E. coli Typing, characterization and nomenclature of VTEC

Transcription

1 E. coli Typing, characterization and nomenclature of VTEC FLEMMING SCHEUTZ STATENS SERUM INSTITUT The International Escherichia and Klebsiella Centre (WHO)

2 DIARRHOEAGENIC E. coli (DEC) & EXTRAINTESTINAL PATHOGENIC E. coli (ExPEC) A/EEC: EPEC: ETEC: EIEC: Attaching and effacing (A/E) E. coli Enteropathogenic E. coli Enterotoxigenic E. coli Enteroinvasive E. coli VTEC: STEC: SLTEC: EHEC: EAggEC: DAEC: ExPEC: Verotoxin producing E. coli Shigatoxin producing E. coli Shiga-like toxin producing E. coli Enterohaemorrhagic E. coli - subgroup EnteroAggregative E. coli Diffusely Adherent E. coli Extraintestinal pathogenic E. coli

3 Clinical detection of E. coli in Denmark - Methods Colony dot blot hybridisation with DNA polynucleotide probes in pools: POOL : vtx, vtx2, ipah VTEC & EIEC eae EPEC & A/EEC POOL 2: LT, ST h, ST p ETEC EC

4 E. coli multiplex-pcr: specificity E. coli group Virulence gene(s) ETEC enterotoxigenic E. coli elti and/or estap / estahe heat-labile enterotoxin heat-stable enterotoxin A/EEC EPEC attaching and effacing E. coli enteropathogenic E. coli eae intimin VTEC verocytotoxin producing E. coli vtx and/or vtx2 verotcytooxin verocytotoxin 2 EIEC enteroinvasive E. coli ipah invasive plasmid antigen 6S - - NEGATIV - VTEC - VTEC - VTEC - VTEC - VTEC - EIEC - VTEC - VTEC - EIEC - NEGATIV - ETEC - ETEC - NEGATIV - ETEC ETEC - ETEC - EIEC - VTEC - ETEC -6S ipah - elti - vtx2 - eae - vtx - esta - -ipah -elti -vtx2 - eae -vtx -esta

5 PHENOTYPIC TYPING of E. coli O:H serotyping Complete serotyping : O:K:H; F Adherence patterns Haemolysin production Haemagglutination patterns Toxin production Phage typing Biochemical reaction patterns

6 vtx, vtx2 or eae positive colonies are live slide agglutinated with OK antisera against common VTEC and EPEC O groups: VTEC/EPEC Pool : O26, O03, O, O45 & O57 Supplemeted by: single antiserum O2 & O45 EPEC Pool 2: O55, O9, O25ac, O27, O28ab Pool 3: O86, O4, O26, O42, O58 All strains should be serotyped and characterised

7 O grouping Boiled culture mixed with O antiserum in microtiterplate or in tubes

8 Serotyping of E. coli O antigen Lipopolysaccharide; ; LPS 8 K antigen Acidic polysaccharide capsules ~ 60 H antigen Protein; Flagella 53 Complete serotype: : O:K:H; F

9 Serotyping of E. coli today.. 2 O antigen Lipopolysaccharide; ; LPS Divided into Antiserum in trough Acidic + - and Neutral

10 Serotyping of E. coli today.. 3 O antigen Lipopolysaccharide; ; LPS Official number is 8 Removed: O3 O47 O67 (Citrobacter) O72 (Citrobacter) O93? (Was an O8 strain) O94 (Citrobacter) O22 (Citrobacter) Actual: : 75 New O antigens: O74 O75 (OX82 - OX86) (= OX3) (= OX7)

11 Serotyping of E. coli today.. 4 K antigen Capsular polysaccharide; ; CPS Divided into OK antiserum Group I & IV + - and Group II & III

12 Serotyping of E. coli today.. 5 K antigen Capsular polysaccharide; ; CPS Group I: Only found in O groups O8, O9, O20 og O0 Amino sugar free; Structural resemblance to from Klebsiella spp. K28 = Klebsiella K54 K30 = Klebsiella K20 K55 = Klebsiella K5 Group IV: CPS Amino sugar containing; No structural resemblance to CPS from other bacteriae K9 K85 K87 = O04 K84 = O93!! = O4 K4?? = O32 K38?=?O78, K43?=?O79 O79?,, K0?=?O74 O74?

