AOAC SPADA Meeting
OCTOBER 16 & 17 2018
S takeholder P anel on A gent D etection A ssays (SPADA)
Stakeholder Panel and Working Group Meetings 2275 Research Boulevard Conference Room #110 Rockville, Maryland, United States
OCTOBER 16 - 17, 2018 AOAC INTERNATIONAL HEADQUARTERS 2275 RESEARCH BLVD., ROCKVILLE, MARYLAND, 20850 Room: 101
OCTOBER 16: 9:00am – 4:00pm ET OCTOBER 17: 9:00am – 4:00pm ET Registration Opens 8:00AM AOAC STAKEHOLDER PANEL ON AGENT DETECTION ASSAYS (SPADA)
A G E N D A
Welcome and Introductions (9:00am – 9:30pm) Dave Schmidt, AOAC Executive Director & Linda C. Beck, PhD, SPADA Chair a. Policies and Procedures b. Introductions
I.
SPADA Background and History (9:30am – 10:30am) Linda C. Beck, PhD, JRAD, SPADA Chair a. SPADA Early Work b. SPADA DoD Initiatives
II.
III. Standards Development at AOAC INTERNATIONAL (10:45am – 11:30pm) Deborah McKenzie, AOAC Sr. Director, Standards Development
IV. Working Groups Launch: Soil Testing Working Group (1:00pm – 4:00pm) a. Working Group Launch Presentation i. Goals, timelines and rosters
b. Background Presentations
i. Linda C. Beck, PhD, JRAD, DUSA‐TE; JPEO JPM Guardian, DBPAO ii. Morgan Minyard, PhD, DTRA iii. Sherry Blight, PhD, Battelle, CBRNE Defense/Aerosol
c. Working Group Discussion
i. Review the proposed outline of the project as presented ii. Review existing practices for preparation and use of soil samples iii. Identify gaps in existing practices iv. Commence process of developing format for standard and parameters to include
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This agenda is subject to change without notice
AOAC INTERNATIONAL Stakeholder Panel on Agent Detection Assays (SPADA) Working Group Sessions – Wednesday, October 17, 2018 9:00 a.m. – 4:00 p.m. Conference Room 101
Agenda
I. Working Group Kickoff Meetings: Bacterial Strain Verification Working Group (9:00am – 12:00pm)
a. Working Group Launch Presentation i. Goals, timelines and rosters
b. Background Presentations
i. Shanmuga Sozhamannan, PhD, Tauri Group, JPEO JPM Guardian, DBPAO ii. David Rozak, PhD, USAMRIID
iii. Ian Gut, PhD, NBACC iv. Katalin Kiss, PhD, ATCC
c. Working Group Discussion
i. Review the proposed outline of the project as presented ii. Identify the minimum requirements needed to verify the species/strain of a culture. With help of the contractor and WG chair, BSVWG will prepare a draft standard for verification of bacterial strains to be presented to SPADA for consideration of approval at the second SPADA meeting
II. Working Group Kickoff Meetings: In‐Silico Analysis Working Group (1:00pm – 4:00pm)
a. Working Group Launch Presentation i. Goals, Timelines and Rosters
b. Background Presentations
i. Shanmuga Sozhamannan, PhD, Tauri Group, JPEO JPM Guardian, DBPAO ii. Jeff Koehler, PhD, USAMRIID iii. Trevor Brown, PhD, JPM MCS/JPEO
c. Working Group Discussion
i. Review the proposed outline of the project as presented ii. Identify the different steps that can be modified/eliminated from wet lab testing by in silico PCR analysis. The group will review and discuss features of the different databases and determine if one or more shall be the focus of the standard setting effort iii. Identify approaches for in silico analyses and potential software to use iv. Determine the optimal settings for each in silico PCR analysis setting v. Discuss and determine how in silico PCR analysis can fit into existing evaluation/validation paradigm and prescribe the procedures of the use of in silico PCR analysis vi. Commence preparation of a draft standard to use in silico PCR analysis to present to SPADA for consideration of approval at the next SPADA meeting
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This agenda is subject to change without notice
AOAC Stakeholder Panel on Agent Detection Assays (SPADA) Program Background
Linda C. Beck, PhD, SPADA Chair Joint Research and Development (JRAD) October 16-17, 2018
AOAC Staff Contacts
• David B. Schmidt, Executive Director, dschmidt@aoac.org
• Jonathan Goodwin, Deputy Executive Director, jgoodwin@aoac.org
• Krystyna McIver, SPADA Project Executive, kmciver@aoac.org
• Deborah McKenzie, Sr. Dir., Standards Development, dmckenzie@aoac.org
• Scott Coates, Sr. Director, AOAC Research Institute, scoates@aoac.org
• Christopher Dent, Standards Development Manager, cdent@aoac.org
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Overview
• SPADA Background and History
• Background on previous standards / SMPRs
• Organization
– Working Groups vs. Stakeholder Panels
• Current Initiative / Meeting Goals
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SPADA Sets Standards 2007 ‐ 2013
• A voluntary consensus standards body originally established via a DHS S&T
SPADA Executive Steering Committee
SPADA
First Responder Working Group
contract with AOAC INTERNATIONAL
B. anthracis Working Group (PCR)
B. anthracis HHA Working Group
• Includes representatives from DHS, CDC, DoD, DoJ, FDA, EPA, USPS, NIST, State & Local Public Health, First Responders, Industry, and Academia • Establishes method performance requirements and panels of reference materials (and validation protocols)
Y. Pestis Working Group (PCR)
Ricin HHA Working Group
F. tularensis Working Group (PCR)
Burkholderia Working Group (PCR)
