Effects of cold temperature and water activity stress on the physiology of Escherichia coli in relation to carcasses
Creators: name: Kocharunchitt, Chawalit
Publisher: name: University Of Tasmania
Year: 2023

description: 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¬¨‚àûC aw 0.985, 14¬¨‚àûC aw 0.985, viii 25¬¨‚àûC aw 0.967, and 14¬¨‚àûC aw 0.967 was compared to that of a reference culture (35¬¨‚àûC 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¬¨‚àûC). 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¬¨‚àûC 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¬¨‚àûC to 14¬¨‚àûC) 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¬¨‚àûC aw 0.993 to 14¬¨‚àûC 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