The release of cells from a biofilm to the surrounding environment is poorly understood and the importance of this stage of biofilm development has only recently been realized. A key part of this process is the generation of phenotypic variants in the biofilm dispersal population. This thesis reports on the characterization of biofilm development of Serratia marcescens MG1, the analysis of the biofilm dispersal population, and the identification of the conditions that trigger phenotypic diversification. Furthermore, it provides an insight into the molecular understanding of how phenotypic variation is being generated, and demonstrates the clinical and environmental implications of phenotypic diversification during bacterial pathogenesis and bacterial persistence. Characterization of the microcolony biofilm development of S. marcescens revealed that the S. marcescens biofilm develops through a process involving microcolony formation, hollowing of mature microcolonies, and a sudden biofilm expansion within a very short period (&lt 24h) resulting in an increase in biofilm biomass with a radiation of biofilm structures at days 3 to 4. The biofilm expansion phase consistently correlated to an increase in the number of dispersal variant morphotypes. Studies of variant induction in planktonic cultures and biofilm flow cells demonstrated that phenotypic diversification in S. marcescens is not only a biofilm-specific phenomenon, but also involves biofilm-specific morphotypes. These morphological variants can only be isolated from the microcolony biofilm morphotype and not from the filamentous biofilms, leading to the hypothesis that there is a strong diversifying selection that is specific to the microcolony biofilms. To further explore how these variants were generated, molecular analyses revealed that exopolysaccharides and lipopolysaccharides are important moieties that are involved in phenotypic variation in S. marcescens biofilms. The etk gene, encoding a tyrosine protein kinase within the exopolysaccharide biosynthesis operon, was found to contain single nucleotide polymorphisms (SNPs) that were present in the 'sticky' variants but not in the 'non-sticky' wild-type or the 'non sticky' small colony variants. Furthermore, infrequent-restriction-site PCR (IRS-PCR), BIOLOG metabolic profiling, and gene sequence analyses, suggest that phenotypic diversification in S. marcescens is likely to involve mutational hotspots in specific genes. The biofilm-derived morphotypic variants differed extensively in cell ultrastructure properties, and exhibited specialized colonization and virulence traits, such as attachment, biofilm formation, swimming and swarming motilities, protease production, and hemolysin production. It was also demonstrated that phenotypic diversification contributed to a varying degree of resistance to protozoan predation, and bacterial pathogenecity in Caenorhabditis elegans, highlighting the complexity of the dispersal populations from S. marcescens biofilms. Furthermore, mixed-culture experiments involving multiple variant isolates (with or without the parental wild-type) showed that the persistence and virulence potential of S. marcescens can be synergistically enhanced in the Acanthamoeba castellanii grazing model and in the C. elegans infection model, respectively. This indicates that the different bacterial morphotypes work in concert to provide S. marcescens with enhanced protection against environmental perturbations and a competitive edge during the infection process. It was proposed that phenotypic diversification is not only an integral part of S. marcescens biofilm life-cycle, but also represents an important strategy for bacteria to greatly enhance its survival and persistence in different environments, ranging from aquatic and soil ecosystems, to those of the infected hosts.

Thesis (M.Sc.)(Microbiology)--University of Pretoria, 2006. / Includes summary. Includes bibliography. Available on the Internet via the World Wide Web.

Vibrio vulnificus is an opportunistic human and animal pathogen that is predominantly found in estuarine waters. In aquatic ecosystems, it colonizes filter-feeders, such as oysters, and has been found to form biofilms on the surface of various marine organisms, including plankton, algae, fish, eels, and crustaceans. The bacterium can spontaneously develop a rugose phenotype, which is associated with the production of polysaccharide(s) that impart a raised, wrinkled appearance to cells, copious biofilm formation, and increased stress resistance. Biofilm and rugose colony development, along with pellicle and aggregate formation, are believed to be crucial for the environmental survival and persistence of V. vulnificus. As the biosynthesis of polysaccharide(s) is a key feature linking these physiological processes, the main objectives of this study were to identify polysaccharide loci contributing to survival phenotypes in V. vulnificus and to gain insight into the regulation of these loci. Two polysaccharide loci (brp and rbd) were found to contribute to biofilm formation. The brp locus is regulated by the second messenger c-di-GMP and by at least two transcriptional regulators BrpR and BrpT. Lesions in glycosyltransferases in the locus or in either of the regulators abated the inducing effects of c-di-GMP on biofilm formation. The rbd locus is regulated not by c-di-GMP, but instead by a response regulator (RbdG) belonging to the TCRS family, which is encoded within the locus. The biofilms associated with the expression of the brp and rbd polysaccharides were structurally unique and simultaneous expression of both loci dramatically enhanced pellicle formation. Each locus also provides unique survival characteristics; the development of rugosity and stress resistance could be attributed to brp expression whereas rbd expression augmented aggregate formation. The ability of V. vulnificus to differentially regulate expression of the brp and rbd polysaccharides may allow the bacterium to “fine tune” its biofilm lifestyle to maximally benefit from the characteristics associated with each locus.