Environmental Factors Working Group (PCR) Public Health Actionable Assay Working Group*
Assay Control Working Group (PCR )
Variola Working Group (PCR)
All SPADA members volunteer their time and expertise
Original Objectives in 2007 - Establish standards to validate Polymerase Chain Reaction (PCR)‐based technologies that detect aerosolized Bacillus anthracis , Yersinia Pestis , or Francisella tularensis - Pilot the validation process with an assay that detects B. anthracis 2009 - Develop standards to validate immunoassay‐based Hand‐Held Assays (HHAs) that detect B. anthracis or Ricin in suspicious powders - Test commercially‐available HHAs 2010 - Develop standards to validate PCR‐based technologies that detect aerosolized Burkholderia psuedomallei and Burkholderia mallei - Develop standards to validate PCR‐based technologies that detect B. anthracis in suspicious powders 2011 - Develop recommendations on controls needed for field‐based assays 2013 - Develop standards to validate PCR‐based technologies that detect aerosolized Variola - Establish First Responder Working Group - Maintain a SPADA Executive Steering Committee SPADA Sets Standards 2007 - 2013
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SPADA Working Groups 2007 ‐ 2013
B. Anthracis Handheld Assay Working Group (BaHHAWG) Marian McKee, BioReliance Corp. Ricin Handheld Assay Working Group (RicinHHAWG) Mark Poli, DoD
B. anthracis Working Group (BaWG) Paul Jackson, LLNL and Ted Hadfield, MRI
Y. pestis Working Group (YpWG) Luther Lindler , DHS
Burkholderia Working Group (Bur WG) Paul Keim, NAU and Alex Hoffmaster, CDC
F. tularensis Working Group (FtWG) Peter Emanuel, DoD Mark Wolcott, DoD
Assay Controls Working Group (ACWG) Christina Egan, NYSDH and Larry Blyn, Ibis
Environmental Factors Working Group (EFWG) Stephen Morse, CDC
Variola Working Group (VWG) Victoria Olson, CDC and Ted Hadfield, MRI
Public Health Actionable Assay Working Group (PHAAWG) Peter Estacio, LLNL
SPADA Sets Standards 2014 ‐ 2016
• A voluntary consensus standards body established via DUSA‐TE sponsored project through JHU/APL • Includes representatives from DHS, CDC, DoD, DoJ, FDA, EPA, USPS, NIST, State & Local Public Health, First Responders, Industry, and Academia • Establishes Standard Method Performance Requirements (SMPRs) that include inclusivity/exclusivity panels
SPADA
VEE Working Group
B. anthracis Working Group
C. burnetti Working Group
Brucella suis Working Group
Burkholderia pseudomallei Working Group Botulinum Neurotoxin A Working Group
SEB Working Group
Y. Pestis Working Group
F. tularensis Working Group
Variola Working Group
All SPADA members volunteer their time and expertise
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SPADA Sets Standards 2014 - 2016
Under Contract with Deputy Undersecretary of the Army‐ Test and Evaluation through The Johns Hopkins University, Applied Physics Laboratory 2014 - Establish standards to validate technologies that detect Venezuelan Equine Encephalitis Virus, Staphylococcus Entertoxin B, and Coxiella burnetti (Q‐fever) with emphasis on the warfighter. 2015 – 2016 - Establish standards to validate technologies that detect Bacillus anthracis , Yersinia Pestis , Francisella tularensis, Brucella suis, Burkholderia pseudomallei, Variola, and Botulinum Neurotoxin A with emphasis on the warfighter. 8
SPADA Working Groups 2014 ‐ 2016
Concluded at September 2015 SPADA Meeting:
Concluded at August 30, 2016 SPADA Meeting:
Venezuelan Equine Encephalitis WG James Samuel, U of Texas, A&M
Burkholderia pseudomallei WG Jay Gee, CDC
C. Burnetti WG Eileen Ostlund, USDA, ARS
Brucella suis WG Frank Roberto, Idaho Natl. Laboratory
SEB WG Sandra Tallent , FDA
Variola WG Victoria Olson, CDC
Approved at March 22 – 23, 2016 Meeting:
B. anthracis WG Paul Jackson, LLNL and Ted Hadfield, Hadeco
Botulinum Neurotoxin A WG Sashi Sharma, FDA, HHS
Y. pestis WG Luther Lindler, DHS
F. tularensis WG Paul Keim, Northern Arizona University
SPADA ‐ 2017
• Tasked with streamlining the Environmental Organisms Panel • EOP is an evaluation of potential cross‐reactivity to the DNA from a wide variety of organisms that are in the environment
SPADA
Environmental Organisms Panel Working Group
• SPADA streamlined this panel for to promote efficiency and modernity
All SPADA members volunteer their time and expertise
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SPADA Working Groups 2017
Concluded at April 2017 SPADA Meeting:
Environmental Organisms Panel Working Group Chair: Linda C. Beck, JRAD (Formerly of NSWC Dahlgren) SPADA voted to accept a revised environmental factors panel appendix containing the final environmental panel as an addition to all existing DoD SMPRs for aerosol samples.
Environmental Organisms Panel E nvironmental Factors Annex (in all SMPRs); three sections: • Environmental Matrix Samples – Aerosol; Soil Testing • Environmental Organisms • Potential Interferents (operational background)
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Publication & Availability • An overview of the JHU APL/DoD/AOAC project and all 10 SMPRs together with the revised Environmental Organisms Panel is being published in the Nov/Dec issue of the Journal of AOAC INTERNATIONAL. • It will be available on the web with a DOI (Document Object Identifier). • URL will be made available to DoD and SPADA members.
