Microbial biofilms in food environments: study approaches and intervention strategies A Ph.D. dissertation presented by Francesca Frigo to the University of Udine for the degree of Ph.D. in the subject of Food Science (Cycle XXVI) Department of Food Science UNIVERSITY OF UDINE Italy March 2014 Coordinator: Mara Lucia Stecchini, Professor Department of Food Science University of Udine, Italy Supervisors: Michela Maifreni, PhD Department of Food Science University of Udine, Italy Marilena Marino, PhD Department of Food Science University of Udine, Italy Reviewers: Giovanni Di Bonaventura, PhD Department of Biomedical Sciences University G. d’Annunzio of Chieti Pescara Barbara Cardazzo, PhD Department of Public Health, Comparative Pathology and Veterinary University of Padova TABLE OF CONTENTS List of tables .................................................................................................................................................................. 1 List of figures ................................................................................................................................................................ 2 Summary ....................................................................................................................................................................... 4 List of Abbreviations .................................................................................................................................................... 7 Chapter 1. General Introduction ........................................................................................................................... 8 1.1 Historical basis of biofilm study ..................................................................................................... 9 1.2 Biofilm structure ........................................................................................................................... 10 1.3 The biofilm matrix and its functions ............................................................................................ 11 1.4 Process of biofilm formation ........................................................................................................ 12 1.4.1 Attachment .............................................................................................................................. 13 1.4.2 Microcolony formation and biofilm maturation ...................................................................... 14 1.4.3 Detachment and dispersal of cells from biofilms .................................................................... 15 1.5 Parameters influencing biofilm formation .................................................................................... 15 1.6 Biofilms in the food industry ........................................................................................................ 16 1.6.1 Foodborne pathogens and spoilage organisms forming biofilm ............................................. 17 1.7 Biofilm control and removal......................................................................................................... 18 1.7.1 Control .................................................................................................................................... 18 1.7.2 Removal and eradication ......................................................................................................... 20 1.8 References .................................................................................................................................... 25 Chapter 2. Evaluation of Culture Conditions and Methods in Studying Microbial Biofilm .............................. 29 2.1 Introduction .................................................................................................................................. 30 2.2 Materials and methods .................................................................................................................. 31 2.2.1 Microtiter plate assay .............................................................................................................. 31 2.2.2 CBR assay ............................................................................................................................... 32 2.2.3 Drop plate method ................................................................................................................... 33 2.2.4 Statistical analysis ................................................................................................................... 34 2.3 Results and discussion .................................................................................................................. 34 2.3.1 Microtiter plate assay .............................................................................................................. 34 2.3.2 CBR assay ............................................................................................................................... 37 2.3.3 Drop plate method ................................................................................................................... 39 2.4 Conclusions .................................................................................................................................. 41 2.5 References .................................................................................................................................... 42 Chapter 3. Biofilm Formation of Food Pathogens and Spoilers as affected by Temperature, pH, Glucose and Sodium Chloride ......................................................................................................................................................... 43 3.1 Introduction .................................................................................................................................. 44 3.2 Materials and methods .................................................................................................................. 45 3.2.1 Bacterial strains and culture conditions ................................................................................... 45 3.2.2 Central Composite Design and statistical analysis .................................................................. 45 3.2.3 Statistical analysis ................................................................................................................... 46 3.3 Results and discussion .................................................................................................................. 46 3.3.1 Staphylococcus aureus ............................................................................................................ 48 3.3.2 Pseudomonas spp. ................................................................................................................... 58 3.3.3 Listeria monocytogenes ........................................................................................................... 63 3.4 Conclusions ................................................................................................................................... 81 3.5 References ..................................................................................................................................... 82 Chapter 4. Effect of Temperature on Biofilm Formation and Sanitizers Sensitivity of Pseudomonas spp. ........ 85 4.1 Introduction ................................................................................................................................... 86 4.2 Materials and methods .................................................................................................................. 87 4.2.1 Biofilm formation by Pseudomonas spp. ................................................................................. 87 4.2.2 Biofilm susceptibility towards disinfectants ............................................................................ 87 4.2.3 Kinetics of adhesion and biofilm formation on stainless steel AISI 304, and resistance to peracid-based sanitizer ........................................................................................................................................ 88 4.3 Results and discussion................................................................................................................... 89 4.4 Conclusions ................................................................................................................................. 101 4.5 References ................................................................................................................................... 102 Chapter 5. Effectiveness of Chemical Sanitizers and Pulsed Light for the Inactivation of Listeria monocytogenes and Pseudomonas fluorescens Biofilms ........................................................................................... 103 5.1 Introduction ................................................................................................................................. 104 5.2 Materials and methods ................................................................................................................ 105 5.2.1 Bacterial strains and culture conditions ................................................................................. 105 5.2.2 Biofilm formation on stainless steel and PTFE...................................................................... 105 5.2.3 Statistical analysis .................................................................................................................