Vibrio vulnificus is an opportunistic human and animal pathogen that is predominantly found in estuarine waters. In aquatic ecosystems, it colonizes filter-feeders, such as oysters, and has been found to form biofilms on the surface of various marine organisms, including plankton, algae, fish, eels, and crustaceans. The bacterium can spontaneously develop a rugose phenotype, which is associated with the production of polysaccharide(s) that impart a raised, wrinkled appearance to cells, copious biofilm formation, and increased stress resistance. Biofilm and rugose colony development, along with pellicle and aggregate formation, are believed to be crucial for the environmental survival and persistence of V. vulnificus. As the biosynthesis of polysaccharide(s) is a key feature linking these physiological processes, the main objectives of this study were to identify polysaccharide loci contributing to survival phenotypes in V. vulnificus and to gain insight into the regulation of these loci. Two polysaccharide loci (brp and rbd) were found to contribute to biofilm formation. The brp locus is regulated by the second messenger c-di-GMP and by at least two transcriptional regulators BrpR and BrpT. Lesions in glycosyltransferases in the locus or in either of the regulators abated the inducing effects of c-di-GMP on biofilm formation. The rbd locus is regulated not by c-di-GMP, but instead by a response regulator (RbdG) belonging to the TCRS family, which is encoded within the locus. The biofilms associated with the expression of the brp and rbd polysaccharides were structurally unique and simultaneous expression of both loci dramatically enhanced pellicle formation. Each locus also provides unique survival characteristics; the development of rugosity and stress resistance could be attributed to brp expression whereas rbd expression augmented aggregate formation. The ability of V. vulnificus to differentially regulate expression of the brp and rbd polysaccharides may allow the bacterium to “fine tune” its biofilm lifestyle to maximally benefit from the characteristics associated with each locus.

This study involves the development of a biofilm reactor that supports growth of a deep biofilm on a gas permeable membrane. The reactor solution is not aerated, and oxygen is supplied through the membrane. The reactor is termed a substratum-aerated-biofilm reactor or SAB. With adequate concentrations of electron-donors and electron-acceptors, a deep biofilm grows on the membrane and is comprised of different layers of bacterial activity. The aerobic layers are near the membrane support, while the anaerobic layers are near the biofilm-liquid boundary. In the SAB, the substrate diffuses from the bulk liquid into the biofilm to react. Oxygen diffuses through the membrane into the biofilm. All products likewise are transported by molecular diffusion through the biofilm and into the bulk liquid. The reactors consisted of a reactor wall made of a plexiglass cylinder with the gas permeable membrane supported on a shallow rotating cup. The cup was designed so that the cup and the membrane function as a flat plate. The flat plate was utilized for support of the biological growth, transfer of oxygen, and mixing of the bulk liquid and the gas phase. The experiments were conducted in completely mixed, continuous-flow reactors maintained at 25°C with a hydraulic detention of 8 hours. Pure oxygen was delivered to a gas compartment under the membrane. All reactors were fed a synthetic waste buffered to pH 7.0. The background solution for the feed solution was made from distilled water combined with adequate inorganic nutrients and vitamins. The background solution was supplemented with acetate and ammonia to obtain the desired substrate compositions. Combined nitrification and heterotrophic oxidation activity resulted when the SABs where fed 5 or 10 mg/1 acetate, and 10 mg-N/1 ammonia. Combined nitrification, heterotrophic oxidation, and denitrification resulted with acetate concentration of 20, 40, and 100 mg/1, and 10 mg- N/1 ammonia. Combined heterotrophic oxidation and fermentation resulted with acetate concentration of 800 mg/1, and 10 mg-N/1 ammonia. A series of mass balances were developed to determine the fate of the nitrogen compounds and acetate. These results showed that the flux values for carbon oxidation, nitrification, denitrification, and fermentation are higher than those reported for competing technologies such as rotating biological contactors. / Graduation date: 1990

Thesis (DTech (Chemical Engineering))--Cape Peninsula University of Technology, 2009. / Includes bibliographical references. Also available online.

