Beef Industry Safety Summit Renaissance Austin Hotel 9721 Arboretum Blvd. Austin, TX March 1-3

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2 USDA, Food Safety and Inspection Service Beef Industry Safety Summit Renaissance Austin Hotel 9721 Arboretum Blvd. Austin, TX March 1-3 Uday Dessai MPH, MS, PhD Senior Public Health Advisor Office of Public Health Science, FSIS, USDA

3 Food Safety and Inspection Service Whole Genome Sequencing FSIS Update FSIS Strategic Plan Whole Genome Sequencing at FSIS Capacity building Current Status Application of WGS in FSIS Investigative Process and AMR WGS Considerations Interagency Collaboration Gen-FS Concluding Remarks 3

4 Projected FSIS Timelines Whole Genome Sequencing and Strategic Planning at FSIS Goal 5: Effectively Use Science to Understand Foodborne Illness and Emerging Trends Outcome 5.1: FSIS continually improves its capacity for and use of cutting-edge science in policy development to better defend against public health risks Outcome 5.2: FSIS increases the application of cutting-edge science across the farm-to-table supply chain to improve public health Traditional Subtyping Healthy People Goals and Targets: 2020 FSIS Strategic Plan Strategic Plan External validation HP: 2030 Strategic Plan Application of Science, Technology and Innovation Continues Upgrade WGS/NGS/other applications Implementation of WGS Infrastructure in routine use at FSIS Continue to develop criteria for the application of WGS in regulatory decision making Serotyping, PFGEs, AST and other... Fit-For-Purpose - Detection system Partner collaborations Pilot studies Large Scale implementation In-field detection, dataanalysis and decision making capability SNP (Kmer, hqsnp) and wgmlst and new tools Decreased reliance on non WGS technologies Explore Utilization of In-Field Real-time Technologies FSIS set of Reference Genomes Unique analytic capability Community/Microbiome Resistome, Virulome etc. FSIS WGS data analysis pipelines Pan-genome (core + variable) type of analysis Less dependence on culture enrichment and isolates (!) Complement CID FSIS WGS

5 Whole Genome Sequencing at FSIS: Superior Resolution and Multiple Uses WGS is a very powerful method that promises superior resolution for identifying and characterizing species, strains, clades etc. WGS technology and analytic tools are evolving rapidly Can help investigate foodborne disease outbreaks to resolve bacterial identity and relatedness in the context of the epidemiological information Can more definitively identify the specific type and sub-type of bacteria involved in outbreak and help reduce incidents of illness or death due to foodborne pathogens WGS can potentially aid in identifying environmental harborage and recurrences of pathogens in FSIS-regulated establishments/products and further support the inspection and verification process 5

6 Whole Genome Sequencing at FSIS: Bacterial Characterization Methods Pulsed-field gel electrophoresis (PFGE) and Multi-Locus VNTR Analysis (MLVA) PulseNet member since start of the network, mid-1990 s Uploads since 2006: 2,106 Lm, 1,435 STEC, 15,266 Salmonella spp., 2,408 Campylobacter spp. MLVA: Salmonella Typhimurium and E. coli O157 isolates Molecular serotyping of Salmonella isolates CDC molecular serotyping method since June 2012 Utilize NVSL for traditional serotyping, as needed Antimicrobial Susceptibility Testing (AST) Salmonella, generic E. coli, Campylobacter, Enterococcus Storage (lyophilization since 1990 s) 6

7 Whole Genome Sequencing at FSIS: WGS Capacity Building FSIS continues to build WGS capacity and expected to be fully functional with 6 Sequencers in operation by FY 2017 At full capacity FSIS intends to sequence at least 5000 isolates per year Two positions hired to engage in informatics work In collaboration with CDC, FSIS uses the WGS information in addition to PFGE and epidemiological information to further understand the suspect pathogen and source products FSIS works with NARMS to understand the occurrence or introduction of AMR genes in pathogens of interest When WGS is fully functional at FSIS, the Agency intends to share WGS information with the establishments, in addition to the serotype, PFGE, and antimicrobial susceptibility results for further considerations and actions 7

8 Concept Overview Diagram For WGS Food Safety and Inspection Service: WGS data flow and in-house analyses Commercial software: BioNumerics version 7, CLC Genomics, Geneious Approved Freeware: FastQC, NCBI Genome WorkBench * Based on current requirements. If bandwidth needs increase, may need to upgrade to faster network infrastructure such as Internet 2 infrastructure, currently used by ARS. 8

