Browsing by Subject "Biofilms"
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Item Analysis of batrachochytrium dendrobatidis gene expression in planktonic versus biofilm life cycle stages(2012-05) Silva, Shalika; San Francisco, Michael; Zak, John; Rumbaugh, Kendra P.Batrachochytriun dendrobatidis(Bd) is a chytrid fungus that has been recognized as the most likely cause for the massive amphibian declines worldwide. Previous studies in our lab have shown the ability of Bd to form a biofilm. Genes, GGDEF, EAL, Zap1, ADH3, CS1, CS2, and Cps1p, that play roles in biosynthesis and hydrolysis of cyclic-di-GMP, inhibition of extracellular matrix production, regulation of chitinous cell wall production, respectively, are known to be involved in the biofilm formation of both fungi and bacteria. Expression profiles of these genes were compared between biofilm and planktonic cells, through reverse transcriptase and quantitative real-time polymerase chain reactions (RT-PCR & qPCR). Analyses were carried out using the 5.8S rRNA gene as an internal standard. A phenotypic microarray (FUNGILOG) was also used to compare carbon utilization differences between biofilm and planktonic cells. Our results indicate that Cps1p, ADH3, Zap1, CS1 and CS2 genes are highly expressed in the biofilm stage being up-regulated 12-fold, 10-fold, 5-fold and 3-fold respectively. Differential expression of Cps1p and ADH were statistically significant (p value = 0.0104 and 0.0016 respectively). The carbon utilization studies indicate that there were no significant differences in carbon utilization between the two life stages of the fungus. These studies will provide good insight about the survival of Bd in the environment without its animal host.Item Biofilm Formation by Moraxella Catarrhalis(2004-05-04) Pearson, Melanie Michelle; Hansen, Eric J.This is the first detailed study of biofilm formation in vitro by the Gram-negative bacterial pathogen Moraxella catarrhalis. Growth of M. catarrhalis in a continuous-culture biofilm system resulted in little detectable change in outer membrane protein production compared to broth-grown M. catarrhalis. Biofilm-grown M. catarrhalis may produce an extracellular polysaccharide and different colony phenotypes when grown in this continuous culture biofilm system. Transmission electron microscopy of biofilm-grown M. catarrhalis cells revealed abundant projections extending from the bacterial cell surface that were identified as the proteins UspA1, UspA2, and Hag, which are all putative members of the autotransporter protein family. The Hag protein of strain O35E was shown to be necessary for hemagglutination, autoagglutination, and binding of human IgD. A crystal violet-based assay utilizing 24-well tissue culture plates was also used to evaluate biofilm formation by M. catarrhalis. The ability of M. catarrhalis strains to form biofilms in this crystal violet-based assay varied considerably, but most strains form little or no biofilm in this system. Screening of M. catarrhalis transposon insertion mutants using the crystal violet-based assay revealed that the UspA1 or the related UspA2H proteins play a strain-dependent, positive role in biofilm formation. Expression of the Hag protein prevented biofilm formation in tissue culture plates by several M. catarrhalis strains. In contrast, there appears to be a positive selection for Hag expression by strain O46E in the continuous-culture biofilm system. Proteins involved in cell wall recycling may also be involved in biofilm formation by M. catarrhalis. Nucleotide sequence analysis, site-directed mutagenesis, and domain swapping experiments indicated that the N-terminal region of UspA1 or UspA2H is likely involved in biofilm formation by M. catarrhalis.Item Characterization of biological hydrogel barriers(2014-12) Kaliki, Srimahitha; Smyth, Hugh D.C.,; Barr, Ronald; Milner, Thomas; Dunn, Andrew; Marek, StephenBiological hydrogel barriers include mucus, bacterial biofilms, fungal biofilms, and others. Biofilms are polysaccharide hydrogels. Biofilms are commonly found in the lungs of cystic fibrosis patients. Cystic fibrosis (CF) patients are susceptible to these types of chronic infections because their mucus barrier is abnormal. A common bacterial infection in these patients is caused by the bacterium Pseudomonas aeruginosa. While it is found that the bacteria can infect CF patients easily, the treatment of such infections by drugs had been found to be quite inefficient due to the structure of the biofilm itself and formidable mucus barrier. Mucus is a hydrogel which protects the gastrointestinal, genitor-urinal and respiratory