Université catholique de Louvain Faculté d’Ingénierie Biologique, Agronomique et Environnementale Louvain-la-Neuve - 2012 TTThhheee uuussseee ooofff lllliiiiqqquuuiiiiddd ccchhhrrrooommmaaatttooogggrrraaappphhhyyy cccooouuuppplllleeeddd tttooo mmmaaassssss ssspppeeeccctttrrrooommmeeetttrrryyy aaasss aaa nnneeeccceeessssssaaarrryyy tttoooooollll fffooorrr ttthhheee aaasssssseeessssssmmmeeennnttt ooofff ccceeerrreeeuuulllliiiidddeee iiiinnn fffooooooddd Présidente : Prof. M.L. de Keersmaecker Thèse présentée par Promoteurs : Prof. J. Mahillon Laurence DELBRASSINNEDELBRASSINNE Ir J. Van Loco en vue de l’obtention du grade de Lecteurs : Dr K. Dierick Docteur en Sciences Agronomiques Dr L. Pussemier et Ingénierie Biologique Prof. Y.J. Schneider ACKNOWLEDGMENTS - ACKNOWLEDGMENTS Writing a thesis is a long journey… Everything started almost four years ago and was a complex intermingling of exciting discoveries but also harder times. And throughout the journey, a lot of people stood by my side who encouraged me and whom I would not miss to thank. In the first line, I would like to warmly thank my promoter Prof. Jacques Mahillon for his constant attention. Despite the geographical distance, he provided me with his helpful advice and always prompt reaction, particularly in the writing process. It was much more than expected and I learnt a lot from this nice collaboration. I am also grateful to my second promoter Ir Joris Van Loco for having initiated this project, for his always interesting suggestions and constructive ideas. The path taken during this thesis was largely under his supervision. Talking about supervision, I could not forget to mention Dr Mirjana Andjelkovic, my direct supervisor who dedicated me a great part of her time and always let the door wide open for me. Warm thank for the helpful discussions and for always ensuring that I am improving myself in scientific communication field. It was a constant and agreeable teaching. My gratefulness goes also to Dr Katelijne Dierick and Dr Luc Pussemier for their advice during annual meetings and for having accepted, together with Prof. Yves-Jacques Schneider, to review this thesis. Being part of two institutions, I had the chance to mix with brilliant and pleasant people from both sides: the IPH (Scientific Institute of Public Health) group and the MBLA team. I could not have been happier to perform my daily work surrounded by nice people. Principally because I was helped by the kindest technician. Thank you for having followed me in all my motivations: Jacques, mille mercis pour tout! My deep appreciation also goes to three persons in particular: Aurore, with whom I shared every moment related to the thesis and thanks to whom the journey was surely softer, Vincent, for his i [Laurence DELBRASSINNE] ASSESSMENT OF CEREULIDE BY LC-MS² talented mind and strong human qualities and Pauline, my thesis student for her enthusiasm and dynamism. Your support was helpful. I am also grateful to all the Food and the Foodborne Pathogens sections who allowed me the work in a so nice atmosphere and to the MBLA team for having adopted me as if I was part of their lab. I would like to extend my thanks to all the people with whom I have had the pleasure to work. Dr Andreja Rajkovic, for excellent suggestions during article redaction, Dr Nadine Botteldoorn and Dr Sarah Denayer for welcoming me in their lab, Dr Tim Reyns for ensuring continuity of cereulide analyses, Dr Claude Biéva for his enthusiasm regarding scientific questions, and Dr Sophie Jan for the nice collaboration. Then my sweet family, far from science but close to me, offering me continuous support and love. To my parents, grand-parents, godmother, adorable sister, and brother-in-law: sorry for the time I spent away from you, while working on this thesis and thanks for believing in me! Sharing with friends is probably the best way to relieve stress. How lucky I am with my dear friends! Les filles, you are an inexhaustible source of happiness; les Agros, how strong is our connection; les Romains, thanks for the perfect times filled with humor and finally, the old ones, thanks for always being there for me. Your friendship is a gift which spices up life and renders it great. Finally, how could I finish these acknowledgments without addressing all my thanks to the most caring man I know ? Olivier, my best friend, your sense of style has made this thesis much more enjoyable to read. I thank you for being this marvellous person and for understanding me so well beyond the words. ii ABSTRACT - ABSTRACT Certain B. cereus strains are able to produce an emetic toxin named cereulide which is at the origin of numerous foodborne outbreaks with even fatal outcomes. This stressed the importance of setting up rapid and reliable