Streptococcus mutans is a secondary colonizer of the dental plaque biofilm and is the primary causative agent of dental caries. Sugar metabolism is central to S. mutans growth and survival. S. mutans produces lactic acid as an end product of sugar metabolism, which results in dissolution of the tooth enamel, leading to dental cavities. Sucrose metabolism also results in the formation of extracellular dextrans that are a key component of the extracellular matrix that encases the bacteria in the biofilm. The availability of sugars is dependent on diet, on competition with other bacteria and on the location of the bacteria within the dental plaque. I hypothesize there are distinct subpopulations of S. mutans within biofilms that respond differently to environmental conditions. I have identified several genetic markers that are helping us identify and characterize some of these subpopulations, and how they react to starvation and to the restoration of nutrients in single species biofilms of S. mutans. Two of the loci that were identified as markers via microarray analysis are rpsT and pdh. rpsT encodes a small ribosomal protein which is strongly expressed during exponential growth, when the cells are producing high levels of ribosomes. The other marker, pdh, is a four-gene operon encoding the pyruvate dehydrogenase complex; pdh is upregulated in late stationary phase. Our laboratory has recently shown that expression of the pdh operon is important for long-term survival in stationary phase, where a subpopulation (~0.5%) is dividing, forms long chains and expresses pdh. In the current studies, rpsT and pdh promoters driving expression of gfp were used to identify the exponential phase subpopulation (rpsT) and a subpopulation capable of surviving in late stationary phase (pdh). In addition, I developed an unstable variant of GFP by fusing a proteolytic tag sequence to the C-terminus of GFP (encoded by ugfp). When the rpsT promoter was inserted upstream, uGFP was produced and subsequently degraded within about 1.5 hours of translation. This behavior allowed us to distinguish exponentially growing cells, as the signal diminishes once the cells entered stationary phase. In biofilms that had been starved for 10 days, there was no expression of PrpsTugfp. I observed that when sucrose was added to these biofilms, some bacteria within the biofilm microcolonies underwent fast exponential-like growth indicated by expression of PrpsT-ugfp. Within 24 hours of the sucrose addition, most growth had ceased and fluorescence had decreased. Using a Ppdh-gfp construct in bacteria in 10-day starved biofilms, fluorescence was observed in long chains of cells within the biofilms indicating slow growth. I hypothesized that the pdh-expressing cells were capable of responding to sucrose restoration and would be one of the principal subpopulations to do so. However, when sucrose was added, these fluorescing chains did not exhibit any growth, while other non-fluorescing bacteria within the biofilm clearly responded to the sucrose by growing. This was unexpected since inactivating the pdh operon leads to drastically reduced survival. It is concluded that pdh plays a role in long term survival, but pdhexpressers do not appear to respond to sugar restoration. This led me to hypothesize that the pdh-expressing population is interacting with other populations of cells in some capacity, enabling them to survive. To determine if this was the case, we performed a mixed culture experiment with wild-type S. mutans and the pdh null mutant. I observed that when these two strains were grown in co-culture, the pdh null mutant survived at low levels, for over 30 days, while this mutant by itself typically did not survive past ten days. This result indicates that the wild-type strain was able to interact with the mutant, leading to increased survival. In biofilms, it seems possible that the pdh-expressing cells secrete a substance or directly interact with other cells, somehow promoting their survival in the starved biofilm. The fluorescent constructs appear to mark distinct populations of cells that respond in different ways to sugar availability, suggesting that S. mutans forms a mixed population of cells able to grow in the presence of sugar or survive prolonged sugar starvation. These studies demonstrate that indeed subpopulations of cells do exist within biofilms, and their interactions may be more complex than previously thought. / Microbiology and Immunology

Salmonella enterica serovar Typhimurium is an important cause of gastroenteritis in the United States and the developing world. Biofilm growth is an significant mechanism, which S. Typhimurium utilizes to contaminate food products and survive in the environment. Biofilms are also an important part of the infectious process for many pathogenic bacteria. As part of the biofilm, S. Typhimurium produces an extracellular matrix consisting of cellulose, extracellular DNA, and most importantly, the amyloid protein curli. Similar to amyloids associated with human diseases, curli is recognized by the innate immune system through Toll-Like Receptors (TLRs). Here, we studied the immune receptors recognizing curli as well as interactions between eDNA and curli during biofilm development in order to glean a better understanding of these complex bacterial communities and the immune response to them. Recently, our lab demonstrated that curli fibers are recognized by the TLR2/TLR1 complex. CD14 has been shown to be a common adaptor protein for TLR2/TLR1 complex in response to one of its ligands, tri-acylated lipopeptide, Pam3CSK4. In order to study the role of CD14 in the immune receptor complex recognizing curli, we utilized HeLa 57A cells, a human cervical cancer cell line that has a stably transfected luciferase reporter for Nf-κB activation. When