Real-Time WGS for Routine Typing, Surveillance, and Outbreak Detection of VTEC. Katrine Grimstrup Joensen
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1 Real-Time WGS for Routine Typing, Surveillance, and Outbreak Detection of VTEC Katrine Grimstrup Joensen
2 Short about me Ph.d project: Application of WGS for Diagnostics, Surveillance and Outbreak Detection of Foodborne Pathogens DTU Food, Division for Epidemiology and Microbial Genomics (Frank Møller Aarestrup and Henrik Hasman) Statens Serum Institut (SSI), Department of Microbiology and Infection Control (Eva Møller Nielsen)
3
4 Overview VTEC Conventional typing WGS typing
5 VTEC Infections Study for 3 months or until reaching 40 isolates
6 Conventional Vs. WGS-Typing Conventional: Serotyping PCR, Hybridisation VCA Haemolysis PFGE... Whole genome sequencing: Determination of the complete DNA sequence of the organism at once
7 Benchtop Sequencing IonTorrent PGM ~200bp reads, 1 Gb output ~18 hours from DNA to sequence data (2.5 normal working days) ~8 hours hands-on time 2-4 E.coli per chip
8 Web-tools for WGS data User-friendly WGS tools Center for Genomic Epidemiology (CGE)
9 Database based on Identibac scheme
10 Virulence Database 76 virulence genes stx1 (14A,18B variants) stx2 (114A,43B variants) eae (45 variants) >stx1_1_a_ab ATGAAAATAATTATTTTTAGAGTGCTAACTTTTTTCTTTGTTATCTTTTCAGTTAATGTGG TGGCGAAGGAATTTACCTTAGACTTCTCGACTGCAAAGACGTATGTAGATTCGCTGAAT GTCATTCGCTCTGCAATAGGTACTCCATTACAGACTATTTCATCAGGAGGTACGTCTTT ACTGATGATGATAGTGGCACAGGGGATAATTTGTTTGCAGTTGATGTCAGAGGGATAG ATCCAGAGGAAGGGCGGTTTAATAATCTACGGCTTATTGTTGAACGAAATAATTTATAT GTGACAGGATTTGTTAACAGGACAAATAATGTTTTTTATCGCTTTGCTGATTTTTCACAT GTTACCTTTCCAGGTAAACAGCGGTTACATTGTCTGGTGACAGTAGCTATACCACGTTA CAGCGTGTTGCAGGGATCAGTCGTACGGGGATGCAGATAAATCGCCATTCGTTGACTA CTTCTTATCTGGATTTAATGTCGCATAGTGGAACCTCACTGACGCAGTCTGTGGCAAGA GCGATGTTACGGTTTGTTACTGTAAGCTGAAGCTTTACGTTTTCGGCAAATACAGAGGG GATTTCGTACAACACTGGATGATCTCAGTGGGCGTTCTTATGTAATGACTGCTGAAGAT GTTGATCTTACATTGAACTGGGGAAGGTTGAGTAGCGTCCTGCCTGACTATCATGGAC AAGACTCTGTTCGTGTAGGAAGAATTTCTTTTGAAGCATTAATGCAATTCTGGGAAGCG TGGCATTAATACTGAATTGTCATCATCATGCATCGCGAGTTGCCAGAATGGCATCTGAT GAGTTTCCTTCTATGTGTCCGGCAGATGGAAGAGTCCGTGGGATTACGCACAATAAAA TATTGTGGGATTCATCCACTCTGGGGGCAATTCTGATGCCGAACTATTAGCAGTTGAG GGGGTAAAATGAAAAAAACATTATTAATAGCTGCATCGCTTTCATTTTTTTCAGCAAGT GCGCTGGCGACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATGATG ACGATACCTTTACAGTTAAAGTGGGTGATAAAGAATTATTTACCAACAGAGGAATCTTC AGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATGACTGTAACCATTAAAACTAATGCC TGTCATAATGGAGGGGGATTCAGCGAAGTTATTTTTCGTTGA Gene Description Variants in database asta Heat-stable enterotoxin 1 11 bfpa Major subunit of bundle-forming pili 5 cba Colicin B 15 cci Cloacin 4 cdtb Cytolethal distending toxin B 14 celb Endonuclease colicin E2 10 cfa_c Colonisation factor antigen I 4 cif Type III secreted effector 4 cma Colicin M 19 cnf1 Cytotoxic necrotizing factor 7 cofa Longus type IV pilus subunit 1 eae Intimin 45 eata SPATE 3 efa1 EHEC factor for adherence 11 ehxa Enterohaemolysin 12 epea SPATE 1 espa Type III secretions system 23 espb