Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and found in drinking water distribution systems and household plumbing. They are opportunistic pathogens of humans, causing lung disease. Their ability to adhere and form biofilm is attributed to a waxy, lipid-rich, cell envelope. This highly hydrophobic envelope also contributes to the characteristic antibiotic-, chlorine-, and disinfectant- resistance of NTM. NTM in household plumbing reside primarily in biofilms and the ability to form biofilm has been linked to virulence. Shedding of cells from biofilm and the subsequent aerosolization of microorganisms through showerheads presents a significant public health risk, particularly to those individuals with associated risk factors. Three species of NTM, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus, were examined for adherence and biofilm formation on surfaces common to household plumbing systems, including glass, copper, stainless steel, polyvinyl chloride, and galvanized steel. All experiments were conducted with sterile, Blacksburg tap water in a CDC Biofilm Reactor. Highest adherence was observed by M. avium on galvanized steel surfaces, reaching 15,100 CFU/cm2 surface at 6 hours incubation at room temperature. After 3 weeks incubation at room temperature, biofilm formation of M. avium was also highest on galvanized steel surfaces, reaching 14,000,000 CFU/cm2 surface. Lowest adherence was observed by M. abscessus on polyvinyl chloride (PVC) surfaces, reaching 40 CFU/cm2. Lowest biofilm formation was observed by M. intracellulare on glass surfaces, reaching 5,900 CFU/cm2. Surfaces, such as galvanized (zinc), on which high adherence and biofilm formation was observed, should be avoided in household plumbing systems of NTM patients and individuals at risk for developing NTM disease. Additionally, surfaces such as copper, harbor fewer NTM and may provide a safer alternative for household plumbing of NTM patients. / Master of Science

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Dans un procédé de filtration, un fluide traverse une membrane (barrière sélective). Une force motrice s’applique entre les deux côtés de la membrane qui peut être un gradient de pression, température ou un potentiel électrique/chimique. Dans les procédés de filtration par un gradient de pression, certains composés du milieu fluide, traversent la membrane alors que d’autres sont retenues sur la surface membranaire. Ces procédés sont très utiles dans différents domaines de l’industrie, notamment en ce qui concerne le traitement des eaux et des effluents, biotechnologie, agroalimentaire et pharmacie. En plus les procédés de filtration offrent des installations plus compactes avec une optimisation des coûts opérationnels comparant avec des procédés traditionnels de séparation notamment distillation et cristallisation. Par ailleurs, ces procédés se réalisent en absence des additifs chimique et changement de la phase. Dans cette étude, on se focalise sur les procédés de microfiltration. L’inconvénient principal de ces procédés est l’accumulation continue de particules/molécules sur la surface de la membrane. Ceci affecte la sélectivité de la membrane, modifie la qualité et la quantité de liquide passant à travers la membrane et conduit à une augmentation des coûts et de l’énergie. Le Colmatage (encrassement) membranaire se produit dans tous les types de procédés membranaires et par conséquent est connu le principal obstacle à l’utilisation répandue de ces procédés. Différentes techniques sont utiles pour surmonter les effets de l’encrassement de la performance de la membrane: le traitement physico-chimique des membranes utilisées, la modification des conditions opératoires (flux tangentiel de la solution d’alimentation sur la surface de la membrane est souvent appliqué pour réduire au minimum l’accumulation de particules), l’utilisation de membranes moins sensibles au colmatage, etc. Tout dépendant de la nature des solutions traitées, les particules déposées sont très variables. Les micro-organismes, des matières organiques naturelles notamment les protéines, les polysaccharides, les substances humides, les oxydes inorganiques et les sels contribuent au colmatage des membranes. Dans les dernières années, un grand nombre d’études expérimentales ont été investis pour comprendre les mécanismes de colmatage. Il a été souligné que les propriétés physico-chimiques de la membrane, la chimie des solutions et les conditions opératoires sont les trois principaux facteurs influant sur les mécanismes de colmatage. En parallèle, les modèles théoriques ont été proposés pour confirmer / décrire les observations expérimentales. La modélisation du colmatage membranaire est un outil essentiel pour évaluer les mécanismes qui le causent. Il permet également prédire la performance du système de filtration et par conséquent trouver des stratégies adaptées pour empêcher la modification de la performance membranaire pendant le procédé de filtration. En général, les modèles de classifient en deux grandes catégories: les modèles de transport de masse qui se concentrent sur le transport de solutés dans le procédé de filtration, et les modèles de colmatage basés sur le blocage des particules/molécules sur la surface ou à l’intérieur de la membrane. Dans la plupart des cas, les modèles dépendent fortement des paramètres empiriques ou semi-empiriques et restent phénoménologique. 