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AOAC Standard Methods Performance Requirements • A standard for analytical methodology. – the traditional standard was a description of a method. – an SMPR specifies the minimum performance requirements for a methodology.
• Documents a community’s analytical needs
• Description of the analytical requirements
• Includes method acceptance requirements
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AOAC Standard Methods Performance Requirements
• Previous SPADA initiatives have developed 19 sets of AOAC Standard Method Performance Requirements® (SMPR®)
Standard Methods Performance Requirements
General Format
– Intended Use – Applicability – Analytical technique – System suitability
– Reference materials – Validation guidance – Maximum time-to-determination – Method performance requirements table – Inclusivity/exclusivity/environmental contaminants
Standard Methods Performance Requirements
Use of SMPRs • Guidance to developers for the development of new assays. • Advance the state-of-the-art in a particular direction. • Address specific analytical needs. • Specifications for acquisition. • Vendor self-qualification. • Basis for method acceptance and AOAC approval.
Organization: AOAC Stakeholder Panels
• Standard adopting bodies for AOAC • Meetings are open to all interested parties. • Oversee working groups • Typically 50+ members. • Voting members are vetted based on: • Expertise • Perspective/sector (government, academia, industry, etc.) • Availability
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October 2018 Kick- Off for New AOAC SPADA Initiative This work is collaboratively funded by: The Department of the Undersecretary of the Army ‐ Test and Evaluation (DUSA‐TE)
Joint Program Executive Office Joint Program Manager – Guardian Defense Biological Product Assurance Office (JPM‐ Guardian DBPAO) 20
October 2018 Meeting Goals
SPADA will launch new working groups on:
Bacterial Strain Verification Co-Chairs Linda C. Beck, JRAD and Shanmuga Sozhamannan, ECBC In-Silico Analysis Co-Chairs Linda C. Beck, JRAD and Shanmuga Sozhamannan, ECBC Soil Analysis Co-Chairs Linda C. Beck, JRAD and Morgan Minyard, DTRA
This work is funded by DUSA‐TE and JPEO JPM Guardian, DBPAO
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Bacterial Strain Verification
Bacteria Strain Verification Working Group It is not an uncommon occurrence for an assay developer, researcher, or evaluator to discover that a bacterial culture is not what it is purported to be in terms of species or strain. There is no consensus on the process to authenticate bacterial strain. This group will work to develop guidelines for the characterization and authentication of bacterial strains to provide confidence in the identification of material being used.
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In-Silico Analysis
In Silico Analysis Working Group
With the advantages of in silico PCR analysis, the building of confidence in the results can been enhanced by establishing standards and recommendations for use as a complimentary tool to wet testing. The goal of the AOAC SPADA Working Group is to draft standard procedures for the use of in silico PCR analysis, so different analysts working across separate laboratories will achieve equivalent results, and thus build confidence in the data from in silico PCR analysis.
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Soil Analysis
Soil Testing Working Group
Increased confidence and reliability in performance of assays that meet the operational needs when deployed in the field requires evaluating inhibition, interference, and cross‐ reactivity of an assay. Currently there is a lack of consensus standards for the preparation and use of soil testing samples. The goal of the AOAC SPADA Working Group is to draft standard procedures for preparation and characterization of soils to be used in the evaluation of candidate biothreat detection assays.
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Questions?
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AOAC Standards Development: An Overview
AOAC SPADA Meeting Rockville, MD USA Tuesday, October 16, 2018
Deborah McKenzie Sr. Director, Standards and Official Methods SM AOAC INTERNATIONAL
AOAC began in Washington, DC as the Association of Official Agricultural Chemists (1884)
• Federal and state departments of agriculture through the USDA Bureau of Chemistry. Initially to standardize methodology to be used for composition of fertilizers by state laboratories Directed by Harvey Washington Wiley who wrote the 1906 law that began the US Food and Drug Administration (FDA)
• By the 1980s AOAC’s membership included microbiologist, food science professionals • In 1991, Association of Official Agricultural Chemists legally changed its name to AOAC INTERNATIONAL • Often referred to as Association of Analytical Communities ‐ used to encompass all of the scientific disciplines involved in AOAC’s work.
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AOAC Leverages the Power of Many
AOAC Leverages Networks to Assemble Stakeholders & Experts
AOAC INTERNATIONAL Headquarters
• Develop international voluntary consensus standards method performance requirements
• Discuss & adopt methods that are published in the Official Methods of Analysis of AOAC INTERNATIONAL using judgment of the world’s leading experts. Providing fit for purpose methods through standards development
GeneralLocationsof AOAC stakeholderpanelparticipants GeneralLocationsof the16 AOAC INTERNATIONALcurrentSections
AOAC ® INTERNATIONAL (AOAC) is an independent third‐party international standards developing organization and AOAC has no vested interest in the development of standards or in the evaluation of methods of analysis.
AOAC Stakeholder Panels
Strategic Food Analytical Methods SPSFAM
Agent Detection
Alternative Methods ISPAM
AOAC Standards Developing Communities
Assays SPADA
Infant Formula and Adult Nutritionals SPIFAN
Dietary Supplements SPDS
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What Are Standards?
Common and repeated use of rules, conditions, guidelines or characteristics for products or related processes .
The definition of terms; classification of components; delineation of procedures; processes, products, systems, services, or practices; test methods and sampling procedures. A Performance standard is a standard that states requirements in terms of required results with criteria for verifying compliance but without stating the methods for achieving required results. Voluntary Consensus standards are standards developed or adopted by voluntary consensus standards bodies , both domestic and international.
