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CFSAN Regulatory Codes: XI
CFSAN Program Priority Codes:
Start Date: 01/01/99    Completion Date: 01/01/04


Statement of Research Problem:
Recognizing that bacterial pathogens will continue to evolve, public health initiatives must include research to understand how these pathogens arise, propagate, and emerge. Bacteria have great ability to adapt rapidly and propagate to fill an existing niche. In research on the emergence of antimicrobial resistance, however, neither the role of genetic diversity in bacterial populations nor the effects of the selective landscape has been adequately addressed. This project investigates the proposition that antimicrobial-resistant strains are emerging from specific "pools" that exist in bacterial populations at large. We have shown previously that a high frequency (1-5%) of methyl-directed mismatch repair (MMR) defective, pathogenic Escherichia coli and Salmonella enterica strains exist and persist among natural populations. We will assess the role of MMR^- mutators in the emergence of antimicrobial resistance by determining the mechanisms by which microbes acquire resistance to traditional food safety barriers, the role(s) that genetic diversity of a bacterial population plays in the emergence of antibiotic resistance in food-borne pathogens, and how recombination and mutation affect the population structure of pathogens that contaminate the food supply.

Statement of Project Objective(s):
The objective of this project is to understand the molecular genesis and emergence of antibiotic and other antimicrobial resistances among bacterial pathogens. Research will focus on the role of mutators, specifically those deficient in methyl-directed mismatch repair, on establishing antimicrobial resistance by genetic change (mutation) and exchange (recombination). Phylogenetic analyses will be used to assess whether clonal theory adequately addresses lineages observed among emerging pathogens. Finally, we will explore if an MMR^- phenotype can be enriched under experimental conditions using an in vivo murine infection model.

Anticipated Impact on FDA Regulatory Program:
The impact of this project includes the development of rapid methods to detect and identify antimicrobial-resistant pathogens in our food supply and to aid in our understanding of how resistance emerges. Moreover, in order for intervention strategies to be effective, it is essential to understand the process of emergence to be able to predict if certain pathogens will be in an unprocessed food and to explore processing parameters that will eliminate them. Finally, by identifying critical bacterial subpopulations (like the mutators) that are more apt to resist antibiotics and antimicrobial treatments, appropriate containment procedures can be implemented before these strains are disseminated globally.

Project Priority Changes During FY2000: None

Administrative Liaison(s): T. A. Cebula: 202/205-4217

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
T. A. Cebula	OPDFB/DMBRE/MBB	0.5, 0.5, 0.5	1
S. Assar	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
E. W. Brown	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
M. L. Kotewicz	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
J. E. LeClerc	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
D. D. Levy	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
B. Li	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
W. L. Payne	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
A. Shifflet	OPDFB/DMBRE/MBB	0.8, 0.8, 0.8	1
	Total FTE:	6.9, 6.9, 6.9

Collaborators: Dr. Richard B. Raybourne, DVA/OPDFB/CFSAN; Dr. Michael J. Myers, DAR/OR/CVM; Dr. Joel Unowsky, DAIDP/ORM/CDER

Component 1 FY 2000 Deliverables:

1. Compare genetic relatedness between mutator strains and representative antibiotic-resistant strains isolated from clinically and agriculturally relevant collections of Salmonella.
   
2. Determine whether mutator strains of Salmonella have an adaptive advantage for survival during infection in a murine model system.
Component 1 FY 2000 Progress:

1. Molecular phylogenetic analyses established that genomic features of the mutS region of the chromosome have been acquired by horizontal exchange between classes of natural E. coli strains and allowed for localization of recombination sites in the region.
   
2. Sequencing of Salmonella mutator strains showed large deletions encompassing the mutS gene; sequence analysis of the 12.6 kb flanking the wild-type mutS gene revealed that discrete segments of DNA are evolutionarily related to disparate species and are the likely products of horizontal transfer events among ancestral lines.
   
3. Competitive infection of C57BL/10 mice using mutator and non-mutator strains of Salmonella Enteritidis showed that the mutator strain can overtake populations of bacteria colonizing the liver and spleen of infected animals.
Technical Barriers to Meeting Component 1 Objectives or Deliverables: None
Component 1 FY 2001 Deliverables:

1. Determine whether strains of pathogenic E. coli and Salmonella have modified genes within or near mutator loci that would signal promiscuous recombinational events.
   
2. Establish whether particular mutator populations of Salmonella can be enriched during serial infection using a murine model system.
Component 1 FY 2002 Deliverables:

1. Examine genes that are modulated by mutator loci for expression in response to environmental stresses and/or antimicrobial treatments.
   
2. Determine if the mutator phenotype accelerates the emergence of antibiotic resistant pathogens during Salmonella infection in a murine model system.

LeClerc, J.E., B. Li, W.L. Payne, and T.A. Cebula (1999) Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome. J. Bacteriol. 181:7614-7617.

LeClerc, J.E. and T.A. Cebula (2000) Pseudomonas Survival Strategies in Cystic Fibrosis. Science 289:391-392. (Letter).

Cebula. T.A. and J.E. LeClerc (2000) DNA Repair and Mutators: Effects on Antigenic Variation and Virulence of Bacterial Pathogens, pp. 143-159. In K.A. Brogden et al. (eds.) Virulence Mechanisms of Bacterial Pathogens, 3^rd ed. American Society for Microbiology, Washington, D.C.

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Hypertext updated by dav 2001-OCT-02