State Sponsored Programs, Bioterrorism, Biocrimeand Evolving Technology - Requires a Responsive Bioforensic Capability
State Sponsored Programs andBioterrorism
Biocrimes
• Amerithrax• Atlanta Ricin • Las Vegas
Ricin
• Texas Ricin• Mississippi Ricin• 20 or more
investigations/yr
Evolving Technology
• “Do it Yourself” Bio• Genetic Engineering• Gene Editing• Custom Made Biological
Threat Agents
Mission Deters and Prevents
NBFAC is located in Frederick, Md close to FBI Headquarters, Wash, DC and FBI Laboratory, Quantico, Va
• Supports federal law enforcement
1
DARPA Friend or Foe program and EPA Needsfor BioThreat Detection from Environmental
Samples
Sanjiv Shah, Ph.D.National Homeland Security Research Center
U.S. Environmental Protection Agency
DARPA Friend or Foe Program Proposers’ DayArlington, VA, USAFebruary 28, 2018
2
EPA Relevance
Detection of Known Biothreat Agents from Complex Environmental and Water Matrices Samples Following Intentional or Accidental Incidents
Air and Water Monitoring For Known and Unknown/Emerging Pathogens (Biosurveillance?)
3
EPA Operational Challenges
No Portable Screening Device to Quickly Detect Viable Biothreat Agent and Map the Extent of Contamination in the Field During a Wide Area Incident
Time Consuming and Laborious Analytical Methods to Detect Viable Biothreat Agents Delay Remediation Efforts
Enhanced Air and Water Monitoring (Biosurveillance)
4
EPA’s Interest and Operational Needs
Biothreat Analysis from Diverse and Complex Sample Types Soil, Air, Water, Runoff, Sewage, Biofilms,
Vegetation (leaves, bark, stems, etc.)
Sample Collection and Processing Methods to Recover/Isolate Viable Biothreat Agents from Such Complex Environmental and Water Matrices Samples
Field-Portable and Fixed-Laboratory Biosensors and Analytical Method(s) for a Quick Determination of Presence of Biothreat Agents in such Samples
Pathogen Discovery: Foodborne Outbreaks of Unknown Etiology*
John Besser, CDC Frank Niagro, USDA/FSIS Chad Nelson, FDA/OFVM
*Naturally occurring or intentional
Outbreaks for Pathogen Discovery
Etiology of foodborne disease• Most agents of foodborne
diseases are unknown• PulseNet cases are <4%• Huge, untapped potential• Current tools inadequate
New agents
2,465 outbreaks of unknown etiology reported to CDC 2007-2016 (~200/year)
DARPA Friend or Foe Program
• Assemble and characterize shared binned reads
New tools needed to improve..• Understanding of matrices• Signal‐to‐noise, phasing• Reference‐free binning • Metagenome comparisons• Agent isolation
“Krona chart” for visualizing species in metagenome analysis
Unbiased Process• Standardized specimen collection to maintain living cell and DNA viability
• Screen for known pathogens and known microorganisms
• Shotgun metagenomics on remaining samples
• Reference‐based read binning and reference free read binning
• Use genetic information to guide culturing approach; assess virulence; modified Koch’s postulates
Shotgun metagenomics for outbreaks….• Highest potential for
pathogen discovery
Pathogen Discovery: Foodborne Outbreaks of Unknown Etiology
• Characterize clinical matrices• Develop wet and dry OUE metagenomic pipelines• Coordinate investigations
• Characterize food matrices• Develop wet and dry OUE metagenomic pipelines
• Characterize food matrices• Develop wet and dry OUE metagenomic pipelines
Academic researchers
Biotech companies
State health and Ag agencies
• Virulence studies/Koch’s postulates• Binning/assembly strategies• Novel culture method development
• Lead outbreak investigations• Coordinate viable specimen collection
• Novel bioinformatics software• High speed cell sorting• Phasing strategies• Signal enrichment
Questions?
Whole Genome Sequencing: Branch WGS Programs and Support
Support CFSAN’s mission to protect and promote public health by addressing critical knowledge gaps through research needed to support regulatory decision making by improving techniques to rapidly and accurately characterize and subtype foodborne pathogens.