US OMB Circular A‐119
AOAC Consensus Standards
Examples: AOAC Standards
Basic Principles
• Transparency • Openness • Balance of Interests • Due Process • Consensus • Appeals
• Performance Requirements • Guidelines
• Sampling Standards •Methods of Analysis
• Best Practices • Operational Documents
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Advisory Panel, Stakeholder Panel & Working Groups
Advisory Panel
• US DUSA-TE, JPEO JPM Guardian, DBPAO
• Community of experts and key stakeholders • Balanced representative subset of vetted voting stakeholders • Anyone with a material interest can participate
Stakeholder Panel
• Consists of technical experts from Industry, Government, CRO’s, and Academia to develop method performance criteria required for needed methods • Present background and history on analytical method needs for stakeholder panel • Members approved by SPADA Chair • Begin drafting standards
Working Groups
Stakeholder Panel Role and Output
• Defines specific analytical issue(s)
1 st
• Forms working groups to draft standard(s) that address the issue(s)
2 nd
• Comments on draft standard(s)
3 rd
• Adopts voluntary consensus standard(s)
4 th
AOAC Voluntary Consensus Standards – Published in Official Methods of Analysis of AOAC INTERNATIONAL – Manuscript published in Journal of AOAC INTERNATIONAL
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Working Groups
• SPADA has had 18 working groups – 19 standards developed across 11 biological threat agents
– 3 new working groups formed
• Soils Testing ‐ (Dr. Beck and Dr. Minyard) • Bacterial Strain Verification – (Dr. Beck & Dr. Sozhamannan) • In Silico Analysis – (Dr. Beck & Dr. Sozhamannan)
– Working groups to begin work during this SPADA meeting on draft standards • Continue over next 8 months
Standards Drafting Overview
Working Group Meets
• Draft
standard
• Public
Post draft standard
Comment Period (≥ 30 days)
Reconcile Comments
• Recommend final draft to SPADA
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Establish AOAC Standards
Establish Standards
• SPADA demonstrates consensus on any standard • Documents a community’s needs • Very detailed description of the analytical requirements • Published by AOAC as a standard
Second SPADA meeting
• Working Groups present draft standard
• Vetted Representative Voting Members selected based on registration and organizational or subject matter perspective – represents balance of perspectives – Demonstrates consensus on behalf of stakeholders – 2/3 vote in favor of a motion establishes an AOAC standard
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Documentation and Communication
• AOAC carefully documents the actions of Stakeholder Panel, Working Groups, and ERPs
• AOAC will prepare summaries of the meetings – Communicate summaries to the stakeholders – Publish summaries in the Referee section of AOAC’s Inside Laboratory Management • AOAC publishes its voluntary consensus standards and Official Methods – Official Methods of Analysis of AOAC INTERNATIONAL – Journal of AOAC INTERNATIONAL • AOAC publishes the status of standards and methods in the Referee section of AOAC’s Inside Laboratory Management
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AOAC Standards Published Since 2011
OTHER 3% AOAC Standards Developed
ISPAM 3%
SPADA 19%
SPSFAM 17%
SPDS 30%
SPIFAN 28%
SPADA SPDS SPIFAN SPSFAM ISPAM OTHER
Questions?
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Soil Standards for Assay Development and T&E
Linda C. Beck, Ph.D. Principal Biological Scientist Joint Research and Development, Inc. Supporting DUSA –TE and JPEO JPM Guardian
This work was funded by DUSA-TE and JPEO JPM Guardian, DBPAO
BACKGROUND
• AOAC Stakeholder Panel on Agent Detection Assays (SPADA) Standards Project: • Standard Methods Performance Requirements: SMPR (10) • Documents community’s analytical needs • Descripts analytical requirements • Includes method acceptance requirements • Panel – consensus standards body: • DoD, DHS, CDC, FDA, EPA, USPS, NIST, ATCC, State and Local PH, Industry, Academia, First Responders
• Most recent panel WG effort: • Environmental Factors Annex (in all SMPRs); three sections: • Environmental Matrix Samples – Aerosol; Soil Testing • Environmental Organisms • Potential Interferents (operational background) Soil testing has always generated the most questions
SOIL TESTING
• Recommendations to evaluate candidate assays using soil samples was added to Part 2 of the Environmental Factors appendix in 2017: • 2.2: Soil Testing • “Airborne soil particles may constitute a significant challenge to the analysis of collected aerosol samples by polymerase chain reaction (PCR) assays. Soils contain genomic materials or nucleic acid fragments of countless archaebacterial, bacterial, and eukaryotic organisms. Some of the more common soil organisms can be anticipated. Soils may also contain unanticipated components that interfere with extraction, denaturation, polymerization, or annealing reactions. Therefore, determining the effect of a variety of representative soils on the robustness of a PCR assay is an important first step.” • However, instructions in the Soil Testing section are extremely limited because there was not a consensus on how to conduct soil testing , nor what kinds of soil samples to use (T&E; assay development; industry/gov’t )
WHY IMPORTANT Currently:
• Lack of uniformity for assay development and comparison of performance across vendors for the USG
• Limited soil representations
• Unknown soil composition as relates to bio assay performance testing
• The community does not evaluate an assays performance using consistent standard soil samples and standard methodology for testing
• Testing for cross reactivity and inhibition limited to the near neighbors
• Testing interferents conducted with identified operational interferents (i.e. diesel exhaust, sea salt, etc.) during DT, DT/OT and O
OBJECTIVE
Develop specific soil standards to support assay development and T&E testing (Bio detection/identification)
Standards for: • Characterization of soil – parameters • Representative soils for testing
PATH FORWARD • Develop the process for a standard method of soil collection • Identify/characterize types of soil needed • Create soil standards for material development • Standardize the characterization of soil • Support the development and archival of test materials
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JRAD Proprietary
QUESTIONS
Soil 101
Morgan Minyard
Soil 101 Soils are very complex and unique They are an important natural resource that is decreasing over time In US alone, over 19,000 soils have been identified The study of soil science includes taxonomy, chemistry, physics and biology in addition to agricultural and conservation practices.
