B01009: An assessment of population changes in Salmonella enteritidis, and the emergence of strains with altered properties during food processing

Study Duration: April 1998 to October 2001

Contractor: University of Reading

The consumer preference for foods that are fresher-tasting and more natural has led to a reduction in the levels of preservatives in some traditional foods, and the development of newer types of product that rely on combinations of mild processes (e.g. mild heat plus refrigerated storage). Great care is taken to ensure the safety of such foods.

However, there is concern that mild treatments which do not kill all bacteria in food may eventually lead to the emergence of more robust strains better able to withstand heat, acid, drying and other processes used in food preservation. If such robust strains were to arise among foodborne pathogens, they would create problems for food manufacturers and might pose a threat to health.

At present there is no evidence to suggest that foodborne pathogens are becoming more resistant to adverse conditions. Nevertheless it has recently become apparent that natural isolates of Salmonella and Listeria monocytogenes show considerable variation in stress resistance and in virulence.

Such strain variation is also relevant to the newer methods of food preservation. For example, some strains of Escherichia coli O157 are much more resistant to inactivation by high hydrostatic pressure than others. The extent and basis of such strain variations in resistance are poorly understood and this has obvious practical implications in setting guidelines for safe preservation processes and in risk assessment.

This project aims to establish if exposure of Salmonella enterica serovar Enteritidis to stresses such as heat or acid treatment gives rise to more resistant types either by selection of robust strains that were already present in mixed populations of natural isolates or by selection of spontaneous mutants arising from �normally resistant� strains.

Work in this project was co-ordinated with a similar project at the Exeter Public Health Laboratory. It was decided that Reading would concentrate on heat stress and Exeter would concentrate on acid. The original workplan at Reading was divided into two areas:

• investigating the selective effect that repeated cycles of stress treatment would have on a mixed culture of many different Salmonella enterica serovar Enteritidis strains
• investigating changes in resistance of single strains under the same conditions. The first part required the development of a high-resolution genetic fingerprinting method, amplified fragment length polymorphism (AFLP), that would enable individual strains within the population to be identified.

Although initial results with this method were very promising, problems of reproducibility were encountered. While this work was in progress, a report appeared in the literature describing the application of AFLP to Salmonella enterica serovar Enteritidis. In view of this, more attention was devoted to investigating the development of enhanced heat resistance in individual strains and in assessing the extent and practical significance of the variation in resistance to heat and other stresses seen among natural isolates of Salmonella enterica serovar Enteritidis.

The major project findings are:

• In natural populations of Salmonella enterica serovar Enteritidis there exist some strains that are significantly more stress resistant than other strains. The more resistant strains outnumbered the sensitive ones by about 2:1.
• This variation in resistance appears to be due to the loss of resistance in some strains caused by mutation in the rpoS gene, rather than to the emergence of more resistant mutant strains (for example as a result of mild food processing conditions). Therefore, this strain variation in resistance among foodborne pathogens does not indicate a net increase in risk to the consumer or increased difficulty for the food processor.
• It is extremely important to ensure that strains used for testing the safety of novel food processes are representative of the resistant types. Various laboratory strains of salmonella and E. coli are known to harbour rpoS mutations and are therefore unsuitable for such purposes.
• A simple colony screening test was developed that distinguished between resistant and sensitive strains.
• Heat cycling can further increase the heat resistance of both rpoS positive and negative strains. (Strains with unusual heat resistance are known to occur among natural isolates of salmonella but appear to be rare).
• The development of enhanced heat resistance did not occur more readily in a strain that had a higher than normal mutation rate.
• Heat cycling does not result in a general increase in resistance to treatments such as high pressure or exposure to a high salt concentration.
• In a liquid medium heated at a constant temperature, the time needed to inactivate heat-cycled strains would be about twice that needed for strains with normal resistance.
• However, studies of inactivation in agar sausages heated in a water bath (representing a solid food heated by conduction from the outside), showed that the processing time only needs to be increased by about 15% (relative to the normal treatment) to ensure safety.

This work has identified a major cause of the natural strain variation in heat resistance in an important foodborne pathogen and has highlighted the importance of strain selection when evaluating the safety of novel food preservation procedures. It has also demonstrated that extra-resistant strains can arise by repeated exposure to heat stress.

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