tracts from pathogens and external environments. In our preliminary studies, topically applied nanoparticles disrupted these hydrogel barriers and resulted in the increase in permeability to solutes. The long term goal of this proposal is to understand and quantify the effects of the interaction between nanoparticles and biological hydrogel barriers. Discovering how nanoparticles disrupt the hydrogel barriers is important for understanding the health risks. The hypothesis of this research is that nanoparticles result in disruption of the hydrogel barrier structure that leads to increased exposures to co-deposited solutes. Quantifying the structural changes and diffusivity of such solutes using different novel techniques is the central object of my thesis. Bulk Rheological studies were performed using mucin samples treated with nanoparticles. It was noticed that the viscosities showed a negative trend with regards to the nanoparticle sizes which seemed to be contradictory to Einstein’s prediction. A possible mechanism of action was explained. Multiple particle tracking was performed to quantify viscosities of nanoparticles in mucin solution. Subsequently, drug diffusion studies were performed on similar samples to provide a relationship between the nanoparticle size and the drug permeability. Atomic force microscopy was performed in liquid cell using force mode on biofilms when treated with different sized nanoparticles. Micro-elasticity of these biofilms was calculated and compared.Item The emergence and divergence of antimicrobial tolerance and resistance in Pseudomonas aeruginosa biofilms(2006-05) Lai, Hoi Yi; Kirisits, Mary JoTo effectively minimize biofilms, which are prevalent in chlorinated drinking water distribution systems, the effect of biofilm age on antimicrobial tolerance and resistance must be investigated. It was our hypothesis that antimicrobial tolerance emerges quickly during biofilm development and that both antimicrobial tolerance and resistance increase as the biofilm ages. We further hypothesized that antimicrobial tolerance and resistance vary among the individual community members. In this study, young and mature biofilms of Pseudomonas aeruginosa, a model biofilm organism, were grown and exposed to antimicrobial agents in several different treatments. Results showed that the increased antimicrobial tolerance of intact biofilms compared to planktonic cells arises early (i.e., within hours) in biofilm development. The short-term tolerance of resuspended mature biofilm cells to antimicrobial agents peaked at a biofilm age of 14 days and subsequently declined; the peak and decline in antimicrobial tolerance may be related to periodic detachment events in the biofilm. The antimicrobial resistance of resuspended mature biofilm cells continuously exposed to antimicrobial agents increased with biofilm age. Furthermore, individual members in mature biofilm communities exhibited variation in antimicrobial tolerance, thereby highlighting divergence of the biofilm community from the original parent strain.Item An evaluation of the use of superparamagnetic iron oxide nanoparticles to overcome extracellular barriers to lung disease for drug delivery(2011-12) McGill, Shayna Lorraine; Smyth, Hugh; Croyle, Maria; McConville, Jason; Wiederhold, Nathan; Roy, KrishnenduPrimary 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.Item Linking gene expression to performance in a fungal vapor-phase bioreactor treating ethylbenzene(2004) Gunsch, Claudia Kneller; Kinney, Kerry A.Item Microbial community structure and nitrogen cycling in limestone biofilms(2015-08) Hancock, Tinisha; Brand, Jerry J. (Jerry Jay), 1941-; La Claire, John W; Leibold, Mathew A; Mehdy, Mona; Nobles, David RBiofilms inhabiting rock surfaces were of particular interest for this study, due to their ubiquity in central Texas and possible role in nitrogen cycling. Biofilm samples from an ornamental pond in Austin, Texas were collected over the course of two and a half years. Microscopic analysis indicated that the general physical structure of the biofilm remained constant, but the organisms inhabiting the biofilm varied. Metagenomic analysis confirmed that taxonomic diversity in biofilm communities is dynamic and variable, but the predicted functional capacities appear to be relatively stable throughout the sampling time. Less than one quarter of the variation in the taxonomic community data is explained by climate variables, indicating that a combination of stochastic and deterministic factors may drive community assembly. Limestone biofilm communities may be colonized from guilds of organisms that