IN VITRO GUT MODEL BACILLUS ANTHRACIS GUT MICROBES ZIHLER ET AL 2011 ASSESSING THE EFFECT OF BIOLOGICAL AGENT INGESTION A SYSTEMS BIOLOGY BIOLOGICAL AGENT MODELING PLATFORM DR AMANDA N PAYNE MICROBIOLOGIST NAVAL SURFACE WARFARE CENTERDAHLGREN ID: 1047113
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1. 500 nm2.00 µmEPITHELIAL CELL MODELIN VITRO GUT MODELBACILLUS ANTHRACISGUT MICROBESZIHLER ET AL. 2011ASSESSING THE EFFECT OF BIOLOGICAL AGENT INGESTIONA SYSTEMS BIOLOGY BIOLOGICAL AGENT MODELING PLATFORMDR. AMANDA N. PAYNE, MICROBIOLOGIST, NAVAL SURFACE WARFARE CENTER-DAHLGRENDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
2. PROJECT SIGNIFICANCE AND RELEVANCE TO NSWCDD“For the life of me, I cannot understand why the terrorists have not attacked our food supply because it is so easy to do.” – 2004 SECRETARY THOMPSON Aerosol agent detection, counter-proliferation and defeat is the major resource allocation area Develop technical focus within critical science and engineering expertise to investigate risks associated with biological agent ingestion Identify agent(s) of interest; Leverage Z21 expertise with Bacillus anthracis Design and implement an in vitro systems biology modeling platform of ingested biological agent(s): Inhalational exposure outcomes homogenous, highly lethal Ingestional outcomes reportedly extremely variable; asymptomatic to fatal reported Vaccination may not prove effective for gastrointestinally-acquired biological agents (e.g. Fransicella tularensis, KuoLee R. et al. 2007. Infect Immun, 75: 1651-1660)Why??DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
3. GASTROINTESTINAL TRACT: ANATOMY AND FUNCTIONSOURCE: CHILDREN’S HOSPITAL COLORADO LARGEST ORGAN IN HUMAN BODY DIVERSE AND DYNAMIC ENVIRONMENT: LARGE pH RANGE OXYGEN STRATIFICATION REGIONS OF HIGH ENZYME ACTIVITY CONSTANT MOVEMENT HIGHLY ENERVATED BIOLOGICAL AGENT DEFENSE PERSPECTIVE: FACILITATES INGESTED AGENT ENTRY SITE OF AGENT-HOST INTERACTION LARGE INTESTINE MOST IMPORTANT DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
4. LARGE INTESTINAL IMPORTANCE IN MAMMALSCONVENTIONAL THOUGHTLARGE TUBE WITH IN/OUTPUTDESTINED FOR NUTRIENT ABSORPTIONREALITY: HIGHLY COMPLEX NETWORK OF CELLULAR AND IMMUNOGENIC ACTIVITY MOST HIGHLY COLONIZED HUMAN SITE: 1014 CFU G-1 LARGEST MICROBIOME: “GUT MICROBIOTA” LUMINAL VS. MUCOSAL NICHESDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
5. GUT MICROBIOTA: AN ORGAN WITHIN AN ORGAN GUT MICROBIOTA BASIC FUNCTIONALITY: DIGESTION COMMUNICATION WITH GI EPITHELIUM COMMUNICATION WITH CNS EDUCATE IMMUNE SYSTEM PATHOGEN EXCLUSION AND DEFEAT GUT MICROBIOTA BASIC COMPOSITION: >1000 SPECIES (BACTEROIDETES, FIRMICUTES, ACTINOBACTERIA, PROTEOBACTERIA) DELICATE BALANCE, HIGHLY INDIVIDUAL ORGANIZED INTO HIERARCHICAL NICHESPERTURBATION: ANTIBIOTICS, VACCINATION, PATHOGENSDYSFUNCTION: CHANGE IN COMPOSITION AND FUNCTIONALITY LOSS OF NICHE LOSS OF IMMUNOGENIC TOLERANCE PATHOGEN INVASION INTO EPITHELIAL CELLS SYSTEMIC DISEASEDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
