Cytotoxicity and cell -based sensors for detection of Listeria monocytogenes and Bacillus cereus

It is evident from the number of recent foodbome outbreaks and fatalities that rapid and sensitive microbial detection tools are required. Cytotoxicity-based detection tools were developed for foodbome pathogens, Listeria monocytogenes and Bacillus cereus. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody than available in commercial systems, MAb-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from varieties of samples and tested for cytopathogenic action on both a murine hybridoma B-lymphocyte, Ped-2E9 and a murine macrophage, RAW 264.7 cell line through an alkaline phosphatase (AP) and lactate dehydrogenase (LDH)-release assay, respectively. The RAW cell line was also grown on interdigitated microsensor electrode (IME)-chips using tripeptide (Arg-Gly-Asp) and fibronection adhesion-promoting peptides. The two-step detection method was found to be specific for L. monocytogenes and could be used in pure culture or food systems. Following enrichment, low levels of L. monocytogenes could be isolated, detected and confirmed as cytopathogenic using the AP or LDH assays in under 4 h using either cell line. While a positive cytotoxic response on-chip was found using the LDH or Trypan blue staining assay, significant differences as measured by impedance spectroscopy were not observed. The Ped-2E9 cell line was also used for sensitive, rapid and quantitative detection of B. cereus enterotoxin. Cell-free toxin preparations produced positive cytotoxicity results in as early as 15 min and a highly cytotoxic strain showed effect at a dilution as high as 1:32. Cytotoxic action was associated with fractions containing greater than 30 kDa. SDS-PAGE analysis confirmed the presence of toxin subunits of 22 to 66 kDa. PCR analysis showed a strong correlation between the quantity of toxin genes (bceT, cytK, nheA, nheB, nheC and hblA) in a strain and its cytotoxic potential. When compared with other cell culture assays and commercial diarrheal toxin kits, the Ped-2E9-based Bacillus enterotoxin assay was found to be a more rapid and sensitive alternative to current methods