Intro to Microbial Forensics

Exam 1

microbial forensics

scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime or inadvertent microorganism/toxin release for attribution purpose

attribution

assignment of a sample of questioned origin to a source of known origin to the highest possible degree of scientific certainty while excluding origination from other sources

trace evidence/sample

any sample that falls below the recommended threshold of at any stage of analysis

• Depends on the instrumentation 

• Can not be defined by a precise picogram amount 

areas microbial forensics can assist

1. biosecurity

2. public health forensics

3. foodborne outbreaks

4. source tracking of wastewater

5. counterfeit medications

6. human identification (microbiome)

7. estimating the postmortem interval (microbiome, PMI)

uses of biological warfare

1. gain strategic advantage (small amounts do a lot of harm)

2. area denial (lethal or nonlethal)

3. can target a single person or group

4. incapacitation of warfighters (take people off the battlefield)

5. same as chemical weapons

current state of microbial forensics

• young field

• developed fields are being examined for utility (diagnostics)

• substantial publications support methods of detection

• validation guidelines have been developed

• increased interest (funding) for development

how can microbial forensics help an investigation

• identify biological threat agents

• understand microbial community at scene

• help build a case for attribution (link sample to its origin)

• understand where we land on biorisk spectrum

Black Plague

• greatest public health disaster recorded in history (14th century)

• reduced population from 475 million to 350-375 million

• first use of biological weapons (Crimea catapulted plague infected corpses over castle walls during siege of Café)

• Yersinia pestis (bacteria generally carried by fleas, can be effectively treated with modern antibiotics)

WWII biological weapons

British government establishes a biological weapons program in response to intelligence reports from Germany

• followed by US, France and Japan

Biological and Toxin Weapons Convention (BWC)

Nixon discontinued the offensive biological program in 1969

• signed in 1972

• prohibits the development, production and stockpiling of biological and toxin weapons

first treaty banning an entire category of weapons

US Government realized we were unprepared for a biological attack (prior to 9/11 and Amerithrax)

created…

1. Hazardous Materials Response Unit (HMRU in FBI)

2. Bioterrorism Preparedness and Response Program (BPRP in CDC)

3. Laboratory Response Network (LRN in CDC)

Hazardous Materials Response Unit (HMRU)

provides evidence access, health and safety, and logistical support for FBI investigators

• now called Technical Hazards Response Unit (THRU)

Bioterrorism Preparedness and Response Program (BPRP)

developed a strategic plan and developed guidelines (safety equipment, protocols, etc.)

Laboratory Response Network (LRN)

complete laboratory network charged w/responding to bioterrorism and other public health emergencies

Amerithrax evidence + testing (culturing)

1. culturing

   1. spores in culture plate was left in incubator for several days

   2. several different morphologies other than Anthrax colonies were observed

   3. observed same thing in all letters

   4. tested stock samples from CDC

also…bacterial contamination of B. subtilis present in 323 of over 1,000 CDC stocks

Amerithrax evidence + testing (WGS)

1. allowed scientists to sequence the entire genome and detect small mutations that might be missed by other methods (SNPs)

   1. SNPs are highly informative because even a single nucleotide change can distinguish between different strains or sub-strains of a pathogen

2. specific SNPs in the anthrax spores used in the attacks matched SNPs found in the RMR-1029 flask at USAMRIID

Amerithrax evidence + testing (VNTR)

1. used to differentiate between different isolates of the Ames strain

2. helped narrow down possible sources of the attack spores

Amerithrax evidence + testing (carbon-dating)

spores were made 1998-2001

Amerithrax evidence + testing (defect analysis of envelopes by Secret Service)

originated at Ivin’s post office in Frederick, MD

Microbial Forensics (positive) outcomes from Amerithrax case

State of the art instrumentation for microbiological identification
• Standardized protocols and reagents
• Established sample custody and chain of command
• Limited access to samples
• LRN available for public health and suspect biological attack
• Department of Defense providing confirmatory analysis
• FBI officially added Microbial Forensics to its repertoire

identifying biological threats

identified by CDC into 3 categories

category A

highest priority agents that pose a risk to national security

• can be easily transmitted from person to person

• result in high mortality rates w/potential for major public health impact

• might cause public panic and social disruption

• require special action for public health preparedness

category B

second highest priority

• moderately easy to disseminate

• moderate morbidity and low mortality rates

• require specific enhancements of CDC’s diagnostic capacity and enhanced disease surveillance

category C

third highest-priority include emerging pathogens that could be engineered for mass dissemination in the future b/c of…

• availability, ease of production and dissemination

• potential for high morbidity and mortality rates and major health impact

microbial forensics tools

• immunoassays

• microscopy (SEM, AFM)

• culturing methods

• nucleic ID methods (restriction enzymes, PCR, sequencing)

• spectrometry (LC/MS, FTIR, etc.)

• instrumentation + detection assays are always under development

Enzyme Linked Immunossorbent Assay (ELISA)

uses antibodies w/an affinity to a specific target

• rapid detection, target specific

• detecting toxins, microbial surface proteins, small molecules

quantitative polymerase chain reaction (qPCR)

nucleic acid method of detection

shotgun sequencing

multiple DNA segments of entire sample

a laboratory technique used to determine the entire DNA sequence of an organism's genome by breaking it into small, random pieces and reassembling them using a computer

• target agnostic

• produces large amounts of data

bioinformatics

analytical tool for sequencing-based approaches

Multiple open-source tools available
• Amphora, EDGE, etc.…
• Under aggressive development
• Database development and curation
• Classifiers

an interdisciplinary field that uses computer science, mathematics, and statistics to store, analyze, and interpret large and complex biological datasets, such as DNA sequences and protein structures

core questions when approaching scenario

1. what is the threat agent?

2. is it probative?

3. can it be linked to a source (attribution)?

4. what are the meaning and weight of the conclusion? what are our confidence levels?