SCIENCE AND TECHNOLOGY BRANCH DISCOVER • DEVELOP • DELIVER

Current Needs for Bioforensics

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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

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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

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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?)

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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)

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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

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Thank You!

Questions?

Sanjiv R. Shah, PhDshah.sanjiv@epa.gov

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

Phyla in a Salmonella Positive Metagenome 

Enriched taxonomic phyla associated with diverse brands of cheese

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…..

United States Department of Agriculture Animal and Plant Health Inspection Service

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