Molecular serotyping of Salmonella - Experiences using the method developed by CDC

Transcription

1 Molecular serotyping of Salmonella - Experiences using the method developed by CDC Gitte Sørensen NRL-Salmonella, Denmark Division of Food Microbiology

2 Molecular determination of serotype in Salmonella Selection of method or appoarch: based on White-Kauffmann-Le Minor scheme back compatible with traditional serotyping methods results Serovar

3 CDC approach Sequence genes responsible for O and H antigens rfb region for O antigens flic and fljb for H antigens Indentify antigen-specifik DNA sequences Develop molecular assays based on antigen-specific sequences (Luminex platform: liquid arrays ) Fitzgerald, C., M. Collins, S. van Duyne, M. Mikoleit, T. Brown, and P. Fields. (2007): Multiplex, bead-based suspension array for molecular determination of common Salmonella serogroups. J Clin Microbiol 45: McQuiston, J.R., Waters, R.J., Dinsmore, B.A., Mikoleit, M.L., Fields P.I. (2011): Molecular Determination of H Antigens of Salmonella by Use of a Microsphere-Based Liquid Array. J Clin Microbiol, 49:

4 Process for implementation Visit CDC in 2008 We buy a Bio-Plex in 2009 first test in lab on primers and probes from CDC In 2011 the H-assay also publiced and basis for implemention is now OK At the end of 2012 the method was accreditated by DANAK in our lab Since 1/ we have used the method as routine method in combination with slide-agglutination

5 O-groups and H-antigens covered by the CDC-method used by NRL-DK O-group antigen H-antigen (H-ag) Probe Probe Probe Group D (O:.9.12 and 9.46) a 1-Complex Group B (O:4.12 and O:4,5.12) b 2 Group C1 (O:7) c 5 Group C2 (O:8, 6,8 and 6,8,20) d 6 Group E (O:3,10,15,34 and 3,19) e,h 7 Group G (O:13,22 and 13,23) i G-Complex k f r m(g,m) z 10 m(m,t) 6 O-groups z p 5 H-complexs 29 H-antigens z 29 z 6 y s t(m,t) L-Complex z 51 v z 28 Z 4 -Complex EN-Complex z 24 x z 15

6 1 2 3

7 A bead-based microarray Each bead set is coupled to a specific oligonucleotide probe Target DNA sequences are amplified by PCR Labeled bead sets are then pooled Biotin label The PCR fragments are denatured and hybridized to the pooled bead sets SAPE (flourophore) Biotin label Bead-probe-PCR product mix. SAPE binds to biotin-labelled PCR fragment Any PCR fragment with homolgy to an oligonucleotide probe will bind to that specifically-labeled bead

8 Bead/Probe/PCR/SAPE sandwich is then detected by Bioplex instrument Raw data: median fluorescent intensity (MFI) O Group Isolate # Para A (30) B (38) Labeled Bead C2 (45) C1 (39) D (34) E (41) G (42) Template DNA used in multiplex PCR E C B Negative control Beads are read one at a time in a flow cytometer

9 O-group B - the method cover the most frequent types in DK Type Antigenic Formulae O-group H1 H2 H3 H4 supplementary Paratyphi B / var Java 4,[5],12:b:1,2 B b 1-complex 2 Tartrate negativ Stanley 4,12,27:d:1,2 B d 1-complex 2 Schwarzengrund 4,12,27:d:1,7 B d 1-complex 7 Typhimurium 1,4,[5],12:i:1,2 B i 1-complex 2 4,5,12:i:- /4,12:i:- 1,4,[5],12:i:- B i O:5 positiv Sandiego 4,5,12:e,h:e,n,z 15 B e,h EN-complex z 15 Chester 4,5,12:e,h:e,n,x B e,h EN-complex x Saintpaul 4,[5],12:e,h:1,2 B e,h 1-complex 2 Reading 4,[5],12:e,h:1,5 B e,h 1-complex 5 Derby 4,[5],12:f,g:- B G-complex f Agona 4,[5],12:f,g,s:- B G-complex f s Brandenburg 4,[5],12:l,v:e,n,z 15 B L-complex v EN-complex z 15 Bredeney 4,12,27:l,v:1,7 B L-complex v 1-complex 7 Heidelberg 4,[5],12:r:1,7 B r 1-complex 2 Coeln 4,[5],12:y:1,2 B y 1-complex 2 Indiana 4,12:z:1,7 B z 1-complex 7 Malonate negativ

