THE RISE OF WHOLE GENOME SEQUENCING AS A SUBTYPING TOOL FOR MICROBIAL SOURCE TRACKING: FROM FUNDAMENTALS TO APPLICATIONS

Transcription

1 THE RISE OF WHOLE GENOME SEQUENCING AS A SUBTYPING TOOL FOR MICROBIAL SOURCE TRACKING: FROM FUNDAMENTALS TO APPLICATIONS STEAK EXPERT MEETING: ANGERS FRANCE JUNE, 2015 Kendra Nightingale, Ph.D. Inter national Center for Food Industr y Excellence Depar tment of Animal & Food Sciences Texas Tech University

2 SUBTYPING OF FOODBORNE PATHOGEN ISOLATES Ability to differentiate isolates belonging to foodborne pathogen beyond the species sub-species level Considerations for interpretation of bacterial subtyping or DNA fingerprint typing data: Goal of human DNA subtyping = identify a single specific individual Human DNA subtypes are unique Bacterial subtypes are not Goal of bacterial DNA subtyping = determine if isolates share a recent common ancestor

3 BIOLOGY 101 Molecular Methods DNA mrna Transcription DNA Replication Translation Phenotypic Methods Protein/Enzymes Toxins and Other Metabolites

4 MICROBIOLOGY 101

5 MICROBIOLOGY 101

6 MICROBIAL GENETICS 101 What types of DNA molecules are present in a bacterial cell? What s the size of the genetic material for a typical bacterial pathogen? How many genes does a bacterial pathogen have? What s the average size of a bacterial gene?

7 MICROBIAL GENETICS 101 Generation 1 Generation 2 Generation 3 Generation N Ancestor Genotype Clones Clones Clones and Divergent Genotypes Mutation? Bacterial mutation rate?

8 MICROBIAL GENETICS 101 Mutations Point mutations Frameshift mutations Inversions Insertions/deletions (indels), including duplications Horizontal gene transfer Horizontal gene transfer of homologous gene sequences Horizontal gene transfer of non-homologous gene sequences Plasmid loss or acquisition

9 SUBTYPING OF FOODBORNE PATHOGEN ISOLATES Subtyping tools to detect disease outbreaks and identify food source Phenotypic subtyping Serotyping, phage typing, biotyping Molecular subtyping Band-based PFGE, ribotyping, REP-PCR DNA sequence-based Allele, multilocus sequence typing (MLST), multiple-locus variable number tandem repeat analysis (MLVA), genome Molecular subtyping methods more discriminatory & reproducible Whole genome sequencing will facilitate disease outbreak detection and outbreak investigations to identify food source

10 SUBTYPING & OUTBREAK INVESTIGATIONS Exposure to Pathogen in Food Human infection Food Storage Isolation from sterile body fluid using nonselective blood plates Isolation using selective enrichment and selective and differential agar media Subtyping of Isolate Subtyping of Isolates

11 ESTABLISHMENT OF SUBTYPING NETWORK PulseNet established in the U.S. in 1996: turning point for routine PFGE typing of bacterial foodborne disease surveillance from clinical cases Initially focused on Escherichia coli O157:H7 but expanded to other pathogens (i.e., Campylobacter jejuni, Clostridium botulinum, Cronobacter, Listeria monocytogenes, Salmonella, and Shigella) Expanded to food and environmental isolates Expanded internationally to PulseNet International Development and implementation of standardized protocols and rapid Web-based exchange of resultant pattern data Facilitated detection of temporally and spatially distributed foodborne disease outbreaks & outbreak investigation PulseNet adapted for whole genome sequences and analyses of resultant data Changing the shape of epidemic curves (i.e., reducing noise and expanding time frame of an outbreak)

12 Image provided by Peter Gerner-Smidt, Centers for Disease Control and Prevention

13 TEMPORALLY AND GEOGRAPHICALY DISPERSED OUTBREAK DETECTION Two more cases of the same illness where investigation shows illness came from same food or drink In 2014, the Centers for Disease Control & Prevention monitored between 20 and 40 potential related clusters of illness weekly and investigated >220 multistate clusters Investigations led to identification of 68 confirmed or suspected vehicles and recall of variety of foods Molecular subtyping and surveillance network to identify clusters of cases caused by the same strain

14 Mid-September, 2006 CDC alerted about clusters of E. coli O157:H7 illness in northwest CDC PulseNet confirmed cases caused by same PFGE type End of September, persons infected with outbreak strain in 26 states 52% hospitalized; 15% HUS; three deaths 95% reported eating fresh spinach within 10 d before illness onset Trace-back investigation E. coli O157:H7 matching outbreak PFGE type isolated from cattle on ranch nearby spinach fields & feral hogs