6. ORGANISMAL SIGNIFICANCE OF BACILLUS ANTHRACIS AS BIOLOGICAL THREAT AGENTSPORESHIGHLY CHEMICAL, ENZYME RESISTANTSURVIVE NUTRIENT STARVATION2. VEGETATIVE CELL WITH POLY-D-GLUTAMYL CAPSULE(ESTABLISHMENT OF INFECTION)TOXIN: LETHAL FACTOR, EDEMA FACTOR, PROTECTIVE ANTIGEN2.00 µM GERMINATION MOST SIGNIFICANT EVENT: TRANSITION TO VIABLE ORGANISM FACTORS DICTATING BACILLUS ANTHRACIS SUCCESS AS FOOD-BORNE AGENT: COMPETITION (GUT MICROBIOTA) HOST CONTACT REQUIREMENT(S) HOST FACTOR REQUIREMENT(S)DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
7. PROJECT OBJECTIVE AND KEY POINTSProject ObjectiveAssess the impact of biological agent ingestion utilizing the commensal intestinal microbial community and related host-microbe interactions a benchmark of the potential risk for pathogenesis of ingested biological agent sporesState-of-the-art technology = animal models (expensive, ethical impedance) and in in vitro gut modelsKEY POINTS GERMINATION: WHERE (GI LUMEN AND/OR MUCOSA), REQUIREMENTS GUT MICROBIOTA COMMUNITY / FUNCTIONALITY IMPACT HOST RESPONSE AGENT RESPONSE (ANTIGENS, TRANSCRIPTOME)DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
8. EVALUATION OF BACILLUS ANTHRACIS AS BIOLOGICAL THREAT AGENT: IN VITRO MODEL DESIGN AND PARAMETERSTempStirPumpI. Gastric Conditions: pH 2 peptone water600 U pepsinFlow rate: 0.5 mL min-1 II. Small Intestinal Conditions: pH adjusted to 6.54% bile salt; 2% bile salt0.2% (w/v) pancreatin for an additional 120 min.Pump flow rate: 1 mL min-1 Three-stage in vitro gut modelSingle-Stage in vitro gut modelEnzyme Solutions Continuous Fermentation* pH control Anaerobic control Temperature control Complex MediumKEY IN VITRO MODEL PARAMETERSPayne et al. 2012. Trends Biotechnol*System retention rate: 1/Dµ = D, where µ is the growth rate and D the dilution factor.D = f/v, where D is the dilution rate, f is the flow rate and v equals the total volumeDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
9. GUT MICROBIOTA GROWTH MEDIUM REQUIREMENTSConstituentPectin (citrus)Xylan (oat spelts)Arabinogalactan (larch wood)Soluble starch MucinCasein acid hydrolysatePeptone waterTryptoneYeast extractL-Cysteine HCl monohydrateBile saltsKH2PO4NaHCO3NaClKClMgSO4 hydrate (120.37 g/mol)CaCl2 . 2H2O (147.02 g/mol)MnCl2 . 4H2O (197.91 g/mol)FeSO4 . 7H2O (278.02 g/mol)Hemin solutionTween 80Vitamins solutionVitaminPyridoxine-HCl4-Aminobenzoic acid (PABA)Nicotinic acid (Vit. B3)* Biotin (Vit H)* Folic acid (Vit B9)Cyanocobalamin (Vit B12)Thiamine (Vit B1 HCl)(-) - Riboflavin* Phylloquinone (Vit K1)* MenadioneD-pantothenic acidDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
