salmonella-specific region of the genome

The 2 flagellins are coordinately expressed—one is off when other is on

H Antigens Designations119 H antigens (Phase 1 & Phase 2)

Typically designated by lower case lettersa; b; c; d; e,h; e,n,x; etc1,2; 1,5; 1,7; et al are the notable exceptions

Ran out of letters … started using numbered z’sz4, z6, z10, z15, … z89Typically, no antigenic relationships between “z” antigens

Some H antigens are antigenically relatedRelated antigens referred to as “complexes”Typically, have one antigen in common plus secondary antigens

1 complex: 1,2; 1,5; 1,6; 1,7, etc.G complex: g,m; g,m,s; f,g,t; f,g,s; etc.

Designation of Salmonella SerotypesDesignated according to the conventions of the Kauffmann-White Scheme

2,541 serotypes in 200210-20 new recognized serotypes each year

Confirmed at CDC and IPSubspecies I serotypes: submitting lab gets to name the serotype

Kauffmann-White Scheme maintained by Institut PasteurPopoff, MY. 2001. Antigenic Formulas of theSalmonella Serovars, 8th edition. Pasteur Institute, Paris, France.

Published every five yearsUpdated annually (last updated 2002 …)

.pdf and MS Access versions available from the CDC

Subspecies I serotypes are designated by a name and a formula

Salmonella Typhimurium“Group O:4” or “Group B”

Subspecies O antigen Phase 1 Phase 2H antigens

I 4, [5], 12 : i : 1,2

Salmonella Typhimurium var. O 5 -or

Salmonella Typhimurium var. Copenhagen

I 4,5,12:i:1,2I 4,12:i:1,2

“[5]” means O antigen 5 may or may not be present.

“Group O:48” or “Group Y”

IV 48 : g,z51 : - *

Subspecies O Phase 1 “monophasic”antigen

Subspecies II through VI serotypes are designated by formula only

* Salmonella IV 48:g,z51:- was formerly known as Salmonella Marina.

Examples of Serotype Designations

Salmonella enterica subspecies houtenae serotype 48:g,z51:-Salmonella enteria serotype IV 48:g,z51:-Salmonella IV ser. 48:g,z51:-Salmonella IV 48:g,z51:-

Salmonella enterica subspecies entericaserotype TyphiSalmonella ser. TyphiSalmonella Typhi

Salmonella enterica subspecies enterica serotype TyphimuriumSalmonella enterica serotype TyphimuriumSalmonella ser. TyphimuriumSalmonella Typhimurium

Monophasic Serotypes and Monophasic Variants

Some serotypes are “naturally” monophasicNo second phase flagellar antigen

Specific subspecies I serotypes: S. Typhi, S. Enteritidis, S. Berta, othersMost subspecies IIIa and IV serotypes

Monophasic variants lack either of the two flagellar antigensI 4,5,12:i:- (likely variant of S. Typhimurium)I 9,12:l,z28:- (likely variant of S. Javiana)I 4,5,12:-:1,2 (could be a variant of Typhimurium, Heidelberg, Saintpaul, Paratyphi B, …)

monophasic: the state of having or expressing only one flagellar antigen when two flagellar antigens might be expected

monophasic variant: variants of serotypes that are typically expected to have two flagellar antigens

Serotype Variants: Unable to detect all serotype antigens

Salmonella I 4,[5],12:i:-

Mucoid strains (capsule blocks O antigen detection)Salmonella I O Mucoid:i:1,2

Nonmotile variantsI 4,5,12:nonmotile (Salmonella I 4,5,12:-:- )

Monophasic variantsSalmonella I 4,5,12:i:-Salmonella I 4,12:i:-Salmonella I 4,5,12:b:-

Rough strains (no longer express O antigen)Salmonella I Rough:i:1,2Salmonella I Rough:nonmotile

All of these strains are fully serotyped

Subspecies I: unable to give a “name” when all antigens not detected, but can still identify by a formula

PulseNet: Keeping the serotyping lab on its toes

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-90.3%]PFGE-XbaI

10908070

PFGE-XbaI

JEGX01.0002

JEGX01.0031

JEGX01.0024

JEGX01.0042

JEGX01.0063

JEGX01.0055

JEGX01.0043

JEGX01.0060

JEGX01.0010

JEGX01.0035

JEGX01.0022

JEGX01.0005

JEGX01.0057

JEGX01.0033

JEGX01.0006

JEGX01.0004

JEGX01.0032

JEGX01.0017

JEGX01.0062

JEGX01.0054

JEGX01.0023

JEGX01.0021

JEGX01.0040

JEGX01.0034

JEGX01.0051

JEGX01.0064

JEGX01.0012

JEGX01.0015

JEGX01.0047

JAXX01.0014

JAXX01.0013

JAXX01.0009

JAXX01.0018

JAXX01.0005

JAXX01.0010

JAXX01.0008

JAXX01.0001

JAXX01.0024

JAXX01.0019

JAXX01.0012

JAXX01.0025

JAXX01.0021

JAXX01.0002

JAXX01.0016

JAXX01.0023

JAXX01.0022

JAXX01.0026

JAXX01.0015

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Enteritidis

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Berta

Salmonella Enteritidis

Salmonella Berta

PFGE pattern can be indicative of serotype

I 9,12:g,m:-

I 9,12:[f],g,[t]:-Dendrogram courtesy of Susan Van Duyne and the PulseNet national database

PulseNet: Keeping the serotyping lab on it’s toes

PFGE pattern can be indicative of serotype

But not always ….

