All natural aquatic systems harbour a vast variety of microorganisms. In the aquatic microbial food web, the larger microorganisms (i.e. protozoa) feed on the smaller microorganisms (i.e. bacteria and phytoplankton). An increase in nutrient availability results in changes of the microbial food web structure, like altered community composition and blooms of toxic phytoplankton. In this thesis work I hypothesised that nutrient-rich aquatic environments, with strong protozoan predation, favour the occurrence of predation-resistant bacteria like F. tularensis, and that the microbial food web may provide a reservoir for the bacterium between outbreaks. By using a size-structured ecosystem food web model it was shown that the protozoan predation pressure on bacteria, defined as protozoan predation per bacterial biomass, increases with increasing nutrient availability in aquatic systems (estimated chlorophyll a 0.2 to 112 μg L-1). This dynamics was caused by increasing growth-rate of a relatively constant number of bacterial cells, maintaining the growth of an increasing number of protozoan cells. The results were supported by meta-analysis of field studies. Thus my results suggest that protozoa control the bacterial community by predation in nutrient-rich environments. In a field study in a natural productivity gradient (chlorophyll a 1.4 to 31 μg L-1) it was shown that intense selection pressure from protozoan predators, favours predation-resistant forms of bacteria. Thus, the abundance of predation-resistant bacteria increases with increasing nutrient availability in lakes. Furthermore, I could demonstrate that the bacterium Francisella tularensis, the causative agent of tularemia, was present in eutrophic aquatic systems in an emerging tularemia area. Isolated strains of the bacterium were found to be resistant to protozoan predation. In a microcosm study, using natural lake water, high nutrient availability in combination with high abundance of a small colourless flagellate predator favoured the occurrence of F. tularensis holarctica. In laboratory experiments F. tularensis strains were able to form biofilm at temperatures between 30-37°C, but not below 30°C. In conclusion, I have shown that the protozoan predation pressure on bacteria increases with increasing nutrient availability in aquatic systems. Predation-resistant forms of bacteria, such as F. tularensis are favoured in nutrient-rich environments. The complexity of the microbial food web and nutrient-richness of the water, influence the transmission of the pathogenic F. tularensis holarctica. However, over long periods of time, the bacterium survives in lake water but may lose its virulence. The temperature-regulated biofilm formation by F. tularensis may play a role in colonization of vectors or for colonization of hosts, rather than for survival in aquatic environments.

La contamination bactérienne et le développement de biofilms marins sont des problèmes récurrents, et terriblement coûteux sur les structures immergées des bâtiments de l'armée. L'emploi de composés bactéricides et nocifs pour l'écosystème est désormais interdit. Les travaux menés lors de cette thèse avaient donc pour objectif de mettre au point des traitements de surface originaux préventifs, permettant de limiter l'adhésion des bactéries marines et, le cas échéant, leur développement sur des matériaux à base de cuivre et de titane. Des surfaces de cuivre ont été fonctionnalisées par des espaceurs sur lesquels ont été greffées des molécules antibactériennes : un peptide, la Magainine I et une enzyme, le lysozyme HEWL. Une deuxième stratégie a consisté en le greffage d'espaceurs, puis des agents antibactériens, sur des surfaces microsctucturées à l'aide de photopolymères dérivés de poly(méthyl méthacrylate). Sur les surfaces de titane, la Magainine I et l'HEWL ont été greffés sur deux types d'espaceurs, un silane et un PEG diamine. Les traitements de surface, antibactériens, ont été optimisés grâce à des caractérisations à chaque étape par IR, XPS, AFM et mesures d'angle de contact. Afin de mettre en évidence les propriétés antibiofilms des surfaces fonctionnalisées, quatre tests ont été mis en place : un test d'adsorption de protéines, un test d'adhésion de bactéries, un test de viabilité des bactéries adhérées et enfin un test de croissance des bactéries après contact avec les surfaces fonctionnalisées. A titre d'exemples, le développement de bactéries sur les surfaces de titane a été réduit de 90% et l'adhésion de bactéries réduite de 80% sur les surfaces de cuivre