these cells were transiently transfected with TLR2 and TLR1 together or with the addition of membrane-bound CD14, NfκB activation was enhanced by the presence of CD14 in response to purified curli, GST-tagged curli subunit (GST-CsgA), and the control lipopeptide Pam3CSK4. Soluble CD14 also increased NfκB activation in response to purified curli. Bone marrow derived macrophages (BMDM) from wild type (C57BL/6) mice produced more IL-6 and nitric oxide in response to stimulation with purified curli, GST-CsgA, and Pam3CSK4, than BMDMs deficient in CD14. Binding assays demonstrated direct binding of curli to all members of this hypothesized trimolecular complex, TLR2, TLR1, and CD14. Utilizing synthetic peptides corresponding to the fourth and fifth repeat of the CsgA monomer, CsgA R4-5, and its modified version, CsgA R4-5N122A deficient in forming amyloid fibers, we also showed that binding to CD14, and CD14 enhancement of IL-6 production required the fibrillar amyloid structure of curli. To study interactions between curli and eDNA in biofilms and the resulting immune response generated to composites formed by these ECM components, we analyzed biofilms of GFP expressing S. Typhimurium using confocal laser scanning microscopy (CLSM). Staining for amyloids with Congo Red revealed the presence of curli in the biofilms and staining with propidium iodide demonstrated the presence of extracellular DNA in the biofilms. Co-staining with TOTO-1, a nucleic acid stain, and Congo Red showed co-localization of the fluorescent signal for these molecules within the biofilms. DNase I treatment of the biofilms produced no significant change in biofilm thickness by confocal microscopy signifying that the biofilm, possibly eDNA, was resistant to DNase treatment. This was further confirmed by the presence of DNA in purified curli fibers, which were treated twice with DNase and RNase. Polymerization assays showed acceleration of amyloid polymerization in the presence of DNA from both bacteria and salmon sperm. CLSM of bone marrow derived dendritic cells demonstrated that DCs are able to sample antigens from biofilms. BMDCs also produced robust quantities of proinflammatory cytokines in response to wild type, msbB, and ΔfliCfljB S. Typhimurium biofilms and purified amyloid/DNA composites as measured by ELISA. Using BMDCs deficient in TLR2 and TLR9, we found that this cytokine production was partially dependent on TLR2, but did not require TLR9. Together, these findings significantly broaden our understanding of S. Typhimurium biofilms and the immune response to ECM components present in its biofilms. We now understand that a trimolecular complex of TLR2/TLR1/CD14 is required for full response to curli by innate immune cells. We also discerned that interactions between biofilm components aid biofilm development and create composites that are highly immunogenic. This new information enhances the need to explore the interaction between composite ligands and the immune system rather than only studying ligands individually. / Microbiology and Immunology

The type IV pilus is a motility organelle found in a range of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The principle structural protein of the pilus is the major pilin, PilA, while a set of low abundance “minor pilins” are proposed to constitute the pilus tip. The minor pilins, FimU and PilVWXE, along with the non-pilin protein PilY1, prime assembly of surface-exposed pili. The fimU-pilVWXY1E operon is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is an adhesin and mechanosensor that, along with PilW and PilX, triggers virulence upon surface attachment. Here, we aimed to uncover the mechanism for PilWXY1-mediated virulence. We hypothesized that loss of PilWXY1 would relieve feedback inhibition on FimS-AlgR, resulting in increased transcription of the minor pilin operon and dysregulation of virulence factors in the AlgR regulon. Caenorhabditis elegans slow killing assays revealed that pilW, pilX, and pilY1 mutants had reduced virulence relative to a pilA mutant, implying a role in virulence independent of pilus assembly. FimS-AlgR were required for the increased promoter activity of the minor pilin operon upon loss of pilV, pilW, pilX, or pilY1. Overexpression or hyperactivation of AlgR by point mutation led to reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression. We propose that PilWXY1 inhibit their own expression at the level of FimS-AlgR, such that loss of pilW, pilX, or pilY1 leads to FimS-mediated activation of AlgR, and reduced expression of acute-phase virulence factors. Accumulation of mutations in the minor pilin operon may represent an evolutionary strategy for P. aeruginosa populations in chronic lung infections, as loss of PilWXY1 would upregulate the expression of AlgR-dependent virulence factors – such as alginate – characteristic of such infections. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes dangerous infections, including lung infections in cystic fibrosis patients. The bacteria use many strategies to infect their hosts, one of which involves a grappling hook-like fibre called the type IV pilus. There are many components involved in assembly and function of the pilus, including five proteins called “minor pilins” and a larger protein called PilY1 that may help the pilus detect surface attachment. We used a roundworm infection model to show that loss of PilY1 and specific minor pilins leads to delayed killing, while loss of other pilus proteins has no effect on worm survival. This effect was due to increased activation of a regulatory system called FimS-AlgR that inhibits expression of other factors used by this bacterium to infect its hosts. By studying how P. aeruginosa causes infection, we can design better strategies to disarm it and reduce the severity of infections.

