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The genes responsible for the emetic toxin were discovered and new tests developed
by Graham Burgess and Paul Horwood
February 2006
RIRDC Publication No 04/049 RIRDC Project No UJC-8A
Executive Summary
Development of Improved Molecular Detection Methods for Bacillus cereus Toxins
RIRDC Project: UJC-8A
Despite improving levels
of hygiene and sanitation in Australia and around the world, the incidence
of food-borne disease is believed to be increasing. The reason for the
increase in reported cases of gastroenteritis is probably due to a number
of factors, including: better reporting and diagnostic techniques, changes
in eating habits and the identification of new human pathogens.
Rice is the most commonly implicated food in cases of B. cereus gastroenteritis. Most samples of rice have low levels of B. cereus present. Fried or cooked rice has been implicated in approximately 95% of cases of B. cereus food poisoning with symptoms of vomiting, indicating that there is a relationship between rice and the production of the toxin known as the emetic toxin by Bacillus cereus. In most cases of emetic toxin food poisoning illness is associated with the heating of precooked food held for too long at unsatisfactory storage temperatures.
An important objective of this project was a study of collection of isolates of the bacterium Bacillus cereus isolated in Australia to determine whether they are capable of producing toxins associated with food poisoning. A large collection of isolates was made including bacteria isolated from food and bacteria that had been isolated from food poisoning cases. In addition an extensive international collection of samples of bacteria with a known profile of toxin production was imported. As the emetic toxin has been associated with rice-based foods, the collection included 18 isolates capable of producing the emetic toxin. The collection of Bacillus cereus isolates was supplemented with several closely related Bacillus species as well as DNA extracted from the anthrax organism, Bacillus anthracis.
This provided us with an extensive reference collection that could be used to develop diagnostic tests and as well as a group of locally isolated organisms that would providers with information about the situation in Australia.
Bacillus cereus has been shown to be capable of producing at least four different toxins and there are two commercial kits that can be used to detect two of the most common toxins that are normally associated with the production of diarrhoea in affected individuals. In addition several papers have recently been published describing the use of the technique polymerase chain reaction (PCR) to detect and identify genes responsible for several of the toxins. We evaluated the two commercial kits and applied the gene detection techniques including PCR using our full collection of organisms.
All of the techniques surveyed have advantages and disadvantages. However, we have concluded that none of the standard existing techniques can be used as the definitive assay for detection of the toxins of Bacillus cereus associated with diarrhoea. The most effective method of identifying bacterial strains producing toxins seems to be a combination approach e.g. screening isolates with PCR or a commercial kit and then following this up by using a cell cytotoxicity assay where the effect of the toxin on cultured animal cells indicates the ability of the toxin to damage cells.
Using published techniques for the detection of the genes associated with diarrhoeal toxins we demonstrated that in groups of organisms the presence of any one diarrhoeal toxin varied from 30% to 90%. The majority of Bacillus cereus strains tested contain the genes for at least one of the diarrhoeal toxins. There is the possibility that the organism may contain the gene for producing the toxin and for some reason this gene may not be active. Therefore the only reliable test to determine whether these bacteria are capable of producing the toxin that can damage living cells is to treat living cells with an extract of the organisms.
Techniques for demonstrating the toxins associated with diarrhoea as well as the genes responsible for these toxins are relatively well established. However, techniques for demonstrating the emetic toxin that is associated with vomiting are cumbersome and relatively insensitive. This project used an important property of the emetic toxin to develop a test that would reliably indicate the production of the toxin with the sensitivity that is significantly better than any method published to date.
Using a rational approach we predicted that type of genes that would be necessary to produce the machinery required for the production of a toxin of this type. Having predicted the nature of these genes we adopted a test developed in Europe for detecting similar genes in other organisms. We applied this test to the strains of Bacillus cereus capable of producing the emetic toxin and confirmed that a set of genes as predicted were present in the strains of organisms producing the emetic toxin and not in the organisms that failed to produce this particular toxin.
Having demonstrated this gene we further characterised the gene from the Bacillus cereus isolates and designed a specific test that would detect the gene in Bacillus cereus and not the closely related genes in other species of bacteria. We confirmed that this test only detected the target genes.
This is the first molecular diagnostic developed for detecting the genes responsible for the production of the emetic toxin. This test is both sensitive and specific in that it detects very small numbers of organisms that contain this gene and it will discriminate between very closely related organisms.
Having carried out this project we are now in a much better position to advise the Australian rice industry on appropriate techniques that can be used for quality assurance.
We have been consulting closely with regulatory authorities throughout Australia assisting them to improve their diagnostic techniques so that they are in a much better position to accurately diagnose food poisoning outbreaks. This work will also benefit organisations that are responsible for regulating food standards. They will now be able to make rational decisions on the standards that the industry should responsibly meet.
Initially it was hoped that we would be able to provide the industry with simple tools that would allow them to determine whether strains of Bacillus cereus will likely to produce toxins associated with diarrhoea. Unfortunately we demonstrated that most if not all isolates of Bacillus cereus were capable of producing these disease problems. It would appear that is unlikely that any isolates of Bacillus cereus can be considered to be of no consequence.
The situation is very different for the emetic toxin. This is the toxin that is most likely to be associated with rice. Only a limited range of organisms are capable of producing this toxin and this project has provided the industry and the regulators with tools that can be used to improve quality assurance.
There is the potential to develop these products as simple commercial kits that will promote their use in less sophisticated laboratories than those currently carrying out this sort of testing.
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