Bovine rectoanal junction in vitro organ culture model system to study Shiga toxin-producing Escherichia coli adherence

Authors: Kudva, Indira; BIERNBAUM, ERIKA - Oak Ridge Institute For Science And Education (ORISE); Cassmann, Eric; Palmer, Mitchell

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/6/2023
Publication Date: 5/15/2023
Citation: Kudva, I.T., Biernbaum, E.N., Cassmann, E.D., Palmer, M.V. 2023. Bovine rectoanal junction in vitro organ culture model system to study Shiga toxin-producing Escherichia coli adherence. Microorganisms. 11(5). Article 1289. https://doi.org/10.3390/microorganisms11051289.
DOI: https://doi.org/10.3390/microorganisms11051289

Interpretive Summary: Cattle harbor Shiga toxin-producing E. coli (STEC) O157:H7, a foodborne pathogen that causes illness in humans but not in cattle. STEC persist in the cattle intestine towards the rectum (recto-anal junction), which is a targeted site to reduce the amount of STEC in cattle to minimize contamination of food products. We sought to develop a cost-effective organ culture model system of analyzing STEC attachment to cattle rectal tissue, in a lab setting, that is more high-throughput and limits animal usage. Multiple conditions were examined to standardize the rectal organ culture model and to produce optimal conditions that closely reflect the results observed when tested in live cattle. The organ culture reproduced the expected adherence patterns of different STEC and non-STEC strains, confirming the optimal conditions. The rectal organ culture system advantages include (i) pre-screening multiple bacterial strains with one piece of tissue to minimize animal testing and (ii) evaluating multiple experimental approaches to reduce STEC adherence. Overall, the model enhances testing efficiency and reduces animal usage for the study of STEC and cattle intestinal tissue interactions.

Technical Abstract: Studies evaluating Shiga toxin-producing Escherichia coli O157:H7 (O157) interactions with the bovine recto-anal junction (RAJ) are restricted to either in vitro analysis of bacteria, cells or nucleic acids at the RAJ yielding limited information or expensive in vivo studies in animals. Hence, we sought to develop a complete RAJ-in vitro organ culture (RAJ-IVOC) system that represents all cell types of the RAJ and permits studies with results as observed in vivo. Pieces of RAJ tissues, collected at cattle necropsies from unrelated studies, were assembled and tested variously to derive optimal conditions for assaying bacterial adherence on a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Cell viability, structural cell markers and histopathology were used to determine tissue integrity, and adherence assessed via microscopy and culture. DNA fingerprinting verified recovered bacteria against the inoculum. Appropriately assembled RAJ-IVOC, in Dulbecco’s Modified Eagle Medium, at 39 degrees Celsius, with 5% CO2 and gentle shaking for 3-4 hours, maintained tissue integrity and reproduced the expected adherence phenotype of the test bacteria. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.