10 O-group C1 the method cover the most frequent types in DK Type Antigenic Formulae O-group H1 H2 H3 supplementary Ohio 6,7,14:b:l,w C1 b L-complex Cholerasuis 6,7:c:1,5 C1 c 1-complex 5 Biochemical test Livingstone 6,7,14:d:l,w C1 d L-complex perhaps H-antigens testing Isangi 6,7:d:1,5 C1 d 1-complex 5 Augustenborg 6,7:i:1,2 C1 i 1-complex 2 Mbandaka 6,7,14:z10:e,n,z 15 C1 z 10 EN-complex z 15 Jerusalem 6,7:z10:l,w C1 z 10 L-complex Braenderup 6,7,14:e,h:e,n,z 15 C1 EN-complex e,h z 15 Rissen 6,7,14:f,g:- C1 G-complex f Montevideo 6,7,14:g,m,[p],s:- C1 G-complex m(gm) s Thompson 6,7,14:k:1,5 C1 k 1-complex 5 Infantis 6,7,14:r:1,5 C1 r 1-complex 5 Virchow 6,7,14:r:1,2 C1 r 1-complex 2 Colindale 6,7:r:1,7 C1 r 1-complex 7 Bareilly 6,7:y:1,5 C1 y 1-complex 5 Tennesee 6,7,14:z 29 :- C1 z 29 Malonate negativ S.Livingstone (6,7:d:l,w) S.Wil (6,7:d:l,z13,z28) Not all H:d strains positive in assay

11 O-group C2 and D supplementary test necessary for many types Type Antigenic Formulae O-group H1 H2 H3 supplementary Manhattan 6,8:d:1,5 C2 d 1-complex 5 O:6 pos,o:20 neg Muenchen 6,8:d:1,2 C2 d 1-complex 2 O:6 pos, O:20 neg Kentucky 8,20:i:z 6 C2 i z 6 Hadar 6,8:z10:e,n,x C2 z 10 EN-complex x O:6 pos Newport 6,8,20:e,h:1,2 C2 e,h 1-complex 2 O:6 pos Kottbus 6,8:e,h:1,5 C2 e,h 1-complex 5 O:6 pos Blockley 6,8:k:1,5 C2 k 1-complex 5 O:6 pos Altona 8,20:r[i]:z 6 C2 r z 6 [i] Bovismorbificans 6,8,20:r:1,5 C2 r 1-complex 5 O:6 pos Albany 8,20:z4,z 24 ;- C2 z 24 O:6 neg,o:20 pos Type Antigenic Formulae O-group H1 H2 H3 H4 supplementary Hillingdon 9,46:g,m:- D G-complex m(gm) H:q neg, O:46 pos Enteritidis 1,9,12:g,m:- D G-complex m(gm) H:q neg, O:46 neg Blegdam 9,12:g,m,q:- D G-complex m(gm) H:q pos, O:46 neg Dublin 1,9,12:g,p:- D G-complex p H:u neg Rostock 1,9,12:g,p,u:- D G-complex p H:u pos Goettingen 9,12:l,v:e,n,z 15 D L-complex v EN-complex z 15 Panama 1,9,12:l,v:1,5 D L-complex v 1-complex 5 O:9 pos,o:46 neg India 9,46:l,v:1,5 D L-complex v 1-complex 5 O:46 pos, O:9 neg

