published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

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This thesis relates to the mathematical modelling of biofilm in two primary areas: biofilm growth, and the effect of microbial immobilisation in biofilm on environmen- tal contaminant transport in the Earth's subsurface. For biofilm growth we construct a model based on polymer solution theory. Parameter estimates motivate a very different model from two published biofilm models also based on polymer solution theory. Analysis of ID solutions provides an expression for growth rate suitable for comparison with experiment. Stability analysis of spatial perturbations to a growing planar front reveals an interfacial instability mechanism similar to that found in a published theoretical study not based on a specific material model. We derive a stability criterion as a critical external nutrient boundary layer thickness, and for the travelling wave solution we identify the finite perturbation wavenumber selected by the instability. For environmental contaminant transport, we identify dissolution of organic phase contaminants and sorption of hydrocarbons onto solid grains as primary lim- itations on bioavailability. We build a pore scale model including both organic phase dissolution and micro- bial uptake and use it to parameterise pore scale Sherwood and Damkohler numbers with respect to pore Peclet number. We illustrate their relation to effective macro- scopic parameters for varying organic phase size relative to pore size. A simple intraparticle diffusion sorption model is extended by considering an external biofilm layer on the particles. A larger scale model considers contaminant transport in a ID flow through a bed of such particles. A physically reasonable pa- rameter regime is suggested, providing analytical solutions for breakthrough curves.

Enterococci are opportunistic pathogens that are frequently a source of nosocomial infections and it is their resistance to antibiotics and their ability to form biofilms that represent important virulence traits. Normally, in healthy individuals it is a harmless commensal that is usually found in the intestine. This thesis firstly studies signal peptidases (SPases), which play an essential role in protein translocation. Interestingly, E. faecium was found to contain three type I SPases. Many proteins that are secreted are virulence factors, and the aim was to delete one or more of the SPases and study the effect of its removal on virulence. Unfortunately no mutants were obtained suggesting that the genes were essential. To establish if the genes were essential an inducible integration vector was constructed, but due to time constraints this could not be tested further. Biofilm formation was studied in both E. faecium and E. faecalis. The presence of the Enterococcal Surface Protein (Esp) in E. faecium was shown to increase hydrophobicity, and therefore also increase biofilm formation. Similarly, E. faecalis isolates that were good biofilm formers were also more hydrophobic in nature. The expression of Esp in E. faecium was studied under different conditions; these studies indicated that the highest level of Esp expression was found in biofilms cells. This growth-dependent manner Esp expression was not observed in E. faecalis BS12297. Surprisingly, Esp in E. faecium was also shown to have a role in ampicillin resistance, which was identified using calorimetry. This method proved to be a sensitive and rapid method to analyse antibiotic resistance. In the gut, bacteria encounter various adverse conditions, such as low pH and the presence of bile salts. Here we investigated the effects of bile salts on biofilm formation in E. faecium and E. faecalis and demonstrated that biofilm formation is induced at physiological concentrations of bile salts. In E. faecium the presence of bile salts caused an increase in initial attachment, microcolony formation and EPS production. Various factors were investigated, including hydrophobicity, cell growth, cell morphology, Esp expression and the production of extracellular polymeric substance (EPS). In E. faecium, only EPS production appeared to play a role, but the stimulation of biofilm formation due to bile salts is still to be fully explained.

Most marine invertebrates have a complex life cycle involving a benthic adult phase, and a planktonic larval phase. The process whereby tiny larvae are able to locate, settle and metamorphose in a habitat where juveniles are then capable of successfully establishing themselves is a key stage in the life cycle and a central theme of current marine research. Bacterial biofilms are an important settlement cue for many larvae, and it appears that particular strains within environmental communities may be responsible for the inducing ability of some biofilms. The focus of this thesis is the importance of biofilms for larval settlement of the blacklip abalone Haliotis rubra and the sea urchin Heliocidaris erythrogramma. Larval development of the sea urchin Centrostephanus rodgersii was also examined, but this species was problematic for a larval settlement study. H. rubra larval settlement occurred in response to several macroalgal species. Biofilmed (but otherwise abiotic) surfaces did not induce settlement of H. rubra larvae and reduction of surface films of bacteria and diatoms on inducing algae did not reduce the settlement response. Macroalgae, particularly green algal species, may play an important role in the recruitment of H. rubra larvae in the field and can be used to induce settlement in hatcheries. H. erythrogramma settled in response to a range of surfaces with highest settlement on coralline algae. Settlement was reduced by autoclaving plants and treating plants with antibiotics. Molecular and culture based analysis revealed a shift in microbial community structure between plants treated with antibiotics and unmanipulated plants. Many bacterial strains, dominated by the genera Pseudoalteromonas, Shewanella and Vibrio, induced larval settlement. Three probes targeting Pseudoalteromonas, Shewanella and Vibrio were developed for CARD-FISH, enabling quantification of these genera in biofilms on algae. The three genera were found in highest numbers on coralline algae and in variable numbers on the surfaces of other algae. Recruitment of H. erythrogramma also occurs in highest numbers on coralline algae, and in low amounts on co-occurring algae. This is the first example demonstrating that bacteria that induce settlement in the laboratory are also present in the juvenile recruitment habitat.

