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Abstract

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.

Item Type:	Thesis - PhD
Authors/Creators:	McQuestin, Olivia
Keywords:	Escherichia coli, Food, Meat
Copyright Holders:	The Author
Copyright Information:	Copyright 2006 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Thesis (PhD)--University of Tasmania, 2006. Includes bibliographical references
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