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Food Security Research at the National Center for Toxicological Research

Food Security Research at the National Center for Toxicological Research. Daniel A. Casciano, Ph.D. FDA/NCTR. NCTR Mission.

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Food Security Research at the National Center for Toxicological Research

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  1. Food Security Research at the National Center for Toxicological Research Daniel A. Casciano, Ph.D. FDA/NCTR

  2. NCTR Mission To conduct peer-reviewedscientific research that supports and anticipates the FDA’s current and future regulatory needs. This involves fundamental and applied research specifically designed to define biological mechanisms of action underlying the toxicity of products regulated by the FDA. This research is aimed at understanding critical biological events in the expression of toxicity and at developing methods to improve assessment of human exposure, susceptibility and risk.

  3. DEPARTMENT OF HEALTH AND HUMAN SERVICESFOOD AND DRUG ADMINISTRATIONNATIONAL CENTER FOR TOXICOLOGICAL RESEARCH Office of the Director Office of Research Office of Management Division of Biochemical Division of Genetic Office of Management Office of Planning, Toxicology and Reproductive Services Finance, and Toxicology Information Technology Division of Biometry Division of Microbiology Division of Facilities, Division of Planning and Risk Assessment Engineering and Maintenance Division of Chemistry Division of Division of Division of Financial Neurotoxicology Administrative Management Services Division of Veterinary Division of Molecular Division of Services Epidemiology Information Technology

  4. Centers of Excellence • The Functional Genomics Center • The Structural Genomics Center • The ToxicoInformatics Center • The Hepatotoxicity Center • The Phototoxicity Center

  5. Outline • BSL3 Laboratory • DNA based tools (Microbiology) • Proteomic Tools (Chemistry)

  6. BSL3 Laboratory • Contract has been awarded with a completion date of summer 2004 • Laboratory will have seven suites for research and testing of select agents • Purchased individual ventilated cages to house rodent model systems

  7. DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004) • Development of a Microarray Chip for the Detection of Multiple Antibiotic Resistance Markers. (PI: S. A. Khan) • Novel Molecular Approach for the Detection and Analysis of the Most Populous Bacterial Species in the Human Gastrointestinal Tract. [PI: R.-F. Wang] • Studies on Mechanism of Fluoroquinolones Resistant Salmonella spp. Isolated from Animal Feeds (Poultry), Animal Production and the Development of Molecular Methods for Screening the Drug Resistance Genes (PI: A.A. Khan) • In Vitro model and Molecular Analysis of Competitive Exclusion Products (PI: R.D. Wagner). • Probiotic Effects on Host Defense Against Enteric Pathogens [PI: R.D. Wagner]

  8. DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004) • Molecular Screening Methods for the Determination of Vancomycin Resistance in Selective Competitive Exclusion Product CF3 (PreemptTM) bacteria. (PI: S.A. Khan). • In Vitro Assay for Perturbation of Colonization Resistance by Antibiotic Residues. [PI: R.D. Wagner] • Determining the Effect of Low Levels of Antibiotic Residues on the Human Intestinal Microflora using an in vitro Continuous Culture System [PI: B.D. Erickson] • Elucidation of the Mechanism of Resistance Development in Anaerobic Bacteria from the Human Intestinal Tract. [PI: F. Rafii] • Studies on the Fluoroquinolone Resistance in Campylobacter sp. Isolated from Poultry (PI: M.S. Nawaz).

  9. DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004) In collaboration with the Chemistry Division: • Evaluation of pyrolysis MAB/Tof MS and MALDI/T of MS for rapid characterization of presumptive bio-terro agent samples. (PI: J. Wilkes). • Combining MAB/MS with Pattern Recognition to Sub-type Bacteria. (PI-J. Wilkes).

