Rapid Ped-2E9 Cell-Based Cytotoxicity Analysis and Genotyping of Bacillus Species

Abstract

Bacillus species causing food-borne disease produce multiple toxins eliciting gastroenteritis. Toxin assays with mammalian cell cultures are reliable but may take 24 to 72 h to complete and also lack sensitivity. Here, a sensitive and rapid assay was developed using a murine hybridoma Ped-2E9 cell model. Bacillus culture supernatants containing toxins were added to a Ped-2E9 cell line and analyzed for cytotoxicity with an alkaline phosphatase release assay. Most Bacillus cereus strains produced positive cytotoxicity results within 1 h, and data were comparable to those obtained with the standard Chinese hamster ovary (CHO)-based cytotoxicity assay, which took about 72 h to complete. Moreover, the Ped-2E9 cell assay had 25-to 58-fold-higher sensitivity than the CHO assay. Enterotoxin-producing Bacillus thuringiensis also gave positive results with Ped-2E9 cells, while several other Bacillus species were negative. Eight isolates from food suspected of Bacillus contamination were also tested, and only one strain, which was later confirmed as B. cereus, gave a positive result. In comparison with two commercial diarrheal toxin assay kits (BDE-VIA and BCET-RPLA), the Ped-2E9 assay performed more reliably. Toxin fractions of >30 kDa showed the highest degree of cytotoxicity effects, and heat treatment significantly reduced the toxin activity, indicating the involvement of a heat-labile high-molecularweight component in Ped-2E9 cytotoxicity. PCR results, in most cases, were in agreement with the cytotoxic potential of each strain. Ribotyping was used to identify cultures and indicated differences for several previously reported isolates. This Ped-2E9 cell assay could be used as a rapid (ϳ1-h) alternative to current methods for sensitive detection of enterotoxins from Bacillus species.

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

“…This difference may be due to strain variation and in agreement with the previous report. 37 In the present study, we also tested lower MOIs, 50:1 and 10:1. With L. monocytogenes strains Scott A and V7 at MOI of 50:1 and 10:1, at 3 h postinfection, the cytotoxicity values obtained from treatments of these pathogenic strains were significantly higher (Po0.05 or Po0.01) when compared with negative control, nonpathogenic L. innocua F4247 at all postinfection time points (3 and 6 h; Figure 3b) but significantly lower (Po0.05 or Po0.01) than MOI of 100:1.…”

“…Also, to asses how the different multiplicity of infections (MOIs) ratio of L. monocytogenes may affect the Ped-2E9 cell cytotoxicity results, two lower MOIs (50:1 and 10:1) were tested along with the 100:1 ratio, at 3 and 6 h postinfection time points. Furthermore, a comparison of two-dimensional (2D) conventional Ped-2E9 cell assay 16,[37][38][39][40] was done with the three-dimensionally encapsulated Ped-2E9 cells in collagen matrix to assess whether 3D encapsulated system produces similar assay results. We also performed cryo-SEM analysis with infected hybridoma cells following the same procedures mentioned above.…”

“…Crude toxins from Bacillus species, prepared from bacterial cell-free culture supernatants as described previously, 37 were used for cytotoxicity assays immediately or stored at 41C for not more than 48 h.…”

“…[34][35][36] Previous studies from our laboratory have shown that a mouse B-cell hybridoma (Ped-2E9)-based cytotoxicity assay can be used to detect the presence of pathogenic Listeria species and the enterotoxins from Bacillus species. 16,37,38 This cytotoxicity assay can also differentiate the presence of viable pathogenic Listeria species from nonpathogenic ones. 16,[39][40][41][42] Adherent and nonadherent mammalian cell lines of various tissue origins were used previously to distinguish differential cytotoxic response of virulent Listeria species from avirulent ones.…”

“…This assay measures the activity of enzymes such as alkaline phosphatase (ALP) or lactate dehydrogenase released by the hybridoma cells infected with pathogen or toxins 16,[37][38][39]46,47 making the Ped-2E9-based assay a robust assay system that can easily be integrated into an automated sensing platform. The rapid detection of viable pathogenic Listeria species and enterotoxin from Bacillus from food, clinical and environmental samples are important from commercial and biosecurity perspectives.…”