13 Serotyping of E. coli today.. 6 K antigen Capsular polysaccharide; ; CPS Many group IV K antigens are bound to lipid A Because they are heat-stable the cover the O antigen, thus making the O determination appear negative. Either you need to autoclave the culture or to isolate K minus forms:

14 Serotyping of E. coli today.. 8 K antigen Capsular polysaccharide; ; CPS K minus form

15 Serotyping of E. coli today.. 9 K antigen Group II: Found in many O groups Capsular polysaccharide; ; CPS Structural resemblance to CPS from Gram-positives 20-50% of the sidechains are lipidbound Subgroup: I/II or III K2a, ac, (K62); K3; K0; K; K9; K54; K96?; K98 (?)

16 Serotyping of E. coli today.. 0 K antigen Group II: Capsular polysaccharide; ; CPS Structural resemblance to CPS from Gram-posistives K2a, ac, (K62) Neisseria meningitidis H K (~K92) Neisseria meningitidis C Pasteurella haemolytica A2 Moraxella nonliquefacius K7, (56) Streptococcus pneumonia type 3 K4 Neisseria meningitidis 29-e K8a, ab ~Haemophilus influenzae type b K92 Neisseria meningitidis C K93 Neisseria meningitidis A Bacillus pumilus K00 ~Haemophilus influenzae type b

17 Serotyping of E. coli today.. K antigen Capsular polysaccharide; ; CPS Official number is 0 Actual number is 60 i. e. IF grouped by backbone polysaccharide structure NOTE: The B antigens are obsolete!

18 Escherichia coli K-determination with phage cross-brush 0 µl of phage suspension Positive control* Bacterial Bacterial culture culture Bacterial Bacterial culture culture2 2 Bacterial Bacterial culture culture3 3 Bacterial Bacterial culture culture4 4 Bacterial Bacterial culture culture5 5 Bacterial Bacterial culture culture6 6 Negative reactions Positive reactions *) Positive control strains: Incubation Original No. for Serotype 4 K U 9-49 O2:K:H4 K5 Bi O0:K5:H4 for 4 hours

19 Serotyping of E. coli today.. 3 H antigen Flagella,, protein structure Official number is 56 Actual number is 53 Removed: H3 H22 H50 (= H0)

20 Serotyping of E. coli today.. 4 H antigen Flagella,, protein structure After passage in semi- solid agar it is important to maintain motility in a rolling broth! 6 hours at 37ºC

21 Typing of pathogenic E. coli strains using flagellar (H)- antigens: : serotyping vs flic- polymorphisms Rita Prager, Ute Strutz, Angelika Fruth and H. Tschäpe

22 PCR-products derived from the flic-gene PCR using different primers S S lanes: (bp) O7:H4 2 O5:H9 3 O92:H33 4 O8:H9 5 O64:H - 6 O57:H2 7 O8:H6 8 O:H2 9 O7:H4 S flic-f flic-f flic-f flic-f flic-f flic-m flic-m flic-m flic-m molecular standard

23 Some RsaI restriction profiles of amplified flic-genes according to Fields et al (bp) lanes: flic-rflp type 2 O26:H 3 O57:H7 2 4 O56:H9 2-5 O57:H O57:H2 4 7 O9:H4 5 8 O46:H2 6 9 O28:H2 7 O8:H6 8 2 Ont:H O22:H8 9 4 O5:H6 0 5 O26:H - 6 O64:H O76:H9 3 8 O2:H9 3-, 0, 9 = molecular standard

24 Summary of flic(f) restriction pattern and their genetic distance flic-f type

25 Diagram flic C V C2 PCR Sequence primer F R+R2 Sequence 450 bp Up to 800 bp

26 E. coli test strains DENDROGRAM based on partial sequencing of 450 bp in the central variable region of flic Test H3 Test H Nucleotide Substitutions (x00) Test H9 Test H9 Test H26 Test H20 Test H46 Test H5 Test H30 Test H32 Test H37 Test H4 Test H4 Test H3 Test H28 Test H23 Test H5 Test H7 Test H Test H2 Test H45 Test H8 Test H6 Test H34 Test H33 Test H39 Test H42 Test H5 Test H56 Test H38 Test H55 Test H29 Test H25 Test H7 Test H4 Test H52 Test H0 Test H43 Test H48 Test H24 Test H2 Test H47 Test H27 Test H8 Test H40 Test H Test H35 Test H2 0 0