9 Whole Genome Sequencing and Bacterial Characterization at FSIS: Where We Are and Where We Want to Be... As Is Sample Recover Isolate Subtype/Characterize Isolates Using Database To Be PFGE AST Mol. Serotyping Other Whole Genome Sequencing Database Note: PFGE, Anti-microbial Susceptibility Testing, Molecular Serology, etc., are different methods that are collectively used to accurately characterize species of pathogens. The WGS technology can generate comparable (Red dashed lines) and much more information to differentiate closely related pathogens. 9

10 Whole Genome Sequencing at FSIS:WGS Milestones and Accomplishments Isolates Sequenced Milestone Dates July, 2014: Salmonella species and Listeria monocytogenes December, 2014: STECs February, 2015: Campylobacter May 15, 2015: Capability to directly upload WGS files into NCBI was acquired 153 isolates uploaded to NCBI in FY2016 Quarter 2 (as of 1/20/2016) 10

11 Whole Genome Sequencing at FSIS: WGS Sequences Uploaded to NCBI USDA-FSIS isolates Uploaded to NCBI by organism Total = 2,588 Salmonella spp. = 1,396 Listeria monocytogenes = 473 Campylobacter = 502 STEC = 217 FSIS Isolates associated with Beef products Total = 676* Salmonella = 209 Salmonella (HACCP, other) = 90 Salmonella NARMS Cecal = 119 Campylobacter = 240 NARMS cecal isolates = 240 Escherichia coli (STECs) = 209 Uploads to NCBI SRA by any lab Salmonella spp. = 52,179 Listeria monocytogenes = 9,901 Campylobacter = 16,346 Escherichia coli = 31,670 Listeria monocytogenes = 18 RTE Products = 18 *Numbers as of 2/17/16 11

12 Whole Genome Sequencing at FSIS: WGS Metadata Shared by FSIS with NCBI Sequence Uploads Product/Source type (Ready To Eat product, raw meat/poultry, environmental swab, etc.) Year sample was collected State where sample was collected Subtyping information when available Salmonella serotype, PFGE STEC O-group info, PFGE Campylobacter species, PFGE PFGE for Lm Metadata is immediately available for upload to NCBI 12

13 Whole Genome Sequencing at FSIS: FSIS Commitment to Real-Time Lm WGS an Interagency Project A multi-agency collaboration to provide Lm WGS data in realtime Support of epidemiologic investigations As part of this collaboration FSIS performs WGS on all isolates (food, contact and non-contact environmental swabs) From August March 2015: sent 249 Lm isolates to FDA CFSAN for WGS From April 2015 Present: sequenced and directly uploaded 184 Lm isolates into NCBI Provides NCBI Accession Number to CDC PulseNet to connect the PFGE data, WGS sequence data and the associated metadata 13

14 Whole Genome Sequencing at FSIS: An Example of a Salmonella Investigation - Retrospective Analysis Primary pattern A Primary pattern B Secondary pattern C Secondary pattern D Secondary pattern E FSIS food and environmental samples from one investigation were compared to clinical isolates with an epidemiological link to the establishment where sampling occurred. The isolates from the investigative sampling had 2 different primary PFGE patterns and 3 different secondary PFGE patterns.. WGS was able to show high similarity (0-5 SNP differences) between differing primary PFGE patterns and primary/secondary combinations. 14

15 Food Safety and Inspection Service Whole Genome Sequencing at FSIS: An Example Salmonella with a Unique AMR gene In 2015, as part of NARMS retail testing, FDA isolated Salmonella with extended spectrum beta-lactamase (ESBL) resistance from a retail poultry product purchased in December, Patients with Salmonella infection expressing this resistance pattern have few treatment options Contained bla CTX-M-65 plasmid based resistance gene Never-before reported in Salmonella from food items in the US FSIS actions Determinations of bla CTX-M-65 ESBL resistance among FSIS isolates Phenotype and genotype (ResFinder) Isolates with a match were investigated for their sources/origin and possible connections to human cases No human illnesses connection from FSIS regulated products was found FSIS promptly notified all corporations with ESBL matches