<p> A novel mathematical model has been developed for biofilms and bioflocs. The model is based on the use of the effectiveness factor and the effect of cell density is included. The key assumption in the model is that cell density decreases in proportion to the substrate concentration within the biofilm or biofloc, reflecting lower rates of cellular metabolism. The equations given by the model were solved numerically for three types of reaction kinetics: Monod, Andrews (substrate inhibition), and multiple-Monod (twolimiting substrates), as well as for two geometries: a slab, as a representation of a biofilm and a sphere, as a representation of a biofloc. The simulations indicate that a decrease of the cell density in the biofilm and biofloc results in a decline of the effectiveness factor. Furthermore, the analytical solutions and approximate analytical versions of the effectiveness factor for the biofilm in two cell growth models: Monod and Andrews, have been derived. The effectiveness factors derived analytically are in agreement with those calculated numerically, and the approximate analytical versions are valid for the Thiele modulus greater than five. This new model was tested using operational data available in the literature, by including the effectiveness factor as a part of the design equations for an upflow anaerobic sludge blanket (UASB) reactor. </p> <p> For any biologically mediated transformation, it is critical to uniquely identify the parameters associated with microbial growth models. In this study, it is proved that the parameters of the integrated Andrews model are identifiable if the experimental data does not contain any random noise based on a criterion proposed by Beck and Arnold [1977]. When noise is present, the parameters may or may not be identifiable, depending on noise levels. A new approach has been developed based on the calculation of dimensionless sensitivity coefficients. Plotting these coefficients provides straightforward visualization of parameter identification. This method was used for quantitative evaluation of the noise level that can be associated with measurements, while still allowing parameter identification. It was demonstrated that an indirect cause of the parameter nonidentification of the integrated Andrews model is the linearization of the Andrews model at a low or high substrate concentration. Robinson [1985] obtained a similar result with the Monod model. </p> / Thesis / Master of Science (MSc)

Thesis advisor: Tim van Opijnen / Thesis advisor: Michelle Meyer / Although bacteria are often studied as planktonic or free-living organisms, they frequently grow in complex surface-attached communities known as biofilms. Biofilms are communities of microorganisms attached to surfaces and embedded in a self-produced extracellular matrix. Biofilms are dynamic structures analogous to human settlements shaped by space and environment. These microbial communities fulfill critical roles in multiple infections in the human body. Streptococcuspneumoniae is a human pathogen that can cause biofilm-associated infections in various tissues and organs. This thesis offers a unique outlook for the study of S. pneumoniae biofilms by combining in vitro, genome-wide, and in vivo experiments to elucidate the complex population dynamics of S. pneumoniae biofilms. Existing methods to cultivate S. pneumoniae biofilms fail to fully capture the complexity of these communities, and most studies are limited to short periods of time. We developed a robust in vitro assay to grow S. pneumoniae biofilms. This assay can be maintained forever rather than days. We then use this robust assay to study their behavior in vivo and monitor disease outcomes. After establishing clear differences in biofilm and dispersal samples, we monitor population dynamics using genome-wide techniques (Tn-seq, RNA-seq and WGS) to provide some insights into this complex mode of growth. This work includes the first global identification of genetic requirements during biofilm establishment in two different S. pneumoniae strains using Tn-Seq. Coupled with our transcriptomic analysis, we found that genes involved in multiple pathways, such as capsule biosynthesis, nucleotide metabolism, and stress response, contributed to biofilm growth. Lastly, we studied the development of antibiotic resistance to three different types of antibiotics under S. pneumoniae biofilm conditions. We revealed common adaptive pathways to achieve biofilm growth and antibiotic resistance (antibiotic target genes), as well as novel routes of adaptation to develop resistance. Our findings add to the growing body of knowledge in the field of bacterial genetics and antimicrobial resistance, paving the way for future research and therapeutic advancement. