OPTIMIZATION OF FLOW CYTOMETRY ASSAYS FOR THE DETECTION OF INJURED FOODBORNE PATHOGENIC BACTERIA Alicia Subires Orenes Facultat de Veterinària Departament de Ciència Animal i dels Aliments Doctoral thesis OPTIMIZATION OF FLOW CYTOMETRY ASSAYS FOR THE DETECTION OF INJURED FOODBORNE PATHOGENIC BACTERIA Alicia Subires Orenes Bellaterra (Cerdanyola del Vallès), 2015 Josep Yuste Puigvert, professor agregat, i Marta Capellas Puig, professora titu- lar del Departament de Ciència Animal i dels Aliments de la Universitat Autò- noma de Barcelona, fan constar que la llicenciada en Ciència i Tecnologia dels Aliments Alicia Subi- res Orenes ha dut a terme, sota la seva direcció, el treball titulat “Optimization of flow cytometry assays for the detection of injured foodborne pathogenic bacteria”, que presenta per optar al grau de Doctor. I perquè així consti, signem el present document a Bellaterra (Cerdanyola del Vallès), el 27 de juliol de 2015. Josep Yuste Puigvert Marta Capellas Puig This project was supported by grant AGL2007-66877/ALI from the Spanish Ministry of Education and Science (MEC). Alicia Subires benefited from a grant by the Commission for Universities and Research of the Ministry of Innovation, Universities and Enterprise (DIUE) of the Autonomous Government of Catalo- nia. to find you in infinity of space and time one must first divide and then combine J. W. von Goethe Agraïments Als meus directors de tesi, la Marta i el Josep, dels qui he après tant durant aquests anys, per confiar en mi i en la meva capacitat per tirar la feina endavant, per la seva paciència i bons consells. A la Manuela Costa, tècnica del servei de citometria de la UAB, per la seva amabilitat en tot moment i per iniciar-me en la citometria de flux. I would like to acknowledge Dr. Tjakko Abee, who gave me the chance to work with his research group, and Dr. Maarten Mols, who gave me so many valuable lab tips. A Manuela Hernández, por acogerme en su despacho estos últimos meses de redac- ción, por su buen humor, y por enseñarme tanto durante las prácticas del “workshop”. A la resta de gent del Departament: becaris, tècnics, professors. Uns ja han marxat, altres encara hi són, però a tots els agraeixo la seva ajuda quan ho he necessitat, la seva amabilitat, el seu suport i els bons moments, tant dins com fora de la feina. Als meus pares i a la meva germana. Quina sort tenir-vos... Publications and presentations related to this thesis Publications Title: Flow cytometry immunodetection and membrane integrity assessment of Esch- erichia coli O157:H7 in ready-to-eat pasta salad during refrigerated storage Authors: A. Subires, J. Yuste, M. Capellas Reference: International Journal of Food Microbiology, 2014, 168-169: 47-56 Scientific communications Title: Immunodetection and membrane integrity assessment of Escherichia coli O157:H7 by three-colour flow cytometry Authors: A. Subires, J. Yuste, M. Capellas Meeting: 5th International Conference on Analysis of Microbial Cells at the Single Cell Level Place and date: Carry-Le-Rouet, France, 5-8 November, 2011 Type of communication: Poster communication Title: Assessment of green-fluorescent nucleic acid stains as bacterial viability indica- tors for flow cytometry analysis Authors: A. Subires, M. Capellas, S. De Lamo, J. Yuste Meeting: 22nd International Symposium of the International Committee on Food Microbiology and Hygiene, Food Micro 2010: Microbial behaviour in the food chain Place and date: Copenhagen, Denmark, 30 August-3 September, 2010 Type of communication: Poster communication Title: Inactivation-associated reactive oxygen species formation in heat- and acid- stressed bacteria Authors: A. Subires, M. Mols, M. Capellas, J. Yuste, T. Abee Meeting: 22nd International Symposium of the International Committee on Food Microbiology and Hygiene, Food Micro 2010: Microbial behavior in the food chain Place and date: Copenhagen, Denmark, 30 August-3 September, 2010 Type of communication: Oral communication Title: Flow cytometry for injured pathogen detection Authors: A. Subires, J. Yuste, M. Capellas Meeting: 30th Annual Rapid Methods and Automation in Microbiology Work- shop/Symposium Place and date: Manhattan, KS, USA, 11-18 June, 2010 Type of communication: Lecture Title: Flow cytometry detection of Escherichia coli O157:H7 stressed by chemical and physical treatments Authors: A. Subires, J. Yuste, M. Capellas Meeting: V European Symposium on Food Safety Place and date: Berlin, Germany, 7-9 October, 2009 Type of communication: Poster communication Contents i Table of contents Abstract .......................................................................................................... 1 Resumen ......................................................................................................... 3 Chapter 1. Literature review ........................................................................ 7 1. Injured bacteria in food .............................................................................................................. 9 1.1. Minimally processed food as pathogen vectors ..................................................... 9 1.2. Stress exposure throughout the food chain ......................................................... 10 1.2.1. Stress factors and cellular targets................................................................ 10 1.2.2. Stress adaptive responses .............................................................................. 13 1.3. Potential public health hazards posed by injured bacteria ............................ 15 2. Detection of injured cells ........................................................................................................ 16 2.1. Culture-based methods ................................................................................................ 17 2.1.1. Differential plating method ........................................................................... 17 2.1.2. Thin agar layer method ................................................................................... 18 2.2. Culture-independent methods .................................................................................. 19 2.2.1. Molecular methods ........................................................................................... 19 2.2.2. Fluorescence-based techniques .................................................................. 21 2.2.2.1. Assessment of physiological parameters by fluorescent probes ................................................................................................... 21 2.2.2.2. Fluorescence detection ................................................................. 34 3. Basic principles of flow cytometry ....................................................................................... 37 3.1. Instrumentation ............................................................................................................... 38 3.1.1. Fluidics ................................................................................................................... 38 3.1.2. Optics ..................................................................................................................... 40 3.1.2.1. Excitation optics................................................................................ 40 3.1.2.2. Collection optics ............................................................................... 42 3.1.3. Signal processing electronics ....................................................................... 43 3.1.3.1. Analog signal processing .............................................................. 43 3.1.3.2. Digital signal processing ............................................................... 46 3.2. Data display and analysis ............................................................................................. 46 ii Contents 3.3. Cell parameters measured by flow cytometry ..................................................... 48 3.3.1. Scattering signals ............................................................................................... 48 3.3.2. Fluorescence signals......................................................................................... 50 3.4. Flow cytometers for bacterial analysis .................................................................... 53 4. Flow cytometry measurements of food-related bacteria .......................................... 55 4.1. Challenges of flow cytometry detection of bacteria in food .......................... 55 4.2. Sample preparation ........................................................................................................ 56 4.2.1. Liquid foods ......................................................................................................... 59 4.2.2. Semi-solid and solid foods ............................................................................. 60 4.3. Applications ....................................................................................................................... 60 4.3.1. Identification of bacteria ................................................................................. 60 4.3.2. Enumeration of bacteria ................................................................................. 69 4.3.3. Physiological state assessment .................................................................... 72 5. References..................................................................................................................................... 74 Chapter 2. Introduction