La plaie tumorale est une infiltration par des cellules tumorales du tissu cutané, des vaisseaux sanguins et/ou lymphatiques. Ces plaies peuvent se présenter sous des aspects très différents accompagnés de symptômes variés. Outre les risques d'infection locale, les odeurs nauséabondes posent des problèmes récurrents d'isolement social, de difficultés conjugales et entrainent une sensation de dégout, de pourriture, voire de mort imminente. Elles influencent même le comportement médical. La colonisation bactérienne, les odeurs et les risques infectieux semblent intimement liés chez ces patients porteurs de plaie, dont l'immunité est le plus souvent affaiblie par l'administration de traitements anticancéreux. Ce projet a consisté à : 1) créer un fichier dynamique de données et prélever les patients porteurs de plaies tumorales, dans le cadre d'un travail de recherche clinique, 2) par une approche expérimentale personnelle et collaborative: caractériser la flore microbienne, la présence éventuelle de biofilm et les composés volatils des plaies, 3) analyser l'ensemble des données en mettant en évidence, si elles existent, les corrélations entre résultats des prélèvements et observations cliniques, 4) proposer de premières hypothèses, des pistes de solution, une adaptation des pratiques de soins.Une flore mixte composée de 54 bactéries différentes a été retrouvée sur les plaies et la présence de biofilm mise en évidence dans 35% des cas. 91 composés volatils ont été identifiés. Aucune infection n'a été relevée durant la période d'évaluation. Les conséquences de l'interactivité entre les bactéries, le biofilm et les composés volatils nous amènent à penser qu'il serait nécessaire de: 1) développer de nouvelles stratégies et topiques, 2) poursuivre les travaux sur le biofilm, 3) construire des projets en liens avec des psycho-oncologues et neuropsychiatres, 4) progresser vers la conception d'outils diagnostics et l'identification de biomarqueurs.

Escapin is an L-amino acid oxidase that produces antimicrobial metabolites collectively called “Escapin Intermediate Products” (EIP-K). EIP-K and H2O2 together were previously shown to be bactericidal towards diverse planktonic bacteria. The present work investigates the ability of EIP-K and H2O2 to antagonize bacterial biofilms, using Pseudomonas aeruginosa as a model. The project had three aims: 1) determine the most effective concentrations of EIP-K and H2O2 necessary to break down existing P. aeruginosa biofilms, using a crystal violet assay; 2) examine the ability of EIP-K + H2O2 to inhibit biofilm formation, using triphenyl tetrazolium chloride dye; and 3) determine the effect of EIP-K + H2O2 on the viability, biomass and structure of biofilms cultivated in flow cells using confocal laser scanning microscopy (CLSM). Results showed that EIP-K + H2O2 significantly reduced biofilm biomass relative to controls and that the compounds are effective at nanomolar concentrations.

Despite extensive preventative efforts, the problem of controlling infections associated with biomedical materials persists. Bacteria tend to colonize on biocompatible materials and form biofilms; thus, novel biomaterials with antibacterial properties are of great interest. In this thesis, titanium dioxide (TiO2)-associated photocatalysis under ultraviolet (UV) irradiation was investigated as a strategy for developing bioactivity and antibacterial properties on biomaterials. Although much of the work was specifically directed towards dental materials, the results presented are applicable to a wide range of biomaterial applications. Most of the experimental work in the thesis was based on a resin-TiO2 nanocomposite that was prepared by adding 20 wt% TiO2 nanoparticles to a resin-based polymer material. Tests showed that the addition of the nanoparticles endowed the adhesive material with photocatalytic activity without affecting the functional bonding strength. Subsequent studies indicated a number of additional beneficial properties associated with the nanocomposite that appear promising for biomaterial applications. For example, irradiation with UV light induced bioactivity on the otherwise non-bioactive nanocomposite; this was indicated by hydroxyapatite formation on the surface following soaking in Dulbecco’s phosphate-buffered saline. Under UV irradiation, the resin-TiO2 nanocomposite provided effective antibacterial action against both planktonic and biofilm bacteria. UV irradiation of the nanocomposite also provided a prolonged antibacterial effect that continued after removal of the UV light source. UV treatment also reduced bacterial adhesion to the resin-TiO2 surface. The mechanisms involved in the antibacterial effects of TiO2 photocatalysis were studied by investigating the specific contributions of the photocatalytic reaction products (the reactive oxygen species) and their disinfection kinetics. Methods of improving the viability analysis of bacteria subjected to photocatalysis were also developed.