27 Comparison of partial flic molecular typing with previous studies phenotype Antigen H H3 H7 H35 H36 H40 H44 H47 H53 H54 H55 flic (F) 2 6 n.a n.a. n.a. 55 Genotypes RFLP a Sequencing flic (M) Entire flic b Partial flic c 2- /2/ n.a. n.a. d Bands e /38 47a 2 2//2/2 53 n.a. n.a. f 54 2 n.a./0/ n.a. no PCR amplification; a Typing of flic by Restriction Fragment Length Polymorphism by Prager et al.; b Sequences obtained by Wang et al.; c This study; d Four strains tested; e Two strains tested; f Three strains tested

28 Non-O57 VTEC causing outbreaks O22:H8 O26:H O03:H2 O03:H25 O04:H2 O:H- / H2 / H8 O3:H2 O7:H4 O8:H2 O9 O2:H9 / H2 O28:H2 O45:H- O?:H9 Citrobacter freundii

29 LT ST p ST h CFA inv ipah saa heat-labile toxin heat-stable toxin (porcine) heat-stable toxin (human) colonization factors enteroinvasive E. coli invasion plasmid antigen STEC autoagglutinating adhesin EAgg aggregative adherence DA lpf cnf VIRULENCE FACTORS IN E. coli - on chromosome (Pathogenicity Islands) - plasmids, transposons & phages diffuse adherence Long Polar Fimbriae Cytotoxin necrotising EAF factor bfpa LEE eae ehxa hlya asta chua EPEC adherence Bundle forming pilus locus for enterocyte effacement genes attaching and effacing enterohaemolysin haemolysin EAST OMP iron uptake vtx verocytotoxin necrotising factor vtx2 verocytotoxin 2

30 GENE SUB-TYPING Specific PCR RFLP: restriction fragment length polymorphism Sequencing eg flic vtx

31 DNA TYPING or FINGERPRINTING PFGE: Pulsed Field Gel Electrophoresis RiboPrinting: Automated rrna typing RAPD: Random Amplified Polymorphic DNA Box-ERIC ERIC: Enterobacterial Repetitive Intergenic Consensus sequence Rep-PCR PCR: Repetitive Extragenic Palindromic sequence PCR VNTR: Variable Number of Tandem Repeats MLVA: Multiple-locus variable number of tandem repeats Analysis MLST: Multi-locus locus Sequence Typing SSR: Simple Sequence Repeats Polymophism Variation at Mononucleotide Repeat Loci

32

33 PFGE types of Danish O57 County (Alle) PFGE-profiles within O group O57 (2 profiles, 55 isolates) Antal mar apr maj jun jul aug sep okt nov dec jan feb mar apr maj jun jul PFGE type EXHX EXHX EXHX EXHX EXHX0.007 EXHX0.006 EXHX0.005 EXHX EXHX0.002 EXHX0.000 EXHX0.000 EXHX EXHX EXHX EXHX EXHX0.003 EXHX EXHX0.008 EXHX0.009 EXHX EXHX0.00 År Date of isolation

34 PFGE profiles of farm-related related VTEC isolates Pearson correlation (Opt:.00%) [0.0%-98.3%] PFGE-XbaI PFGE-XbaI C O 2 H M C O 5 H K C C O57. O57 H- H- EXHX0.00.DK EXHX DK M K C O57 H 7 EXHX0.009.DK K C O57 H 7 EXHX DK K C O57 H 7 EXHX0.009.DK K goats_a2a O57 EXHX0.009.DK goats_8. O57 EXHX0.009.DK C O57 H 7 EXHX0.003.DK K C C O57. Oru H 7 H 8 EXHX DK K M goats_7

35 Multiple Locus Variable number repeats Analysis (MLVA) Repetetive DNA sequences Linked to modulation of gene expression in bacteria From bp repeats Number of repeat units varies from strain to strain because of mispairing during replication that results in introduction or deletion of repeat units Areas where there is variation called Variable Number of Tandem Repeats (VNTR) Combined analyse of several VNTR areas called MLVA MLVA developed for O57 using 7 VNTR areas Lindstedt et al

