Food Security Research at the National Center for

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

NCTR Mission To conduct peer-reviewed scientific 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.

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

Centers of Excellence • • • The Functional Genomics Center The Structural Genomics Center The Toxico. Informatics Center The Hepatotoxicity Center The Phototoxicity Center

Outline • BSL 3 Laboratory • DNA based tools (Microbiology) • Proteomic Tools (Chemistry)

BSL 3 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

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]

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 CF 3 (Preempt. TM) 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).

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).

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

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 BSL 3 laboratory functions and operation.

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

Microarray Detection of Multiple Antibiotic Resistance Markers In Salmonella typhimurium DT 23 (ACSSu. T-Type) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Cml. A bac. A ole. B tet. A tet. B lmr. B sat. G bla. TEM 1 D Amp aac. A aad. B 8 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. npt. I ole. B ole. C qac. H Spa sul. I tlr. B Tet tet. A tet. O Ddl van. A van. B

PFGE of Fluoroquinolone Resistant Strains of Campylobacter spp. 1 2 3 kb 437 243 97 48. 5 23. 1 Sal. I+Sma. I 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Detection of Multidrug-resistant Salmonella typhimurium by multiplex PCR bp 1 2 3 4 5 6 7 8 9 2072 600 200 584 -bp (flo) 392 -bp (spv. C) 321 -bp (inv. A) 265 -bp (int)

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

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

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. Peptostreptococuss spp. 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

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

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

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. ).

Future Research (Cont. ) • Establish baseline data that may provide information on the • • development of fluoroquinolone resistance 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.

Division of Chemistry Goal: Rapid bacterial characterization by mass spectrometry 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

17" 26" 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 24" MALDI Tof MS • Protein profiles • Not-portable • Auto-sampler • ~ 500, 000 cells/spectrum • < 15 secs/spectrum 4' 8' 3'

Current Major Issues/Questions Characterize Py MAB MS and MALDI MS time, 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: H 7, 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)

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

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) Salmonella spp. 16 PCs, 80% X-Val

Raw Vs. Simulated +1 Charge State, MALDI MS Raw spectrum with mixed charge states Charge separation and transformation to (+1) V. parahaemolyticus O 3: K 6 tdh+ (2030) CHCA matrix, positive ion mode

Automated Charge State Assignment, Deconvolution

Preliminary Comparisons Py. MS Time/analysis Capital Investment $/Analysis Taxonomic Power Spectral Database Suitability Practicality Use for chemical agents + MALDI MS + + +++ + Preliminary Conclusions The Py. MAB 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.

Summary • BSL 3 Laboratory • DNA based tools (Microbiology) • Proteomic Tools (Chemistry)

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