B14006: Assessment of the ability of Clostridium perfringens strains to produce enterotoxin after exposure to defined environmental conditions

Study Duration: November 2002 to November 2005

Contractor: Health Protection Agency

C. perfringens is a common cause of food poisoning usually involving foods such as meat or poultry that have been stored incorrectly after cooking or reheated insufficiently. Such practices allow the organism to grow to high levels and when the food is eaten the bacteria produce a toxin in the human gut which causes the disease symptoms of diarrhoea, severe stomach pain and nausea. The illness develops 6-24 hours after eating food containing high numbers of C. perfringens and, although recovery normally occurs within 2 days, it can be serious in the elderly or very young.

This type of food poisoning is most often associated with large scale catering such as occurs in residential homes, day care centres, canteens, restaurants, wedding receptions etc., where large volumes of foods are often cooked well ahead of serving and then reheated. The C. perfringens toxin is encoded by a toxin gene and this is usually present on the bacterial chromosome in those strains that cause food poisoning.

C. perfringens food poisoning is caused by a toxin and currently there is little information about what affects toxin production by this microorganism. The overall aim of this project is to obtain data on the behaviour of a range of strains of C. perfringens in terms of their ability to produce toxin and subsequently cause food poisoning. The outcome of the research will increase our understanding of enterotoxin production in strains of C. perfringens. It will also provide information that could be used to predict the risks of toxigenic organisms causing food poisoning in particular situations and consequently there is the potential to generate strategies to minimise the risk for the population.

The principal objectives of the project are to determine if heating and cooling regimes, similar to those used in the catering industry, affect the amount of toxin produced by C. perfringens and to try and identify particular heating and cooling practices that maximise toxin production by C. perfringens.

In order to investigate the effect of heat and cooling treatments on enterotoxin production a range of different food poisoning strains of C. perfringens will be studied. In addition, a method for measuring C. perfringens toxin is going to be developed and the heating and cooling regimes commonly used in food catering identified.

An in vitro experimental system simulating the situation in C. perfringens food poisoning will be devised and the heating regimes assessed using pure spores from strains of C. perfringens and also using spores inoculated into beef slurry. After heating it may be necessary to enable surviving spores to germinate and then to induce spore formation in order for enterotoxin production to occur. The number of spores and the amount of enterotoxin produced are measured at the end of spore induction. In addition genetic studies will be performed on all strains of C. perfringens to see if the heating had any effect on the gene that encodes enterotoxin.

The initial phase of the project investigated sporulation (asexual reproduction by the production and release of spores), in a range of C. perfringens strains in four different sporulation media to determine which medium produced the maximum yield of spores for subsequent heat treatments.

Thirty-one strains of C. perfringens were selected to represent a range of potential toxin producers, 16 of which had been associated with food poisoning and 15 with cases of sporadic diarrhoea. All strains were inoculated into each of the four sporulation media and, following incubation, spore counts were performed and the amount of enterotoxin (CPE) in spore culture supernatant determined.

Among 22 of the 31 strains tested, yields of greater than 104 spores/ml in at least one of the sporulation medium used were obtained. Only seven strains were unaffected by the sporulation medium used and produced high spore yield of more than 104 spores/ml in all four sporulation medium. Thus the medium for optimum sporulation varied with each strain and the same strain did not necessarily produce a high yield of spores in all four media.

Attempts were made to produce high yield spore crops from a representative of 22 strains from the culture collection for the heat treatment experiments. The sporulation methods used for each strain was the same as in a related FSA-funded project using a common set of C. perfringens strains. Of the 22 strains selected, 16 produced spore yields greater than 104, three strains produced spore crops of 103 while three strains did not sporulate. Heating experiments were performed on the 19 strains that sporulated.

Six different heat treatments were studied in this project, ranging from heating at 63ºC to 95ºC followed by holding and cooling to 4ºC over periods ranging from 7 hours to 24 hours. All strains were tested in triplicate for each of the six heat treatments; toxin detection and spore and vegetative cell counts were performed at various intervals throughout the heat treatments.

The results from the different heat treatments show that the amount of toxin produced after these treatments varied between strains but was significantly greater when exposed to the first treatment, which involved heating to 75ºC over 3 hours and cooling to 4ºC over 7 hours. However, a similar effect on enterotoxin production by this heat treatment was not detected in a selected group of strains tested in beef slurry.

Molecular typing was performed on C. perfringens strains before and after heating. Genotyping included detection of alpha toxin and enterotoxin genes by real time PCR, genomic location (chromosomal or plasmid) of the enterotoxin gene (cpe) by PCR and subtyping by amplified fragment length polymorphism (AFLP).

All the strains of C. perfringens were found to possess both alpha and cpe genes, apart from the negative control strain. The cpe gene was shown to be located on a plasmid in 30% of the strains from food poisoning incidents, and on the chromosome in 70%. A plasmid location for the cpe gene in food poisoning strains has not been previously reported in Europe or the US. Data generated from a related FSA-funded project confirmed that those isolates with a chromosomally located cpe gene were usually more heat resistant than those with a plasmid borne cpe gene, except for one strain with a plasmid cpe gene that had heat resistant spores.

After the heat treatments, 70% of strains that had the chromosomal cpe retained the cpe gene in the same location and data from AFLP analysis supported this finding. All the strains with plasmid encoded cpe genes showed changes in cpe location after at least one of the heat treatments. As all but one of the plasmid cpe strains were more heat sensitive it seems most likely that the observed differences in cpe location are due to cross contamination with spores possessing a chromosomal cpe gene.

In conclusion, careful consideration should be given to the risk associated with holding times and temperatures for food preparation, cooking, manufacture and cooling. All of these stages can affect the ability of C. perfringens to survive and may influence the extent of enterotoxin production. This study has demonstrated that temperatures such as 75ºC with hold times up to three hours followed by slow cooling, may affect enterotoxin production following sporulation.

Final report is available from the Agency's Information centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk).

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email: science@foodstandards.gsi.gov.uk