assemble based on the functional requirements prescribed by environmental conditions and resource availability. Natural biofilm samples were collected from other distinct patches of limestone in green spaces near Austin. Natural biofilms were thicker and more complex than the ornamental pond biofilms, yet they were not significantly different from each other in terms of their taxonomic community composition. The functional diversity of the natural biofilms was nearly identical to that seen in the ornamental pond biofilms. Taxonomic and functional diversity in natural biofilms were strongly correlated and significantly dissimilar. A strong negative correlation between actinobacteria abundance and bacteroidetes abundance was found, indicating that these organisms might be competitors. Cyanobacteria abundance was positively correlated with both humidity and precipitation, indicating that cyanobacteria might require more water than other organisms in the biofilm community. Organisms within limestone biofilm communities were capable of fixing atmospheric nitrogen, but the rate of nitrogenase activity was highly variable throughout the sampling period. Nostoc was the most abundant and active nitrogen-fixing organism. The abundance of cyanobacteria capable of fixing nitrogen was prone to fluctuation, whereas the abundance of non-photosynthetic nitrogen fixers remained relatively constant. Nitrogenase activity in the light reflects a combined effort between cyanobacterial and other nitrogen fixers and a consortium of other nitrogen fixers may be solely responsible for nitrogenase activity in the dark.Item Natural Organic Matter (NOM) in Aquatic Systems: Interactions with Radionuclides (234Th (IV), 129 I) and Biofilms(2011-10-21) Zhang, SaijinA series of laboratory and field investigations were carried out to elucidate the importance of natural organic matter in aquatic systems, i.e., trace element scavenging (e.g., 234Th) by exopolymeric substances (EPS), formation of biofilms, as well as interactions with 129I. A method involving cross flow ultrafiltration, followed by a three-step cartridge soaking and stirred-cell diafiltration, was developed for isolating EPS from phytoplankton cultures, especially in seawater media. EPS isolated from a marine diatom, Amphora sp. was then subjected to semi-quantitative (e.g., carbohydrate, proteins) and quantitative analysis (e.g., neutral sugars, acidic sugars, sulfate). It appeared that Th (IV) binding by EPS was dominated by the acidic polysaccharides of fraction. For EPS of biofilms collected from polluted streams, hydrophobic proteins were the most abundant components in EPS, followed by more hydrophilic carbohydrates. However, chemical composition of carbohydrates or proteins, i.e., monosaccharides and amino acids, respectively, varied with environmental conditions and substrata applied, which suggests that the formation of biofilms on different substrates is regulated by specific properties of microorganisms, environmental conditions and nature of substratum. No correlation between relative hydrophobicity of substratum and development of biofilm was found in this study. A sensitive and rapid GC-MS method was developed to enable the determination of isotopic ratios (129I/127I) of speciated iodine in natural waters. At the F-area of the Savannah River Site (SRS), iodine species in the groundwater consisted of 48.8 percent iodide, 27.3 percent iodate and 23.9 percent organo-iodine. Each of these iodine species exhibited vastly different transport behavior in the column experiments using surface soil from the SRS. Results demonstrated that mobility of iodine species depended greatly on the iodine concentration, mostly due to the limited sorptive capacity for anions of the soil. EPS, especially enzymes (e.g., haloperoxidases) could facilitate the incorporation of iodide to natural organic carbon. At high input concentrations of iodate (78.7 ?M), iodate was found to be completely reduced and subsequently followed the transport behavior of iodide. The marked reduction of iodate was probably associated with natural organic carbon and facilitated by bacteria, besides inorganic reductants (e.g., Fe2 ) in sediments and pore water.Item Probing topographical influences on biofilm formation using dynamic-mask multiphoton lithography(2011-05) Fox, Michelle Ann; Shear, Jason B.; Whiteley, MarvinIt 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.Item Regulation of biofilm formation and outer membrane protein expression in Vibrio cholerae by iron(2008-08) Craig, Stephanie Anne; Payne, Shelley M.Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT.