Application of Viability Real-time PCR in Detecting Shiga Toxin-producing 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪  From Clinical Stool Samples
Creators: Stokowski, Taryn
Publisher: name: University of Alberta Library
Year: 2023

<p>Shiga toxin-producing 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 (STEC) is a food-borne pathogen that causes acute gastroenteritis. It has a low infectious dose and children have an elevated risk of developing severe complications (e.g., hemolytic uremic syndrome). To prevent secondary spread, Alberta’s microbiological clearance exclusion protocols prohibit patients with active STEC infections from working in healthcare, handling food, or attending daycare. Such patients submit specimens until they produce two consecutively culture negative stools at least 24 hours apart. This testing is typically conducted using CHROMagar™ STEC. However, some non-O157 STEC strains are unable to grow on the media so samples are instead enriched in tryptic soy broth before being tested by real-time PCR (RT-PCR) for the 𝘴𝘵𝘹 genes. This thesis investigates whether a novel direct-from-stool viability RT-PCR assay can serve as an alternative testing method. This involves applying a membrane-impermeable dye which, when photoactivated, irreversibly binds DNA and prevents amplification. If successful, this assay will determine with high sensitivity if a sample contains live STEC without an overnight growth period. Currently, few studies have attempted viability RT-PCR in stool, none of which have used STEC as the target organism.</p>

<p>Viability RT-PCR conditions were first optimized using cell suspensions in phosphate buffered saline (PBS). The multiplexed 𝘴𝘵𝘹₁ and 𝘴𝘵𝘹₂ primer-probe sets had a limit of detection of 10³ CFU/mL, did not cross-react with DNA from other organisms (including four Shigella strains), and detected all of the 𝘴𝘵𝘹 subtypes tested except for 2f. The final viability RT-PCR protocol involved treating samples with 100 μM PMAxx™ and 1% dimethyl sulfoxide (DMSO), a 15 min photoactivation, and transferring the suspension to a new tube prior to DNA extraction. While this protocol successfully eliminated the detection of a high concentration of heat-killed cells (10⁹ CFU/mL), its performance was inconsistent and produced some false negative results at a low concentration (10⁴ CFU/mL) of live cells.</p>

<p>The conditions above were used to develop viability RT-PCR assays for direct-from-stool (DFS) and rectal swab (RS) applications. The major modification made when applied to stool was the elimination of the tube transfer step because it did not improve the detection of live cells. Both the DFS and RS viability RT-PCR protocols successfully eliminated the detection of heat-killed cells spiked into stool at a high concentration, but consistently produced false negative results when live STEC cells were spiked at a low concentration (10⁵ CFU/mL stool). The DFS protocol was tested using clinical microbiological clearance specimens from four patients, and was found to perform inconsistently and to produce false negative results. That is, despite being positive by post-enrichment RT-PCR, several stool samples were negative by DFS viability RT-PCR. </p>

<p>In conclusion, all of the viability RT-PCR protocols developed in this thesis were successful at selectively detecting live cells at high bacterial loads when found alongside a high concentration of heat-killed STEC. This suggests that PMAxx™ was successfully activated in stool and bound DNA from heat-killed STEC. However the protocols were not consistently successful at detecting live cells at low bacterial loads. Thus, the hypothesis that viability RT-PCR would be as sensitive as conventional RT-PCR was not supported. This means that the DFS viability RT-PCR assay is unsuitable for microbiological clearance testing because such specimens often have a low load of live bacteria. Instead, the results of this study endorse the use of post-enrichment RT-PCR because it was the most sensitive assay tested and was suitable for strains which did not grow on CHROMagar™ STEC.</p>