Salmonella Serotyping - APHL...2006/04/05 · Salmonella Enteritidis Salmonella Berta ÃPFGE...
Transcript of Salmonella Serotyping - APHL...2006/04/05 · Salmonella Enteritidis Salmonella Berta ÃPFGE...
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