Secreted protein B 14 espc SPATE 3 espf Type III secretion system 13 espi SPATE 2 espj Prophage-encoded t3ss effector 2 espp Putative exoprotein precursor 4 etpd Type II secretion protein 3 f17-a Subunit A of F17 fimbrial protein 7 f17-g Adhesin subunit of F17 fimbriae 9 fana Involved in biogenesis of K99/F5 fimbriae 1 fasa Fimbriae 987P/F6 subunit 1 feda Fimbrial protein F107 subunit A 3 fedf Fimbrial adhesin AC precursor 6 fim41a Mature Fim41a/F41 protein 2 gad Glutamate decarboxylase 70 hlye Avian E.coli haemolysin 1 iha Adherence protein 19 ipad Invasion protein Shigella flexneri 9 ipah9.8 Invasion plasmid antigen 8 irea Siderophore receptor 4 iron Enterobactin siderophore receptor 13
11
12
13 Output example
14 Comparing typing results Serotype Routine Typing VirulenceFinder MLST Isolate O5:H- vtx1c vtx1c ST-447 C O26:H11 eae, vtx1a eae, vtx1a ST-21 C O27:H30 vtx2b vtx2b ST-753 C (replicate) O36:H C O55:H12 vtx1a vtx1a ST-101 C O76:H- vtx1c vtx1c ST-675 C904-12A O103:H2 O103:H- O rough:h2 eae, vtx1a eae, vtx1a ST-17 ST-1967 ST-17 C C C O115:H- eae, vtx2a eae, vtx2a ST-333 C885-12B C896-12A O117:K1:H7 vtx1a vtx1a ST-504 C C C O121:H19 eae, vtx2a eae, vtx2a ST-655 C O128ab:H2 vtx2b vtx2b ST-25 C vtx1c, vtx2b vtx1c, vtx2b ST-811 C O128abc:H- eae, vtx2f eae, vtx2f ST-20 C C O145:H- eae, vtx2a eae, vtx2a ST-32 C C C C O146:H21 vtx1c, vtx2b vtx1c, vtx2b ST-442 C eae eae C896-12B C885-12A O157:H7 (O157:H-) eae, vtx1a, vtx2c eae, vtx1a, vtx2c ST-11 C C697-12A (replicate) C697-12B eae, vtx1a C eae, vtx1a, vtx2a eae, vtx1a, vtx2a C C C C C C eae, vtx2a eae, vtx2a C eae, vtx2c eae, vtx2c ST-2966 C O165:H- eae, vtx1a, vtx2a eae, vtx1a, vtx2a ST-119 C905-12
15 Phylogeny -SNPtree
16 Phylogeny -NDtree
17 VTEC Outbreak Denmark faced an outbreak of verocytotoxin-producing E. coli (VTEC) O157:H7 infections in autumn Thirteen cases were diagnosed of which eight had haemolytic uraemic syndrome (HUS). Epidemiological investigations suggested ground beef as the vehicle of the outbreak. The outbreak strain had a rare toxin gene subtype profile: eae, vtx1a and vtx2a, and a high proportion of HUS (62%) among cases, a finding previously linked with the outbreak subtype profile. Toxin subtyping can be useful to identify high risk VTEC strains
18 Outbreak Serotype Routine Typing VirulenceFinder MLST Isolate O5:H- vtx1c vtx1c ST-447 C O26:H11 eae, vtx1a eae, vtx1a ST-21 C O27:H30 vtx2b vtx2b ST-753 C (replicate) O36:H C O55:H12 vtx1a vtx1a ST-101 C O76:H- vtx1c vtx1c ST-675 C904-12A O103:H2 eae, vtx1a eae, vtx1a ST-17 C O103:H- ST-1967 C O rough:h2 ST-17 C O115:H- eae, vtx2a eae, vtx2a ST-333 C885-12B C896-12A O117:K1:H7 vtx1a vtx1a ST-504 C C C O121:H19 eae, vtx2a eae, vtx2a ST-655 C O128ab:H2 vtx2b vtx2b ST-25 C vtx1c, vtx2b vtx1c, vtx2b ST-811 C O128abc:Heae, vtx2f eae, vtx2f ST-20 C C O145:H- eae, vtx2a eae, vtx2a ST-32 C C C C O146:H21 vtx1c, vtx2b vtx1c, vtx2b ST-442 C O157:H7 (O157:H-) eae eae C896-12B C885-12A eae, vtx1a, vtx2c eae, vtx1a, vtx2c ST-11 C C697-12A (replicate) C697-12B eae, vtx1a C eae, vtx1a, vtx2a eae, vtx1a, vtx2a C C C C C C eae, vtx2a eae, vtx2a C eae, vtx2c eae, vtx2c ST-2966 C O165:H- eae, vtx1a, vtx2a eae, vtx1a, vtx2a ST-119 C O180:H- eae eae C641-12A ST-301 eae, vtx2a a eae C641-12B O181:H16 vtx1a b vtx1c - C OX187:H28 vtx2d c vtx2g ST-200 C O rough:- vtx2a vtx2a ST-678 C a Re-typed to lack vtx2 at SSI b Re-typed to vtx1c at SSI c Re-typed to vtx2g at SSI