  10. Suite of Methods Used to Identify Animal Pathogens by the Surveillance/Diagnostic Program Division of Microbiology VITEK Biochemical Substrates BIOLOG Carbon Source Utilization Molecular Biology DNA Sequencing PCR MIDI Cellular Fatty Acids

  11. Relevance to Food Safety/Security Issues • Expertise and experience in diagnostic microbiology and microbial identification. • Experience and expertise in the use of automated microbial identification instrumentation. • Expertise and experience in BSL3 laboratory functions and operation.

  12. Research Methods Used In Monitoring Antibiotic Resistance In Food-borne Pathogens 2 4 8 16 Broth Dilution PFGE PCR Sequencing DiskDiffusion Microarray

  13. Microarray Detection of Multiple Antibiotic Resistance Markers In Salmonella typhimurium DT23 (ACSSuT-Type) 8 13. nptI 14. oleB 15. oleC 16. qacH 17. Spa 18. sulI 19. tlrB 20. Tet 21. tetA 22. tetO 23. Ddl 24. vanA 25. vanB 1. CmlA 2. bacA 3. oleB 4. tetA 5. tetB 6. lmr 7. lmrB 8. satG 9. blaTEM1D 10. Amp 11. aacA 12. aadB

  14. PFGE of Fluoroquinolone Resistant Strains of Campylobacter spp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 kb 437 243 97 48.5 23.1 SalI+SmaI

  15. bp 1 2 3 4 5 6 7 8 9 2072 600 584-bp (flo) 392-bp (spvC) 321-bp (invA) 200 265-bp (int) Detection of Multidrug-resistant Salmonellatyphimurium by multiplex PCR

  16. In Vitro Intestinal Model Evaluation of antimicrobial drug resistance transfer Testing efficacy of competitive exclusion (probiotic) products Intestinal cell and bacteria co-culture Detection of pathogen effects on intestinal ecology Study innate immune responses to intestinal bacteria

  17. Human Flora Associated Mouse Model Evaluation of antimicrobial drug resistance transfer Testing efficacy of probiotic products Detection of pathogen effects on intestinal ecology Study immune responses to intestinal bacteria

  18. The Most Representative Bacterial Species Isolated from the Healthy Adult Human Gastrointestinal Tract Bacteroides spp. Bacteroides vulgatus Bacteroides uniformls Bacteroides stercoris Bacteroides fragilis Bacteroides ovatus Bacteroides caccae Bacteroides distasonis Bacteroides thetaiotaomicron Bacteroides capillosus Bacteroides merda Fusobacterium spp. Fusobacterium prausnitzii Fusobacterium russii Bifidobacterium spp. Bifidobacterium adolescentis Bifidobacterium longum Bifidobacterium catenulatum Bifidobacterium infantis Bifidobacterium angulatum Eubacterium spp. Eubacterium aerofaciens Eubacterium rectale Eubacterium biforme Eubacterium eligens Eubacterium lentum Eubacterium ventriosum Clostridium spp. Clostridium perfringens Clostridium butyricum Clostridium ramosum Clostridium indolis Peptococcus sp. Peptostreptococussspp. Peptostreptococuss anaerobius Peptostreptococuss productus Peptostreptococuss parvulus Peptostreptococuss micros Peptostreptococussprevotii Prevotella sp. Ruminococcus spp. Ruminococcus bromii Ruminococcus obeum Ruminococcus gnavus Ruminococcus callidus Ruminococcus torques Ruminococcus albus Enterococcus spp. Enterococcus faecium Enterococcus fecalis Enterococcus siraeum Lactobacillus spp. Lactobacillus acidophilus Lactobacillus fermentum Escherichia coli Propionibacterium acnes

  19. DNA Microarray Results for the 40 Predominant Human Intestinal Bacteria 1 12 13 24 25 36 37 48 49 60 61 72 84 73 96 85 97 108 109 120

  20. DNA Microarray of Eleven Human Fecal Samples 1 2 4 3 5 6 7 8 9 10 11

  21. Future Research • Application of the microarray chip in detecting the antimicrobial resistance markers in food-borne pathogens and bioterror agents. • Understanding the role of various genes in resistance development. • Development of microarray methods for the detection of Salmonella spp., and Vibrio spp. in seafood. • Study the intracellular signaling mechanisms on mammalian cells by food-borne pathogens (Salmonella spp., Campylobacter spp., and Vibrio spp.).