1. Avoir une meilleure compréhension des mécanismes du colmatage membranaire lors de la filtration d’un milieu liquide contenant les micro-organismes en suspension. Il est important de souligner que des eaux industrielles et des eaux usées dans plusieurs domaines appartiennent à ce type d’effluents. 2. Proposer un modèle macroscopique décrivant les mécanismes de colmatage observés. [...] / During a membrane filtration process, a liquid medium is filtered through a membrane(selective barrier). The applied driving force between two sides of the membrane can be a gradient of pressure, temperature or a chemical/electrical potential.In pressure driven filtration processes (application of a pressure gradient as driving force between two sides of the membrane), certain components of the liquid medium pass through the membrane, while others are retained at the membrane surface. These processes are widely used as separation techniques in different industrial fields like waste water treatment, biotechnology, food and pharmacy. Compared to conventional techniquesof separation (distillation, crystallization, ...), membrane processes offer more compact installations with more optimized operational costs. Moreover, membrane processes are mainly performed in absence of chemical additives and phase change. In this work we focus on the pressure-driven microfiltration membrane processes.The main disadvantage of these processes is the continuous accumulation of particles on the membrane surface. This affects the membrane selectivity, modifies the quality and the quantity of the liquid passing through the membrane and leads to an increase of energy costs. Membrane fouling occurs in all types of membrane processes and therefore is known as the major obstacle for widespread use of these processes. Different techniques are used to overcome the effects of fouling on the membrane performance : physical-chemical treatment of used membranes, modification of the operational conditions (tangential flow of the feed solution to the membrane is often applied for minimizing the particle accumulation to the membrane surface), use of membranes less susceptible to fouling, etc. Depending on the nature of the treated solutions, the deposited particles are highly variable. Microorganisms, natural organic matter such as proteins, polysaccharides, humid substances, inorganic oxides and salts contribute notably to membrane fouling.It should be noted that membrane fouling problem is a multi-physics (hydrodynamics,mass transport, physics, chemistry), multi-scale (different length scales are involved:molecules, pores and membrane surface) and time dependent (evolution of the membrane microstructure and the molecule-surface interactions) phenomena.In the last decades, a huge number of experimental studies have been invested to understand fouling mechanisms. It has been pointed out that membrane physicochemical properties, solution chemistry and operational conditions are the three major factors affecting the fouling mechanisms. In parallel, theoretical models have been proposed to confirm/describe the experimental observations.Modeling of membrane fouling is an essential tool for assessing the fouling mechanisms. It helps predicting the membrane performance and consequently finding adapted strategies to prevent their modification during the filtration process.In general, the models can be classified into two main categories: mass transport models which focus on solute permeation during the filtration process, and fouling models based on particle or solute blocking within the membrane porous structure. In most of the cases, models depend strongly on the empirical or semi-empirical parameters and thus remain phenomenological. Two main objectives have been set for the present work: 1. Get a better understanding of the membrane fouling mechanisms during filtration of a liquid medium containing suspended microorganisms. It should be pointed out that several Industrial streams and wastewaters belong to this kind of effluents.2. Propose a macroscopic model describing the observed fouling mechanisms. [...]