Mission Statement
DM in conjunction with the Agency's field staff, is responsible for promoting and protecting the public's health by ensuring that the nation's food supply is safe, sanitary, and wholesome, by rapidly characterizing and subtyping foodborne pathogens
Vision Statement
Whole Genome Sequencing: WGS Programs and SupportMission / Objectives
Personnel• Main Participants: Allard, Timme, Payne, Muruvanda,
Kanstanis, Hoffman, Melka, Wang, Curry, Payne• ORISE fellows: Pirone‐Davies, Sanchez, C., Ryan,
Lindley• Commissioner fellow: Stevens
• Develop high‐throughput methods for ORA labs and other stakeholders
• Develop information management system for all phases of WGS
• Develop approaches for detecting microbial contaminants in food
• Support program stakeholders method development needs
• Support stake holders by validating NCBI new web interface methods for rapid SNP clustering and visualization for real‐time bacterial characterization
• Provide SME to Office of Criminal investigation for drafting legal documents for use in bring WGS data to the courts and for specific regulatory actions
• SME support of other offices and agencies to include: ORA, CVM NARMS, CARB, WHO and FAO
Stakeholders
• OFS• ORA• State health labs• University centers• CFVM
Resources• Illumina WGS platform• PacBio WGS platform• SLIMS and GIMS
Whole Genome Sequencing: Environmental and produce
Projects
• Phenotypic, Genotypic and Comparative Genomic Characterizations for Detection of SalmonellaIsolated from Tomato‐Related Agricultural Environments (Becky, FTE = 0.55 Christina)
• Effects of Cultivar and Postharvest Practices on Cantaloupe Colonization by Listeria monocytogenes (Dumitru/Yi 0.65; Antonio, Jie)
• Limit of Detection for Metagenomic Methods (Andrea 1.0 Rachel, Padmini)
• Large‐Scale Comparison of Food and Clinical Listeria moncytogenes Genomes to Identify Genetic Determinants for Virulence and Persistence in Diverse Environment (Gina 0.55; Cary)
• Development and Inter‐Laboratory Validation of a Real Time PCR kit for Detection of Salmonella enteritidis in eggs (Narjol on hold)
• Analysis of Adaptive Changes in Salmonella entericaGrown in Acidic Foods and Low Water Activity Foods using RNA‐Seq Technology (Jie 0.335; Jamie, Liz)
• Evaluation of Biocontrol Agent Paenibacillus alveiStrain TS‐15 on Reducing Major Foodborne Pathogens in Selected Produce Crops Including Tomatoes, Sprouts, and Cantalopues (Jie 0.55; Liz)
Non Enrich
Enriched Firmicutes Proteobacteria
Enriched Firmicutes
BMC Res Notes. 2012 Jul 27;5:378. doi: 10.1186/1756‐0500‐5‐378.Using metagenomic analyses to estimate the consequences of enrichment bias for pathogen detection.
Pettengill JB1, McAvoy E, White JR, Allard M, Brown E, Ottesen A.
FDA Cultural Methods Battle Indigenous Microflora Constantly
GOAL = <5 years have first 25 mapped and rapid detection assay developed
Adaptations of particular interest to food safety specialists:
(1) Thermal tolerance(2) Dessication resistance(3) Osmotic/Ionic tolerance(4) Quat resistance(5) Chlorine resistance(6) Biofilm persistence(7) Surface adherence(8) Antibiotic resistance(9) Antimicrobial resistance(10) Ecological fitness(11) Heavy metal resistance(12) Metabolic persistence(13) Enhanced hydrophobic fitness(14) Produce invasiveness(15) Flower invasiveness(16) Root system invasiveness(17) Acid resistance
(18) Surface water fitness(19) In vivo plant migratory fitness(20) Soil fitness(21) Capsaicin resistance (22) Swarming(23) Trans‐ovarian poultry colonization(24) Fecal persistence (poultry)(25) Yolk content invasion(26) Multidrug resistance(27) External amoeba harborage(28) Internal amoeba harborage(29) Acyl‐homoserine lactone (AHL) (30) KatE stationary‐phase catalase(31) In vivo migratory fitness(32) RDAR phenotype(33) The ‘Weltevreden’ type(34) Persistence within the tomato**
Better understanding of adaptive change in Salmonella and Lmmay provide more accurate risk assessment as well as enhanced preventive control
measures on the farm and in the processing plant.
Functional Assays for SNPs
Species Resistance
Virulence Subtype
Serotype Adaptations
ONE MICROBIOLOGICAL WORKFLOW: ONE MICROBIOLOGICAL TOOL BOXAll AT YOUR FINGERTIPS
IN THE NOT SO DISTANT FUTURE…..
Animal and Plant Health Inspection Service (APHIS)
• Mission: Protect and promote food, agriculture, natural resources
• APHIS protects and promotes U.S. agricultural health, regulates genetically engineered organisms, administers the Animal Welfare Act and carries out wildlife damage management activities.
• APHIS has diagnostic laboratories for animal, plant, and wildlife diseases
APHIS Diagnostic Sampling• Diagnostic sampling from a variety of tissues
and species (animal and plant) and the environment
• Examples of samples: – Blood, tissue (respiratory, enteric, reproductive, brain,
gill), lesion swabs, cloacal swabs,– Complex environmental samples (manure pits and
lagoons, water sources, soil)
Bacterial Diagnostics
• Brucella• Leptospira• Salmonella • Campylobacter• Taylorella• Mycoplasma• Mycobacteria• Shiga toxin producing E. coli • Emerging bacterial diseases and antimicrobial resistance
Diagnostic Interests• During disease outbreaks want high throughput
diagnostic capabilities• Want rapid field detection-based systems to expedite
point of care diagnostic screening • Also interested in rapid genomics analysis to facilitate
epidemiological investigations• Ability to rapidly identify pathogens from complex
environmental samples• Bacteriophage based pathogen recognition