10. NSWCDD IN VITRO GUT MODEL OPERATIONAL SETUPEPPENDORF BIOFLO 115 BIOREACTORSINGLE STAGE MODELPROXIMAL COLON (pH 5.7)GUT MICROBIOTA +GASTRIC AND SMALL INTESTINAL-TREATED B. ANTHRACIS SPORESFRESH MEDIUMSPENT MEDIUM (REACTOR OUTFLOW)CO2 regulator (ANAEROBIC CONTROL)SODIUM HYDROXIDE(pH CONTROL)CAVEAT: MODEL DESIGN PRECLUDES INCLUSION OF HOST FACTORS; MODELS ARE OF LUMINAL GI PROCESSESDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
11. IN VITRO GUT MODELIN VITRO HUMAN CELL MODELS+1. MONOLAYERCOLON CELL LINES: CACO-2, HT-29, T842. M-CELL MODELCACO-2 B CELL2. 3-D CELL MODELEPITHELIAL CELL MACROPHAGEDENDRITIC CELLSCELL MODEL DIVERSITYINFLAMMATIONPATHOGEN ADHESIONINVASIONGENE EXPRESSIONCHANGESCOMBINED IN VITRO GUT AND IN VITRO HUMAN CELL MODELS:SYSTEMS BIOLOGY APPLICATIONDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
12. IN VITRO GASTRIC AND SMALL INTESTINAL-TREATMENT INDUCES PHYSICAL CHANGES IN B. ANTHRACIS SPORES2.00 µmTEM Images of Treated B. anthracis Sterne Spores2.00 µm1.00 µmControl (Untreated)Gastric TreatedGastric and Small Intestinal TreatedSignificant exosporium wrinkling100 nm100 nmControl (Untreated)Gastric and Small IntestinalEffect disappears after small intestinal treatment (resembles control again)500 nmGastric TreatedTREATMENT DOES NOT IMPACT SPORE VIABILITYSEM Images of Treated B. anthracis Sterne SporesDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
13. IN VITRO GUT MODEL RESULTS:B. ANTHRACIS IN CULTURE FAIL TO GERMINATELACK OF GERMINATION: COMPETITION (GUT MICROBIOTA)pHGERMINATION REQUIREMENTSPeriodDAY 1DAY 2DAY 3DAY 4DAY 5DAY 6DAY 7DAY 8DAY 9DAY 10DAY 11DAY 12DAY 13DAY 14Microflora AdditionStartStabilizationExperimentationEXPERIMENT 1EXPERIMENT 2GUT MICROBIOTA: CHANGE IN COMMUNITYCHANGE IN FUNCTIONALITYDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
14. B. ANTHRACIS GASTRIC AND SMALL INTESTINAL-TREATED SPORES ALSO FAIL TO GERMINATE WITHOUT GUT MICROBIOTADISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
15. B. ANTHRACIS GASTRIC AND SMALL INTESTINAL-TREATED SPORES ALSO FAIL TO GERMINATE UNDER AEROBIC CONDITIONS IN FERMENTATION MEDIUMDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
16. B. ANTHRACIS GERMINATION IS WILD-TYPE WITHOUT ENZYMATIC TREATMENTCONDITIONS:37°C4 HOURDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.
17. PROJECT OUTLOOK CONTINUE EFFORT TO UNDERSTAND B. ANTHRACIS GERMINATION IN GI TRACT: IN VITRO M-CELL MODEL EXPERIMENTS IN VITRO GUT MODEL AT DIFFERENT pH INVESTIGATE IMMUNE RESPONSE OF B. ANTHRACIS SPORES CYTOKINE/CHEMOKINE ASSAYS ELECTRON AND CONFOCAL MICROSCOPY OF CELLS IN CONTACT WITH SPORES CORRELATE RESULTS WITH INHALATIONAL DATA EXAMINE DIFFERENT B. ANTHRACIS STRAINS (PASTEUR) COMBINE EFFORT WITH NRL TO DEVELOP FOOD-BORNE AGENT DETECTION PLATFORMDISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited.