Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Abstract

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food‐poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET‐RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.

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

“…Similar analysis was also done with the Caco-2 cell line (24,47). A CHO-based assay was developed for detection of both the emetic and the diarrheal toxin through cytotoxic staining and measurement of metabolic activity (5,25,40). The drawback of these cell-based assays is the length of time required to complete the assays, 24 h to 72 h (5,38,54).…”

“…BcET has either an unknown type of enterotoxic action or none at all (1,12) but is generally found in more than half of outbreak-associated strains. EntFM, like BcET, has not been implicated as the enterotoxin responsible for a food poisoning outbreak, but EntFM is present in most outbreak-associated strains and is actually the most prevalent enterotoxin gene for all B. cereus strains (25,52). Unlike EntFM and BcET, CytK has been implicated in B. cereus-related deaths due to necrotic enteritis (32).…”

“…The PCR is generally used as a tool for toxin and strain characterization (18,22,25,29,(41)(42)(43). PCR-based detection gives a high level of accuracy but does not reveal pathogenic potentials of strains, since phenotypic expression of genes is not always warranted.…”

Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

“…Polymorphism similar results in the hbl or nhe operon were reported by Guinebretière, Broussolle and Nguyen-The (2002) among diarrheal B. cereus strains. Hsieh et al (1999) investigated the virulence profiles of B. cereus s.l. group bacteria, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks.…”

OECD Issue Paper on Microbial Contaminants Limits for Microbial Pest Control Products

“…Most methods for isolating and enumerating Bacillus cereus require additional confirmatory tests for distinguishing presumptive isolates from other species in the Bacillus cereus-group, including For nonspecific detection of enterotoxins, Vero cell or Chinese hamster ovary cells have been used to assess cytotoxicity of strains (Damgaard et al 1996, Hsieh et al 1999. Furthermore, several PCR methods have been developed to detect the genes involved in enterotoxin production (Stenfors Arnesen et al 2008).…”