Strategy for developing a molecular subtyping tool for a foodborne bacterial pathogen using a whole genome analysis approach: the case of Salmonella Enteritidis.

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Presentation transcript:

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

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

Improving food safety 3

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

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

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 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 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 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

Salmonella Enteritidis PFGE analysis Data source: PulseNet Canada Analysis: CFIA 10 PFGE PATTERNSFrequency % Rank PRIMARY: SENXAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI SEN XAI

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

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

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 BMC Genomics 2014, 15: 713

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

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 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

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

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

SNP PCR test is highly discriminatory 21 SNP CLADES IIIIIIIVVVIVIIVIIIIXXXIXII PFGEFrequency Rank XAI XAI st XAI nd 5 1 XAI XAI XAI XAI XAI rd 5 XAI XAI

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

20 S. Enteritids P 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

Number and sequence lengths of Salmonella Enteritidis prophages 25 Salmonella Enteritidis genomes (Reference P and SENT 1-11) Prophage P ,007 8,0068, ,168 8,1678,4238,168 8, ,042 42,04142,04242,041 42,04042, ,877 14,87614, , ,9873,994 3,987 3,9943,9873,9933,994 4, ,705 36,20935,70535, ,502 11,501 11,50211,501 11,50011,50311,50211, ,6098,5518, , , , ,544 TOTAL (bp) 137,069124, ,896132,838133,535132,947124,289125,051152,403163,369132,905

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

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

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 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

10052pt8_00dpt2300dpt810supt13a09pt13a08 pt2310s u pt P PT pt8_00d pt2300d pt810su pt13a pt13a pt2310su pt Pairwise SNP counts 31

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