36 Minimum Minimum spanning spanning tree tree AA AB AC AD AE AF AG AH AI AJ AK AL AM AN AO AP AQ AR AS AT AU AV AW AX AY AZ A0 A A2 A3 A4 A5 A6 A7 A8 A9 BA BB BC BD BE BF BG BH BI BJ BK BL BM BN BO BP BQ BR BS BT BU BV BW BX BY BZ B0 B B2 B3 B4 B5 B6 B7 B8 B9 CA CB CC CD CE CF CG CH CI CJ CK CL CM CN CO CP CQ CR CS CT CU CV CW CX CY CZ C0 C C2 C3 C4 C5 C6 C7 C8 C9 DA DB DC DD DE DF DG

37 PFGE XbaI MLVA Vhec Vhec2 Vhec Vhec4 Vhec5 Vhec6 Vhec7 Isolate# Purification A B A B A B A B A B A B A B A B C A B C A B C A B C A B C A B A B Patient Patient 2 PFGE and MLVA results from siblings involved in the milk associated O57:H- outbreak MLVA types that differ from the epi-type are indicated with colours. PFGE types that differ are shown with coloured numbers.

38 PFGE XbaI MLVA Vhec 577 Vhec2 47 Vhec3 Vhec4 Vhec5 Vhec6 Vhec7 Isolate# Purification A B C A B C D A B C D A B C D A B C D PFGE and MLVA results for five isolates from one patient with an O57:H7 infection. Different MLVA alleles are shown in colours PFGE types are shown in coloured numbers

39 Nomenclature of Verocytotoxins vtx s & VTEC

40 Stx/VT and VT2 Main groups Subtypes Suffixed with small Arabic letters Stx: Shiga toxin from Shigella stxa VT: E. coli Verocytotoxins vtxb b to vtxd VT2: vtx2a a to vtx2g Variant designations Subtype - first published O group - strain No. Phylogenetic sequence based nomenclature for the combined A- and B-subunitsB SP A A 2 SP B

41 4 vtx genes 408 vtx2 genes

42 Stx slt- or VT slt-2 or VT2 slt-iiv or e stx2c or VT2v stx2vha and vtx2d2 stx2d-o stx2d-ount stx2f stx2g stxa vtx2a vtx2c vtx2d vtx2d vtx2g vtx2e vtx2f vtx2b vtx2b SP A A 2 SP B vtxb vtxc vtxd

43 slt-2 or VT2 vtx2a-o57-edl933 stx2c or VT2v vtx2c-o57-e325 stx2vha and vtx2d2 vtx2d-o9-b-b2f vtx2d-o9-a-b2f stx2d-ount stx2d-o vtx2b-o8-eh250 vtx2b-o-ph slt-iiv or e vtx2g-o2-7v vtx2e-o39-42 stx2g Stx Stxa-S. dysenteriae-388t vtx2f-o28-h.i.8 stx2f slt- or VT vtxb-o57-edl933 SP A A 2 SP B vtxc-o74-dg3-3 vtxd-ont-mhi83

44 8 vtx variant designations stxa-s. dysenteriae-388t stxa-s. sonnei-cb7888 vtxb-o-ph vtxb-o57-edl933 vtxb-o48-94c vtxb-o-cb68 vtxc-o74-dg3-3 vtxd-ont-mhi83

45 vtx2a-o48-94c vtx2a-o vtx2a-o57-sf vtx2a-o57-edl933 vtx2a-o26-fd930 vtx2c-o57-fly6 vtx2c-o vtx2c-o57-c vtx2c-o57-e325 vtx2c-o74-b-03 vtx2d-o8-c466-0b vtx2d-o9-b-b2f vtx2d-o vtx2d-o9-a-b2f vtx2d-o74-ec720a vtx2d-o6-nv206 vtx2d-o73-c65-02 vtx2b-o96-s-6 vtx2b-o-s-3 vtx2b-o-ph vtx2b-o74-a-03 vtx2b-o vtx2g-o2-7v vtx2g-o2-s86 vtx2g-out-s-8 vtx2e-o39-42 vtx2e-o0-e-d43 vtx2e-o vtx2f-o28-h.i.8 vtx2a-e cloacae-95mv2 vtx2b-o vtx2b-ont vtx2b-o8-eh vtx2 vtx2 variant variant designations designations vtx2d-c freundii-lm76 vtx2a vtx2c vtx2d vtx2b vtx2e vtx2g vtx2f

