Browsing by Subject "bacteria"
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Item Bacteria in Ballast Water: The Shipping Industry's Contributions to the Transport and Distribution of Microbial Species in Texas(2010-10-12) Neyland, Elizabeth B.The transportation of organisms in the ballast water of cargo ships has been recognized as a source of invasive species despite current control measures. Pathogenic bacteria in the ballast tank have been studied but the total diversity of the ballast tank bacterial community has not been examined. This study is the first to characterize the total bacterial community within a ballast tank by constructing a clone library from a ballast water sample from a cargo ship in the Port of Houston, amplified ribosomal rDNA restriction analysis (ARDRA) and phylogenetic analysis. Bacterial communities in Texas ports and bays were also examined using denaturing gradient gel electrophoresis (DGGE), looking at both temporal and spatial variations for effects of deballasting activity. This ballast tank bacterial community had a high level of diversity (95%) with the clone library only representing 40% of the total community of the tank. Most probable originating habitats of the ballast bacteria were: marine pelagic (40%), estuarine (37%), coastal (6%), freshwater (3%) and other (14%), even though this ballast tank was exchanged with pelagic water. Predominate groups were alpha- and gammaproteobacteria, a few betaproteobacteria and bacteriodetes, and one each of verrucomicrobia, planctomycetes and actinobacteria, but no pathogens were detected. The data reveals a ballast tank that consists of half marine-pelagic, half port bacteria, revealing a low efficacy of exchange control methods and potentially invasive bacteria. The bacterial communities of five ships that exchanged ballast water in the Pacific Ocean shared on average 50% similarity. Two ships that exchanged ballast water in temperate latitudes were more similar than three other ships that exchanged in tropical latitudes, showing a correlation between location of exchange and community similarity. The bacterial communities of the Ports of Houston and Galveston exhibit stable, seasonal successions over one year. The port and bay systems of Texas exhibited spatial variations in bacterial communities related to salinity levels. Both experiments did not show evidence of community disruption by deballasting activities. This study shows that ballast water is a viable vector for invasive bacterial transport, although impact on Texas estuarine systems seems minimal.Item Design, testing and optimization of a microfluidic device for capture and concentration of bacteria(Texas A&M University, 2006-10-30) Cherla, SrinivasEffective detection of bacterial pathogens in large sample volumes is a challenging problem. Pre-concentration routines currently in practice before the actual detection process are cumbersome and hard to automate. An effort is made to address the problem of volume discrepancy between day-to-day samples and the concentrated samples needed for analysis. Principles of conceptual design are used in formulating the ??????Need Statement??????, ??????Function Structure?????? and in identifying the ??????Critical Design Parameters?????? and ??????Design Constraints??????. Electrokinetic phenomena are used to exploit the surface charges on bacteria. Electrophoresis is used to transport the bacteria to electrode surface and ??????Electrostatic trapping?????? is then used to capture these microbes on the electrode surface. The captured microbes can then be concentrated in a concentrator unit. A prototype microfluidic device is fabricated for showing the proof of concept. Optimization is done to minimize hydraulic power consumption and wetted volume. Observations from the initial prototype device along with the optimization results are used in building a new prototype device. Operation of this device is demonstrated by capture of bacteria from flow. Qualitative studies are conducted and preliminary quantification is also done.Item The effect of nutrient limitations on the production of extracellular polymeric substances by drinking-water bacteria(2013-05) Evans, Ashley Nichole; Kirisits, Mary JoBiological filtration (biofiltration) of drinking-water is gaining popularity due the potential for biodegradation of an array of contaminants not removed by traditional drinking-water processes. However, previous research has suggested that biomass growth on biofilter media may lead to increased headloss, and thus, greater energy and water requirements for backwashing. Research has suggested that the main cause of headloss might be due to extracellular polymeric substances (EPS) rather than the bacterial cells themselves. As EPS production has been shown to increase under nitrogen- and phosphorus-limited or -depleted conditions, the goal of this research was to add to the body of knowledge regarding biofiltration by studying the relationship between EPS production and nutrient limitations in drinking-water. Batch experiments with a synthetic groundwater were run with a mixed community of drinking-water bacteria under nutrient-balanced (a molar carbon to nitrogen to phosphorus ratio [C:N:P] of 100:10:1), nutrient-limited (e.g., C:N:P of 100:10:0.1), and nutrient-depleted conditions (C:N:P of 100:0:1 or 100:10:0). After 5 days, growth was measured as the optical density at 600 nanometers (OD600), and the concentrations of free and bound carbohydrates and proteins, the main components of EPS, were measured. In batch experiments with 2.0 and 0.2 g/L as carbon (mixture of acetic acid, mannitol and sucrose) increases in EPS production per OD600 and decreases in growth were noted under