16 Food Safety and Inspection Service Whole Genome Sequencing at FSIS: Current and Future WGS Considerations Case Definitions: FSIS depends on its public health partner (CDC/States) for case definitions, the descriptions of the outbreak strain(s) and the subtyping method used to define the strain(s) Single/Multiple Strain: Isolates from clinical specimens and food samples collected during investigations are not always found to have a single strain by PFGE analysis, WGS, and/or other subtyping method Higher Resolution and Changing Strains: The clinical specimens and food and environmental samples collected over time during an investigation or through routine surveillance may yield the pathogen(s) of interest but not the outbreak strain as characterized by WGS Case Definitions and Surveillance: FSIS will need to explore how to interpret and apply the case definitions based on WGS analyses by our public health partners to FSIS surveillance results - as WGS continues to become a prominent tool for microbial surveillance 16

17 s Interagency Workgroup Gen-FS Interagency Group Name: Interagency Collaboration on Genomics and Food Safety (Gen-FS) Agency partners: CDC, FDA, NCBI/NIH and FSIS (Agencies with mostly Public Health and Regulatory Focus) Nature of Work: WGS technology is rapidly evolving and Gen-FS provides the participating agencies a governance structure for collaborative decision making Gen-FS will focus on the development and implementation of WGS tools, data analysis pipelines and methods for the in-depth analyses of pathogens from food, feed, environmental, clinical, and animal sources Gen-FS will harmonize the planning and implementation of WGS activities including surveillance, detection, investigation and research activities Gen-FS will further strengthen outbreak cluster detection and response, and the detection/transmission of antimicrobial resistance Charter Signatories: Agency Heads/Leaders Director/Commissioner/Administrator 17

18 Whole Genome Sequencing: Concluding Remarks FSIS is committed its Healthy People 2020 Pathogen Reduction Goals and beyond To accomplish the pathogen reduction goals, FSIS works closely with its public health partners FSIS utilizes the best available science to understand the sources of food pathogens and their prevention and control FSIS is fully committed to utilizing the analytic power and resolution of WGS in its pathogen reduction public health protection pursuit FSIS is rapidly developing WGS capacity to be able to undertake real-time WGS on all the FSIS isolates FSIS continues to collaborate with the CDC, FDA and NCBI to further understand the scope and applicability of WGS findings in FSIS s regulatory context In its investigative decision making process FSIS utilizes WGS findings/interpretations as a part of the totality available evidence 18

19 Projected FSIS Timelines Whole Genome Sequencing and Strategic Planning at FSIS Goal 5: Effectively Use Science to Understand Foodborne Illness and Emerging Trends Outcome 5.1: FSIS continually improves its capacity for and use of cutting-edge science in policy development to better defend against public health risks Outcome 5.2: FSIS increases the application of cutting-edge science across the farm-to-table supply chain to improve public health Traditional Subtyping Healthy People Goals and Targets: 2020 FSIS Strategic Plan Strategic Plan External validation HP: 2030 Strategic Plan Application of Science, Technology and Innovation Continues Upgrade WGS/NGS/other applications Implementation of WGS Infrastructure in routine use at FSIS Continue to develop criteria for the application of WGS in regulatory decision making Serotyping, PFGEs, AST and other... Fit-For-Purpose - Detection system Partner collaborations Pilot studies Large Scale implementation In-field detection, dataanalysis and decision making capability SNP (Kmer, hqsnp) and wgmlst and new tools Decreased reliance on non WGS technologies Explore Utilization of In-Field Real-time Technologies FSIS set of Reference Genomes Unique analytic capability Community/Microbiome Resistome, Virulome etc. FSIS WGS data analysis pipelines Pan-genome (core + variable) type of analysis Less dependence on enrichment and isolates (!) Complement CID FSIS WGS

20 WGS: Questions/Discussion Questions 20

21 Reserved Slides for Discussion Slides for Discussion 21

22 WGS and Changes in Incidence and Outbreaks: Ex. Lm (CDC Slide) (Will Other Pathogens Show a Similar Trend!) isolates in 2010 compared to 9,640 in

23 For Discussion - Some Questions/Concerns on the use of WGS Sequencing and data quality issues Data storage and transmission Metadata How to determine closeness (SNPs vs wgmlst etc.) Trace back/forward Harborage/Persistence Time period Cost Lab Informatics 23

24 Subtyping Methods at FSIS BAX speciation Campylobacter Molecular Serotype Salmonella Pulsed-Field Gel Electrophoresis Salmonella Campylobacter STEC and E. coli O157:H7 Listeria monocytogenes Antimicrobial Susceptibility Testing Salmonella Campylobacter STEC,O157:H7 and generic E. coli Enterococcus Potential to be replaced with a single WGS workflow