Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as a novel tool for detection, identification and typing of spoilage microbiota in the brewing industry Anneleen D. Wieme Dissertation submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Biochemistry and Biotechnology Promotors: Prof. Dr. Peter Vandamme Prof. Dr. Anita Van Landschoot In Sweet Memory of Beloved Diane De Sutter, Our Guardian Angel Wieme, A.D. - Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as a novel tool for detection, identification and typing of spoilage microbiota in the brewing industry. © 2014 Anneleen D. Wieme. ISBN-number: 978-94-6197-197-5 All rights reserved. No part of this thesis protected by this copyright notice may be reproduced or utilised in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage or retrieval system without written permission of the author. Printed by University Press | http://www.universitypress.be Cover design by Els Dufourmont Ph.D. thesis, Faculty of Sciences, Ghent University, Ghent, Belgium Publicly defended in Ghent, Belgium, May 28th, 2014 This Ph.D. work was supported by the research fund of the University College Ghent, Belgium. Author’s email address: [email protected] EXAMINATION COMMITTEE Prof. Dr. Savvas SAVVIDES (Chairman) L-Probe: Laboratory for Protein Biochemistry and Biomolecular Engineering Faculty of Sciences, Ghent University Prof. Dr. Peter VANDAMME (Promotor) LM-UGent: Laboratory of Microbiology Faculty of Sciences, Ghent University Prof. Dr. Anita VAN LANDSCHOOT (Promotor) Laboratory of Biochemistry and Brewing Faculty of Bioscience Engineering, Ghent University Prof. Dr. Anne WILLEMS LM-UGent: Laboratory of Microbiology Faculty of Sciences, Ghent University Prof. Dr. Jørgen LEISNER Department of Veterinary Disease Biology Faculty of Health and Medical Sciences, University of Copenhagen Prof. Dr. Yves GUISEZ Laboratory of Molecular Plant Physiology & Biotechnology Department of Biology, University of Antwerp Dr. Nele VANBENEDEN Product Developer in Global Supply Chain Technology & Policies at Heineken, The Netherlands Dr. Koenraad VAN HOORDE Laboratory of Biochemistry and Brewing Faculty of Bioscience Engineering, Ghent University TABLE OF CONTENT EXAMINATION COMMITTEE ..................................................................................... 5 TABLE OF CONTENT .............................................................................................. 6 LIST OF ABBREVIATIONS ....................................................................................... 10 LIST OF GENES ................................................................................................... 11 OUTLINE OF THE THESIS ........................................................................................ 13 PART I | BACKGROUND & OBJECTIVES ...................................................... 15 BACKGROUND .................................................................................................... 16 OBJECTIVES ....................................................................................................... 17 PART II | LITERATURE STUDY ................................................................. 19 1 The brewing process and beer spoilage bacteria ........................................... 20 1.1 Microbiological threats during the brewing process ......................................................... 20 1.2 Microbiological stability of beer ..................................................................................... 24 1.3 Hop resistance ............................................................................................................. 25 1.4 Thriving beer spoilage bacteria – a current overview ........................................................ 28 1.4.1 Gram-stain positive beer spoilage bacteria ............................................................................. 30 1.4.2 Gram-stain negative beer spoilage bacteria ............................................................................ 34 1.4.2.1 Enterobacteriaceae .......................................................................................................... 34 1.4.2.2 Acetic acid bacteria .......................................................................................................... 35 1.4.2.3 Strict anaerobic beer spoilage bacteria .............................................................................. 35 1.4.2.4 Zymomonas .................................................................................................................... 37 2 Current approaches for the detection and/or identification of beer spoilage bacteria .................................................................................................... 39 2.1 Detection of beer spoilage bacteria via growth on selective and non-selective media ......... 40 2.2 Phenotypic features for identification of isolated spoilage bacteria .................................. 40 2.3 ATP bioluminescence .................................................................................................... 41 2.4 Immuno-based approaches ........................................................................................... 41 2.5 Genotypic detection and/or identification methods ......................................................... 42 2.5.1 Oligonucleotide probe-based approaches ............................................................................... 42 2.5.2 PCR-based detection and/or identification methods ............................................................... 43 2.5.3 Fingerprinting techniques ....................................................................................................... 45 2.6 The ongoing search for improved, alternative methods ................................................... 46 3 The application of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in microbiology ............................................................. 48 3.1 From culture to mass spectrum ...................................................................................... 51 3.2 Commercially available MALDI-TOF MS identification databases ..................................... 52 3.3 Current applications of MALDI-TOF MS in microbiology .................................................. 53 3.4 Beyond species level identification ................................................................................ 54 3.4.1 Infraspecific level discrimination ............................................................................................. 54 3.4.2 Direct MALDI-TOF MS detection and identification of bacteria from specimen ....................... 55 3.4.3 MALDI-TOF MS analysis of specimen-containing mixed populations ...................................... 56 PART III | EXPERIMENTAL WORK ............................................................ 71 PREAMBLE ........................................................................................................ 73 4 The effects of the growth medium on matrix-assisted laser desorption/ionisation time-of-flight mass spectra: a case study of acetic acid bacteria .................................................................................................... 75 4.1 Introduction ................................................................................................................. 76 4.2 Material and Methods ................................................................................................... 77 4.2.1 Strains and growth media ....................................................................................................... 77 4.2.2 MALDI-TOF MS sample preparation and data acquisition ....................................................... 77 4.2.3 MALDI-TOF MS data analysis ................................................................................................. 78 4.3 Results and Discussion .................................................................................................. 78 4.3.1 Media that do not sustain optimal growth can influence the SSP strongly ............................... 78 4.3.2 The culture medium used affects strain level differentiation ................................................... 83 4.3.3 A growth medium-dependent core set of peak classes decreases with increasing number of strains ..................................................................................................................................... 87 4.3.4 Species level differentiation is growth medium-independent and is not biased by similarity coefficient used ....................................................................................................................... 88 5 Identification of beer spoilage bacteria using MALDI-TOF MS ........................ 97 5.1 Identification of bacteria isolated from spoiled beer and brewery samples using MALDI-TOF MS .............................................................................................................................. 97 5.1.1 Introduction ............................................................................................................................ 98 5.1.2 Materials and Methods ........................................................................................................... 99 5.1.2.1 Bacterial strains