Cell Culture Detection and Conditions for Production of a Bacillus cereus Heat-Stable Toxin

Szabo, Richard A.; Speirs, Joan I.; Akhtar, M. Kalim

doi:10.4315/0362-028x-54.4.272

Cited by 52 publications

(32 citation statements)

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“…The activity of the P 1 promoter was strongly dependent on the nutrients provided. Thus, the lux reporter system described here visualizes the findings of previous studies reporting substantial variances in cereulide production on different growth media for a certain strain (50,59). Media routinely used for B. cereus detection and experimental procedures (MYP, PC medium, LB, and BCM) led to higher P 1 activities than protein-enriched media such as Columbia blood, TSA, and BHI agar (see Fig.…”

Section: Discussionsupporting

confidence: 68%

“…S1 in the supplemental material). However, blood, TSA, and BHI agar are still frequently used to study cereulide production by emetic B. cereus, though it was reported earlier that less toxin was detected than with, e.g., skim milk-or rice-based media (26,59). Correspondingly, high promoter activity was detected in the carbohydrate-rich cooked rice, while only intermediate promoter activity was observed in the proteinaceous Camembert cheese ( Fig.…”

Section: Discussionmentioning

confidence: 90%

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Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods

Dommel

Frenzel

Strasser

et al. 2010

Appl Environ Microbiol

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Cereulide, the emetic Bacillus cereus toxin, is synthesized by cereulide synthetase via a nonribosomal peptide synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and biochemical characterization of the ces gene locus encoding cereulide synthetase; however, detailed information about the transcriptional organization of the ces genes was lacking. The present study shows that the ces-PTABCD genes are transcribed as a 23-kb polycistronic transcript, while cesH, encoding a putative hydrolase, is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located upstream of the cesP coding sequence, encoding a 4-phosphopantetheinyl transferase. This promoter drives transcription of cesPTABCD. In addition, intracistronic promoter regions in proximity to the translational start sites of cesB and cesT were identified but were only weakly active under the chosen assay conditions. The identified main promoter was amplified from the emetic reference strain B. cereus F4810/72 and fused to luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring system to assess cereulide production in different types of foods. ces promoter activity was strongly influenced by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter system for monitoring of ces gene expression in complex matrices.Bacillus cereus is the causative agent of two types of food poisoning: diarrhea and emesis. The toxicoinfection referred to as diarrheal disease occurs after consumption of B. cereus spores or vegetative cells, most likely due to the action of heat-labile enterotoxins, which are produced by cells multiplying in the small intestine (4,22,30,39). Contrarily, the emetic type of food-borne illness is caused by intoxication with the heat-stable peptide cereulide, which is preformed in foods and elicits vomiting a few hours after ingestion (18,51,58).Regulation of enterotoxin expression of B. cereus has been studied in some detail (e.g., references 16, 29, and 46; for a review, see reference 58), revealing a major role of the pleiotropic transcription regulator PlcR in activation of enterotoxin genes and other virulence factors (1,29,45). Cereulide synthesis in emetic B. cereus, in contrast to synthesis of B. cereus enterotoxins, is not controlled by PlcR but by the Spo0A phosphorelay. The global transition state factor AbrB was identified as one factor repressing cereulide production in early exponential phase (38). Although B. cereus enterotoxins and the emetic toxin cereulide seem to belong to completely different regulatory networks, production of both types of toxins depends substantially on nutritional and environmental factors (3,46,55), and it is expe...

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 90%

Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods

Dommel

Frenzel

Strasser

et al. 2010

Appl Environ Microbiol

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“…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).…”

mentioning

confidence: 99%

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

et al. 2005

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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.

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“…Szabo et al (1991) addressed this and determined that the optimum range for the formation of the toxin of B. cereus is from 25°C to 30°C. The minimum recorded temperature of the toxin formation was 15°C.…”

Section: Dry Heat Inactivation Of Bacillus Cereus In Ricementioning

confidence: 99%

Dry heat inactivation of Bacillus cereus in rice

Houška¹,

Kýhos²,

Landfeld³

et al. 2007

Czech J. Food Sci.

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Houška M., Kýhos K., Landfeld A., Průchová J., Schlemmerová L., Šmuhařová H., Špelina V., Novotná P. (2007): Dry heat inactivation of Bacillus cereus in rice. Czech J. Food Sci., 25: 208-213.The aim of this work was to validate the method of decontamination of rice at the temperature of 120°C (determined as optimal in previous experiments). Bacillus cereus was selected as the marker micro-organism for the monitoring of decontamination. The spores of Bacillus cereus are moderately heat resistant. In order to show the efficacy of our decontamination process, we artificially contaminated the rice under study with B. cereus. Decontamination was carried out in a homogenising steriliser about 20 h after contamination. The sample was first heated to 90°C and held at this temperature for 70 minutes. Then the temperature was increased to 120°C and held for 3 hours. Five samples were taken for microbiological analyses as follows: before the experiment, on reaching 120°C, and then after 1, 2, and 3 h of decontamination. Decontamination of rice from vegetative forms and spores of B. cereus present at the level of 400 CFu/ml was effected by heating to 120°C and holding for 1 hour.

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Cell Culture Detection and Conditions for Production of a Bacillus cereus Heat-Stable Toxin

Cited by 52 publications

References 0 publications

Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods

Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods

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

Dry heat inactivation of Bacillus cereus in rice

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