12 O-group E and G supplementary test necessary for many Type Antigenic Formulae O-group H1 H2 H3 H4 H5 supplementary Liverpool 1,3,19:d:e,n,z 15 E d EN-complex z 15 O:19 pos O:10 neg Lexington (var) 3,10:z 10 :1,5 E z 10 1-complex 5 O:10 pos O:19 neg, O:15/34 Meleagridis (var) 3,10:e,h:l,w E e,h L-complex O:10 pos O:19 neg, O:15/34 Anatum (var) 3,10:e,h:1,6 E e,h 1-complex 6 O:10 pos O:19 neg, O:15/34 Regent 3,10:f,g,[s]:[1,6] E G-complex f [s] [1-complex] [6] O:10 pos O:19 neg Westhampton (var) 3,10:g,s,t:- E G-complex s O:10 pos O:19 neg, O:15/34 Senftenberg 1,3,19:g,[s],t:- E G-complex s O:19 pos O:10 neg London (var) 3,10:l,v:1,6 E L-complex v 1-complex 6 O:15/34 Give (var) 3,10:l,v:1,7 E L-complex v 1-complex 7 O:10 pos O:19 neg, O:15/34 Weltevreden (var) 3,10:r:z 6 E r z 6 O:15 Krefeld 1,3,19:y:l,v E y L-komplex v O:19 pos O:10 neg Type Antigenic Formulae O-group H1 H2 H3 supplementary Putten 13,23:d:l,w G d L-complex Grumpensis 13,23:d:1,7 G d 1-complex 7 Kedougo 13,23:i:l,w G i L-complex Idikan 13,23:i:1,5 G i 1-complex 5 Havana 13,23:f,g,[s]:- G G-complex f [s] Worthington 13,23:z:l,v G z L-complex v Poona 13,22:z:1,6 G z 1-complex 6 O:22 pos O:23 neg Cubana 13,23:z29:- G z 29 O:23 pos O:22 neg

13 EURL-Salmonella typing Study 2013 use of Molecular Serotyping Reference Results Molecular Serotyping - NRL Denmark Strain O-antigens H-antigens H-antigens Serovar (phase 1) (phase 2) O-group H-ag H-ag H-ag H-ag supplementary S1 13,23 d e,n,z15 Telelkebir G d EN-Comp z15 O:22,O:23 S2 6,7 r 1,7 Colindale C1 r 1-Comp 7 S3 1,4,[5],12 e,h e,n,z15 Sandiego B eh EN-Comp z15 S4 9,46 d z6 Plymouth D d z6 O:9,O:46 S5 6,7,14 r 1,2 Virchow C1 r 1-Comp 2 S6 1,4,[5],12,[27] g,s,t [1,2] Kingston B G-Comp s (H:t) S7 1,13,23 f,g,[s] - Havana G G-Comp s S8 6,8 z10 e,n,x Hadar C2 z10 EN-Comp x O:6,O:8 S9 6,7,14 k 1,5 Thompson C1 k 1-Comp 5 S10 1,13,23 z l,w Worthington G z L-Comp S11 3,{10}{15}{15,34} e,h 1,6 Anatum E eh 1-Comp 6 O:10,(15,34), O:19 S12 1,9,12 l,v 1,5 Panama D L-Comp v 1-Comp 5 O:9, O:46 S13 1,9,12 l,z13 e,n,x Napoli D L-Comp EN-Comp x O:9,O:46, H:z13,H:w S14 8,2 i z6 Kentucky C2 i z6 S15 6,7,14 z10 e,n,z15 Mbandaka C1 z10 EN-Comp z15 S16 1,4,[5],12 b 1,2 Paratyphi B var Java B b 1-Comp 2 biochemical test S17 6,7,14 r 1,5 Infantis C1 r 1-Comp 5 S18 1,9,12 g,m - Enteritidis D G-Comp m(gm) H:q,(H:t) S19 1,4,[5],12 i - 1,4,[5],12:i:- B i O:5 S20 1,4,[5],12 i 1,2 Typhimurium B i 1-Comp 2 S21 42 g,t - 42:g,t:- - G-Comp O- and H-typning

