Horwood, Paul Francis (2005) Detection and further characterisation of the toxins and associated genes of Bacillus cereus. PhD thesis, James Cook University.

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Abstract

The food poisoning bacterium Bacillus cereus produces a large array of potentially pathogenic substances including four haemolysins, three different types of phospholipase C, the emetic toxin (cereulide) and at least five enterotoxins. The relative importance of these metabolites to the pathogenicity of B. cereus strains has not been fully elucidated. The major goals of this project were to evaluate existing toxin detection methods, develop improved methods of detecting pathogenic strains of B. cereus and to characterise the genes associated with the production of cereulide. A large number of foodborne and clinical strains of B. cereus were tested for diarrhoeal toxin production using previously reported methods, including polymerase chain reaction (PCR), gel diffusion haemolysis, Vero cell cytotoxicity and two commercially available diarrhoeal toxin detection kits. The genes for all five of the diarrhoeal toxins (haemolysin BL, enterotoxin T, non-haemolytic enterotoxin, enterotoxin FM and cytotoxin K) have been characterised and subsequently PCR primers have been designed to detect these genes. The PCR methods for three of these toxins (HBL, enterotoxin T and enterotoxin FM) were utilised to determine the prevalence of toxin genes in B. cereus. The gene for enterotoxin FM was the most commonly detected with 86.8% of the isolates containing this gene, followed by haemolysin BL (50%) and enterotoxin T (42.6%). The Vero cell cytotoxicity assay was deemed to be the most useful detection method due to its ability to detect actual toxicity, regardless of which of the five diarrhoeal toxins the strain in question was able to produce. Currently there are no simple and reliable methods available for detection of emetic strains of B. cereus. The most commonly used method of detecting emetic strains of B. cereus is the HEp-2 cell cytotoxicity assay. Cereulide causes vacuolation of the HEp- 2 cell mitochondria. This effect is transitory and often difficult to identify. Finlay et al. (1999) improved this method by utilising the tetrazolium salt MTT (3-(4,5- dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide). Although this method was sensitive and removed the subjectivity inherent in the original method, the MTT assay produces an insoluble, crystalline formazan end product that requires an additional step to solubilise the product before the absorbance readings can be taken. This method was improved by replacing MTT with the next generation tetrazolium salt, MTS (3-(4,5- dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). The advantages of MTS over MTT include the rapidity of colour development, the storage stability of MTS and the ability to return the sample to the incubator to await further colour development. A large number of B. cereus strains were tested using the HEp-2/MTS assay. The results correlated exactly with the HEp-2 mitochondrial assay. However, the sensitivity of the assay was increased and the visualisation of results relied upon observing a colour change reaction that negated the subjectivity of the original method.

Cereulide bears a close resemblance to metabolites produced by non-ribosomal peptide synthetases (NRPS) from the genera Bacillus and Streptomyces. Turgay and Marahiel (1994) developed universal primers to detect a 500 base pair (bp) region that has been highly conserved in all of the NRPS genes sequenced thus far. These primers were utilised to determine if production of cereulide is linked to peptide synthetases. Two previously reported emetic strains of B. cereus were tested using the NRPS primers, resulting in 497 bp products, which were subsequently cloned and the nucleotide sequence determined. The nucleotide and translated amino acid sequences showed a high degree of homology with other peptide synthetases, such as surfactin, gramicidin, bacitracin, tyrocidine and lichenysin. Primers were designed from variable regions of the NRPS consensus sequence to be specific for the B. cereus NRPS gene sequence. A PCR-ELISA detection system was also developed to increase the specificity of the assay. Analysis of a large number of emetic and non-emetic strains of B. cereus showed that the PCR primers distinguished between emetic and non-emetic strains. This PCR method will greatly improve food laboratories' abilities to detect strains of B. cereus capable of causing emesis and also enable a preventative approach to be applied to the control of emetic food poisoning.

A wide variety of other Bacillus species were tested for toxin genes using previously published enterotoxin PCRs and the novel cereulide PCR developed in this study. One strain of B. thuringiensis (BT1) contained all three of the enterotoxin genes that were targeted. One strain of B. circulans (2715) contained the gene for enterotoxin FM. A strain of B. licheniformis (BL1) and a strain of B. subtilis (BS1) contained the gene for cereulide.

Item ID:	1301
Item Type:	Thesis (PhD)
Keywords:	Food poisoning, Bacillus cereus, Toxin detection methods, Methods of detecting pathogenic strains, Gene for cereulide, Polymerase chain reaction, PCR, Gel diffusion haemolysis, Vero cell cytotoxicity, Diarrhoeal toxin detection kits, Haemolysin BL, Enterotoxin T, Non-haemolytic enterotoxin, Enterotoxin FM, Cytotoxin K, Prevalence of toxin genes, Tetrazolium salt, HEp-2 cell mitochondria, MTS, Primers, Non-ribosomal peptide synthetases, Nucleotide sequence, PCR-ELISA detection system, Bacillus thuringiensis, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis
Related URLs:	http://eprints.jcu.edu.au/6334/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Horwood, Paul F., Burgess, Graham W., and Oakey, H. Jane (2004) Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus. FEMS Immunology & Medical Microbiology, 236 (2). pp. 319-324.
Date Deposited:	19 Dec 2006
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110801 Medical Bacteriology @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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