15 November, 2006 State Health Departments detected elevated incidence of illness due to Salmonella Tennessee Three closely related PFGE types rarely reported before October, 2006 February, 2007 Case-control study conducted Strong association with Peter Pan & Great value peanut butter produced at the same plant Outbreak PFGE types isolated from opened & unopened peanut butter produced from August, 2006 to January, 2007 > 600 people infected with outbreak PFGE types

16 PERSISTENCE OF L. MONOCYTOGENES IN FOOD PROCESSING PLANTS Listeria contamination patterns in six RTE small and very small RTE meat processing plants for two years 1,743 samples collected bi-monthly Year 1 Non-food contact surfaces In-plant training for all employees General knowledge on Listeria ecology, transmission, and control Testing and molecular results from Year 1 Year 2 Non-food contact surfaces, food contact surfaces, finished product for some plants

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18 DNA SEQUENCE-BASED STRAIN TYPING/IDENTIFICATION METHODS Allelic typing Multi-locus sequence typing (MLST) Multi-locus variable number tandem repeat analysis (MLVA) Single nucleotide polymorphism (SNP) typing Clustered regular spaced palindromic repeat (CRISPR) typing Whole genome sequencing CDC, FDA and regulatory agencies in other countries routinely performing whole genome sequencing of foodborne pathogen isolates from human clinical cases of foodborne disease

19 HISTORY OF GENOME SEQUENCING Sequencing of DNA molecules began in the late 1970s Chemical degradation, followed by the Sanger chain termination method, known as the gold standard of DNA sequencing Shutgun sequencing, based on the Sanger method, cloning fragments into Escherichia coli for amplification First bacterial genome sequence completed (Haemophilus influenzae; 1.8 million bp) completed in 1995 E. coli O157:H7/K-12 comparative genomes study; First Listeria monocytogenes genome published in 2001 The first finished human genome sequence (3 billion bp) completed in 2003 Project took 13 years to complete and cost $2.7 billion

20 THE RACE FOR THE $1,000 GENOME 2003 The J. Craig Venter Science Foundation promised $500,000 to the first group to produce a technology capable of sequencing a human genome for $1,000 The X Prize Foundation promised an additional $5-20 million to the winner 2004 The National Institutes of Health (NIH) launched $70 million program to support researchers working to sequence complete mammal-sized genomes initially for $100,000 and ultimately for $1,000

21 NEXT GENERATION SEQUENCING (NGS) Generate vast amounts of sequence data quickly and relatively inexpensively Unique chemistry and platforms template preparation, sequencing/imaging & data analyses) Eliminate need for shot-gun cloning & amplification Template preparation Clonally amplified template originating from single molecule Single DNA molecule template Sequencing/imaging Cyclic reversible termination Single nucleotide addition Sequencing by ligation Real-time Sequencing Metzker, 2010

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23 APPLICATIONS OF NEXT- GENERATION SEQUENCING ChIP-Sequencing Methylation patterns Whole genome sequencing Development of detection kits, better subtyping tools, detection of outbreaks, identification of food source, microbial ecology & evolution Expression tags Metagenomics & microbial diversity Microbiome, culture independent diagnostics Targeted resequencing Small RNA analysis Transcriptome sequencing Expression of genes under defined experimental conditions, niche adaption

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25 Solexa/Illumnia

26 Solexa/Illumnia

27 RAPID WHOLE GENOME SEQUENCING (WGS) BASED SUBTYPING 3 days DNA extraction Library prep 24 h Sequencing on Bench top sequencer (e.g., MiSeq, Ion Torrent) 12 h De novo assembly Rapid classification to subpopulation using pairwise distances based on average nucleotide identity values (BLAST) Inference of subpopulation structure based on SNP calling.

28 APPLICATIONS OF WGS IN FOOD MICROBIOLOGY/FOOD SAFETY Facilitate development of improved detection assays Identification of genes/markers unique to pathogens or outbreak strains Allow for development improved molecular subtyping methods Pathogens that are difficult to differentiate by PFGE (e.g., certain Salmonella serotypes) Provide new insight into biology of food-associated microorganisms (pathogens, spoilage organisms, beneficial microorganisms) Taxonomy (Five new Listeria spp., new Salmonella serotype), transcriptional profile and niche adaptation Allow for large scale population based studies of food associated microorganisms, environmental microorganisms and intestinal microbes Metagenomics to probe whole microbial community (i.e., culturable and non-culturable) sequencing total microbial DNA

29 WGS FOR OUTBREAK SPECIFIC DETECTION Background: Very large outbreak associated with non-o157:h7 strain Unusual clinical characteristics Serotype O104:H4