Thesis (Ph.D.)(Botany))--University of Pretoria, 2003. / Includes bibliographical references.

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: Michael Franklin. Includes bibliographical references.

Primary barriers to drug delivery include mucus and biofilms, which can hinder drug and gene delivery by several orders of magnitude, preventing effective therapeutic effects. By understanding the physical and chemical properties of these ubiquitous barriers, one may employ drug delivery approaches, such as design of nanoparticle and microparticle systems, to attempt to overcome the transport barriers. Nanoparticles are a growing interest in drug delivery, specifically as drug carriers, though most will become entrapped within these extracellular barriers further limiting their desired affects. Previous studies have generally manipulated the surface chemistries or size of these nanoparticles to allow for nearly a 2-fold increase in passive diffusion through barriers. To expand the current ideas of overcoming these barriers, studies in presented in this dissertation were performed using a type of active nanoparticle, superparamagnetic iron oxide nanoparticles. It was first investigated whether these particles would disrupt extracellular barriers under an oscillating magnetic field, which resulted in a 2-fold increased diffusion of particles upon biopolymer breakage. Secondly, influences of an external static magnetic field on diffusion of these nanoparticles through model barriers were determined. Both of these methods resulted in higher fold increases, reaching up to 28-fold compared to 2-fold as described in the literature. Next an examination of drug permeation enhancement in models of extracellular barriers by nanoparticle interactions was performed, using a passive mechanism as found in the literature. With a range of different nanoparticles including diesel particulate matter, barrier function was disrupted resulting in a 5-fold increase in drug permeation. To further manipulate drug diffusion an assisted delivery systems was observed, where magnetic nanoparticles could influence un-associated drug diffusion, resulting in 4-fold increase in drug diffusion. Finally formulations of nanosuspensions were created for aerosol delivery and their performance evaluated in vitro. A dry powder formulation containing drug and nanoparticles was formulated using a spray-drying technique. Upon barrier deposition studies using the dry powder formulation, permeation rates were determined resulting in a 2-fold increase for nanoparticle permeation. When drug diffusion was determined up to a 54-fold increase in drug was seen when co-delivered with nanoparticles, compared to controls containing only drug. / text

It has only been within recent decades that the complexity and heterogeneity of the biofilm mode of bacterial existence has been widely appreciated. Biofilms have persisted for billions of years as social communities of cells aggregated and attached on surfaces, and today they are both necessary and harmful within the human body and our surrounding environment. They show extremely high antibiotic resistance relative to planktonic cells and are sources of persistent infections. Biofilms are also the most common cause of failure for indwelling biomedical devices and implants. As a result, research efforts and commercial developments are focusing on creating better biomaterials that prevent bacterial attachment to surfaces leading to biofilm formation. While chemical methods to combat bacterial infections have been around for over a century in the form of antimicrobials, relatively little is known about how topographical methods can prevent bacterial attachment to surfaces. The reason for this is that micro- and nano-scale fabrication technologies (which are needed to produce topographies on size scales that might be expected to influence bacterial attachment) are fairly recent developments. In this thesis work, microscale topographies were developed for probing and influencing bacterial attachment to surfaces using dynamic-mask multiphoton lithography. Multiphoton lithography is an inherently three-dimensional fabrication technique. When combined with the dynamic-mask-based technology developed in the Shear laboratory, it allows for rapid prototyping of 3D structures of arbitrary complexity with submicron resolution in the radial dimension. A variety of topographical approaches for influencing bacterial attachment of Pseudomonas aeruginosa cells were explored within this work. P. aeruginosa was selected as a model organism for biofilm formation and because it is commonly isolated from infections associated with biomedical implant devices. Topographical approaches included the design of topographies based on microscale surfaces of naturally-antifouling leaves and mathematical functions, pillars, and surfaces containing various sizes and geometries of holes. Challenges relating to an imaging artifact caused by light scattering induced by the surfaces shed light on issues associated with assessing bacterial attachment levels on microscale topographical surfaces. Finally, future directions for this work are presented with ideas that extend into the nanoscale regime. / text