1. LLNL-PRES-658976This work was performed under the auspices of theU.S. Department of Energy by Lawrence LivermoreNational Laboratory under contract DE-AC52-07NA27344.Lawrence Livermore National Security, LLCSahar El-Etr, Biomedical Scientist

2. Develop better methods for transport ofpathogens from the field to the laboratory at roomtemperature without loss of viability to enable livepathogen characterization. Existing transport devices are modification of aswab with a transport medium. Storage containersare usually cryogenic vials frozen at -80oC. Sampleclean up and enrichment currently occurs usingpathogen-specific transport/culture media.Lawrence Livermore National Laboratory LLNL-PRES-6589762 3. Environmental amoeba exist as trophozoites (active feeding form)under favorable conditions and encyst (make cysts) under stress. Technology takes advantage of the fact that many microbialpathogens are known to survive in amoeba cysts for many yearsand to emerge after the amoeba excyst. Decided to make use of mother nature and developed first livingsystem for the transport and storage of pathogens.Lawrence Livermore National Laboratory LLNL-PRES-6589763Trophozoites Cysts 4. Add nutrientrich mediacyststransport tolabencystmentreplicationsurvivalin cysts: Pathogen of Interest: Skin/ EnvironmentalFloraexcystmenttrophozoitesexcystmentLab: pure pathogenrecovery and IDFieldInoculateDeviceSwab woundSampleDecontaminationand PathogenEnrichment inAmoebaLawrence Livermore National Laboratory LLNL-PRES-658976 4 5. We have demonstrated the ability of the system to detect as few as10 bacterial colony forming units (CFUs) after 6-weeks ofinoculation at temperatures ranging from 4-42oC with no loss ofviability. We can detect Gram-negative (e.g. A. baumannii), Gram-positive(e.g. MRSA) fastidious bacterial pathogens, select agents andgastroenteritis causing viruses. Emerging microbes do not show enhanced resistance to antibioticsor biocides and do not show mutations due to growth in amoeba. May not work for anaerobic bacteria or enveloped viruses. We are currently building a prototype device for field deployment.Lawrence Livermore National Laboratory LLNL-PRES-6589765 6. System can be kept at ambient temperatures with no requirementof freezing or cold chain. Bacterial pathogens maintain their clinical phenotype with no lossof virulence, since they demonstrate similar rates of survival inmammalian cells as macrophage grown strains. Since Amoeba naturally feed on bacteria, samples contaminatedwith patient or environmental flora are cleaned up duringtransport. System enriches for the pathogen of interest.Lawrence Livermore National Laboratory LLNL-PRES-6589766 7. ApplicationDescriptionSetting Target Customers Current Practice#1 Samplecollection deviceField and hospitaldeploymentMilitary Medical ServicepersonnelLawrence Livermore National Laboratory LLNL-PRES-658976Use of traditional media-containing swabs at ambienttemperatures, major problem of samplecontamination or loss of genetic mobile elements#2 Samplecollection deviceClinic/hospitaldeploymentCivilian physicians andnursesUse of traditional media-containing swabs at ambienttemperatures, major problem of samplecontamination or loss of genetic mobile elements#3 Sample cleanupand enrichmentdeviceDiagnostic Militaryand civilianlaboratoriesDiagnostic laboratorypersonnelAttempting to culture samples and isolate pathogenof interest#4 Storage Device ResearchLaboratoriesResearch Personnel Long-term storage at -80 oC. Requires freezing anddoes not protect sample from contamination and lossof virulence7 8. Kevin Christopherchristopher6@llnl.govLawrence Livermore National Laboratory LLNL-PRES-658976 8