Les biofilms sont des édifices macromoléculaires qui résultent de l'adhérence de microorganismes à une surface. Ils sont constitués de cellules enchâssées dans un réseau d'exopolymères d'origine biologique qui forment une matrice extracellulaire. Les biofilms posent des problèmes technologiques et sanitaires dans de nombreux domaines, aussi bien agroalimentaire, médical, ou industriel. Comprendre les mécanismes de formation de ces structures est donc un enjeu majeur. Malgré une grande diversité de la structure des biofilms, de grands principes semblent en régir la composition. Ainsi, la présence de polysaccharides comme l'alginate et la cellulose joue un rôle majeur dans leur formation et dans la définition de leurs propriétés mécaniques. Si la présence de polymères protéiques comme les fibres amyloïdes semble avoir un caractère universel au sein des biofilms, leur rôle dans la formation de la matrice et dans ses propriétés mécaniques restait à définir. Lors de cette étude, nous avons caractérisé la structure et la composition de trois biofilms monobactériens issus de trois espèces différentes P. aeruginosa NK 125502, S. enterica CIP 58.58 et S. epidermidis CIP 53.124. Nous avons décrit la formation de fibres amyloïdes par différents peptides issus de protéines bactériennes impliquées dans la formation de biofilm et montré par différentes techniques qu'ils sont capables de former des fibres amyloïdes. Nous avons notamment identifié un peptide amyloïde, suggérant la présence de ce type de fibre au sein des biofilms de Staphylococcus, mais aussi plus généralement dans les biofilms des espèces exprimant une protéine de type Bap (Biofilm associated protein). Enfin, nous avons analysé les propriétés mécaniques de différentes matrices synthétiques à base d'alginate et de méthyl-cellulose, en présence et en absence de protéines et de peptides amyloïdes afin de mieux comprendre l'apport qu'a ce type de fibre sur les propriétés de ces structures. Ainsi, les fibres amyloïdes modifient les propriétés mécaniques des gels synthétiques, permettant d'augmenter la déformation sous contrainte. En conclusion, ce travail apporte de nouveaux éléments pour la compréhension du rôle des fibres amyloïdes dans le renforcement de la matrice du biofilm. La capacité à former des fibres amyloïdes par un peptide issu de la protéine Bap de S. epidermidis suggère que cette propriété est plus largement présente au sein de cette famille de protéines. Les travaux menés au cours de cette thèse, et l'ensemble des techniques utilisées, avec notamment la mise au point de l'observation de la biréfringence du rouge Congo par microscopie confocale permettront de développer les études sur cette famille de protéines amyloïdes ainsi que sur les matrices complexes de type biofilm

The waterborne bacterial pathogen Vibrio cholerae uses a process of cell-to-cell communication called quorum sensing (QS) to coordinate transcription of four sRNAs (Qrr1-4; quorum regulatory RNAs) in response to changes in extracellular QS signals that accumulate with cell density. The Qrr sRNAs are predicted to negatively control translation of several mRNAs, including hapR, which encodes the master QS transcription factor that controls genes for virulence factors, biofilm formation, protease production, and DNA uptake. The Qrr sRNAs are also predicted to positively control vca0939, which encodes a GGDEF family protein that promote biofilm formation by elevating intracellular levels of the second messenger molecule c-di-GMP. Using complementary in vivo, in vitro, and bioinformatic approaches, I showed that Qrr sRNAs base-pair with and repress translation of the mRNA encoding HapR. A single nucleotide mutation in Qrr RNA abolishes hapR pairing and thus prevents cholera toxin production and biofilm formation that are important in disease, and also alters expression of competence genes required for uptake of DNA in marine settings. I also demonstrated that base-pairing of the Qrr sRNAs with vca0939 disrupts an inhibitory structure in the 5' UTR of the mRNA. Qrr-activated translation of vca0939 was sufficient to promote synthesis of c-di-GMP and early biofilm formation in a HapR-independent manner. Thus, these studies define the non-coding Qrr sRNAs as a critical component allowing V. cholerae to sense and respond to environmental cues to regulate important developmental processes such as biofilm formation.