Soil are: •Plant growth medium •Regulator for water supply •Recycler of raw materials •Habitat for Soil Organisms •Engineering Medium •Environmental interface
https://www.nrcs.usda.gov/wps/portal/nrcs/detail/nj/soils/?cid=nrcs141p2_018867
Soil horizons: •Consist of the O, A, E, B and C horizon •Not all horizons are present in a soil •Each horizon has different properties •The soil horizons and their properties identify the soil •Soil on top of another soil is possible, usually due to flooding
The 12 Soil Orders
• Entisol – Youthful soil with little profile development • Andisol – Not a well developed clay layer but prominent organic‐rich A • Gelisol – Permafrost within 1 m of soil surface • Histosol – Organic accumulation of great depth due to anaerobic conditions • Inceptisol – More well developed clay layer or B horizon • Aridisol – Arid to semi‐arid ecosystems of shrub and short grasses • Vertisol – Highly‐active and swelling clay • Mollisol – Semi‐arid or moist grassland ecosystem and deep organic‐rich A • Alfisol – Clayey B horizon with low acidity • Spodosol – Humic B horizon with high acidity and coarse texture (sandy)
• Ultisol – Clayey B horizon with high acidity • Oxisol – Oxic B hoizon with low‐activity clay
https://en.wikibooks.org/wiki/High_School_Earth_Science/Soils
https://www.ctahr.hawaii.edu/mauisoil/a_profile.aspx
5 Soil forming factors
• Climate – Weather regime • Parent Material – includes bedrock and deposited sediments • Organisms – biota includes bacteria to trees to animals • Relief – topography makes a deference • Time – weathering time not actual
Soil profile includes the Picacho inceptisol that is underlaid by 4 ‐ 8 m of saprolite
One of the fastest weathering granodiorite in the world. Forms a unique soil profile Quick percolation so a well drained soil
Granodiorite exposed road cut demonstrates that bedrock undergoes spheroidal weathering to saprock
Bedrock Minerals: Quartz (20‐24%) Plagioclase (50%) Biotite (9%) Hornblende (6%) K‐feldspar (2%) Fe, Ti oxides (2%) Apatite (0.6%)
Saprolite Minerals: Mica Iron oxides – Goethite and Hematite Halloysite Kaolinite Quartz
Saprock (Cr)
White et al. 1998; Buss et al., 2008
Soil Texture
Soil Texture
Soil Texture: • Critical to understanding soil behavior • Varies little with time • Impacts water retention • Impacts gas exchange • Impacts nutrient retention • active fraction of soil (clay) • Silt includes bedrock material and minerals • Sand lest active and less surface area
h ttp://www.soils4teachers.org/files/s4t/texture.pdf
Habitat for Soil Organisms
• Primary Producers • Plants • Moss • Algae • Lichens • Primary Consumers • Herbivores
• Photosynthetic Micobes
• Detritivores (dead leaves) • Saprophytic (dead tissue)
• Secondary Consumers
• Consume Primary Consumers
• Tertiary Consumers • Predators • Microbial feeders • Ecosystem Engineers • Earthworms • Burrowing animals
• Certain beetles – dung beetle
https://www.nrcs.usda.gov/Internet/FSE_MEDIA/nrcs142p2_049822.jpg
Habitat for Soil Organisms
Organisms Microflora Bacteria
Number per m 2
Number per gram soil
10 13 ‐10 14 10 12 ‐10 13 10 10 ‐10 11 10 9 ‐10 10 10 9 ‐10 10 10 6 ‐10 7 10 3 ‐10 6 10 3 ‐10 6 10‐10 3 10 2 ‐10 4
10 8 ‐10 9 10 7 ‐10 8 10 5 ‐10 6 10 4 ‐10 5
Actinomycetes Fungi Algae
Fauna
Protozoa Nematodes Mites Collembola Earthworms Other fauna
10 4 ‐10 5 10‐10 2
1‐10 1‐10
KSU image
Soil porosity and soil drainage
• Soil porosity impacts:
• Water flow and therefore drainage • Gas exchange • Mineral weathering • Root growth
Anaerobic microbial processes, reduced iron
Oxic conditions, oxidation of iron to iron oxides
Soil pH • Impacts nutrient availability • Acidic soils Al3+ exchangeable and heavy metals • Neutral and basic soils Cations like Calcium and potassium exchangeable • Biota diversity • Soil horizons and structure
pH range Ultra acidic < 3.5 Extremely acidic 3.5–4.4 Very strongly acidic 4.5–5.0 Strongly acidic 5.1–5.5 Moderately acidic 5.6–6.0 Slightly acidic 6.1–6.5 Neutral 6.6–7.3 Slightly alkaline 7.4–7.8 Moderately alkaline 7.9–8.4 Strongly alkaline 8.5–9.0 Very strongly alkaline > 9.0
Red more acidic Blue more basic Yellow neutral
Soil Internet Resources
• Web Soil Survey • https://websoilsurvey.sc.egov.usda.gov/App/HomePage.htm
• Critical Zone Exploration Network and Critical Zone Observatories • http://criticalzone.org/national/about/partner/critical‐zone‐exploration‐ network/
• Soil Testing Labs • https://agsci.psu.edu/aasl/soil‐testing
Soil experiment guidelines • OECD 106 Guideline • Provides Guidance on: • Chemical properties that should be known • Soil characteristics • Soil sampling • Soil storage Soil type
pH range (in 0.01 M CaCl2)
Organic carbon content (%)
Clay content (%)
Soil texture *
1 2 3 4 5 6 7
4.5‐5.5
1.0‐2.0 3.5‐5.0 1.5‐3.0 3.0‐4.0
65‐80 20‐40 15‐25 15‐30
clay
> 7.5
clay loam silt loam
5.5‐7.0 4.0‐5.5
loam
< 4.0‐6.0
< 0.5‐1.5 < 0.5‐1.0
< 10‐15
loamy sand
> 7.0 < 4.5
40‐65 < 10
clay loam/clay
> 10
sand/loamy sand
Protein Extraction from Soils
Sherry Blight, Ph.D. Principal Research Scientist Battelle Memorial Institute October 2018
Distribution Statement D: Distribution authorized to Department of Defense and their contractors in order to protect technical data or information from automatic dissemination. Other requests for this document shall be referred to the Joint Project Manager Guardian, 2800 Bush River Road, Aberdeen Proving Ground, MD 21010-5424.
