# Presentation: 899070

## 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

**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 Research**

**Division of Facilities****,**

**Engineering and**

**Maintenance**

**Division of**

**Administrative**

**Services**

**Office of Management**

**Services**

**Division of Planning**

**Division of Financial**

**Management**

**Division of**

**Information**

**Technology**

**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

## DIVISION OF MICROBIOLOGY PROJECTS RELATED TO  FOOD SECURITY/COUNTERTERRORISM INITIATIVE (FY-2003/2004)

**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)

**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)

**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)**

## Suite of Methods Used to Identify Animal Pathogens by the Surveillance/Diagnostic Program Division of Microbiology

**VITEK**
**Biochemical Substrates**

**BIOLOG**
**	****Carbon Source Utilization**

**MIDI**
**Cellular 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.**

## Research Methods Used In Monitoring Antibiotic Resistance In Food-borne Pathogens 

**Microarray**

**Disk**** ****Diffusion**

**Research Methods Used In Monitoring Antibiotic Resistance In Food-borne Pathogens**** **

**Sequencing**

## Microarray Detection of Multiple Antibiotic 

**    ****1. *****CmlA***
**  ****2.  *****bacA***
**  ****3.  *****oleB***
**  ****4.  *****tetA***
**  ****5.  *****tetB***
**  ****6.  *****lmr***
**  ****7.  *****lmrB***
**  ****8.  *****satG***
**  ****9.  *****blaTEM1D***
**10.  *****Amp***
**11.  *****aacA***
**12.  *****aadB***

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

**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***

## PFGE of Fluoroquinolone Resistant Strains of Campylobacter spp.

***Sal*****I+*****Sma*****I**

**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 

## In Vitro Intestinal Model

**Evaluation of**
**antimicrobial drug**
**resistance transfer**

**Study innate**
**immune responses**
**to intestinal bacteria**

**Intestinal cell and**
**bacteria co-culture**

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

**Detection of**
**pathogen effects on intestinal ecology**

## Human Flora Associated Mouse Model 

**Testing efficacy of **
**probiotic products**

**Evaluation of**
**antimicrobial drug resistance transfer**

**Detection of**
**pathogen effects**
**on intestinal**
**ecology**

**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**

**25**

**37**

**49**

**24**

**36**

**48**

**60**

**72**

**84**

**96**

**108**

**120**

**61**

**73**

**85**

**97**

**109**

## DNA Microarray of Eleven Human Fecal Samples

**1**

**2**

**5**

**4**

**9**

**7**

**10**

**11**

**3**

**8**

**6**

**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 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

## 

****

## 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: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)

## Slide 27

## Slide 28

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

***V. parahaemolyticus  ***
**O3:K6***** tdh*****+ (2030) **
**CHCA matrix, **
**positive ion mode**

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

## Automated Charge State Assignment, 

**Automated Charge State Assignment, **
**Deconvolution**

## Preliminary Comparisons

**Preliminary 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.