30 GENOME SEQUENCING Goals: Characterize strain to possibly gain insights into reason beyond unique epidemiological and clinical features of outbreak Use genome sequence data to develop PCR assays for detection Performed, using the Ion Torrent system, both in Europe (Life Technologies, Darmstadt Training Center in collaboration with Münster University) and in China (BGI-Shenzen) Results: Strain lacks intimin and encodes for a number of genes that confer resistance to different antimicrobials Strain seems to be a hybrid that has properties of EHEC and enteroaggregative E. coli

31 FROM GENOME TO ASSAY Ion Torrent Instrument approx. $50,000 Reagents costs per isolate about $100 Initial sequencing of one isolate in < 1 week

32 WGS FOR IMPROVED SUBTYPING Background Salmonella Montevideo is very clonal Large number of unrelated isolates have same PFGE type, e.g., isolates from pistachio outbreak and salami outbreak Similar issues with a number of other Salmonella serotypes (i.e., Newport and Enteritidis)

33 Xbal SpeI Den Bakker et al AEM.

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35 WGS BIOLOGY OF FOOD-ASSOCIATED MICROBES Sanger method (Nelson et al., 2004) F6854 (food 1988) 133 contigs (2.97 MB combined length) Pseudomolecule 8X coverage Roche/454 Pyrosequencer (Orsi et al., 2008) F6900 (human ) 35 contigs (2.96 Mb combined length) 26X coverage J0161 (human 2000) 49 contigs (2.97 MB combined length) 2 contigs (82,678 bp combined length) extra-chromosomal plasmid (not used for analyses purposes) 29X coverage J2818 (food 2000) 38 contigs (2.97 Mb combined length) 24X coverage

36 RESULTS Full refined alignment: 2,922,773 bp (98.4 %) of F6854 chromosome sequence (2,971,285 bp) 3 sub-alignments Backbone alignment Prophage 1 alignment (inserted into comk) Prophage 2 alignment (inserted into trna-thr-4) SNPs (backbone and prophage 2): 44 SNPs 42 singletons (observed in a single isolate) 2 differentiate 1988 isolates from 2000 isolates Possible problems: Assembly Alignment Sequencing errors

37 DISTRIBUTION OF SNPS Re-sequencing (Sanger method) confirmed 12 SNPs 8 SNPs specific to J2818 (2000 food) 3 in intergenic regions 1 synonymous 4 nonsynonymous phosphoenolpyruvate synthase, putative similar to ethanolamine utilization protein EutE RDD family (transport?) AddB 2 SNPs unique to J0161 (2000 human) 2 in intergenic regions 1 not found in the J0161 isolate in the FSL collection 1 SNP unique to F6900 (1988 human) Intergenic region 1 SNP differentiated 1988/2000 isolates 1 nonsynonymous Putative phage tail protein

38 RECOMBINATION Several recombination events involving a lineage I isolate, J1194, were identified by GENECONV All events fall within one of the prophages, inserted into comk F685 4F690 0J281 8J016 FSL J1-194 ER 1 ER 2 R 1 Prophage inserted in comk R 2 R 3 Lysogeny control Cell lysis Tail and base structures Head structural components & assembly DNA packaging Lysogeny control DNA replication, recombination, modification and gene expression modulation

39 12 YEARS OF EVOLUTION Ancestor A 1 F6900 (human) F685 4 (food) comk prophage recombination trna prophage mutation or recombination Ancestor B 8 1 J281 8 (food) J0161 (human)

40 WHOLE GENOME SEQUENCING OF PAIRED PERSISTENCE STRAINS FRO THE SAME SITE 6 paired isolates corresponding to 3 different strains (ribotype or sigb allelic types) isolated from three persistently colonized sites W Sep-07, B Nov-13, L. monocytogenes W Sep-08, B Sep-11, L.monocytogenes W Aug-07, B Jan-11, L. innocua 253 SNPs between W1-215 and B3-276, mostly in prophage recombination region 17 SNPs between W1-527 and B SNPs between W1-179 and B2-365

41 WGS AND POPULATION STUDIES Culture independent diagnostics Sequencing of all DNA found after enrichment is feasible and is being done by FDA Reduces bias that is inherent in traditional detection Creates data on all DNA found; huge potential for creating incriminating data in the absence of public health hazards WGS based characterization of microbial diversity found in a given sample Massively parallel 16S rdna sequencing Potential of source tracking

42 OVERALL SUMMARY AND CONCLUSIONS While next generation genome sequencing is making real world contributions to food safety Improved subtyping over PFGE Identification of better target genes for detection Translation of transcriptomics, metabolomics etc. findings to improved prevention and treatment is in the early stages Still a bottleneck with assembly, annotation and analyses of WGS data Bioinformatics pipelines are rapidly catching up with hardware, making application by public health labs feasible WGS will also impact pathogen detection approaches