1. Strategy for developing a molecular subtyping tool for a foodborne bacterial pathogen using a whole genome analysis approach: the case of Salmonella Enteritidis Dele Ogunremi DVM, PhD Ottawa Laboratory Fallowfield Dele.Ogunremi@inspection.gc.ca 343-212-0235

2. Why a new strategy? What is so important about Salmonella Enteritidis? What is the new strategy? Why SNPs? Conclusions Acknowledgment 2 OUTLINE

3. Improving food safety 3

4. Salmonella is an important foodborne pathogen Highest burden of illness ranking among foodborne pathogens Wide distribution Many serovars of Salmonella (Total number of serovars = 2,579 serovars) Salmonella enterica serovar Enteritidis: most common serovar contaminating food, mainly poultry products. 4

5. Whole genome analysis: a mastertool Diversity can still be found in a highly clonal organism such as Salmonella Enteritidis - single nucleotide polymorphism, SNPs - prophages 5 Two key messages

6. Definition means of identifying types of organisms within a species = evaluating relatedness An ideal subtyping test: inexpensive rapid reproducible discriminatory epidemiologically concordant 6 Microbial subtyping

7. 7 Past: Subtyping technology negligible or unavailable 7 The Evolution of Molecular Subtyping of Bacteria Present: PFGE, phage typing New: Whole genomes + PFGE + phage typing

8. 8 Current Microbial subtyping tools Subtyping ApproachAssay PhenotypicBiotyping Serotyping Phage typing MolecularPulse-Field Gel Electrophoresis Variable number tandem repeats (MLVA) Multi-locus sequence typing (MLST) PCR-RFLP Random Amplified Polymorphic DNA typing Sequence typing

9. 9 PulseNet: PFGE technology PFGE technology has been used to build an international molecular subtyping network, PulseNet, in order to utilize DNA fingerprinting to detect outbreaks of foodborne diseases at the earliest possible stage PulseNet Canada serves as a rapid communications platform that has the ability to link laboratories both nationally and internationally when investigating sporadic and outbreak cases as well as identifying and confirming the original source of contamination A large outbreak in one place may be obvious A dispersed outbreak may be difficult to detect without molecular subtyping network-PulseNet

10. Salmonella Enteritidis PFGE analysis Data source: PulseNet Canada Analysis: CFIA 10 PFGE PATTERNSFrequency % Rank PRIMARY: SENXAI SEN XAI 0001 43413.14 SEN XAI 0002 260.8 SEN XAI 0003 112233.81 SEN XAI 0004 581.7 SEN XAI 0006 63519.22 SEN XAI 0007 1374.1 SEN XAI 0008 2648.0 SEN XAI 0009 80.2 SEN XAI 0013 30.1 SEN XAI 0016 30.1 SEN XAI 0019 30.1 SEN XAI 0021 10.0 SEN XAI 0025 10.0 SEN XAI 0035 20.1 SEN XAI 0038 44313.43 SEN XAI 0040 10.0 SEN XAI 0041 571.7 SEN XAI 0053 10.0 SEN XAI 0054 20.1 SEN XAI 0060 80.2 SEN XAI 0062 80.2 SEN XAI 0066 40.1 SEN XAI 0068 441.3 SEN XAI 0069 10.0 SEN XAI 0074 20.1 SEN XAI 0075 80.2 SEN XAI 0076 20.1 SEN XAI 0077 100.3 SEN XAI 0084 30.1 SEN XAI 0088 40.1 SEN XAI 0092 10.0 SEN XAI 0093 50.2 SEN XAI 0094 20.1 SEN XAI 0110 20.1 SEN XAI 0111 10.0 SEN XAI 0114 90.3 3315100.0

11. Microbial genome sequencers 11 Ion Torrent PGM Illumina MiSeq 3730 Genetic analyzer

12. Approaches Entire genomes Targeting specific parts of the genomes Advantages 1. Accuracy 2. Cost-efficiency 3. Comprehensive 4. Extensible 12 Strategy for developing a subtyping tool for Salmonella Enteritidis: genomics-based

13. Whole genome analysis: at least two different platforms 454, Illumina, Ion Torrent, PacBiosciences Assemble genome: Reference assembly De novo assembly Optical mapping Strategy for developing a subtyping tool for Salmonella Enteritidis

15. 15 BMC Genomics 2014, 15: 713

16. Pan genome: entire set of the genes within the species or serovar Core genome: common to all members of the species or serovar Accessory genome: genes present in at least two strain but absent in at least one strain 16 Pan genome of Salmonella Enteritidis

17. Genomes: core vs accessory Number of genes (%) Salmonella Enteritidis (Ogunremi et al., 2014 BMC Genomics 15: 713) Listeria monocytogenes (Deng et al., 2010 BMC Genomics 11:500) Core genome 4,600 (95%) 2,456 (61%) Accessory genome 233 (5%) 1,596 (39%) Total genome 4,833 4,052 17