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Al igual que muchos ecosistemas costeros del mundo, diversos estudios realizados en el estuario de Bahía Blanca reflejan que este ambiente se encuentra impactado por el aporte de metales, originados por diversas actividades antropogénicas que se desarrollan en sus inmediaciones. Las planicies de marea estuarinas son ecosistemas costeros de gran importancia debido a su extensión, a los importantes servicios ecosistémicos que brindan y a que se encuentran dentro de los ecosistemas más productivos de la Tierra. La alta productividad biológica se basa principalmente en la actividad de las comunidades microfitobentónicas que, en las planicies de marea del estuario de Bahía Blanca, están representadas por biofilms y matas microbianas que colonizan su superficie. Varios autores han indicado la capacidad de estas comunidades para secuestrar potenciales contaminantes, entre ellos, los metales. El objetivo de esta tesis es evaluar la influencia de estas comunidades microbianas presentes en el área media (Puerto Rosales) e interna (Almirante Brown) del estuario de Bahía Blanca, en los procesos de captación y distribución vertical de estos contaminantes inorgánicos. Para ello, se analizó la distribución y dinámica de metales esenciales como Cr, Cu, Fe, Mn, Ni y Zn, y otros no esenciales como el Cd, Hg y Pb. Además, se analizaron variables secundarias como la influencia de las mareas, el contenido de materia orgánica, tamaño de grano y, en el caso de la mata microbiana, los parámetros fisicoquímicos temperatura, pH y potencial redox. El análisis de la información obtenida mostró que las matas microbianas fueron sensibles a la presencia de metales, dado que fueron capaces de concentrarlos aun cuando estuvieron en bajas concentraciones. Asimismo, las matas microbianas mostraron una mayor eficacia en el secuestro de metales respecto al biofilm que cubre los primeros milímetros de la misma. La influencia de las matas microbianas en la presencia de metales fue más notoria en la zona alta de Puerto Rosales, donde se registraron mayores concentraciones respecto a las capas ubicadas por debajo de la comunidad microbiana activa. Esto podría ser explicado por el mayor contenido de materia orgánica encontrado en las matas microbianas de esta zona, en conjunto con las características fisicoquímicas atribuibles a los períodos de exposición a las cuales se ven sometidas. La secreción de sustancias poliméricas extracelulares por parte de diatomeas y cianobacterias móviles como respuesta a un período de exposición mayor a 6 días registrado en ésta zona, sumado a la afinidad para unir metales que presenta la pared de las cianobacterias dominantes de estas matas microbianas epibentónicas, favorecería el secuestro de los mismos. La mayoría de los metales analizados presentaron mayores concentraciones en las capas subyacentes a la mata microbiana. viii Esto podría atribuirse a la influencia de los microorganismos presentes en esa profundidad (1 - 5 cm), así como a la presencia de sedimento de tamaño de grano fino. En cuanto al ambiente fisicoquímico de las matas microbianas, se encontraron diferencias entre los sitios evaluados, presentando Almirante Brown un pH alcalino, una mayor temperatura y un ambiente más reductor respecto a Puerto Rosales, lo cual podría implicar diferencias en la actividad microbiana de ambos sistemas. Por último, se encontró que Almirante Brown se encuentra bajo una mayor influencia por parte de los metales seleccionados para su evaluación en la presente tesis doctoral, dado que posee las mayores concentraciones promedio en seis de los nueve metales analizados (Cr, Cu, Fe, Ni, Pb y Zn). Ello podría estar relacionado a su proximidad a un ex basurero y la influencia de la descarga de efluentes cloacales con escaso tratamiento. / As in many coastal ecosystems in the world, diverse studies carried out in the Bahía Blanca estuary (referred to in this document as EBB, from its name in Spanish) reflect that this environment is impacted by the supply of metals originated in a variety of anthropogenic activities that are carried out in its surroundings. The tidal flats of estuarine tides are coastal ecosystems of great importance given their extension, the important ecosystemic services they provide, and because they are among the most productive ecosystems on Earth. The high biological productivity is mainly based in the activity of microphytobenthic communities that, in the tidal flats of the EBB, are represented by biofilms and microbial mats that colonize its surface. Various authors have indicated the capacity for these communities to sequester potential contaminants, such as metals. The goal of this thesis is to evaluate the influence of these microbial communities present in the middle (Puerto Rosales) and inner (Almirante Brown) areas of the EBB on the capture and vertical distribution processes of these inorganic contaminants. Towards this end, the distribution and dynamics was analyzed for essential metals like Cr, Cu, Fe, Mn, Ni and Zn, and other non-essential ones like Cd, Hg, and Pb. Furthermore, secondary variables were also analyzed, such as the influence of tides, the organic matter content, grain size and, in the case of the microbial mat, the physicochemical parameters of temperature, pH, and the redox potential. The analysis of the data showed that the microbial mats