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Common Analytical Laboratory System (CALS) • Since 2015 • ALS since 2012 • MSD PR2-1800 is the toxin detector used
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National Guard Response Sectors
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Soil Testing on the PR2 • 1X Phosphate buffered saline + 0.1% Triton X-100 (PBST) • Purified botulinum neurotoxin type A (BoNT/A), ricin, and Staphylococcus aureus enterotoxin B (SEB) • NIST Soils: Montana I, New York/New Jersey Waterway Sediment, Flint Clay • 4 extraction protocols/buffers were tested Current CST 10X PBST MSD proprietary buffer (PBST with BSA and azide) MO BIO NoviPure TM Soil Protein Extraction Kit (garnet beads + vortex)
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Results – BoNT/A (ng/mL)
2500
2000
1500
NY/NJ Flint Clay Montana 1
1000
Average ECL
500
0
10X PBST MSD Buffer MO BIO Buffer CST (PBST)
Extraction Method
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Results – SEB (ng/mL)
700
600
500
400
NY/NJ Flint Clay Montana 1
300
200 Average ECL
100
0
10X PBST MSD Buffer MoBio Buffer CST (PBST)
Extraction Method
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Results – Ricin (ng/mL)
7500
6000
4500
NY/NJ Flint Clay Montana 1
3000
1500
Average ECL
0
10X PBST MSD Buffer
MoBio Buffer
CST
Extraction Method
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Other Soils Tested on the PR2
• Sigma - clean loam (RTC) • Sigma also has a few other “clean” soils (RTC) Clay loam, clay, sandy loam, sand
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Naval Medical Research Center (NMRC) PR2 Confidence Testing • 98 different soil samples were purchased from the North American Proficiency Testing (NAPT) Program of the Soil Science Society of America • One potting soil purchased locally • Same CST PBST extraction utilized • Toxin LOD determination possible, but at some cost to sensitivity (but specificity gained)
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Final Thoughts • Clean soil background (or “why we are here”) CSTs currently using PBST to determine thresholds
Storage conditions? How do we obtain? • Potentially a standardized protein extraction protocol PBST is not the ideal extraction buffer Is this possible? Instrumentation, procedural issues Varies from protein to protein of interest • National Ecological Observatory Network (NEON) (National Science Foundation [NSF]) – collect field data from 81 sites (47 terrestrial)
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Sponsor
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800.201.2011 | solutions@battelle.org | www.battelle.org
Defense Biological Product Assurance Office Presented to SPADA on October 17, 2018 SPADA- Strain Verification Standards for Assay Development
Shanmuga Sozhamannan, Technical Coordinator, DBPAO Joint Program Executive Officer for Chemical, Biological, Radiological and Nuclear Defense This work was funded by DUSA-TE and JPEO JPM Guardian, DBPAO
UNCLASSIFIED Distribution Statement A:Approved for public release; distribution isunlimited.
Why do we need a Standard for strain verification and authentication; i.e., characterization?-
There is no such consensus standard agreed upon by the community
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Defense Biological Product Assurance Office (The amazon.com of DoD) Dugway Proving Ground anthracis debacle CRP/DBPAO Major Milestones
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DBPAO Product portfolio
Ordering System For Critical Assays and Reagents (OSCAR)- “Amzon.com” for Biodefense products
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All hell broke loose in the summer of 2015 (May 22 nd to be precise)
News about anthrax mailings
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The Solution to the problem- Risk Mitigated Reference Materials (RM2)
SPARK: Guaranteed Risk free because the organism does not have the ability to cause disease
This effort is unique and to the best of our knowledge does not exist in JPEO or other agencies
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Lesson from Genome Sequencing Standards • Genomics Standards Consortium • MIGS • Minimum Information about a Genome Sequence- captures information about a genome sequence- e.g., depth and breadth of coverage, sequencer type, organism information etc (Chain et al: Science 2009 326 (5950:236-237); Ladner et al. Mbio 2014 Jun 17;5(3):e01360-14) • MIMS • Minimum Information about a Metagenomic Sequence/Sample; metadata on samples and sequences (Kottmann et 1l 2008. OMICS 12 (2):115-121). • Why not a standard for microbial strains? • MIBS • Minimum Information about a Bacterial Strain. What are the information to be captured? • MIVS (not the focus now) • Minimum Information about a Viral Strain. What are the information to be captured?