The combination of moving bed biofilm reactors and membrane bioreactors (MBBR-MR) can compensate for the drawbacks of both of these systems and further increase their acceptance and application in wastewater treatment industries. Despite the potential benefits of a MBBR-MR technology there has only been limited study of this configuration. The present study consisted of an overall assessment of the performance of a combined MBBR-MR system under high and low loading rates. Since colloidal matter in mixed liquor suspended solid (MLSS) is considered as one of the important contributors to membrane fouling, pre-treatment of membrane feed by coagulation was investigated for improving membrane performance. The performance of the MBBR-MR was assessed based on its chemical oxygen demand (COD) removal efficiency and membrane fouling mechanisms. The study was carried out using pilot-scale MBBR and bench-scale batch membrane filtration setups (Millipore Inc. Bedford, MA). The pilot MBBR had a working volume of 1.8 m3 and a 30% carrier fill fraction. The MBBR was operated with loading rates of 160 ± 44 g/m2/d (hydraulic residence time (HRT) of 4.6 h) and 223 g/m2/d (HRT of 2.6 h). The MBBR feed was obtained from a starch recovery line in a potato chip processing factory. The carriers were mixed by coarse bubble aeration and the dissolved oxygen (DO) was maintained above 2 mg/l. Preliminary jar test trials (based on turbidity removal) were performed in order to obtain an optimal dosage of coagulants for subsequent ultrafiltration (UF) tests. The efficiency of three coagulants (alum, ferric chloride and a blend of polyaluminum chloride and polyamine) was evaluated. The membranes were composed of polyethersulfone (PES) and had a pore size of 0.05 microns. The results of this study indicate that the combination of MBBR with membrane filtration can be operated at relatively high loading rates to yield a constant high quality permeate that is suitable for water reuse purposes. Fouling of the membrane by the wastewater was found to be substantially reduced by treatment with the MBBR. The reversible and irreversible fouling of the MBBR effluent were 56 and 63%, respectively, of that observed with the raw wastewater. The MBBR Loading-rate was found to affect treatment efficiency of the MBBR-MR and membrane performance. Operation under the elevated loading-rate conditions HRT = 2.6 hours) resulted in an increase in the irreversible fouling of the membranes (60% on average). The addition of all the coagulants in this study was found to decrease the fouling of the membrane. However, the extent of the pre-coagulation effect on membrane fouling was found to strongly depend on the type and dosage of the coagulant and the MBBR effluent characteristics. All the coagulants were effective in decreasing membrane fouling at their optimal dosages which was determined in preliminary jar tests. Ferric chloride performed the best as a pretreatment coagulant compared to alum (Aluminum sulfate) and the coagulant blend with reductions in both reversible and irreversible fouling (43-86% and 51-71%, respectively) and increased consistency (in decreasing fouling) as compared to the other coagulants. Alum had no effect on irreversible fouling and the coagulant blend significantly increased irreversible fouling in some trials (up to 196% or by a factor of 3 when overdosed). Additionally, alum and the blend were, on average, 29% and 7%, less effective than ferric chloride in reducing reversible fouling under the conditions and dosages tested.

<p>This thesis evaluates the performance of a deammonification system designed as a two-step tech-nology consisting of an initial partial nitritation followed by an Anammox process. Operation of a technical-scale pilot plant at the Himmerfjärden Wastewater Treatment Plant (Grödinge, Swe-den) has been assessed. Oxygen Uptake Rate (OUR) to evaluate the respiration activity of nitrifi-ers in the system and batch tests to assess reaction rates have also been applied in the study. It was found that the total inorganic nitrogen elimination strongly depended on the nitrite-to-ammonium ratio in the influent to the Anammox reactor, which was correlated with the per-formance of the partial nitritation phase. Therefore, a control strategy for oxidation of ammo-nium to nitrite has been proposed. Controlled oxygen supply to the partial nitritation reactor is obligatory to obtain a proper pH drop indicating oxidation of ammonia to nitrite at the adequate ratio. A very high nitrogen removal efficiency (an average of 84%) and stable operation of the system have been reached. Conductivity measurements were also used to monitor the system influent nitrogen load and the nitrogen removal in the Anammox reactor. The data gathered from the operation of the pilot plant enabled the use of multivariate data analysis to model the process behaviour and the assessment of the covariances between the process parameters. The options for full-scale implementation of the Anammox systems have been proposed as a result of the study.</p>

Bicyclic dihydrothiazolo fused 2-pyridones have been studied as a new class of antibacterial agents, termed pilicides, that target the formation of adhesive bacterial surface organelles (pili) in pathogenic bacteria. Synthetic methods to further functionalize the bicyclic 2-pyridone scaffold have been developed in order to increase water-solubility and thereby facilitate biological evalua-tions. This was accomplished by introducing aminomethylenes at the open position C-6. Tertiary amines were introduced via a microwave–assisted Mannich reaction and a synthetic route based on a formyl intermediate gave access to primary, secondary and tertiary amines, but also to other interesting functionalities. Biological evaluation confirmed that several of the function-alized compounds inhibited pili formation in uropathogenic E. coli., as dem-onstrated by assays of hemagglutination, biofilm formation and adherence to bladder cells. Co-crystallizing one of the pilicides with the target protein gave information about the binding site and based on this a mechanism of action was proposed, which was supported experimentally by surface plas-mon resonance and single point mutations in the protein. Furthermore, the previously developed acylketene imine reaction used to prepare bicyclic thiazolo fused 2-pyridone pilicides has been developed to allow preparation of other ring-fused 2-pyridone systems. Benzo[a]quinolizine-4-ones and indolo[2,3-a]quinolizine-4-ones could be prepared in a fast and simple manner starting from dihydroisoquinolines and a β-carboline. Finally, this method could also be applied for the preparation of heteroatom analogs of the previously studied sulfur containing pilicides. Biological evaluations established that the sulfur atom can be replaced by oxygen and still maintain the ability to prevent pili assembly.