|BIERNBAUM, ERIKA - Oak Ridge Institute For Science And Education (ORISE)|
Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 2/24/2022
Publication Date: N/A
Technical Abstract: Shiga toxin-producing Escherichia coli O157:H7 (O157) preferentially colonize the bovine rectoanal junction (RAJ) located at the terminal end of the gastrointestinal tract. The RAJ is characterized by the columnar epithelial cells towards the distal colon that abruptly transitions to the stratified squamous epithelial cells towards the anus. A number of in vitro and in vivo studies have been conducted utilizing the bovine RAJ to better understand the dynamics between the site and O157. While such studies have provided some insights into O157 attachment and the host cell responses, these are usually restricted to in vitro evaluation of cells or nucleic acids from specific regions of the RAJ, or in vivo evaluation in live animals. This limits the information garnered from in vitro studies and increases reliance on in vivo studies that can be expensive especially when evaluating multiple bacteria. Hence, the goal of this study was to develop a complete RAJ-in vitro organ culture (RAJ-IVOC) model system with optimal conditions that would permit bacterial adherence studies without loss of tissue integrity and yield adherence results similar to that observed in vivo. The RAJ tissues, collected at necropsies of cattle in unrelated studies, were cut into rectangular pieces, each piece encompassing both cell types. The pieces were assembled variously and tested at different incubation times to determine conditions maintaining tissue integrity long enough to run adherence assays. Tissue integrity was evaluated by staining tissue sections for structural cell markers and histopathological examination. Bacterial strains (O157 strain EDL933 and E. coli K12) with known adherence differences, in vitro on the RAJ squamous epithelial cells were used, at various concentrations, to standardize the RAJ-IVOC adherence assay. Adherence was evaluated by immunofluorescent staining of tissue sections and culture of tissues for bacteria. Our results demonstrated that an appropriately assembled RAJ-IVOC, in Dulbecco’s Modified Eagle Medium broth and agarose, can maintain tissue integrity and be used to evaluate bacterial adherence at 39oC, with 5% CO2 and gentle shaking for 3-4 hours. Using these conditions, we were able to reproduce differences in adherence between the tested bacteria and are presently applying this system to study a wider panel of O157 strains. The RAJ-IVOC model developed here provides a convenient pre-screening system to evaluate interactions of multiple bacteria with the RAJ, prior to in vivo experiments, allowing for reduction of animal usage.