Serotype# isolates

1999-2003 Ssp O Group Phase 1 Phase 2Baildon 109 I 9,46 a e,n,xLomalinda 59 I 9 a e,n,xOranienburg 2922 I 7 m,t -Othmarschen 95 I 7 g,m,t -

Serotype# isolates

1999-2003 Ssp O Group Phase 1 Phase 2Saintpaul 2858 I 4 e,h 1,2Reading 416 I 4 e,h 1,5Chester 149 I 4 e,h e,n,xSandiego 632 I 4 e,h e,n,z15

PulseNet: Helping solve old questionsSalmonella Paratyphi B

vsSalmonella Paratyphi B var. L (+) tartrate+ (aka var. Java)

Paratyphi B is invasive, typhoidalParatyphi B var. L (+) tartrate + is less invasive, GI pathogenTo date, tartrate+ vs tartrate- phenotype has been the only way to differentiate

Likely that these two variants are not very accurately tracked in the national surveillance database

Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%]PFGE-XbaI

100

908070

PFGE-XbaI

CDC__00-0306

CDC__04-0421

CDC__02-0531

CDC__05-0051

CDC__00-0216

CDC__96-0583

CDC__98-0328

CDC__97-0146

CDC__03-0662

CDC__00-0267

CDC__01-0078

CDC__496

CDC__6

CDC__02-0303

CDC__AM00582

CDC__AM01136

CDC__00-0391

CDC__04-137

CDC__04-0615

CDC__04-0137

CDC__8

CDC__AM09823

CDC__AM01811

CDC__AM06652

CDC__AM11088

CDC__3

CDC__7

CDC__01-0290

CDC__01-0586

CDC__02-0234

CDC__00-0301

CDC__05-0044

CDC__05-0049

CDC__AM13085

CDC__04-0126

CDC__04-0332

CDC__00-0171

CDC__02-0104

CDC__03-0451

CDC__01-0399

CDC__00-0043

CDC__01-0516

CDC__03-0147

CDC__00-0295

I 4,5,12:b:- var. L(+) tartrate

I 4,5,12:b:- var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

I 4,5,12:b:z33 var. L (+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

I 4,5,12:b:-

Paratyphi B var. L(+) tartrate

I 4,5,12:b:-

I 4,5,12:b:-

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

I 4,5,12:b:- var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

Paratyphi B var. L(+) tartrate

I 4,5,12:b:- var. L(+) tartrate

Paratyphi B var. L(+) tartrate

I 4,5,12:b:- var. L(+) tartrate

I 4,5,12:b:- var. L(+) tartrate

SerotypeTartrate

FermentationTartrate

PCRsopE PCR

avrA PCR Isolate

I 4,5,12:b:- var. L(+) tartrate + + + - + 00-0306I 4,5,12:b:- var. L(+) tartrate + + + - - 04-0421Paratyphi B var. L(+) tartrate + + + - + 02-0531Paratyphi B var. L(+) tartrate+ +, delayed + - - 05-0051I 4,5,12:b:z33 var. L(+) tartrate + + + + + 00-0216Paratyphi B var. L(+) tartrate + + + - + 96-0583Paratyphi B var. L(+) tartrate + + + - + 98-0328I 4,5,12:b:- var. L(+) tartrate + + + - + 97-0146Paratyphi B var. L(+) tartrate + + + - + 03-0662I 4,5,12:b:- - + - - 00-0267I 4,5,12:b:- - + - - 01-0078Paratyphi B - - + - 496Paratyphi B - - + - 6Paratyphi B - - + - 02-0303Paratyphi B - - + - AM00582Paratyphi B - - + - AM01136Paratyphi B - - + - 00-0391Paratyphi B - - + - 04-0137Paratyphi B - - + - 04-0615Paratyphi B - - + - 04-0137Paratyphi B - - + - 8Paratyphi B - - + - AM09823Paratyphi B - - + - AM01811Paratyphi B - - + - AM06652Paratyphi B - - + - AM11088Paratyphi B - - + - 3Paratyphi B - - + - 7Paratyphi B var. L(+) tartrate+ +, delayed + - - 01-0290Paratyphi B var. L(+) tartrate + + + - - 01-0586Paratyphi B var. L(+) tartrate + + + - - 02-0234Paratyphi B var. L(+) tartrate + + + - - 00-0301Paratyphi B var. L(+) tartrate+ +, delayed + - - 05-0044Paratyphi B var. L(+) tartrate+ +, delayed + - - 05-0049Paratyphi B var. L(+) tartrate+ + + - + AM13085Paratyphi B var. L(+) tartrate+ + nd nd nd 04-0126Paratyphi B var. L(+) tartrate + + + - + 04-0332I 4,5,12:b:- var. L(+) tartrate + + + - - 00-0171I 4,5,12:b:- var. L(+) tartrate + + + + + 02-0104Paratyphi B var. L(+) tartrate + + + - + 03-0451Paratyphi B var. L(+) tartrate + + + - - 01-0399I 4,5,12:b:- var. L(+) tartrate + + + + + 00-0043Paratyphi B var. L(+) tartrate + + + - + 01-0516I 4,5,12:b:- var. L(+) tartrate + + + + + 03-0147I 4,5,12:b:- var. L(+) tartrate + + + + + 00-0295

Disclaimer: PFGE done by Salmonella reference lab and NOT PulseNet!

The Future: NextGen Serotyping Methods

Serotyping is easyPreparing serotyping reagents in hard

Hundreds of antisera requiredTypically use rabbit antisera—multiple absorptions to get desired specificity

Solution: Use DNA-based methods to ID serotypeBase on genes responsible for serotype to make it compatible with serotypes determined by traditional methods

To date, have probes for about 20 O groups and 25 H antigens

Currently adapting to the Luminex platform

The National Salmonella Reference Lab

Linda GheeslingLonnie BryantSarah DudaMatt MikoleitCollette FitzgeraldJohn McQuistonMarcus Collins