Current Needs for Bioforensics SCIENCE AND TECHNOLOGY BRANCH DISCOVER DEVELOP DELIVER
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1 Current Needs for Bioforensics SCIENCE AND TECHNOLOGY BRANCH DISCOVER DEVELOP DELIVER 1
2 State Sponsored Programs, Bioterrorism, Biocrime and Evolving Technology - Requires a Responsive Bioforensic Capability State Sponsored Programs and Bioterrorism Amerithrax Atlanta Ricin Las Vegas Ricin Biocrimes Texas Ricin Mississippi Ricin 20 or more investigations/yr Evolving Technology Do it Yourself Bio Genetic Engineering Gene Editing Custom Made Biological Threat Agents
3 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
4 DARPA Friend or Foe program and EPA Needs for 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 Day Arlington, VA, USA February 28,
5 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?) 2
6 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) 3
7 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 4
8 Thank You! Questions? Sanjiv R. Shah, PhD 5
9 Pathogen Discovery: Foodborne Outbreaks of Unknown Etiology* DARPA Friend or Foe Program John Besser, CDC Frank Niagro, USDA/FSIS Chad Nelson, FDA/OFVM Outbreaks for Pathogen Discovery New agents 2,465 outbreaks of unknown etiology reported to CDC (~200/year) *Naturally occurring or intentional Etiology of foodborne disease Most agents of foodborne diseases are unknown PulseNet cases are <4% Huge, untapped potential Current tools inadequate
10 Shotgun metagenomics for outbreaks. Highest potential for pathogen discovery 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 Assemble and characterize shared binned reads Use genetic information to guide culturing approach; assess virulence; modified Koch s postulates
11 Pathogen Discovery: Foodborne Outbreaks of Unknown Etiology Characterize clinical matrices Develop wet and dry OUE metagenomic pipelines Coordinate investigations Lead outbreak investigations Coordinate viable specimen collection Virulence studies/koch s postulates Binning/assembly strategies Novel culture method development State health and Ag agencies Characterize food matrices Develop wet and dry OUE metagenomic pipelines Academic researchers Biotech companies Characterize food matrices Develop wet and dry OUE metagenomic pipelines Novel bioinformatics software High speed cell sorting Phasing strategies Signal enrichment Questions?
12 Whole Genome Sequencing: Branch WGS Programs and Support 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 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.
13 Whole Genome Sequencing: WGS Programs and Support Mission / Objectives 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 Personnel Main Participants: Allard, Timme, Payne, Muruvanda, Kanstanis, Hoffman, Melka, Wang, Curry, Payne ORISE fellows: Pirone Davies, Sanchez, C., Ryan, Lindley Commissioner fellow: Stevens
14 Whole Genome Sequencing: Environmental and produce Projects Phenotypic, Genotypic and Comparative Genomic Characterizations for Detection of Salmonella Isolated 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 enterica Grown in Acidic Foods and Low Water Activity Foods using RNA Seq Technology (Jie 0.335; Jamie, Liz) Evaluation of Biocontrol Agent Paenibacillus alvei Strain TS 15 on Reducing Major Foodborne Pathogens in Selected Produce Crops Including Tomatoes, Sprouts, and Cantalopues (Jie 0.55; Liz)
15 FDA Cultural Methods Battle Indigenous Microflora Constantly Non Enrich Enriched Firmicutes Proteobacteria Enriched Firmicutes BMC Res Notes Jul 27;5:378. doi: / 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.
16 Phyla in a Salmonella Positive Metagenome
17 Enriched taxonomic phyla associated with diverse brands of cheese
18 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** GOAL = <5 years have first 25 mapped and rapid detection assay developed
19 Functional Assays for SNPs Better understanding of adaptive change in Salmonella and Lm may provide more accurate risk assessment as well as enhanced preventive control measures on the farm and in the processing plant.
20 IN THE NOT SO DISTANT FUTURE.. Species Resistance Virulence Subtype Serotype Adaptations ONE MICROBIOLOGICAL WORKFLOW: ONE MICROBIOLOGICAL TOOL BOX All AT YOUR FINGERTIPS
21 United States Department of Agriculture Animal and Plant Health Inspection Service
22 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
23 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)
24 Bacterial Diagnostics Brucella Leptospira Salmonella Campylobacter Taylorella Mycoplasma Mycobacteria Shiga toxin producing E. coli Emerging bacterial diseases and antimicrobial resistance
25 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
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