Effects of cold temperature and water activity stress on the physiology of Escherichia coli in relation to carcasses Chawalit Kocharunchitt Bachelor of Biotechnology with Honours Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy University of Tasmania June, 2012 DECLARATION OF ORIGINALITY I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any tertiary institution and, to the best of my knowledge and belief, contains no copy of material previously published or written by another person, except where due reference is made in the test of the thesis. This thesis does not contain any material that infringes copyright. Chawalit Kocharunchitt June, 2012 i STATEMENT ON ACCESS TO THE THESIS I understand that the University of Tasmania will make this thesis available for use within the University Library and, via the Australian Digital Theses network, for use elsewhere. This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Chawalit Kocharunchitt June, 2012 STATEMENT ON PUBLISHED WORK The publisher of the paper comprising Chapter 2 hold the copyright for that content, and access to the material should be sought from the respective journal. The remaining non published content of the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Chawalit Kocharunchitt June, 2012 ii STATEMENT ON THE CONTRIBUTION OF OTHERS I acknowledge the works done in collaboration with the Commonwealth Scientific and Industrial Research Organization (CSIRO) Food and Nutritional Sciences (North Ryde, Australia). The major contribution of Dr. Thea King from (CSIRO) Food and Nutritional Sciences toward the transcriptomic works, including the analysis of transcriptomic data and the interpretation based on them is acknowledged. I also acknowledge the technical support provided by Mr. Edwin Lowe from Central Science Laboratory (University of Tasmania, Hobart, Australia) for the proteomic works. Finally, I acknowledge the assistance of Dr. Olivia McQuestin from the Department of Health and Human Services (Hobart, Australia) with general laboratory works. Chawalit Kocharunchitt June, 2012 iii STATEMENT OF CO-AUTHORSHIP This thesis includes work, which has been published or submitted for publication in a peer-reviewed journal. More details for each chapter are described in the section of “Publications Arising from the Thesis”. Proportionate co-author contributions were as follows: Chapters 2-4 Chawalit Kocharunchitt (30%), Thea King (30%), Kari Gobius (10%), John P. Bowman (10%), Tom Ross (20%) Chapter 5 Chawalit Kocharunchitt (60%), John P. Bowman (10%), Tom Ross (30%) Details of the Authors roles: Mr. Chawalit Kocharunchitt (candidate) made key contribution to most part of the research, data analysis, interpretation of results, and led the preparation and refinement of initial and successive manuscripts. Dr. Thea King from CSIRO Food and Nutritional Sciences (Australia) contributed to data analysis, interpretation of some results and development of the manuscripts. Dr. Kari Gobius from CSIRO Food and Nutritional Sciences (Australia) provided general guidance Assoc. Prof. John P. Bowman from Food Safety Centre, University of Tasmania (Australia) provided directional advice as supervisor, and contributed to the idea and manuscript preparation. iv Assoc. Prof. Tom Ross from Food Safety Centre, University of Tasmania (Australia) provided directional advice as supervisor, and contributed to the experimental design, implementation of the research program and manuscript preparation. We the undersigned agree with the above stated “proportion of work undertaken” for each of the above published or submitted peer-reviewed manuscripts contributing to this thesis: (Assoc. Prof. Tom Ross) (Prof. Holger Meinke) Supervisor Head of School School of Agricultural Science School of Agricultural Science University of Tasmania University of Tasmania June, 2012 v ACKNOWLEDGEMENTS I would like to express my gratitude to the following people for their contributions to this project and to whom I am most grateful. My academic supervisors, Assoc. Prof. Tom Ross and Assoc. Prof. John Bowman for their guidance, support and assistance during supervision of this project. The University of Tasmania and the Meat and Livestock Australia (MLA) for generous financial support. Dr. Thea King, Mr. Edwin Lowe, Dr. Kari Gobius, Dr. Olivia McQuestin, Dr. David Ratkowsky and Prof. Tom McMeekin for their advice and help during this project. Dr. Carlton Gyles from the University of Guelph (Guelph, Canada) for supplying Escherichia coli O157:H7 strain Sakai to be used in this project. Dr. Lyndal Mellefont, Mrs. Lauri Parkinson, Mr. Andrew Measham and friends within the School of Agricultural Science for their friendship, advice and help during this research. My family in Thailand, as well as my wife (Ji Li) and son (Jacob Kocharunchitt) for giving me with constant support and encouragement over the course of this work. vi 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, vii 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, viii 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 up- regulate 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