Low water activity-induced inactivation of Escherichia coli : kinetics, processes and applications
Creators: name: McQuestin, Olivia
Publisher: name: University Of Tasmania
Year: 2023

description: Serious illness caused by Escherichia coli and associated with the consumption of foods characterised by low water activity (e.g. uncooked, comminuted fermented meat) has clearly demonstrated the ability of this microbe to survive adverse environments. The consequences of such disease outbreaks have included the loss of life, serious and sometimes long-term illness in other victims, and obviously a detrimental impact on the food industry that is implicated. The prevention of further outbreaks relies upon an improved understanding of the capacity of E. coli to survive inimical conditions. This thesis describes the kinetics of low water activity-induced inactivation of E. coli, including consideration of the combined effects of low pH, and examines the cell death processes involved. In addition, this study attempts to define whether cells that exhibit enhanced survival in conditions of low water activity do so by inducing a specific stress response. Materials and methods commonly used to analyse bacterial viability and injury were initially evaluated to enable a more accurate description of E. coli inactivation in response to osmotic stress. The composition of the medium was observed to strongly influence the inactivation of E. coli. Traditional, culture-based methods were found to overstate the level of injury in low water activity-treated populations of E. coli and a modified procedure was employed subsequently. Methods were also developed to improve the level of reproducibility between experiments. Initial studies using culture-based methods demonstrated that the inactivation of E. coli in response to low water activity consisted of three distinct phases of inactivation. The ability to induce the final, rapid phase of inactivation would be of considerable benefit during food manufacture processes to better reduce pathogenic loads. Because the use of low water activity as a preservation method in food manufacture is often in combination with acid stress, the kinetics of inactivation of E. coli in response to low water activity and low pH were also investigated. Experiments showed that the order in which these non-thermal stresses were applied influenced the inactivation of E. coli That is, cells were more sensitive to osmotic stress when first exposed to low pH conditions. These findings are of relevance to food manufacturing processes that use multiple stresses to ensure microbiological safety. The level of injury in osmotic or acid treated E. coli populations suggested that the processes responsible for the effect of these stresses were different. The above knowledge was used to develop a broth-based model that mimicked the rates of inactivation of E. coli observed in uncooked, comminuted fermented meat products. This system allowed for the systematic generation of a large amount of data to define the response of E. coli to specific conditions without the limitations of in-product trials. The data generated in this work were incorporated into a predictive model that has since been used by food manufacturers and regulators to assess the ability of uncooked, comminuted fermented meat manufacturing processes to inactivate E. coli. In addition, this study highlighted the need to confirm patterns of microbial responses derived from broth-based systems with that from in-product trials. Although water activity was shown to influence the rate of inactivation of E. coli using the broth-based system, comparisons with in-product trials reported in the literature suggested that this was not the case in the actual food product. Having characterised the kinetics of low water activity-induced inactivation of E. coli, subsequent work attempted to develop knowledge relating to the processes involved in that system. It was hypothesised that the mechanism of inactivation of E coil in response to low water activity involves specific genetic modules shown to mediate the death of bacterial cells in some situations. Using E. coli mutants deleted for one of these systems (mazEP), the involvement of this component could not be conclusively shown but nor could it be ruled out. The mazEF module might mediate the death of a proportion of E coil cells that are killed immediately following exposure to some low water activity environments. The implications of this self-mediated cell death pathway on the present understanding of bacterial inactivation are discussed. Finally, investigations aimed at identifying a specific stress response that enhances the survival of E coli in lethal water activity environments provided no direct evidence for such a response. Provision of the compatible solute betaine did not alter the survival characteristics of osmotically stressed E coli and proteomic experiments indicated that four stress-related proteins do not form a specific response to lethal osmotic stress in E. coli. The use of proteomic techniques further provided a general overview of the physiology of E coli that are able to survive osmotic challenges, which may be of considerable value when coupled with genomic studies that more comprehensively assess stress physiology in E coli. Overall, the work presented throughout this thesis develops the present understanding of the response of E coil to inimical conditions relevant to the manufacture of uncooked, comminuted fermented meat products.