OPTIMIZATION OF RAPID CYTOTOXICITY ASSAYS FOR SCREENING BACILLUS CEREUS GROUP ISOLATES
Open Access
- Author:
- Mukherjee, Manjari
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 14, 2019
- Committee Members:
- Jasna Kovac, Thesis Advisor/Co-Advisor
Joshua D Lambert, Thesis Advisor/Co-Advisor
Robert F Roberts, Committee Member - Keywords:
- Bacillus cereus
cytotoxicity - Abstract:
- The Centers for Disease Control and Prevention estimates 63,400 cases of foodborne illness occurring annually in the U.S. due to Bacillus cereus. Additionally, the number of cases is likely underreported, as the disease usually resolves within 24 hours, although deadly cases have been reported. The B. cereus group of microorgansms is composed of 18 phylogenetically related species. Some representatives of the B. cereus group species can cause one of the two types of foodborne illnesses – emetic and diarrheal. Foodborne pathogenic strains are capable of secreting several known and putative diarrheal toxins, including hemolysin BL (encoded by hbl), non-hemolytic enterotoxin (encoded by nhe), and cytotoxin K (encoded by cytK). However, the mechanism of the diarrheal syndrome caused by B. cereus is still not well understood. One of the approaches to studying B. cereus group isolates’ pathogenic potential is through immunoassays and cytotoxicity assessment using human cell lines. With an objective of developing a rapid and reliable screening technique, we evaluated and optimized colorimetry-based cytotoxicity assays to screen B. cereus group isolates and compared the results with those obtained using immunoassays. We found that the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was not appropriate for assessment of the cytotoxic effects of B. cereus supernatants on HeLa human cervical cancer cells due to cell detachment that created error in the readout. We alternatively optimized the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay and used it to characterize 9 novel B. cereus group species validated in 2017, and the 2016 foodborne outbreak-associated isolates. We showed that B. cereus sensu stricto (s.s.) type strain ATCC 14579 was highly cytotoxic towards HeLa cells, in agreement with previous studies. We further conducted comparative analyses of 15 clade IV isolates (13 dairy-associated isolates, and the type strains B. cereus s.s. ATCC 14579 and B. thuringiensis ATCC 10792) in terms of their cytotoxicities towards HeLa, undifferentiated and differentiated Caco-2 cells using both WST-1 and lactate dehydrogenase (LDH) assays. We applied 15% volume/volume (v/v) supernatants on Caco-2 cells and 5% v/v supernatants on HeLa cells to optimally differentiate among isolates with high and low cytotoxicity. We found the LDH assay results from undifferentiated Caco-2 cells to be concordant (P < 0.05) with those from HeLa and differentiated Caco-2 cells, which may indicate a similar mode of action against all tested cells. Further, results of the LDH and WST-1 assay were found to be concordant with each other for HeLa and undifferentiated Caco-2 cells (P < 0.05). This may indicate that for HeLa and undifferentiated Caco-2 cells, the pore-forming nature of the toxins could be the main contributor to the reduced viability of the cells. We additionally saw that the immune-based detection of non-hemolytic enterotoxin B positively correlated with the LDH assay results on differentiated Caco-2 cells (P < 0.05). The assays optimized in the present study will be used for high-throughput screening of larger sets of B. cereus group isolates to elucidate the underlying mechanisms of isolates’ virulence. Furthermore, the growth and cell harvesting conditions determined through this study will be used for the secretome characterization of the different B. cereus isolates through proteomics.