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The impetus for this work (DoD specific) • May 2015 DPG debacle; In its aftermath, many committees were established and investigations were carried out to understand and fix DPG like problems. • Moratorium on Select agent work within DoD; Sec Army Directive to fortify safety of BAST activities within DoD and implementation of recommendations. Establishment of BBPO (BSAT Bio Safety Program Office). • Prevent indiscriminate transfer of iBSATs or dBSATS (derivative) that are not 100% guaranteed for inactivation. • Uncertainty of 100% guarantee of iBSATs safety and security • Need for materials BSAT derivatives across the biodefense enterprise; however, DBPAO divested BSAT business. • But leadership wanted DBPAO involvement in assurance of and confidence in materials from other sources by other means across the enterprise. So, setting the standard for strains used in assay development, test and evaluation
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The impetus for this work (Interagency specific)
• Bio Alliance • Interagency collaboration for a network of repositories for various bacterial and viral reference materials • Networking different repositories • Common standards a must for reliability and confidence in materials • The materials are used for various applications: assay development, Vaccines and therapeutics development
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• Blindly trusting the information provided by the donor of a strain • Discrepancies in genotypes; end up storing in the wrong containment spaces (PAK1 BACI 224 in BSL3 and YERS 003 in BSL2); CDC is not happy about this. • Lab specific growth conditions may affect the strain’s properties and genetic make up (surface antigen expression in Yp is heavily influenced by growth temperature); major issues: wrong classification of BSATs • The sources for strains having the same designations are many. But they may not have the same genotypic/phenotypic characteristics due to different pedigrees, laboratory propagation and growth conditions (Sterne, BG examples) • Set the standards so that those that are authorized to use BSATs can follow and make sure the strain meets the standards and adds confidence to the identity and authenticity of strains. DBPAOs assurance role !! • Once you characterize the strain the stock is aliquoted, frozen and further distributed from the repository or any other source. You can always trace it back to the stock. Further growth is according to the same protocol. [QR coding vials and electronic tracking] The impetus for this work (Global Issues)
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Blindly trusting the information provided by the donor of a strain
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Error in published sequence
• Error discovered in published sequence
Multilocus sequence typing ‐ seven housekeeping genes ‐ evolutionary history generated ‐ Neighbor‐joining method, MEGA7
Johnson 2015 Genome Announc 3, e00151 Johnson 2018 Genome Announc 6, e01144
Previous researchers given wrong sample Current sample is correctly identified as Al Hakam
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FOUO – not approved for public distribution
Blindly trusting the information provided by the donor of a strain • BACI224 • PAK-1: supposed to be wild type Ba with both plasmids. Customer (assay developer) spent a bit of time trying to figure out the reason for PCR failure against one of the plasmids. Turned out, the stock had either lost one of the plasmids or a mixed population. Had been stored in BSL3 • YERS003 • CO92: Based on a PCR result, deemed to have a missing plasmid and was stored in BSL2. Other PCRs showed weak positive for the target plasmid. Sequencing could unequivocally resolve it. Again a problem of mixed population of both genotypes? Growth conditions (media and temperature can make a big difference)
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Strains with the same designations from different labs are not the same (phenotypically and genotypically) • Bacillus anthracis Sterne (different SNPs in WG sequences) • Phage gamma sensitivity is different for different Sterne strains and growth conditions • Bacillus globigii
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Genomic changes in same strains from different sources
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Metabolic changes in same strains from different sources
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• There is no standard on the level/granularity of strain characterization needed to ensure the confidence on the identity of a bacterial or viral strain used in assay development. Each repository/laboratory has their own set of procedures- or they don’t or they do “ad hoc” what is needed. • The cost of extensive characterization may vary widely depending on what assays are done and exorbitant cost can be prohibitive; so need minimum standard, to start with. Do we create tiered standards analogous to sequencing? • Is WGS sequencing good enough? • No- Knowing the genetic make up (genotype) without the phenotype is no use. • The jury is still out there on level of genome sequencing – to finish or not to finish the genome? The impetus for this work (General issues)
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Microbiological analyses essential for strain verification and validation- the B. anthracis like bacteria- B. cereus biovar anthracis B. Ce var anthracis Classic anthracis Klee et al 2006. J Bac 188: 5333-5344
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Even Whole Genome Sequencing Cannot Resolve Some Genomic Rearrangements
Tandem Repeats in Vibrio cholerae Strains
Optical Mapping showed tandem repeats of (~160 kb), read mapping showed mixed results, PCR could not identify the junctions unequivocally
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Cost of Characterization
Strains in CRPµTIC full characterization: ~$100 k/strain
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Why SPADA for this?
• SPADA is an ideal forum to develop a standard because of its history in developing standards and developing a consensus thorough brining in SMEs from across the domains (Sponsor, government, industry, academia- national and international). • SMPR: • 6 Validation Guidance • AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures (Official Methods of Analysis of AOAC INTERNATIONAL, 2016, 20th Ed., Appendix I). • Inclusivity and exclusivity panel organisms used for evaluation must be characterized and documented to truly be the species and strains they are purported to be.
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What is the objective for the WG • Evolve a standard for bacterial strain characterization • Set minimal information needed for such a strain verification and validation • Write the document and publish • What are the broad categories of information to be captured? Process and not the specific method/instrument • What is the outline of the document- this is not the usual run of the mill SMPR. • What is the forum/journal to publish? • Developed many SMPRs for agent specific assays and user scenarios that help the assay developers and stakeholders who use the assays
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What would be an ideal strain characterization? e.g., rBaSwAT
VEGETATIVE CELLS • Phenotypic assays Microbiological tests
Phage sensitivity ( and AP50c) Antimicrobial resistance Spore formation Immunological tests (LFI, PR2, Magpix and Toxin Assay) • Genotypic assays Toxin deletion verification by PCR Optical mapping and Whole genome sequencing Molecular tests for signatures (PCR) SPORES • Production, purification and irradiation inactivation (using new protocol established by the working group) Physical properties Molecular and Immuno assays and bridging studies Animal studies Tab: ~$800K/~8 strains
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Critical Reagents Program Microbial Threat Information Center (CRPµTIC)
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Grades of Characterization Gold- all types of applications including vaccines and therapeutics reference materials Silver- detection assay development Bronze- minimal information for making sure it is the right strain
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What is the format of the standards document Standards Guidelines Opinion document Manuscript What is the forum for publication?
