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 SERVICESFOOD AND DRUG ADMINISTRATION

NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH

Division of BiochemicalToxicology

Division of Geneticand Reproductive

Toxicology

Division of Biometryand Risk Assessment

Division of Microbiology

Division of Chemistry Division ofNeurotoxicology

Division of VeterinaryServices

Division of MolecularEpidemiology

Office of Research

Division of Facilities,Engineering and

Maintenance

Division ofAdministrative

Services

Office of ManagementServices

Division of Planning

Division of FinancialManagement

Division ofInformationTechnology

Office of Planning,Finance, and

Information Technology

Office of Management

Office of the Director

Centers of Excellence

• The Functional Genomics Center• The Structural Genomics Center• The ToxicoInformatics Center• The Hepatotoxicity Center• The Phototoxicity Center

Outline

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

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

• 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)

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).

DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD

SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004)

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).

DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD

SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004)

DIVISION OF MICROBIOLOGY PROJECTS RELATED TO FOOD

SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004)

Suite of Methods Used to Identify Animal Pathogens by the Surveillance/Diagnostic

Program Division of Microbiology

VITEKBiochemical Substrates

BIOLOGCarbon Source Utilization

MIDICellular Fatty Acids

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 BSL3 laboratory functions and operation.

Microarray

PCR

Disk Diffusion

2 4 8 16

Broth Dilution

Research Methods Used In Monitoring Antibiotic Resistance In Food-borne

Pathogens

PFGE

Sequencing

8

1. CmlA 2. bacA 3. oleB 4. tetA 5. tetB 6. lmr 7. lmrB 8. satG 9. blaTEM1D10. Amp11. aacA12. aadB

Microarray Detection of Multiple Antibiotic Resistance Markers In Salmonella typhimurium DT23 (ACSSuT-Type)

13. nptI14. oleB15. oleC16. qacH17. Spa18. sulI19. tlrB20. Tet21. tetA22. tetO23. Ddl24. vanA25. vanB

SalI+SmaI

243

97

48.5

23.1

437

kb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

PFGE of Fluoroquinolone Resistant Strains of Campylobacter spp.

Detection of Multidrug-resistant Salmonella

typhimurium by multiplex PCR

2072

600

200

bp 1 2 3 4 5 6 7 8 9

392-bp (spvC)321-bp (invA)

584-bp (flo)

265-bp (int)

In VitroIn Vitro Intestinal Model Intestinal Model

Evaluation ofEvaluation ofantimicrobial drugantimicrobial drugresistance transferresistance transfer

Study innateimmune responses

to intestinal bacteria

Intestinal cell andbacteria co-culture

Testing efficacy of Testing efficacy of competitive exclusioncompetitive exclusion (probiotic) products(probiotic) products

Detection ofpathogen effects

on intestinal ecology

Human Flora Associated Mouse Human Flora Associated Mouse ModelModel

Testing efficacy of probiotic products

Evaluation ofantimicrobial drug resistance transfer

Detection ofpathogen effectson intestinalecology

Study immune responses to intestinal bacteria

The Most Representative Bacterial Species Isolated from the Healthy Adult Human

Gastrointestinal TractBacteroides spp.Bacteroides vulgatusBacteroides uniformlsBacteroides stercorisBacteroides fragilisBacteroides ovatusBacteroides caccaeBacteroides distasonisBacteroides

thetaiotaomicronBacteroides capillosusBacteroides merdaFusobacterium spp.Fusobacterium prausnitziiFusobacterium russiiBifidobacterium spp.Bifidobacterium

adolescentisBifidobacterium longumBifidobacterium

catenulatumBifidobacterium infantisBifidobacterium angulatum

Eubacterium spp.Eubacterium aerofaciensEubacterium rectaleEubacterium biformeEubacterium eligensEubacterium lentumEubacterium ventriosumClostridium spp.Clostridium perfringensClostridium butyricumClostridium ramosumClostridium indolisPeptococcus sp.Peptostreptococuss spp.Peptostreptococuss

anaerobiusPeptostreptococuss

productusPeptostreptococuss

parvulusPeptostreptococuss microsPeptostreptococussprevotii

Prevotella sp.Ruminococcus spp.Ruminococcus bromiiRuminococcus obeumRuminococcus gnavusRuminococcus callidusRuminococcus torquesRuminococcus albusEnterococcus spp.Enterococcus faeciumEnterococcus fecalisEnterococcus siraeumLactobacillus spp.Lactobacillus

acidophilusLactobacillus

fermentumEscherichia coliPropionibacterium

acnes

DNA Microarray Results for the 40 Predominant Human

Intestinal Bacteria

1 12

13

25

37

49

24

36

48

60

72

84

96

108

120

61

73

85

97

109

1 2

5

4

9

7

10 11

3

86

DNA Microarray of Eleven Human Fecal Samples

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.

Goal: Rapid bacterial characterization by mass spectrometry

Division of ChemistryDivision 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 RecognitionStrategy: 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

26" 24"

17"

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

3'8'

4'

MALDI Tof MS•Protein profiles•Not-portable•Auto-sampler•~ 500,000 cells/spectrum •< 15 secs/spectrum

Current Major Current Major Issues/QuestionsIssues/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 PathogensVibrio, 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)

PFGE Patterns, 29 PFGE Patterns, 29 SalmonellaSalmonella aa

ZZ

C

C

C

B

B

bb

bb

bb

B

B

bb

bb

B

D

dd

95% genetic homology

4 Extra bandsResistant toTe,Er,B,No,Ri

+St,Ge,ToEr,B,No,RiEr,B,No,Ri

KEYa=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-ValSalmonella spp.

m/z

2000 4000 6000 8000 10000 12000 14000 16000

m/z2000 4000 6000 8000 10000 12000 14000 16000

Raw spectrum with mixed charge states

Charge separation and transformation to (+1)

V. parahaemolyticus O3:K6 tdh+ (2030)

CHCA matrix, positive ion mode

Raw Vs. Simulated +1 Charge Raw Vs. Simulated +1 Charge State, MALDI MSState, MALDI MS

Automated Charge State Automated Charge State Assignment, Assignment,

DeconvolutionDeconvolution

Preliminary ComparisonsPreliminary Comparisons

Preliminary ConclusionsPreliminary Conclusions

PyMS MALDI MS Time/analysis + ++ +Capital Investment ++$/Analysis ++Taxonomic Power ++++++Spectral Database Suitability ++Practicality ++Use for chemical agents ++++++

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.

Summary

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

Acknowledgement

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