Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex

Authors: Rasooly, Reuven; Do, Paula; Hernlem, Bradley - Brad

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/10/2020
Publication Date: 9/30/2020
Citation: Rasooly, R., Do, P.M., Hernlem, B.J. 2020. Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex. PLoS ONE. 15(9): Article e0238153. https://doi.org/10.1371/journal.pone.0238153.
DOI: https://doi.org/10.1371/journal.pone.0238153

Interpretive Summary: Bacillus cereus is a bacterium that can be found in food and which makes a toxin that causes vomiting and diarrhea. Most tests for this toxin are not able tell if the toxin is inactive or whether it is active with the ability to make people sick. The only existing test which can discern active toxin uses live rabbits. Ethics and policy disfavor live animal testing and so we developed a method using cultured cells combined with a simple camera device. The cells we used produce an enzyme that catalyzes a reaction that creates light. Active toxin prevents the cells from making that enzyme and so we were able to measure the toxin down to a concentration of 7 ng/mL by the amount by which it reduced the light given off by the reaction. This test method is a simple and inexpensive way to check for the presence of active B. cereus toxin in food to promote food safety and protect consumers.

Technical Abstract: Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. In this study we constructed a low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit the synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml and 125 ng/ml and with a limit of detection of 7 ng/ml Nhe.