Identification and understanding the roles of biofilm formation-related genes in Listeria monocytogenes isolated from seafood : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology, Institute of Food Science and Technology, Massey University, Palmerston North, New Zealand

Listeria monocytogenes is a foodborne pathogen that can lead to severe bacterial infections in immunocompromised people, the elderly and pregnant women and their unborn. Seafood is one of several contamination sources and as the seafood industry is of high economic value to New Zealand, this pathogen needs to be controlled. The main route for contamination is thought to be from biofilms in the seafood-processing environment and their persistence through cleaning and sanitation. Persistent and sporadic strains of L. monocytogenes isolated from mussel-processing facilities were compared using phenotypic assays. Biofilm formation was greater for persistent strains compared to sporadic strains (30°C, 48h) using cell counts and crystal violet staining (CV). The persistent isolate 15G01 exhibited greatest biofilm formation and was therefore chosen to be studied for biofilm formation using transposon mutagenesis. A screen of the transposon library for biofilm-forming ability using the crystal violet assay identified 27 genes to be associated with biofilm formation. Three low biofilm formers (33E11, 39G5, 44D3) and one high biofilm formers (34F11) were analysed with the fluorescent LIVE/DEAD stain and the scanning electron microscope revealing coccoid-shaped cells and long chains for 33E11 and 44D3, respectively. The four mutants and a previously identified fifth (6B4) were investigated for their biofilmforming ability, the surface characteristics of the cells and the influence of cations on biofilm formation. Three different biofilm formation assays were used to assess the composition of the biofilm. The CV assay was used to determine the whole biofilm mass, cell enumeration was applied to calculate viable cells in the biofilm and a formazan based assay (XTT) measured metabolic activity. All three assays showed a significant correlation, however, no correlation with cell surface characteristics was observed. Confocal laser scanning microscopy revealed a unique sandwich structure for the biofilm formed by 44D3, which has not been reported before, and was reversed at higher magnesium concentrations. Magnesium influenced biofilm formation at a concentration of 5 mM resulting in enhanced biofilm formation for the wild-type and the mutant 44D3 and in reduced biofilm formation in 39G5 whereas calcium showed no gene-specific effect on biofilm formation. The research presented in this study provides useful data for the prevention and control of biofilm formation by L. monocytogenes in seafood-processing plants

The Burgundian « visage ». Seals between tradition and innovation

Les grandes étapes de l’histoire du royaume de Bourgogne ainsi que les transformations que ce royaume subit pendant près de cent vingt ans se reflètent dans plusieurs sources, notamment dans les sceaux. Ces derniers permettent, en autres, d’en comprendre l’orientation politique. C’est le cas, par exemple, de Rodolphe Ier qui tient énormément à s’inscrire dans la continuité des Carolingiens de la Francia orientalis pour renforcer sa légitimité et consolider son règne. Par ailleurs, de nombreuses évolutions se lisent dans les sceaux, oscillants entre traditions et innovations, comme lorsque Rodolphe II tente d’agrandir le royaume en focalisant son attention sur la Souabe et sur le regnum Italiae, puis lors de la fusion de la Transjurane et du royaume de Provence sous Conrad et, enfin, au moment où les Ottoniens étendent leur emprise sur Rodolphe III

Introduction

Évoquer la « Bourgogne », c’est mettre l’eau à la bouche des gourmets et des gourmands qui commencent à rêver du Kir, du Nuits-Saint-Georges, du Vosne-Romanée, du coq au vin, de la moutarde dijonnaise et des repas si délicieux préparés à la bourguignonne. Pour l’historien de l’art, c’est songer à Cluny, Tournus, Paray-le-Monial ou Autun, s’extasier au vu des œuvres de Rogier van der Weyden, de Jan van Eyck ou de Claus Sluter. Et qu’en est-il de ceux qui s’intéressent à l’histoire médiévale ? ..

Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafoodprocessing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.Peer reviewe

... the value of princes is judged by the quality of the men they send forth – the Diplomatic gents of Maximilian I

i può probabilmente acconsentire a quel giudizio di Francesco Guicciardini che le capacità, il talento e la saggezza del principe si specchiano anche nella scelta dei suoi ambasciatori. È perciò un aspetto molto affascinante di questo libro il mettere a fuoco gli attori ed esecutori dei potentati ed esaminare la loro interazione e comunicazione con il reggente. Sembra particolarmente interessante studiare gli incaricati di missioni diplomatiche di Massimiliano I visto che queste diedero il via all’ascesa della sua casata – non solo nei regni iberici ma anche in Boemia ed in Ungheria – non per mezzo della guerra, ma mediante la diplomazia, attraverso le trattative e le negoziazioni matrimoniali. Gregor Metzig che dichiara di volersi distaccare, con la sua tesi di dottorato, dalla «storiografia diplomatica convenzionale» e dalla tendenza classica a considerare la «politica europea» di Massimiliano come una «semplice catena di avvenimenti alternanti tra guerre, tregue e riprese delle ostilità tra le case rivaleggianti» (2), si mette sulle tracce di queste persone abili e valenti, gli ambasciatori, che operarono con, per e all’ombra di Massimiliano I e che sono spesso cadute vittime dell’oblio. ..