were sensitive to the presence of metals, given that they were capable of concentrating them even when they were present in low concentrations. Likewise, the microbial mats showed a greater efficacy in the sequestering of metals relative to the biofilm that covers its first millimeters. The influence of the microbial mats on the presence of metals was most significant in the high zone of Puerto Rosales, where greater concentrations were registered in relation to the layers situated below the active microbial community. This could be explained by the greater content of organic matter found in the microbial mats in this zone, in conjunction with the physicochemical features attributable to the exposure periods to which they are submitted. The secretion of extracellular polymeric substances by mobile diatoms and cyanobacteria as a response to a period of exposure greater than 6 days, recorded in this zone, as well as the affinity to bind metals exhibited by the wall in the cyanobacteria that dominate these epibenthic microbial mats, may favor their sequestering. The majority of the analyzed metals presented greater concentrations in the underlying layers in the microbial mat. This could be attributed to the influence of the microorganisms present at that depth (1 - 5 cm), as well as to the presence of sediment of fine grain size. As to the x physicochemical environment, differences were found between the evaluated sites, where Almirante Brown presents an alkaline pH, greater temperature, and a more reductive environment in relation to Puerto Rosales. That fact would imply differences in the microbial activity of both systems. Finally, Almirante Brown was found to be under a greater influence of the metals selected for evaluation. There is high mean concentrations in six of the nine analyzed metals (Cr, Cu, Fe, Ni, Pb, and Zn). This may be related to its proximity to a former garbage dump and the influence of the discharge of sewer discharges with low treatment.

Bacterial biofilms are communities of micro-organisms encased a self-produced extracellular matrix. While they form readily in a nature, biofilm formation in man-made systems have economic and health consequences. Prior research demonstrated that topographical features comprised of uniform, micro-meter sized particles hindered the biofilm formation of Pseudomonas aeruginosa (P. aeruginosa), an opportunistic human pathogen. The goal of the present work is to 1) further develop a potential anti-biofilm coating by improving its robustness and 2) study the mechanism(s) by which surface topography hinders biofilm formation. The robustness of a topographical coating comprised of an array of silica particles is improved by the introduction of silica bridges through a sol-gel reaction. To study the mechanism(s), specifically, we hypothesized that the motion, or surface motility, of P. aeruginosa is hindered by the presence of micro-meter scale obstacles via physical obstruction. To test this, we analyzed the behavior of single P. aeruginosa cells at micron-scale spatial resolutions using time-lapse fluorescence microscopy, image analysis, and particle tracking techniques. We fabricated various types of micron-scale topography with curvature (particle arrays) and recti-linear features (vertical steps) and varied the critical dimension within the range of 0.5 – 10 µm which spans the dimensions of a typical P. aeruginosa cell. We found that there was a threshold feature size of 1-2 µm at which bacterial surface motility is drastically impacted. On positively curved topography (particle arrays), we found that the frequent obstacles reduced the average speed of a bacterium from 6.2  0.3 µm per 5 min on a flat surface to 2.1  0.3 µm per 5 min on an array of 2 µm particles. Furthermore, we observed that bacteria often move in-between particles, suggesting that bacteria have difficulty climbing over tall obstacles. To further investigate P. aeruginosa's ability to cope with topography, we examined the effect of recti-linear features (vertical steps) on surface motility. We found that step heights > 0.9 µm drastically reduced the probability of crossing and that the average speed when approaching the step is reduced by a factor of 2. Interestingly, we find that bacteria have a slight preference to traverse down which is against the direction of gravity in our system. In summary, these results offer insights into how a surface motile bacterium copes with a topographical surface. Our data indicate that the topography of a surface can impede the surface motility of bacterium and thus, may be an important mechanism by which topography prevents biofilm formation. / Doctor of Philosophy / Bacteria and other micro-organisms can grow on surfaces such as medical devices and cause infections. Other examples of where bacteria can grow are on drains and pipes causing clogging, and on the hulls of ships, thus increasing drag. The goal of the current work is to investigate material coatings that resist the attachment and growth of bacteria on surfaces. We demonstrate that changing the roughness of the surface can reduce the number of bacteria found on the surface. More specifically, we have made surfaces covered with spheres that are approximately the same size as a bacterium, about 1 micrometer (10x smaller than the diameter of hair). We find that the spheres act as physical obstacles that block bacteria from moving on a surface. These results suggest that changing the micro-scale geometry of a surface may reduce the rate of infections on medical devices or hinder the growth of bacteria in other systems