AOAC Journal ASM journals Science Other Microbiology Journals
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CONTACT US
Bruce Goodwin Joint Product Leader bruce.g.goodwin4.civ@mail.mil Bryan Necciai
Assistant Product Manager bryan.d.necciai.civ@mail.mil Shanmuga Sozhamannan, Ph. D Technical Coordinator email: shanmuga.sozhamannan.ctr@mail.mil https://www.ncbi.nlm.nih.gov/pubmed/?term=Sozhamannan
This work was funded by DUSA-TE and JPEO JPM Guardian, DBPAO
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Bacterial Strain Verification in the Unified Culture Collection David Rozak USAMRIID 17 October 2018
The Unified Culture Collection
Bacteriology Section
Escherichia, 71 Clostridium, 57
Staphylococcus, 55 Pasteurella, 45
Brucella, 104
Streptococcus, 26 Coxiella, 19
Mycoplasma, 8
Yersinia, 107
Listeria, 6
Enterococcus, 11 Lactobacillus, 11 Neisseria, 11 Enterobacter, 9 Haemophilus, 9
Ralstonia, 6
Burkholderia, 194
Salmonella, 6 Arcanobacterium, 5 Klebsiella, 5 Mannheimia, 5 Mycobacterium, 5 Pseudomonas, 5
Other, 51
Bacteroides, 8 Corynebacterium, 8
Francisella, 241
Bacillus, 382
Numbers of accessioned strains
ISO‐accredited BSAT, exempt BSAT, and near‐neighbor bacterial reference materials for the biodefense community: • Fully characterized bacterial inoculum • Quantified animal challenge doses • Antigen and nucleic acid reference standards
Key Assertions
Production and Characterization
Source
• Untouched original
• Single colony pick
• Colony morphology • Gram Stain • Vitek • Biolog • PCR • Riboprinter • MLST • MiSeq • Colony morphology • Gram Stain • Phenotypic and genotypic assay
Master
Seed
Production
Assay Accuracies
Genus Bacillus Brucella
Inconclusive
Wrong Genus
Wrong Species
Correct
n
5% 3% 2% 9% 7% 8% 1% 0% 0%
4%
37% 21% 46% 12% 69% 41%
54% 256 62% 34 51% 191 78% 97 21% 29 14% 138 6% 104 10% 52 44% 32 36% 105 94% 17 17% 103 43% 132 55% 40 52% 127 45% 73 57% 14 67% 36 95% 20 0 0
15%
Burkholderia
2% 0% 3%
Francisella
Yersinia Bacillus Brucella MLST MIDI Ribotype Biolog Vitek Next Gen Sequencing Procedure • Extract DNA using EZ1 • Measure concentration with Qubit • Nextera DNA Flex Library prep kit • Run library prep on MiSeq using the v2 500 cycle kit • Run FASTQ files through EDGE bioinformatics pipeline 37% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 88% 87% 6% 4% Francisella Yersinia Bacillus Brucella 9% 47% 27% 23% 14% 6% 0% 2% 0% 8% 2% 0% Burkholderia 40% 51% 33% 18% 42% Francisella 6% 5% Yersinia Bacillus Brucella 28% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 0% 4% 51% Francisella 21% 21% 25% 0% 6% 5% Yersinia Bacillus Brucella 3% 0% 0% ‐‐ ‐‐ ‐‐ ‐‐ Burkholderia 12% 0% 2% Francisella ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ ‐‐ Yersinia
0
86% 103
0 0
EDGE Bioinformatics Pipeline • Developed by LANL and Navy • Quality trim and filter • Host removal • Map reads to reference • Assembly • Contig‐based taxonomy classification • MetaPhlAn • Kraken mini • Phylogenetic analysis
• Reads‐based taxonomy classification • GOTTCHA bacterial and viral • BWA
• Result reported from consensus of BWA, Kraken mini, and contig‐based
Validation Results
• 15/16 organisms correctly identified • Inter‐assay repeatability was 100% • Intra‐assay reproducibility was 100%
Accuracy of Platform (%)
UCC Number
Reference Sequence ID
EDGE Result
BACI002 BACI229 BACI232 BRUC106 BRUC076 BURK109 FRAN002 FRAN004 YERS086 YERS114 ACIN001 BACI012 STAP014 PSEU001 SALM001 BURK001
Bacillus anthracis
Bacillus anthracis
100
Bacillus thuringiensis
Bacillus thuringiensis
99.82
Bacillus cereus Brucella abortus Brucella canis
Bacillus cereus Brucella abortus Brucella canis
100 100 100 100 100 100 100 100 100 100 100 100 100
Burkholderia thailandensis Francisella philomiragia
Burkholderia thailandensis Francisella philomiragia
Francisella tularensis
Francisella tularensis
Yersinia pseudotuberculosis Yersinia pseudotuberculosis
Yersinia pestis
Yersinia pestis
Acinetobacter baumannii
Acinetobacter baumannii
Bacillus anthracis
Bacillus anthracis
Staphylococcus aureus
Staphylococcus aureus
Pseudomonas aeruginosa Pseudomonas aeruginosa
Salmonella enteritidis Burkholderia cepacia
Salmonella enteritidis
Burkholderia lata
99.98
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