Les bactéries sont des microorganismes capables de se développer et de proliférer indépendamment les uns des autres en milieu liquide. Mais dès qu’une surface se présente, biotique ou abiotique, les bactéries privilégient un « mode de vie en communauté » pour se protéger des agressions externes et survivre aux environnements hostiles. Ces biostructures, appelées biofilms, sont présentes dans tous les environnements naturels, y compris chez l’Homme où elles peuvent être à l’origine d’infections chroniques lorsqu’elles hébergent des germes pathogènes. Il est aujourd’hui admis que de tels édifices biologiques perdurent sous l’action des antibiotiques. Outre le très médiatique phénomène de résistance qui trouve son origine dans des mutations génétiques bactériennes, la tolérance, quant à elle, provient des spécificités de la structure et de la physiologie des bactéries organisées en biofilms. C’est dans ce contexte que s’inscrit ce travail de thèse qui vise à mieux comprendre les mécanismes sous-jacents au manque d’efficacité d’antibiotiques (vancomycine,daptomycine, rifampicine) vis-à-vis des biofilms de S. aureus, en s’appuyant notamment sur des techniques innovantes d’imagerie à résolution micro-nanométrique. Nous avons mis au point un modèle d’infections sur prothèse vasculaire implantable chez la souris qui a permis une toute première visualisation par imagerie de fluorescence de biofilms formés in vivo et soumis à l’action des antibiotiques mais aussi de montrer leur activité limitée. Nous nous sommes ensuite attachés à une meilleure compréhension de la tolérance aux antibiotiques de biofilms bactériens de S. aureus. Pour ce faire, nos études ont porté, d’une part, sur le rôle de la matrice extracellulaire et, d’autre part, sur le rôle de la physiologie des bactéries incluses en biofilm. Il a ainsi été mis en évidence le rôle crucial de la fluidité membranaire. Ces travaux nous ont alors permis de dégager des pistes pour améliorer l’antibiothérapie disponible mais aussi développer des alternatives à ce type de traitement. / Bacteria are microorganisms capable of growing independently in liquid media. However, as soon as they encounter a surface, either biotic or abiotic, bacteria favour a "community living" to protect themselves from external aggressions and survive in hostile environments. These bacterial communities, named biofilms, are present in all natural environments, including humans where they can cause severe infections when hosting pathogenic germs. It is now accepted that such biological edifices persist under antibiotics action. In addition to antibiotic 'resistance', which is associated with genetic mutations of bacteria, 'tolerance' is related with the specific structure and physiology of bacteria organized in biofilms. In this context, we took benefit from innovative high-resolution imaging techniques to better understand the mechanisms underlying antibiotics (vancomycin, daptomycin, rifampicin) (in)efficacy within S. aureus biofilms. In addition, we developed a model for prosthetic vascular graft infections in mice that allowed the visualization by fluorescence imaging of biofilms formed in vivo and subjected to the action of antibiotics. Considering the very limited antibiotics efficacy observed, we then focused on a better understanding of S. aureus bacterial biofilms tolerance towards antibiotics. To this purpose, our work was focused on the role of both the extracellular matrix and the physiology of bacteria included in biofilms. The crucial role of membrane fluidity was then demonstrated. This work allowed us to identify paths for the improvement of antibiotic therapy and to develop alternatives to this type of treatment.

published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

There is a need to develop a mathematical expression capable of describing the removal of particulate chemical oxygen demand (PCOD) from wastewaters in biological film systems. In this context, organic particles that are maintained in suspension (i.e., not removed during normal settling) are the focus of experimentation, modeling, and discussion. The goal of this research project is to study the kinetics of PCOD removal from wastewaters by bacterial films, or biofilms. To achieve this objective, a bench-scale rotating disc biofilm reactor (RDBR) was operated using methanol (dissolved substrate), Min-U-Sil 10 (inorganic particulates), and Maizena corn starch (organic particulates) dissolved/suspended in the influent stream. The effect of the ratio of biofilm area to volumetric flow rate passing through the RDBR on the concentration of substrate remaining in the final effluent was determined, and the kinetic relationship was established for both dissolved substrate and particle removal. Exocellular polymeric substances (EPS) were extracted and quantified in order to explain the role of biological flocculation, or bioflocculation, in particulate removal. In the literature, Fick's first law and zero-order kinetics have described the diffusion and biochemical reaction of soluble substrate within the bacterial film matrix (when completely penetrated), respectively. The present study confirms this kinetic behavior for various influent methanol concentrations. On the other hand, the removal of particulates, organic and inorganic, adheres to first-order reaction kinetics. These findings, coupled with the identification of EPS, attribute bioflocculation as the primary removal mechanism of particulates. A mass balance on the biofilm reactor allowed for the development of a comprehensive rate expression for substrate consumption by biofilms when both dissolved and particulate substrates are available. Total chemical oxygen demand (TCOD) is comprised of dissolved chemical oxygen demand (DCOD) and PCOD, each of which can be readily determined through laboratory analysis. An equation was developed that accurately describes the disappearance of TCOD by the bioflocculation of PCOD and consumption of DCOD in the bench scale RDBR.