46 + Majority E S N T A A A F L N R K S Q F L Y T T G K vtx2a-o48 O48-94C 838 E S N T A A A F L N R K S Q F L Y T T G K vtx2a-o57 O57-SF 838 E S N T A A A F L N R K S Q F L Y T T G K vtx2a-o26 O E S N T A A A F L N R K S Q F L Y T T G K vtx2a-o57 O57-EDL E S N T A A A F L N R K S Q F L Y T T G K vtx2a-o26 O26-FD E S N T A A A F L N R K S Q F L Y T T G K vtx2a-e E cloacae-95mv2 838 E S N T A A A F L N R K S Q F L Y T T G K vtx2c-o57 O57-E E S N T A A A F L N R K S Q F L Y T T G K vtx2c-o57 O57-C E S N T A A A F L N R K S Q F L Y T T G K vtx2c-o57 O57-FLY6 838 E S N T A A A F L N R K S Q F L Y T T G K vtx2c-o57 O E S N T A A A F L N R K S Q F L Y T T G K vtx2c-o74 O74-b E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o9 O9-a-B2F 838 E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o9 O9-b-B2F 838 E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o57 O E S N T A A A F L N R K S Q S L Y T T G E vtx2d-c C freundii-lm E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o74 O74-EC720a 838 E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o6 O6-NV E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o8 O8-C466-0B 838 E S N T A A A F L N R K S Q S L Y T T G E vtx2d-o73 O73-C E S N T A A A F L N R R A H S L N T S G E vtx2b-o8 O8-EH E S N T A A A F L N R R A H S L N T S G E vtx2b-o22 O E S N T A A A F L N R R A H S L N T S G E vtx2b-ont ONT E S N T A A A F L N R R A H S L N T S G E vtx2b-o96 O96-S E S N T A A A F L N R R A H S L N T S G E vtx2b-o O-S E S N T A A A F L N R R A H S L N T S G E vtx2b-o O-PH 838 E S N T A A A F L N R R A H S L N T S G E vtx2b-o74 O74-a E S N T A A A F L N R R A H S L N T S G E vtx2b-o6 O E S N T A A A F L N R K S Q S L Y T T G E vtx2g-o2 O2-7v 838 E S N T A A A F L N R K S Q S L Y T T G E vtx2g-out Out-S E S N T A A A F L N R K S Q S L Y T T G E vtx2g-o2 O2-S E S N T A A A F L N R K S Q S L Y T T G E vtx2e-o39 O E S N T A A A F L N R K S Q S L Y T T G E vtx2e-o0 O0-E-D E S N T A A A F L N R K S Q P L Y T T G E vtx2e-o22 O E A N T I A A L L N R K P Q D L T E P N Q vtx2f-o28 O28-H-I.8 vtx2d Activatable tail

47 vtx2a a variant designations Non-O57 VTEC vtx2a-o48-94c vtx2a-o O57- Sorbitol fermenting vtx2a-o57-sf If nucleotide sequence is used: vtx2a-o57-sf (ns) vtx2a-o48-94c (ns) for nucleotide sequence

48 2006 Enter-net net ringtrial results Serotype vtx 32 laboratories O38:H26 O28ab:H2 O54:H3 O57:H7 O45:H4 O3:H4 vtxc vtxc vtxd vtx negative vtx negative vtx negative 2 negative neagtive 9 negative positive positive 2 positive

49 2006 Enter-net net ringtrial results Serotype vtx2 32 laboratories O03:H25 O38:H26 O28ab:H2 O54:H3 O57:H7 O45:H4 O3:H4 O5:H49 vtx2a-o57 O57-EDL933 vtx2b-o O-PH vtx2b-o O-PH vtx2b-o8 O8-EH250 vtx2 negative vtx2a-o57 O57-EDL933 vtx2c-o57 O57-FLY6 vtx2a-o48 O48-94C 5 negative 5 negative 3 negative 3 negative 4 positive 4 negative 4 negative vtx2d-o57 O negative vtx2e-ont ONT negative

50 vtx2 subtyping vtx2a vtx2b PCR + RFLP PCR + sequencing 620 bp fragment 49 nucleotide sequences translated into aminoacids were used for comparison and phylogenetic trees 225 VT2A VT2B 95 AA 64 AA Søren Persson s Ph.D.