14 Internal validation (1) Establish a collection of control strains cover al antigens detected by the molecular serotyping Used during implementation and education of the method Used for batch control of primer and bead pools Used as daily control Two EURL-Salmonella typing Study 2009 and 2010 all strains typed right

15 Collection of control strains Strain. no. Salmonella O group H ag H ag H ag H ag 1 Typhimurium(4.5,12:i:1,2) B i 1-complex 2 2 Agona (4,5,12:f,g,s:-) B G-complex f s 3 Cholerasuis(6,7:c:1,5) C1 c 1-complex 5 4 Oranienburg (6,7:g,m,t:-) C1 G-complex m(m,t) t (m,t) 5 Dublin (9,12:g,p:-) D G-complex p 6 Anatum (3,10:e,h:1,6) E e,h 1-complex 6 7 Senftenberg (1,3,19:g,s,t:-) E G-complex s 8 Liverpool (1,3,19:d:e,n,z 15 ) E d (negativ) EN-complex z 15 9 Hadar (6,8:z 10 ;e,n,x) C2 z 10 EN-complex x Establish a collection of control strains cover al antigens detected by the molecular serotyping: 10 Bredeney (4,12:l,v:1,7) B L-complex v 1-complex 7 11 Albany (8,20:z 4 ;z 24 :-) C2 Z4-complex z Bergedorf (9,46:e,h:1.2) D e,h 1-complex 2 13 Krefeld (1,3,19:y:l,w) E y L-complex 14 Ceyco (9,46:k:z 35 ) D k 15 Cubana (13,23:z 29 :-) Used as daily G control z Altona (8,20:r:z 6 ) C2 r z 6 17 Epinay (11:a:l,z 28 ) a L-complex z Hvittingfoss (16:b:e,n,x) b EN-complex x 19 Wayne 30:g,z 51 :-) Follow the current G-complex quality z 51 of the method 22 Sll 50:z:enx z EN-complex x 23 Strassburg(9,46:d:1,7) D d 1-complex 7 24 Enteritidis (9,12:g,m:-) D G-complex m(g,m) Used during implementation and education of the method Used for batch control of primer and bead pools for each strip (8 isolates) is one control strain included

16 Internal validation (1) Establish a collection of control strains cover al antigens detected by the molecular serotyping Used during implementation and education of the method Used for batch control of primer and bead pools Used as daily control Two EURL-Salmonella typing Study 2009 and 2010 all strains typed right

17 Internal validation (2) Results In the periode 23. Feb 2. Marts 2011 all isolates were type both by slide-agglutination and Molecular Serotyping (MS) 153 isolates are typed twofold: 23 different serotypes were detected 107 isolates (70 %) gave at once same result with both methods (need for malonate test for 7 strains by both metods) 27 isolates (18 %) need for supplementary test after MS (most O- testing) 134 (88 %) gave same result with both methods 84 % got a correct serotype by MS only (some re-testing) 98,7 % serotyped correct then MS is used in combination with slide-agglutination 9 isolates typed by MS but not by slide-agglutination (rough strains) 1,3 10 % (2 (6,5 isolates) %) isolates the same gave type strange was not result found by by MS: the two methods (variation in H-antigen) 8 OK after re-testing by MS 2 (1,3 %) isolates gave wrong H-result by MS

18 Flow in our lab Approx isolates per year Start Molecular Serotypning (MS) 2-3 times weekly (10-40 isolates each time): day 1 : DNA-extraction and DNA-amplification (3 hours work) day 2 : Hybridization and Detection on BioPlex (4 hours work) day 3 : Sometimes test H-antigen on swarm ager and biochemical test 90 % or more of the isolates have a type after less than 48 hours