18. 18 Microbial subtyping tools: old and new CurrentNew Generation BiotypingSNP-based (whole genomes or PCR) SerotypingProphage Phage typingCRISPR Pulse-Field Gel ElectrophoresisPseudogenes Variable number tandem repeats (MLVA) Multi-locus sequence typing (MLST) PCR-RFLP Random Amplified Polymorphic DNA typing Sequence typing

19. Whole genome analysis: at least two different platforms 454, Illumina, Ion Torrent, PacBiosciences Assemble genome: Reference assembly De novo assembly Optical mapping SNPs Strategy for developing a subtyping tool for Salmonella Enteritidis

20. Whole genome analysis: at least two different platforms 454, Illumina, Ion Torrent, PacBiosciences Assemble genome: Reference assembly De novo assembly Optical mapping SNP Adapt for routine testing: SE-SNP-PCR Strategy for developing a subtyping tool for Salmonella Enteritidis

21. SNP PCR test is highly discriminatory 21 SNP CLADES IIIIIIIVVVIVIIVIIIIXXXIXII PFGEFrequency Rank XAI.0001 1 1 XAI.0003 281st 7 151541 13 XAI.0006 62nd 5 1 XAI.0007 1 1 XAI.0009 4 1 2 1 XAI.0025 2 2 XAI.0026 1 1 XAI.0038 53rd 5 XAI.0076 3 3 XAI.0214 4 22 55

22. Whole genome analysis: at least two different platforms 454, Illumina, Ion Torrent, PacBiosciences Assemble genome: Reference assembly Optical mapping SNP identification Prophage analysis Adapt for routine testing SE-SNP=PCR Strategy for developing a molecular typing tool for Salmonella Enteritidis

23. 20 S. Enteritids P125109 phage type 4 (Windhorst, 2010; Thomson et al., 2008) 23 Prophages of Salmonella Enteritidis Prophage designationSize (bp) Φ SE 108,186 Φ SE 12/12A17,753 Φ SE 1412,642 Φ SE 2040,664 Total79, 245

25. Number and sequence lengths of Salmonella Enteritidis prophages 25 Salmonella Enteritidis genomes (Reference P125109 and SENT 1-11) Prophage P1251091234567891011 1 8,007 8,0068,007 2 8,168 8,1678,4238,168 8,677 3 42,042 42,04142,04242,041 42,04042,042 4 14,877 14,87614,877 5 48,486 6 3,9873,994 3,987 3,9943,9873,9933,994 4,001 7 35,705 36,20935,70535,704 8 11,502 11,501 11,50211,501 11,50011,50311,50211,757 9 8,6098,5518,660 10 9,241 11 28,108 12 19,137 13 43,544 TOTAL (bp) 137,069124,295 132,896132,838133,535132,947124,289125,051152,403163,369132,905

26. Length of prophage (kb) Genome # 26 Prophages of Salmonella Enteritidis

27. Why a new strategy? What is so important about Salmonella Enteritidis? What is the new strategy? Why SNPs? Conclusion 27 SUMMARY

28. Diversity in the Pan genome of Salmonella Enteritidis Pan genome: entire set of the genes within the species or serovar Core genome: SNPs Accessory genome: prophages 28

29. 29 Acknowledgements CFIA: Ontario Laboratory NetworkCFIA Lethbridge Public Health Agency, NML Andree Ann Dupras Kingsley Amoako Morag Graham Hilary Kelly Kristen Hahn Gary Van Domselaar Sebastien Belanger Noriko GojiShaun Taylor Linru WangMatt Gilmour (Health Services) John Devenish Susan Nadin-Davis McGill University Public Health Agency, Guelph Burton Blais Ken DewarRoger Johnson Olga Andrievskaia Kim Ziebell OLF PFGE Unit Genome Quebec University of Guelph Ray AllainAlfredo Straffa Shu Chen Jennifer Hazelwood Frederick Robidoux Saleema Saleh-Lakha Chris GrenierMathieu Bourgey OLN Management Public Health Agency, Lethbridge National Research Council Karen Jessett Ed Taboada Luke Masson Ray Theoret Stephen Norman University of Saskatchewan Musangu Ngeleka

30. 30

31. 10052pt8_00dpt2300dpt810supt13a09pt13a08 pt2310s u pt13100 819100581021710012 P125109-PT4589596 604 602614586591597886 10052307304311 30428210170289826 pt8_00d27207200201247243269246900 pt2300d196193192240238262238900 pt810su656911724827078904 pt13a091260245270112903 pt13a0859242263105898 pt2310su221241113886 pt1310081960258864 10058252855 10217913 Pairwise SNP counts 31

33. Pan genome: entire set of the genes within the species or serovar Core genome Accessory genome 33 Pan genome of Salmonella Enteritidis