19 Comparing Time WGS-typing was faster Same Isolate Price: ~430
20 Conclusion WGS faster than Routine WGS gives more information e.g.: more virulence genes, resistance genes, MLST, phylogeny etc. Same price Will work for other pathogens as well Easy-to-use bioinformatic tools are necessary
21 Real-Time Surveillance of VTEC using WGS Reviewer comment: It appears that VTEC serotype was determined solely by conventional agglutination methods. Could the investigators add "molecular serotyping" to the WGS output by using the flagellar and O-antigen associated genes?
22 Construction of the SerotypeFinder For user-friendly (O:H) serotype determination from WGS data CGE tools ( Flemming Scheutz Head of WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella Dep. of Microbiology & Infection Control, SSI Anna Maria Malberg Tetzschner Master student DTU/SSI
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24 O-antigen and O-antigen encoding region In Silico O-typing 134 L. Wang et al. LPS periplasmic space O antigen (polysaccharide) Core polysaccharide LipidA outer membrane inner membrane 187 known E. coli O-antigens ori O1-O187 (now the new O188 is being added) chrom osom e wzx and wzy (O-antigen flippase and polymerase) wca his wzm and wzt (ABC transporter) O -a n tig e n b io s y n th e s is g e n e c lu s te r (O -A g c lu s te r ) 134 L. Wang et al. Debroy et al, 2011 Wang et al, 2010
25 In Silico H-typing flic (flagellin) flka, flna, flma, flla
26 SerotypeFinder database construction Collection of genes with serotype information from NCBI O-antigen cluster sequences from Dr. Atsushi Iguchi Few E. coli reference isolates sequenced at SSI
27 O Database All known O-types represented (some represented by wzm/wzt) wzx wzy
28 H Database flic genes All 53 H-types represented
29
30 Output SerotypeFinder
31 Validation of the SerotypeFinder Validation on 682 E. coli genomes (601 for O-typing and 509 for H-typing) 249 genomes from PulseNet STEC genome reference library (PRJNA218110) 130 genome sequences from a previous study on comparative genome analysis of E. coli (+4 Shigella) 44 E. coli genome assemblies from our own Real-Time typing and surveillance of VTEC 146 E. coli reference strains (Illumina 2x100bp) obtained from CDC (will be deposited in PRJNA186441) 109 E. coli sequenced from the SSI strain collection
32 Validation of the SerotypeFinder No. (%) of genomes: With consistent WGS and For validation With detected genes Typing conventional results O a ( 95%) 560 b ( 98%) H ( 100%) 504 ( 99%) a In 51 genomes, genes were found by reference mapping, and in 21 genomes, only one gene was used for prediction. b Eleven predictions were ambiguous between the two O-processing genes [O118/O151(7), O164/O124, O134/O46, O90/O127, and O162/O101].