51 Phylogenetic tree of vtx2 2 subgroups found in Danish patients by alignment of the 59 aa (49 bp) ) fragment 95 amino acids of C-terminal part of subunit A 64 amino acids of the N-terminal part of subunit B vtx2a vtx2c vtx2d vtx2e vtx2g vtx2b Søren Persson s Ph.D.

52 The 2 vtx2 genes found in Danish patients New variants First time in humans B2 (III) 0.0 II B (I) C (IV) vtx2a-o48 O48-94C vtx2a-o57 O57-EDL933 vtx2c-o57 O57-C vtx2c-o57 O57-FLY6 vtx2d-o73 O73-C65-02 vtx2d-o8 O8-C466-0B vtx2d-o57 O vtx2e-o0 O0-E43 vtx2e-ont ONT vtx2g-o2 O2-7v vtx2b-o O-PH vtx2b-o8 O8-EH250 Søren Persson s Ph.D. vtx2a vtx2c vtx2d vtx2e vtx2g vtx2b

53 Prevalence of vtx subtypes & vtx2 variants in 02 O57 vtxb b + vtx2c-o57 O57-FLY6 27 vtxb b + vtx2c-o57 O57-C vtxb b + vtx2a-o57 O57-EDL933 vtxc c + vtx2c-o57 O57-FLY6 vtx2a-o57 O57-EDL933 + vtx2c-o57 O57-FLY6 23 vtx2a-o57 O57-EDL933 7 vtx2c-o57 O57-FLY6 8 vtx2a-o57 O57-SF + vtx2c-o57 O57-FLY6

54 Prevalence of vtx subtypes & vtx2 variants in 83 eae positive non-o57 vtxb 29 vtx2a-o48 O48-94C 20 vtx2a-o57 O57-EDL933 6 vtxb b + vtx2a-o48 O48-94C 5 vtxb b + vtx2a-o57 O57-EDL933 3 vtx2c-o57 O57-FLY6 3 vtx2a-o57 O57-EDL933 + vtx2c-o57 O57-FLY6 2 vtxc vtxd vtx2d-o8 O8-C466-0B vtx2e-ont ONT vtx2g-o2 O2-7v

55 Prevalence of vtx subtypes & vtx2 variants in 44 eae negative non-o57 vtxb 32 vtx2b-o O-PH 8 vtxc 7 vtxc c + vtx2b-o8 O8-EH250 5 vtxb b + vtx2b-o8 O8-EH250 0 vtxc c + vtx2b-o O-PH 8 vtxd 6 vtxb b + vtx2b-o O-PH 5 vtx2a-o48 O48-94C 4 vtxc c + vtx2b-o O-PH + vtx2b-o8 O8-EH250 4 vtx2c-o57 O57-FLY6 4 vtx2d-o57 O vtxb b + vtx2b-o8 O8-EH250 EH250 + vtx2b-o O-PH 2 vtx2e-ont ONT vtxb b + vtx2d-o57 O vtxb b + vtx2c-o57 O57-FLY6 vtxb b + vtx2a-o48 O48-94C vtxb b + vtx2a-o48 + vtx2b-o O-PH vtxc c + vtx2a-o57 O57-EDL933 + vtx2-o8 O8- EH350 vtx2a-o48 O48-94C + vtx2d-o57 O vtx2e-o0 O0-E-D43 vtx2b-o8 O8-EH250 vtx2b-o O-PH + vtx2b-o8 O8-EH250 vtx2c-o57 O57-FLY6 FLY6 + vtx2d-o8 O8-C4660B vtx2d-o73 O73-C65-02 vtx2a-o48 O48-94C + vtx2d-o8 O8-C466-0B vtx2d-o8 O8-C466-0B vtx2g-o2 O2-7v