nutrient-depleted conditions. When the same experiments were conducted with a pure culture of Bacillus cereus, bound polysaccharides normalized to OD600 increased under nitrogen- and phosphorus-depleted conditions. Since previous research suggested that Bradyrhizobium would be an important player in EPS production in drinking-water biofilters, similar batch experiments were conducted with Bradyrhizobium. However, due to experimental challenges with Bradyrhizobium japonicum USDA 110, differences in EPS production under nutrient limitations could not be reliably assessed. Additional work is required with Bradyrhizobium. Recommendations for future work include the replication of these batch conditions in steady-state chemostats containing biofilm attachment media and in bench-scale columns. Additionally, future work should include experiments at carbon concentrations as low as 2 mg/L to match typical carbon concentrations in drinking-water biofilters.Item Evaluation of Ships' Ballast Water as a Vector for Transfer of Pathogenic Bacteria to Marine Protected Areas in the Gulf of Mexico(2013-05-10) Morris, Theresa LAn average of three to five billion tons of ballast water (BW) is transported globally per year; 79 million tons of which is released into U. S. waters. Ballast water is necessary for large ships to maintain balance and stability while loading and unloading cargo as well as during transit. As a ship unloads it?s cargo at a port, it takes on ballast water and then when it loads cargo at the next port, it discharges the ballast water, thus transferring water from port to port. Aquatic organisms are transported around the globe via ballast water. These organisms can potentially cause serious environmental and human health impacts. Historically, transfer of macro-organisms such as fish, snails, and vascular plants via ballast water have been the focus of ballast water research. More recently, microorganisms such as toxin producing dinoflagellates and diatoms i.e. ?harmful algal blooms? and pathogenic bacteria (e.g. Vibrio cholerae), have been found to survive in ballast tanks for several weeks. These organisms have moved to the forefront of ballast water management (BWM) trepidations because they compose serious threats to human health as well as Marine Protected Area (MPA) ecosystems such as coral reefs. Ballasting activities of ships calling at the Port of Houston were used as a model for other major ports in U.S. Gulf States. Ballast water management reports, submitted to the U.S. Coast Guard by all overseas vessels calling at the Port of Houston in 2010 were examined for the ballast management strategies utilized prior to docking. Currently, all ?coastwise? ships are not required to conduct ballast water exchanges (BWE) if they do not transit beyond the 200nm EEZ. Close inspection of BW management report forms for ?overseas? ships determined that the degree of completeness was variable. By comparing the frequency of vessels entering the Port of Houston, ballast water capacities, the management types utilized, and the locations of ballast water exchanges and discharges, it can be concluded that larger vessels present the highest risk of bacterial transfer to coral reefs. Five coral diseases were discovered to be prevalent throughout the ballast management areas their presence has significantly increased in these areas over the past thirty years. The combination of this disease growth along with an increase in shipping and ballast exchanges suggests that the vector at which pathogens are being displaced must lie within the shipping lanes.Item Microfluidic Systems for Investigating Bacterial Chemotaxis and Colonization(2011-02-22) Englert, Derek LynnThe overall goal of this work was to develop and utilize microfluidic models for investigating bacterial chemotaxis and biofilm formation - phenotypes that play key roles in bacterial infections. Classical methods for investigating chemotaxis and biofilm formation have many limitations and drawbacks. These include being unsuitable for investigating the effect of chemorepellents, non-quantitative readouts, and not accounting for interaction between hydrodynamics and biofilm formation. The novel microfluidic model systems for chemotaxis and biofilm formation developed in this study addresses these drawbacks. Chemotaxis model system development was done in three stages. We first developed two static chemotaxis model systems - the two fluorophore chemotaxis agarose plug assay and the mu Plug assay - for rapidly determining the extent of chemotaxis in a qualitative manner. A key feature of these model systems was the incorporation of dead cells and differential labeling with green and red fluorescent proteins for partitioning the effects of movement due to fluid flow from chemotaxis. The static systems were used to rapidly screen a wide range of conditions for use in the flow-based mu Flow chemotaxis model system. The effect of four major variables - cell preparation method, gradient strength, flow rate in the device, and imaging position - that influence the chemotactic response in the mu Flow was characterized using the repellent taxis from Ni^2 gradients as the model chemoeffector. Using the mu Flow chemotaxis device, we investigated the chemotaxis of Escherichia coli RP437 to different signals that are present in the human gastrointestinal tract and are likely to be mediators of infection through their effect on chemotaxis. Our data show that the bacterial signal indole is a repellent, while the signals autoinducer-2 (AI-2) and isatin are attractants for E. coli RP437. However, cells exposed to a competing gradient of indole and either AI-2 or isatin, attracts E. coli. The ?Flow device was also used to