33 Discrepancies Isolate Serotype SerotypeFinder ID Comment RN587/1 O157:H8 H45 Difficult to type according to authors (29) W27 a O115:K :H18 H19 Original transcription error suspected; now the strain is nonmotile and cannot be confirmed; three of 116 motile O115 strains in the WHOCC database are H19, and none are H18 U19-41 O51:K :H24 H49 Original transcription error suspected; the isolate agglutinates only the H pool containing H49, and not the H pool containing H24 Prediction based on flic; H4 and F O15:K14:H4 H17 H17fliC are very similar in sequence; unresolved O144 O124:H30 Suspected transcription error or typing mistake ( ) b O129:K :H11 O13/O135 Re-tested and confirmed O129 C O111:H7 O96 Unresolved F b O62:K :H30 O13/O135 Isolate is still subjected to O-antigen testing, as the antigen is rough F (O19a) c O133 Suspected mix-up; the isolate was subjected to retyping of O antigen, and many cross-reactions occurred (O26, O50 and O133); final O-antigen determination was not possible HW35 O11:Kne:H33 O99 Suspected original transcription error; the isolate was confirmed as O99, with no cross-reaction with the O11 antisera SE11 O152:H28 O173 Unresolved SSI_AA017 O41:H48 O60 Unresolved; poor sequence data and assembly SSI_AA037 O20:K67:H17 O128ac The O128 detected is suspected to be due to K67 SSI_AA099 O43:H46 O131 Retesting confirmed O131 SSI_AA095 O44:K53,93:H46 O17 Cross-reactions between O17, O44, O73, and a The O115 reference strain was sequenced both at CDC and SSI with the same results in SerotypeFinder. O77 make it difficult to determine these O b Variant sequences for the reference strains for O129 [178-54( )] and O62 (F ) were represented in the O database, and therefore, groups resequencing phenotypically; of these retesting strains was confirmed not considered as part of the validation set. O44 c The parentheses National indicate Food that Institute, final O-antigen Technical determination University was not possible. of Denmark
34 O groups and H types covered in the validation of SerotypeFinder No. of genomes in agreement with conventional results (O total; H total) O groups H types 1 (71; 0) O4, O10, O18, O21, O22, O23, O24, O29, O30, O33, O34, O35, O37, O38, O42, O43, O44, O48, O49, O50, O53, O54, O58, O65, O73, O76, O82, O83, O84, O87, O89, O95, O102, O105, O108, O109, O110, O116, O120, O130, O135, O136, O137, O138, O139, O140, O143, O144, O147, O149, O150, O151, O154, O155, O159, O162, O163, O164, O167, O168, O170, O172, O173, O175, O176, O177, O178, O179, O182, O183, O186 2 (82; 0) O1, O5, O20, O25, O28, O32, O39, O40, O46, O60, O70, O78, O79, O80, O81, O88, O92, O96, O98, O99, O101, O114, O123, O126, O131, O132, O134, O141, O153, O156, O158, O160, O161, O165, O166, O171, O177, O181, O184, O185, O187 3 (33; 63) O27, O75, O107, O112, O113, O125, O133, O142, O148, O169, O180 4 (48; 44) O2, O3, O9, O16, O17, O36, O51, O69, O104, O115, O127, O174 5 (25; 25) O11, O91, O117, O119, O146 H1, H9, H40, H43, H56 6 (30; 12) O6, O7, O15, O86, O118 H18, H30 7 (7; 14) O8 H28, H34 8 (8; 0) O45 10 (324; 345) O103 (17), O55 (22), O128 (18), O145 (24), O121 (30), National Food Institute, O26 Technical (43), O111 University (48), O157 of Denmark (122) H3, H5, H12, H14, H15, H17, H20, H26, H27, H31, H36, H37, H38, H41, H45, H46, H48, H49, H51, H52, H53 H23, H24, H29, H32, H35, H39, H42, H44, H47, H54, H55 H33 (9), H10 (10), H16 (12), H25 (11), H21 (13), H4 (14), H8 (14), H6 (17), H2 (34), H19 (39), H11 (45), H7 (127)
35 SSI strain collection covered by validation (N=45947 isolates) 1 genome 17% Non-validated O-groups 4% Other 1% 2 genomes 19% 3 genomes 59% 3 genomes 2 genomes 1 genome Non-validated O-groups Other O-groups not validated: O12, (O12/O16), O13, O14, O19, O41, O52, O59, O61, O62, O63, O64, O66, O68, O71, O74, O77, (O77/O106), O85, O93, O100, O106, O124, O129, O152
36 Conclusion Results obtained from in silico serotyping using WGS are already superior to the conventional methods in terms of reproducibility and discriminatory and definitive power Since January 2015 SSI has performed WGS-based ROUTINE typing and surveillance of VTEC In use at PHE and CDC
37 Acknowledgements Anna M. M. Tetzschner Previous Master student DTU/SSI Flemming Scheutz and Eva M. Nielsen Department of Microbiology and Infection Control, SSI Center for Genomic Epidemiology (CGE): Atsushi Iguchi Department of Animal and Grassland Sciences, Japan Rebecca L. Lindsey Centers for Disease Control and Prevention, Atlanta, Georgia, USA Frank M. Aarestrup Henrik Hasman Ole Lund and Rolf S. Kaas
38 Questions?
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