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Thomas S. Livinghouse. Includes bibliographical references (leaves 76-82).

La mucoviscidose reste une maladie génétique grave sans traitement curatif. L’amélioration de la prise en charge de la maladie s’est accompagnée d’une augmentation importante de l’espérance de vie qui était de moins de un an dans les années 1950 et dépasse en moyenne 40 ans pour les enfants nés aujourd’hui. La mucoviscidose se caractérise au niveau pulmonaire par un mucus anormalement épais qui est difficilement évacué. Le mucus s’accumule alors dans les voies respiratoires et les bactéries y persistent et s’y multiplient. 80 à 95% des patients décèdent des suites de l’insuffisance respiratoire causée par les infections chroniques bactériennes concomitantes aux inflammations des voies aériennes. Les patients souffrent particulièrement d’infections à P. aeruginosa qui est la bactérie la plus fréquemment rencontrée dans la mucoviscidose. Lors des premières infections à P. aeruginosa les bactéries sont de phénotype non mucoïde et sont relativement sensibles aux antibiotiques. Les bactéries finissent par s’adapter dans les voies aériennes des patients atteints de mucoviscidose et forment des biofilms multiresistants aux antibiotiques. Les patients sont alors colonisés par P. aeruginosa de façon permanente. Les infections chroniques à P. aeruginosa sont responsables du déclin de la fonction respiratoire, d’exacerbations pulmonaires nécessitant une hospitalisation et de la mortalité des patients atteints de mucoviscidose. Il est donc urgent d’améliorer les traitements symptomatiques existants dans le but d’augmenter la qualité et l’espérance de vie des patients en attendant de nouveaux traitements curatifs visant à corriger l’anomalie génétique. Ce travail s’est donc orienté vers l’étude de l’efficacité in vitro de différentes combinaisons d’antibiotiques vis-à-vis des biofilms bactériens. Parmi l’ensemble des associations d’antibiotiques testées une synergie d’activité a été observée entre la tobramycine et la clarithromycine sur des biofilms âgés de 24 heures mais aussi de 12 jours. Notamment parmi les 26 souches testées une synergie d’action est observée chez 11 souches après une coadministration pendant 28 jours de tobramycine et clarithromycine en doses répétées sur un biofilm « mûr » âgé de 12 jours. Parmi ces 11 souches, 7 biofilms sont entièrement détruits à la fin du traitement. Sur l’ensemble des souches étudiées le traitement avec l’association est soit supérieur ou équivalent à celui de la tobramycine seule et a permis d’obtenir une action dans 53.8% des cas pour la combinaison versus 30.8% pour la tobramycine. Cependant aucune corrélation n’est trouvée entre le profil génétique, le phénotype mucoïde ou non des souches et la présence de synergie d’action ou la destruction entière du biofilm. Cette dernière étude s’inspire du traitement actuel de référence TOBI® qui s’administre par cycle de 28 jours, 2 fois par jour. Les différents travaux de cette thèse ont permis tout d’abord de développer un modèle in vitro proche des conditions rencontrées in vivo chez les patients atteints de mucoviscidose. Ce modèle pourra également être utilisé pour une série d’autres études et mises au point de nouveaux médicaments sous forme de molécule isolée ou en combinaison. Ce travail a également permis d’envisager le développement d’un nouveau médicament dans le traitement d’infections chroniques à P. aeruginosa chez les patients atteints de mucoviscidose. Cependant des études futures in vivo sur souris mais aussi toxicologiques et cliniques seront indispensables pour confirmer le profil d’efficacité et de sécurité de cette association.