refute a long-standing view on how the repellent Ni2 is sensed in E. coli. Our data show that only the Tar chemoreceptor is needed for sensing Ni^2 and the nickel binding protein, NikA, and the Ni^2 transport system proteins, NikB and NikC, are not required for repellent taxis from nickel. A microfluidic biofilm model was also developed in this study and used in conjunction with a mathematical model to investigate biofilm formation and quorum sensing in closed systems (where biofilm growth and hydrodynamics are interdependent). The mathematical model predictions were experimentally validated using Pseudomonas aeruginosa PA14 in a microfluidic biofilm system at various flow rates.Item Roles of Naturally Occurring Bacteria in Controlling Iodine-129 Mobility in Subsurface Soils(2012-10-19) Li, Hsiu-Ping129I is of major concern because of its biophilic nature, excessive inventory, long half-life (~16 million yrs), and high mobility in the natural environment that depends on its chemical speciation. Iodide (I-) has the highest mobility than iodate (IO3-) and is the predominant species in the terrestrial environment due to prevailing pH and Eh conditions. In order to transform I- to less mobile organo-iodine (OI), strong oxidants are necessary to activate the first electron transfer step from I- to reactive intermediates. The aim of this study was to determine the influence of naturally occurring aerobic bacteria isolated from an 129I contaminated aquifer (F-area of the Savannah River Site, SC) on I- oxidation and OI formation. It was demonstrated that 3 of 136 strains accumulated I- (0.2~2%) in the presence of H2O2, when incubated in the presence of an environmentally relevant concentration of I- (0.1 microM). The accumulation was likely through electrophilic substitution resulting in the iodination of cellular constituents. The results indicated that culturable I--accumulating bacteria are not directly responsible for the high fraction of oxidized iodine species (IO3- and OI, >50% of total I) present in the SRS F-area. Several bacterial strains were found to be capable of stimulating I- oxidation through excretion of oxidants and enzymes. Organic acids in spent liquid medium from 27 of 84 aerobic bacterial cultures enhanced H2O2-dependent I- oxidation 2-10 fold. Organic acids enhanced I- oxidation by (1) lowering the pH of the spent medium and (2) reacting with H2O2 to form peroxy carboxylic acids, which are strong oxidizing agents. In the absence of H2O2, spent medium from 44 of 84 bacteria cultures showed I- oxidizing capacities. One I- oxidizing bacterium was studied to characterize its extracellular I- oxidizing component(s). The I- oxidizing capability from the spent medium was inactive by treatments with heat and H2O2 and absent under anaerobic conditions. Conversely, NADH, NADPH and FMN additions stimulated I- oxidation in the spend medium. These results indicate an oxidase(s) catalyzed I- oxidation. Understanding the bacterial activities involved with I- oxidation and OI formation is expected to help reduce 129I mobility in water-soil systems.Item Study of Midgut Bacteria in the Red Imported Fire Ant, Solenopsis invicta B?ren (Hymenoptera: Formicidae)(2011-08-08) Medina, FrederAnts are capable of building close associations with plants, insects, fungi and bacteria. Symbionts can provide essential nutrients to their insect host, however, the development of new molecular tools has allowed the discovery of new microorganisms that manipulate insect reproduction, development and even provide defense against parasitoids and pathogens. In this study we investigated the presence of bacteria inside the Red Imported Fire Ant midgut using molecular tools and transmission electron microscopy. The midgut bacteria were also characterized by their morphology, biochemical activity, and antibiotic resistance profile. After isolation, culture, and characterization of these bacteria, the molecular analysis revealed ten unique profiles which were identified to at least the genus level, Enterococcus sp./durans, Klebsiella ornithinolytica, Kluyvera cryocrescens, Lactococ-cus garvieae, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Bacillus pumilus, Listeria innucua, Serratia marcescens, and an uncultured bacterium from the Entero-bacteriaceae. New SEM and TEM techniques revealed a possible functional association of endosymbiotic bacteria with the insect host, and it also showed the absence of bacteriocytes in the epithelial cells of the midgut. The PCR results, from the bacteria abundance and distribution studies, showed that Enterococcus sp., Kluyvera cryocres-cens and Lactococcus garvieae are the most abundant species, but they are not consistently found in all sites throughout the southeastern United States. Kluyvera cryocrescens, Serratia marcescens, and an uncultured bacterium (isolate #38: Enterobacteriaceae) were genetically modified with the plasmid vector pZeoDsRed and successfully reintroduced into fire ant colonies. Strong fluorescence of DsRed was detected up to seven days after introduction. The transformed bacteria can still be rescued after pupal emergence; however most were passed out in the meconium. We further demonstrated that nurses contributed to the spread of the transformed bacteria within the colony by feeding the meconium to naive larvae. Although the role of midgut bacteria in the fire ant is still unknown, we have no indication that they cause any pathology. Studies emphasizing the role of these bacteria in fire ant physiology are still ongoing. These results are the foundation for a fire ant biological control program using endosymbiotic bacteria as vectors to introduce foreign genes that express proteins with insecticidal properties.