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Effects of cold temperature and water activity stress on the physiology of Escherichia coli in relation to carcasses

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Kocharunchitt, C (2012) Effects of cold temperature and water activity stress on the physiology of Escherichia coli in relation to carcasses. PhD thesis, University of Tasmania.

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Abstract

Enterohemorrhagic Escherichia coli (EHEC) has emerged as an important food-borne pathogen
of considerable public health concern. The majority of food-borne illnesses caused by EHEC,
particularly serotype O157:H7, appear to be associated with undercooked meat and meat
products. Although several intervention strategies are already in use to control carcass
contamination, no single intervention is 100% effective in eliminating E. coli from carcasses.
This indicates the need for developing an effective intervention. The Australian meat industry
has a particular interest in evaluating the potential effect of combined cold and osmotic stresses
on E. coli, as occurs during carcass chilling. To this end, the present thesis aims to provide a
comprehensive understanding of the growth kinetics and cellular responses of E. coli O157:H7
strain Sakai subjected to conditions relevant to low temperature and water activity conditions
experienced during meat carcass chilling in Australia where cold air chilling, rather than spray
chilling, is routinely employed and leads to a temporary reduction in temperature as well as
reduction in water activity during chilling. Despite that those temperature and water activity
conditions are not lethal to E. coli, other studies have reported a decline in E. coli viability during
cold air carcass chilling that could be exploited to deliberately reduce the prevalence of E. coli
on meat.
An initial study employed an integrated transcriptomic and ‘shotgun’ proteomic approach (using
cDNA microarray and 2D-LC/MS/MS analysis, respectively), to characterize the genome and
proteome profiles of E. coli under steady-state conditions of cold temperature and water activity
stress. Expression profiles of E. coli during exponential growth at 25°C aw 0.985, 14°C aw 0.985,
viii
25°C aw 0.967, and 14°C aw 0.967 was compared to that of a reference culture (35°C aw 0.993).
Gene expression and protein abundance profiles of E. coli were more strongly affected by low
water activity (aw 0.967) than by low temperature (14°C). Predefined group enrichment analysis
revealed that the universal response of E. coli to all low temperature and/or low water activity
conditions included activation of the master stress response regulator RpoS and the Rcs
phosphorelay system involved in the biosynthesis of the exopolysacharide colanic acid, as well
as down-regulation of elements involved in chemotaxis and motility. Characterizing the
physiology of E. coli under chilling and water activity stresses provided a baseline of knowledge
to better interpret, and potentially exploit, this pathogen’s responses to dynamic environmental
conditions that occur during carcass chilling.
To gain deeper insight into the potential mechanisms controlling population responses of E. coli
under dynamic low temperature and water activity conditions, a series of studies were
undertaken to investigate the growth kinetics and time-dependent changes in its global
expression upon a sudden downshift in temperature or water activity. Growth response of E. coli
to a temperature downshift below 25°C was assessed. All downshifts induced a lag phase of
growth before cells resumed growth at a rate typical for the temperature experienced. By
contrast, shifting E. coli to low water activity (below aw 0.985) caused an apparent loss, then
recovery, of culturability. Exponential growth then resumed at a rate under characteristic for the
water activity imposed. In transcriptomic and proteomic studies, E. coli responded to cold shock
(from 35°C to 14°C) and hyperosmotic shock (from aw 0.993 to aw 0.967) by changing
expression of genes and proteins involved in several functional groups and metabolic pathways.
A number of these changes were found to be common for both cold and osmotic stresses,
ix
although stress-specific responses were also observed. The adaptive strategies adopted by cells
generally included accumulation of compatible solutes and modification of the cell envelope
composition. Growth at cold temperature and low water activity, however, appeared to upregulate
additional elements, which are involved in the biosynthesis of specific amino acids. The
present findings also highlighted the robust ability of E. coli to activate multiple stress responses
by transiently inducing the activity of RpoE regulon to repair protein misfolding and aid the
proper folding of newly synthesized proteins in cell envelope, while simultaneously activating
the master stress regulator RpoS to mediate long-term adaptation under the stress conditions of
low temperature and water activity.
A further study was carried out to examine the growth kinetics and molecular response of E. coli
subjected to simultaneous abrupt downshifts in temperature and water activity (i.e. from 35°C aw
0.993 to 14°C aw 0.967). Exposure of E. coli to combined cold and osmotic shifts resulted in a
complex pattern of population changes. Based on enumeration, this repeatable growth behaviour
could be divided into three successive phases: (i) an initial decline in bacterial numbers followed
by a period in which numbers increased rapidly; (ii) a second decrease in culturable cells and
subsequent ‘exponential-like’ growth; and (iii) a constant population level, similar to the starting
density before imposition of the combined shocks (i.e. analogous to a ‘lag’ phase), until ‘true
exponential’ growth was (re)-established. From the proteomic analysis, it was found that the
response of E. coli to downshifts in temperature and water activity involved a highly complex
regulatory network, including changes in abundance of different groups of proteins. This
regulatory network was mediated through a transient induction of the RpoE-controlled envelope
stress response and activation of the master stress regulator RpoS and the Rcs system-controlled
x
colanic acid biosynthesis. Increase in abundance of several proteins with diverse functions was
also observed, including those involved in the DNA repair system, the degradation of proteins
and peptides, the amino acid biosynthetic pathways and the major processes of carbohydrate
catabolism and energy generation.
Overall, the results presented in this thesis, and the interpretations based on them, provide a
detailed understanding of the physiological responses of E. coli to dynamic changes experienced
during carcass chilling under Australian conditions. Such knowledge will aid the development of
more targeted, and less invasive approaches, for the meat industry to eliminate or control this
pathogen

Item Type: Thesis (PhD)
Keywords: E.Coli physiology, carcass chilling, stress response, cold stress, water activity stress
Copyright Holders: The Author
Copyright Information:

Copyright 2012 the Author

Additional Information:

Chapter 2 Published as: Kocharunchitt, C., King, T., Gobius, K., Bowman, J.P., and Ross, T. (2011) Integrated
transcriptomic and proteomic analysis of the physiological response of Escherichia coli O157:H7
Sakai to steady-state conditions of cold and water activity stress, Mol. Cell. Proteomics,
doi:10.1074/mcp.M1111.009019

Chapter 3 eventually published as: King, T., Kocharunchitt, C., Gobius, K., Bowman, J.P., and Ross, T. Global genomic and proteomic responses of Escherichia coli O157:H7 Sakai during dynamic changes in growth kinetics induced
by an abrupt temperature downshift, PLoS One, 2014; 9(6): e99627.

Chapter 4 eventually published as: Kocharunchitt, C., King, T., Gobius, K., Bowman, J.P., and Ross, T. Global
genomic and proteomic responses of Escherichia coli O157:H7 Sakai during dynamic
changes in growth kinetics induced by an abrupt downshift in water activity, PLoS One. 2014 Mar 3;9(3): e90422.

Date Deposited: 17 Aug 2012 04:52
Last Modified: 15 Sep 2017 01:06
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