Evaluation of the microbial safety of commercially produced tomatoes in South Africa and the development of a novel enrichment broth for the identification of Escherichia coli O157:H7 By Brigitte Nelle Van Dyk Submitted in partial fulfilment of the requirements for the degree MSc (Agric) Plant Pathology In the Faculty of Natural and Agricultural Sciences Department of Plant Sciences University of Pretoria Supervisor: Prof. L. Korsten Co-supervisor: Dr. E. M. du Plessis October 2015 DECLARATION I hereby certify that this thesis, submitted herewith for the degree of MSc (Agric) Plant Pathology to the University of Pretoria, contains my own independent work, except where duly acknowledged. This work has hitherto not been submitted for any other degree at any other University Brigitte Nelle van Dyk October 2015 ii In Loving Memory of Jaco iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................. 1 PREFACE ......................................................................................................................... 3 CHAPTER 1 ..................................................................................................................... 6 Abstract ............................................................................................................................. 7 1 Introduction .................................................................................................................. 8 2 Plants as alternative hosts of human enteric pathogens ............................................ 12 2.1 Introduction of foodborne pathogens into the plant environment ............................... 12 2.2 Survival in the plant environment ............................................................................. 13 2.3 Overcoming plant host immunity and establishment ................................................. 24 3 Conventional methods for the detection of foodborne pathogens ............................. 28 3.1 Enrichment ............................................................................................................... 29 3.2 Conventional Detection Methods of Foodborne Pathogens ....................................... 34 4 Application of foodomics for ensuring safe produce ................................................. 41 4.1 Introduction to Foodomics ........................................................................................ 41 4.2 Genomics and Transcriptomics ................................................................................. 42 4.3 Metabolomics ........................................................................................................... 44 4.4 Proteomics with specific reference to MALDI-TOF MS ........................................... 46 5 Conclusions ................................................................................................................. 48 6 Objectives Of This Project ......................................................................................... 49 7 References ................................................................................................................... 51 iv CHAPTER 2 ................................................................................................................... 88 Abstract ........................................................................................................................... 89 1 Introduction ................................................................................................................ 91 2 Materials and Methods ............................................................................................... 94 2.1 Study areas and sampling design............................................................................... 94 2.2 Sample collection ..................................................................................................... 96 2.3 Sample processing .................................................................................................... 98 2.4 DNA extraction, multiplex polymerase chain reaction, and sequencing..................... 99 2.5 MALDI-TOF MS identification of coliforms .......................................................... 101 2.6 Food safety risk assessment .................................................................................... 101 2.7 Statistical analysis .................................................................................................. 102 3 Results ....................................................................................................................... 103 3.1 Indicator organisms from tomatoes ......................................................................... 103 3.2 Indicator organisms from water .............................................................................. 104 3.3 Indicator organisms from soil ................................................................................. 105 3.4 Indicator organisms from contact surfaces .............................................................. 105 3.5 Human pathogens in the tomato supply chain ......................................................... 106 3.6 MALDI-TOF MS identification of dominant coliform species ................................ 106 3.7 Food safety risk assessment .................................................................................... 108 4 Discussion and Conclusions ...................................................................................... 109 5 References ................................................................................................................. 118 v CHAPTER 3 ................................................................................................................. 148 Abstract ......................................................................................................................... 149 1 Introduction .............................................................................................................. 150 2 Materials and Methods ............................................................................................. 153 2.1 Materials ................................................................................................................ 153 2.2 Heat-injury of Cells ................................................................................................ 154 2.3 Solid media for resuscitation and enumeration of Escherichia coli O157:H7 .......... 155 2.4 Isolation of Escherichia coli O157:H7 from spiked surface water samples .............. 155 2.5 Growth of Escherichia coli O157:H7-spiked water samples in different broths ....... 155 3 Results ....................................................................................................................... 156 3.1 Axenic growth of Escherichia coli O157:H7 in different enrichment broths............ 156 3.2 Growth of Escherichia coli O157:H7 on SPEB ....................................................... 156 3.3 Resuscitation and growth of injured Escherichia coli O157:H7 on SPEB................ 157 3.4 Isolation of Escherichia coli O157:H7 from spiked surface water samples .............. 157 3.5 Growth of Escherichia coli O157:H7-spiked water samples in different broths ....... 157 4 Discussion and Conclusions ...................................................................................... 158 5 References ................................................................................................................. 162 SUMMARY................................................................................................................... 175 vi LIST OF TABLES CHAPTER 2 Table 1. Comparison of the cultivation- and processing practices between the three farms assessed in this study ........................................................................................ 131 Table 2. Sampling and processing procedures used during this study. ............................ 132 Table 3. Multiplex primers used for the detection of Salmonella Typhimurium and Escherichia coli O157:H7 ................................................................................ 132 Table 4. Coliform counts from the packinghouse contact surfaces of Farm 2 and 3. ....... 133 Table 5. MALDI-TOF identification of isolates prevalent in the South African tomato supply chain ..................................................................................................... 134 vii LIST OF FIGURES CHAPTER 2 Figure 1. Map of South Africa indicating tomato farm locations ………………………. 136 Figure 2. Mean coliform counts of 117 tomato samples collected from Farm 2 and Farm 3 between 2013 and 2014………………………………………………………… 138 Figure 3. Mean levels of Escherichia coli and coliforms in 99 irrigation water samples collected from all three farms between 2012 and 2014 ………………………. 140 Figure 4. Mean levels of Escherichia coli and coliforms in 63 wash water samples collected from all three farms’ packinghouses between 2012 and 2014 ………………… 142 Figure 5. Spider diagram representing the results fo the diagnostic tool………………… 144 Figure 6. Dominant coliforms identified using MALDI-TOF MS……………………….. 146 CHAPTER 3 Figure 1. Cell densities obtained with different enrichment broths after 24 and 48 hours of cultivation………………………………………………………………………. 169 Figure 2. Recovery of heat-injured Escherichia coli O157:H7 on selective media……… 171 Figure 3. Growth of Escherichia