56 O57 Attack rate of vtx2 variants associated with HUS vtx2a-o57 O57-EDL933 + vtx2c-o57 O57-FLY6 6/23 26% vtx2a-o57 O57-EDL933 3/7 8% vtx2c-o57 O57-FLY6 /8 6% vtx2a-o57 O57-SF + vtx2c-o57 O57-FLY6 / - Non-O57 vtx2a-o48 O48-94C 6/20 30% vtxb b + vtx2a-o57 O57-EDL933 /3 - vtx2a-o57 O57-EDL933 + vtx2c-o57 O57-FLY6 /2 - vtxb b + vtx2a-o48 O48-94C /5 20% vtxb /29 0.8%

57 00% 80% 60% 40% Virulenceprofile of 0 Danish eae negative non-o57 VTEC and clinical manifestation Other D PD BD 20% PBD 0% vtx vtxc vtx2- O- PH vtx + vtx2- O8 vtxc + vtx2- O8 vtxc + vtx2- O- PH vtxd vtx + vtx2- O- PH Other: Asymptomatic carriers and non-diarrhoeal symptoms D: Diarrhoea PD: Persistent diarrhoea (more than 2 weeks) BD: Bloody diarrhoea PBD: Persistent bloody diarrhoea (more than 2 weeks)

58 Conclusions There is no definitive typing method for VTEC Standard methods: O grouping O:H serotyping Virulo-typing Sero-pathovar typing PFGE MLVA Are these adequate?

59 Conclusions Subtyping of vtx2 genes has shown that: Two vtx2a variants associated with HUS: vtx2a-o57 O57-EDL933 (& vtx2-o57 O57-FLY6) vtx2a-o57 O57-SF vtx2a-o48 O48-94C in NSF O57 in SF O57 Non-O57 in Non New Paradigm: May VTEC be categorised differently? Should prevention and control focus on the reservoir(s) ) and transmission of these particular vtx2a subtypes?

60 Conclusions Certain virulence Cocktails including eae positive or negative, and vtx subtypes/variants seem to be associated with specific clinical manifestations Should management and treatment be adjusted according to virulence cocktail? How is detection and surveillance scewed by the lack of specific detection and typing of variants?

61 Is EHEC obsolete? Wide clinical spectrum regardless of serotype Evident in person-to to-person transmission There is increasing evidence that even within the same serotype, O57:H7/NM included, there are differences in the clinical outcome. This seems to be much closer related to the virulence profile than to the serotype. Animals will often carry types that have been referred to as EHEC without any clinical symptoms. The clinical course of disease is therefore host associated and the term EHEC misleading. Furthermore, there is no internationally accepted definition of EHEC and it is therefore used very differently in many countries

62 New nomenclature. HUS inducing and outbreak associated VTEC (HI/OA VTEC/STEC) - eae and vtx2a - eae negative and vtx2d d (activatable( activatable) - eae and vtx in this group are less common but O03:H2, O:[H8], O5:H0, O8:H6, O45:[H28], O26:H, O2:H44, O5:NM, O8:NM, O55:H? and O9:NM have been associated with HUS. We have published Significant HUS-inducing capacity plus significant epidemic outbreak potential or capacity 2. HUS inducing, NOT outbreak associated (HI VTEC/STEC) VTEC with significant HUS-inducing, but low epidemic inducing capacity! No outbreaks detected with these many heterogeneous types. 3. Diarrhoea inducing (in humans!) VTEC (DI VTEC/STEC) Many different virulence profiles and by far dominated by vtx strains. There is no one common denominator for this group except the capacity acity to produce VT and observations of clinical cases of diarrhoea in humans. 4. Animal-associated associated VTEC (AA VTEC/STEC) VTEC that are widely spread out and exclusively found in the animal reservoir; yet they don't show up in human cases. Seems to be their natural habitat.

63 Acknowledgements Statens Serum Institut, Copenhagen Joan Neverman Jensen Søren Persson Katharina E. P. Olsen Mia Torpdahl Eva Møller Nielsen Co-authors Lothar Beutin Denis Piérard rard, Academisch Nancy A. Strockbine, National Beutin, Federal Institute for Risk Assessment, Berlin Academisch Ziekenhuis Vrije Universiteit, Brussel National Center for Zoonotic, Vector-borne and Enteric Diseases (CDC), Atlanta