quantification methods for cereulide in food products. An optimized LC-MS² method was developed in the first part of this thesis and an in-house validation was performed according to the Commission Decision 2002/657/EC. The method was compared with other concurrent methods in terms of specificity, sensitivity and time consumption. It guaranteed low detection limits in rice (LOD of 0.5 ng/g) with a good repeatability and reproducibility. The robustness of the method was tested in other food matrices coming from industry (red beans, chili con carne, and spices) or from foodborne intoxications ( i.e. white and fried rice, and bean soups) in which cereulide was detected at various levels (from 1.5 ng/g up to 13 g/g). Besides, the actual concentration of cereulide was quantified in the pasta leftovers of a recent Belgian lethal case due to B. cereus and it helped to explicitly incriminate cereulide as the most probable cause of the death. The study of critical conditions (temperature and time of storage, type of strain) influencing cereulide production revealed that the correlation between toxin production and bacterial counts is more complex than proportional. The actual role of cereulide for its bacterial producer was also assessed. The emetic toxin displays a fungistatic activity against nine fungal species (Alternaria alternata, Botrytis cinerea, Cladosporium cucumerinum, Magnaporthe grisea, Monographella nivalis, Mortierella isabellina, Sclerotinia minor, Stemphylium vesicarium and Verticillium dahliae ). Finally, the presence and concentrations of cereulide in rice dishes were assessed in Asian restaurants of Brussels and Wallonia. The prevalence of cereulide (7.4 % of the dishes analysed on the day of consumption contained cereulide) was higher than previously studied for the cereulide-producing strains and encourages to further characterize the risk of B. cereus emetic food poisoning. iii [Laurence DELBRASSINNE] ASSESSMENT OF CEREULIDE BY LC-MS² iv PREAMBLE - PREAMBLE This work was realised in the frame of an Ylieff Project at the Scientific Institute of Public Health (IPH) in Brussels, in collaboration with the Laboratory of Food and Environmental Microbiology (MIAE-MBLA) at UCL. This thesis was dedicated to the study of cereulide, the emetic toxin produced by Bacillus cereus . In this context, an LC-MS² method was developed and validated for the quantification of cereulide in food. During the method development, various conditions (time and temperature of storage, type of strain) influencing cereulide production were explored. A complete validation of the LC-MS² method was performed based on several pre-experiments. As direct applications of the validated method, analyses were carried out to quantify cereulide in various food samples that were part of HACCP monitoring or intoxication cases. In parallel, experiments were designed to evaluate the prevalence of cereulide in rice dishes available in 54 restaurants in Brussels and in Wallonia. Besides, the potential biological role of cereulide was explored by growth inhibition tests of fungi. Finally, a lethal B. cereus food poisoning case was thoroughly analysed and, thanks to the LC-MS² quantification, cereulide was clearly incriminated as the cause of the intoxication. A graphical outline of the thesis is displayed on the following page. The first chapter relates the state-of-the-art on cereulide, while the second chapter presents the specific objectives of this thesis. A series of publications that resulted from the experiments described above constituted the core of this thesis (Chapter 3) and are articulated as Method Development (Chapter 3.1), Method Validation (Chapter 3.2) and Method Applications (Chapters 3.3, 3.4 and 3.5). The five sections related to the results are discussed in their globality in Chapter 4. Finally, the concluding considerations on the performed study are given in Chapter 5 together with perpsectives for the future research on cereulide. v [Laurence DELBRASSINNE] ASSESSMENT OF CEREULIDE BY LC-MS² Graphical outline of the thesis vi TABLE OF CONTENTS - TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................... ............................................................................................. ................ III ABSTRACTABSTRACT................................................................................................................................................................................................................... ..............................................................III............ IIIIIIIII PREAMBLE ................................................................................... ...............................................................................................