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The central hypothesis of this proposal is that E. coli O157:H7 forms biofilms on colonic epithelial surfaces in the ruminant intestinal tract. While it is known that E. coli O157:H7 forms biofilms on abiotic surfaces under limiting growth conditions, the formation of biofilms in vivo could contribute significantly to the persistence of E. coli O157:H7 in cattle populations and could explain the intermittent shedding of organisms from infected animals. <P>
This hypothesis will be tested by completing the following objectives:
<OL> <LI> Compare gene expression profiles of E. coli O157:H7 during planktonic growth, biofilm formation on an abiotic surface and growth on a biotic surface of epithelial cells. The hypothesis that we will test in this specific aim is that biofilm-associated genes of E. coli O157:H7 are expressed during interaction with three dimensional T-84 colonic epithelial cell structures lending credence to the central hypothesis. (Cornick, Minion) <LI> Determine if biofilm mutants differ from wild type E. coli O157:H7 in their interactions with 3-dimensional T-84 colonic epithelial cells and their gene expression profiles. A random transposon library of E. coli O157:H7 mutants will be screened for phenotypes that do not form biofilms on an abiotic surface. These mutants will then be examined for their growth characteristics on T-84 epithelial cells, characterization of the specific defect and their global gene expression profiles as compared to wild type. (Cornick, Minion) <LI>Determine if E. coli O157:H7 strains associated with human disease (EHEC) have a greater capacity to form biofilms than ruminant isolates. Biofilm formation from strains of E. coli O157:H7 isolated from children with gastrointestinal disease will be compared to strains isolated from healthy cattle. (Bohach)
NON-TECHNICAL SUMMARY: E. coli O157:H7 and other serogroups of STEC have emerged over the last 20 years as a significant cause of food-borne illness in the U.S. Cattle are considered to be the primary reservoir for STEC and little is known regarding the ecology of these organisms in the gastrointestinal tract. Since STEC colonization of ruminants is ubiquitous, understanding the succession of the ruminant microflora and the ecological role of STEC in the ruminant gastrointestinal tract may suggest rational approaches to alter this relationship. Virtually no work has been done with regards to the role of biofilms on the susceptibility of an individual to colonization by E. coli O157:H7. Understanding the role that biofilms play in the survival of E. coli O157:H7 on both biotic and abiotic surfaces has the potential to greatly expand our knowledge base and may suggest novel intervention strategies that could be employed to combat the survival of this organism.
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PROGRESS: 2004/10 TO 2005/06<BR>
During this period of the grant we have learned to grow T-84 epithelial cells as 3-dimensional aggregates. Currently, we are evaluating the temporal aspects of cell differentiation in this culture system to determine the optimal time frame in which to inoculate these cells. The 3-D epithelial cells will be used in both objectives 1 and 2. We have created a transposon library in E. coli O157:H7 strain 933 and are in the process of screening the library for biofilm mutants which will be evaluated in objective 2. We are also performing biofilm assays on a collection of ruminant isolates which is part of objective 3. We are making progress on all 3 objectives at this time.
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IMPACT: 2004/10 TO 2005/06<BR>
Understanding the role that biofilms may play in the colonization of ruminants and/or the environment may suggest new strategies for intervention.