  22. Future Research (Cont.) • Establish baseline data that may provide information on the development of fluoroquinoloneresistance in chicken and turkey intestinal microflora • Continue to monitor using Pulsed Field Gel Electrophoresis profiles the relatedness of bacterial DNA isolated from poultry and human sources. • Evaluation of drug resistance transfer in the in vitro model of colonization resistance • Evaluate contribution of probiotics toward resistance of food-borne pathogens • Continue to collaborate with investigators in the Division of Chemistry on the rapid identification of bacteria by mass spectrometry.

  23. Division of Chemistry • Tools • Pyrolysis MS + Tof MS analyzer + Pattern Recognition: • Strategy: Heat the bacteria and distinguish bacteria by patterns of ions from all biochemical constituents • MALDI Tof MS + Pattern Recognition • Strategy: Use a laser to ionize proteins and distinguish bacteria by the pattern of protein masses detected, or possibly also by how much of each protein the bacteria produce Goal: Rapid bacterial characterization by mass spectrometry

  24. Py MS + MAB MS • Metastable atom bombardment • Semi-portable • Auto-sampler (not shown) • Reproducible spectra • < 50,000 cells to produce spectrum • < 2 minutes per spectrum 17" 24" 26" • MALDI Tof MS • Protein profiles • Not-portable • Auto-sampler • ~ 500,000 cells/spectrum • < 15 secs/spectrum 4' 8' 3' 

  25. Current Major Issues/Questions Characterize Py MAB MS and MALDI MStime, specificity, reliability, practicality, unit analysis cost Compare performance to standard & novel taxonomic methods (that take days): (PFGE, serotyping, antibiotic resistance profiles) Evaluate Food-borne Pathogens Vibrio, Salmonella, E. coli 0157:H7, Shigella, Listeria, Clostridium, etc. Evaluate Two Patent-Pending Discoveries • Method for spectral drift correction (library assembly) • MALDI charge state de-convolution for greater peak reproducibility (evaluate protein expression levels, not just unique biomarkers)

  26. PFGE Patterns, 29 Salmonella a Z C C C B B b b b B B b b B D d 4 Extra bands Resistant to Te,Er,B,No,Ri +St,Ge,To Er,B,No,Ri 95% genetic homology

  27. KEY a=1 &Z=2 (S. anatum) B=6,8,13,14,19 & b=4,5,7,9,12 (S. heidelberg) C=17,26,27(S. worthington) d=28 &D=29 (S. muenster) 16 PCs, 80% X-Val Salmonellaspp.

  28. Raw spectrum with mixed charge states Charge separation and transformation to (+1) Raw Vs. Simulated +1 Charge State, MALDI MS V. parahaemolyticus O3:K6 tdh+ (2030) CHCA matrix, positive ion mode

  29. Automated Charge State Assignment, Deconvolution

  30. Preliminary Comparisons PyMS MALDI MS Time/analysis + + Capital Investment + $/Analysis + Taxonomic Power +++ Spectral Database Suitability + Practicality + Use for chemical agents +++ Preliminary Conclusions The PyMAB Tof MS has a good chance of meeting needs for rapid characterization in counter-bioterror, clinical, and ordinary public health contexts. Quantitative MALDI MS has potential in differential protein expression research.

  31. Summary • BSL3 Laboratory • DNA based tools (Microbiology) • Proteomic Tools (Chemistry)

  32. Acknowledgement • Jon Wilkes, Ph.D. • Carl Cerniglia, Ph.D.

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