19 O-group H-ag H-ag H-ag H-ag suppl. testing Serotype Negative control Control 1 B i 1-comp 2 Typhimurium B L-comp v EN-comp z15 Brandenburg B i 1-comp 2 Typhimurium B i O:5 4,5,12:i:- Typical routine test in lab B i O:5 4,5,12:i: B i O:5 4,5,12:i: B G-comp f Derby Description O-grp O-grp O-grp O-grp O-grp O-grp L- EN- 1- G- D B C1 C2 E G a b c d eh i k r z10 Control z z29 4 z6 y Comp v z28c1comp xg-comp z15 Comp 2m(mt) 5 6 t(mt) 7 Comp f m(gm) m(mt) p s t(mt) Oranienburg z51 Z4 z24 Blind 67, , , ,5 63, , , , , B i 1-comp 2 Typhimurium Kontrol , , , ,5 4279,5 83, ,5 42, ,5 110, , , , , , , B 82 i , comp 39, ,5 Typhimurium , , , , , , , , , ,5 2842,5 54, , , , ,5 23, , , C1 r 1-comp 5 Infantis , ,5 1799,5 37, , , ,5 37,5 64, , , , , , , ,5 0 60,5 50,5 54,5 69,5 46B 76 G-comp 32 72, ,5 f ,5 62,5 57,5 63,5 68,5 Derby , , , , , , , , ,5 53,5 Control 4 94,5 78, ,5 76,5 52, , ,5 107,5 46, , , ,5 44, ,5B 74,5 i , , , O: , ,12:i: , ,5 64, , , , , ,5 48, , , , , , ,5 7 40, ,5 52,5B 54,5 G-comp 60 65, f , Derby , , , , , , , ,5 63, , , ,5B 44 i ,5 92, O: ,5,12:i: , , , , , , , , , ,5 78,5 51,5 53, ,5 29 Control , E 74 50,5 eh 62 32,5 1-comp 90,5 47,5 40, ,5 Anatum , ,5 27, , , , , , , , , ,5 90, Control B G-comp f Derby 92 57, , ,5 69 2, , , ,5 44, , , , ,5 81, , ,5 47, C1 y 1-comp 5 Bareilly , , , , , , , , , ,5 31, ,5 65, , , , ,5 13, , , C1 r 1-comp 5 Infantis , , , , , , ,5 40, , , , , B i O:5 4,12:i: , , ,5 54, ,5 46,5 56, , , ,5 48, , , , , , ,5 C1 54 r 56,5 53,5 55,5 55, comp , , Infantis 75 Control , ,5 57,5 46, ,5 87, , ,5 43, , ,5 0 1, , , B i comp ,5 244, , , Typhimurium 11 45, , ,5 40, , , , , ,5 43,5 60, , , ,5 60, , , , ,5 42, , B 55 G-comp f94,5 64, , Derby , , , ,5 42, ,5 84,5 55,5 41,5 45, , , , ,5 56, , , Control , E 70 EN-comp z15 89,5 35, d 19 missing Liverpool , , , ,5 64, , , , , B 56,5 34,5 G-comp f86 37,5 51, , Derby 64,5 87, B i O:5 4,5,12:i: B i O:5 4,5,12:i: B G-comp f Derby B G-comp f Derby B G-comp f Derby D G-comp m(gm) O:46, H:q Enteritidis

20 Conclusions - summary (1) Faster if many isolates Very good if many common types Result under 5 hours (few isolates) Type for rough isolates

21 Conclusions - summary (2) Rare types not detected delay New types can be overlooked Still need for slide-agglutination (and sera) Start problems - many re-test or use of extra test of slide-agglutination - SAPE quality and quantity important Bad probes - H:d - O-group D Delivery issues - Beads - we will like to make a pig-kit

22 S.Typimurium DT41 - in the Danish broiler production Problem in many years ( ) In the whole production chain breeding flocks, hatcheries, broiler flocks, slaughter house Outbreak in nov herds and 2 slaughter houses no humane cases Phage typing profile: reaction in phage 6 can be weak or negative MLVA-profil in the 2013 outbreak: , , Do you see this type or problem in your country?