Microbes in nature live collaboratively in adherent communities, known as biofilms. Biofilms can be contextually beneficial or detrimental. In medical implants, biofilms cause infections leading to additional healthcare costs of billions of dollars. Studies have found that micro/nanoscale surface topography can significantly alter (i.e., promote or hinder) the process of biofilm formation. The formation of biofilm starts with planktonic microbes attach to the surface. To further understand the biophysical underpinning of this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. To this end, aligned nanofiber coating with controlled fiber diameter and edge-to-edge spacing were manufactured using the Spinneret-based Tunable Engineered Parameters (STEP) techniques. The effect of surface topography on bacterial near-surface motility was studied. The experimental results showed that the bacterial attachment and near-surface motion can be greatly impacted by surface topography. Furthermore, the finding was applied to ureteral stents. The results showed that the aligned nanofiber can significantly reduce the biofilm formation process on ureteral stents. / Master of Science / Many microbes in nature live in adherent communities called biofilm. Biofilms contain individual microbes inside polymeric matrix which protect them from environmental stressors such as antibiotics. Biofilms are a significant contributor to the infection of implantable medical devices, which leads to additional healthcare costs of billions of dollars annually in the U.S. alone. Studies have found that sub-micron scale surface topography can significantly promote or hinder biofilm formation; however, the exact mechanism remains poorly understood. To further understand this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. The formation of microbial biofilm starts with swimming bacteria sensing the liquid-solid interface and attaching to the surface. Microbes are more likely to settle on a surface if a surface is favorable to attach. However, the decision-making process has not been fully understood. Our experimental results showed that the bacterial attachment and near-surface motion can be greatly influenced by surface topography. Furthermore, the finding was applied to ureteral stents, which is a type of medical implants used to maintain the flow of urine in the urinary tract. Ureteral stents serve great for medical purposes, but as foreign bodies, they also lead to urinary tract infection. The results showed that some types of aligned fiber coating increased microbial attachment density, while other types of aligned fiber coating reduced the bacterial surface coverage by up to 80%, which provides directions for future studies.

Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms. / Master of Science

A silent pandemic, chronic, non-healing wounds are a major cause of morbidity, with treatment and management representing significant health burdens. The opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa are the most common species isolated from chronic wounds. Polydimethylsiloxane (PDMS), a biocompatible and, inexpensive to fabricate polymer, can undergo various modifications. The ability of the produced polymers to attract S. aureus and P. aeruginosa, either from the planktonic state, or while sessile in biofilms on ex vivo skin, was investigated using flat (FL) or patterned (PT) PDMS with or without 1% or 10% triclosan Patterned PDMS + 10% triclosan (PT 10%) attracted significantly more live S. aureus and P. aeruginosa, as determined using Colony Forming Unit (CFU) analysis (*p<0.01), Scanning Electron Microscopy (SEM) (*p<0.01) and Confocal Scanning Laser Microscopy (CSLM) (*p<0.01). The released triclosan was not cytotoxic against either bacteria or primary cultures of human dermal fibroblasts using Water Soluble Tetrazolium Salts (WST-1) assay. High performance liquid chromatography analysis highlights low level of triclosan release from the PDMS. Bacterial infection in co-culture using the Boyden chamber assay increased fibroblast viability in the presence of PDMS (*p<0.05). PT 10% demonstrated superior biofilm transfer from epidermis (*p<0.05), in comparison to all other analysed polymers. In summary, the unique topography of PDMS combined with triclosan attracted bacteria most efficiently. This promising data suggests potential for engineering a patterned polymer to physically transfer biofilms from wounds, and importantly lacks bactericidal properties which is vital in the quest to combat antimicrobial resistance.