Browsing by Subject "Bacteria"
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Item Antimicrobial activities of saponin-rich guar meal extract(2009-05-15) Hassan, Sherif MohamedThree saponin-rich extracts (20, 60, 100% methanol), four 100% methanol subfractions and seven independently acquired fractions (A-G) from guar meal, Cyamopsis tetragonoloba L. (syn. C. psoraloides), were evaluated for antimicrobial and hemolytic activities. These activities were compared against quillaja bark (Quillaja saponaria), yucca (Yucca schidigera), and soybean (Glycine max) saponins in 96-well plates using eight concentrations (0.01 to 1.0 and 0.1 to 12.5 mg extract/mL). Initial guar meal butanol extract was 4.8 ? 0.6% of the weight of original material dry matter (DM). Butanol extract was purified by preparative reverse-phase C-18 chromatography. Two fractions eluted with 20, and one each with 60, and 100% methanol with average yields of 1.72 ? 0.47, 0.88 ? 0.16, 0.91 ? 0.16 and 1.55 ? 0.15% of DM, respectively. Further purification of the 100% methanol fraction using normal-phase silica gel preparatory high pressure liquid chromatography eluted 4 peaks at 16, 39, 44 and 46 min. Only the 100% methanol fraction, its 16 min peak, F and G fractions, and quillaja saponin, exhibited both hemolytic and antibacterial activities against Staphylococcus aureus, Salmonella Typhimurium and E. coli, but 20 and 60% methanol fractions stimulated Lactobacillus spp. growth. Guar meal (0 or 5%) was added to diets fed to chicks from 1 to 21 days of age. Chicks fed both diets were unchallenged or challenged with 5 x 103 Eimeria tenella sporulated oocysts at 10 days. Guar meal diets reduced oocysts shed per gram of feces, body weight, and feed efficiency. Adding 2.5% guar meal, 1% guar gum, or 0.125% saponin-rich guar meal extract to diets fed to chicks to 21 days of age showed that guar meal increased the cfu concentrations of digesta more than controls following a challenge with 107 cfu of Clostridium perfringens at 14 days. Body weights of chicks fed guar meal and saponin-rich extract were significantly lower than control body weights at 21 days of age, whereas the weekly feed to gain ratio of chicks fed saponin-rich extract was higher than controls. Guar meal reduced severity of Eimeria tenella infection and guar saponin-rich extract exhibited antimicrobial activity against several common poultry pathogens.Item The bacterial and fungal microbiome of retail stores(2013-05) Hoisington, Andrew James; Kinney, Kerry A.; Siegel, Jeffrey A.Microorganisms found in the indoor environment of retail stores are not well studied, despite potentially serious human exposure implications. In this study, filters from central heating, ventilation, and air-conditioning (HVAC) units were used to collect a time-integrated sample of the airborne indoor microbiome in 14 U.S. retail stores. The microbial communities recovered from the filter dust samples were analyzed with pyrosequencing to characterize the fungal and bacterial microbiome present. The objectives of this work were to: (1) characterize the microbial communities present in retail stores, (2) delineate relationships between the indoor microbiome and building or environmental parameters, and (3) evaluate the use of HVAC filter dust as a sample location for indoor microbial investigations. The microbiome in retail stores was observed to be diverse with 4,771 and 1,577 unique operational taxonomic units for bacteria and fungi, respectively. The diverse microbial community detected over time in the same store could not be fully explained by seasonal trends. That indicates that even when utilizing a long-term sampling approach like HVAC filter samples, the indoor microbiome cannot be completely characterized by a single sampling event. The bacterial community in retail stores was influenced by the outdoor microbiome and microbiota commonly associated with human skin. Physical location of the retail stores had some influence on the bacterial microbial community present and strongly impacted the fungal community recovered. Air exchange rate did not influence the observed bacterial or fungal communities. In a comparison to five other indoor air samplers, passive samplers including HVAC filter dust and settled dust captured a higher diversity of the microbial community. The collection of a more diverse sample may allow detection of potentially pathogenic microorganisms. However, there was significant difference in the microbial community structures recovered between samplers in the same sampling event which suggests sampling methodology has an impact on the inferred microbiome recovered. The overall results from this study indicate that retail stores harbor a diverse microbial community that varies over time.Item The c-di-GMP binding protein, YcgR, is the primary inhibitor of motor function in Salmonella and Escherichia coli.(2013-12) Nieto, Vincent Michael; Harshey, Rasika M.E. coli and Salmonella enterica have multiple c-di-GMP cyclases and phosphodiesterases. Absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di- GMP-binding protein, for motility inhibition. This study demonstrates that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, with the primary interaction site located within FliM. Interaction of YcgR with these proteins induces a CCW motor bias and reduces the efficiency of torque generation, thus inhibiting both chemotaxis and the speed of movement. In collaboration with David Blair’s group at the University of Utah, we propose a "backstop brake" model showing how both effects of YcgR on the motor can result from an initial disruption of the FliM/FliG interface, followed by destabilization and disorganization of the FliG C-terminal domain, which interacts with the stator protein MotA. Support for this order of events i.e. induction of a CCW bias followed by reduction of torque, is provided for S. enterica motors. Data from single motor analysis show that E. coli and S. enterica motors have inherently different properties, but that YcgR is solely responsible for disruption of motor function in both bacteria. This study also finds that E. coli and S. enterica employ c-di-GMP in additional and different pathways to impede motility. Inhibition of motility and chemotaxis may represent a bacterial strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.Item Characterization of beneficial mutations in unsaturated fatty acid biosynthesis that are recurrent dead-ends in a long-term evolution experiment with Escherichia coli(2014-05) Wolf, Lindsey Nan; Barrick, Jeffrey E.; Bull, JamesMicrobes provide an invaluable tool for watching evolution in action. Throughout more than 55,000 generations, lineages of Escherichia coli cells in a long-term evolution experiment (LTEE) grew in a minimal glucose environment and explored different mutational paths to higher fitness. Genome sequencing identifies genes that accrue mutations early in evolution across the twelve evolving populations. These parallel mutations typically provide a significant fitness benefit and often fix in the population. However, some mutations seem to lead to evolutionary dead-ends. In 7 of the 12 LTEE populations, lineages with mutations in the gene coding for the lipid synthesis repressor, fabR, gain traction within the population, but always eventually go extinct. To parse out the fitness benefits and downstream effects, strains with these mutations were constructed. These mutations increase the growth rate and may affect the length of lag phase after each daily transfer. Another mutation that often fixes within eventually successful clades is within the stress response global regulator spoT. A connection between spoT and fabR mutations could be the key to understanding the eventual outcomes within these lineages. Decreased fatty acid synthesis (repressed by FabR) during glucose starvation activates the global repressor SpoT to produce the cellular "alarmone" (p)ppGpp, inhibiting cell growth during the stringent response. Thus, it is possible that fabR mutations that prolong fatty acid synthesis and spoT mutations that alter the production of (p)ppGpp may both benefit cells by affecting the stringent response. In addition, when these two mutations are combined in a single strain they confer nearly an identical increase in fitness as the single mutations alone, strengthening the argument that they may target the same cellular pathway. Preliminary gene expression analyses of fabR mutants confirmed an expected increase in unsaturated fatty acid synthesis and also found signs that membrane damage responses were activated. It is possible that fabR mutants are near a stability cliff that makes them unable to access otherwise beneficial further mutations. Ultimately, this work will elucidate how interactions between the physiological effects of mutations on evolutionary paths to higher fitness may lead to differences in evolvability that ultimately determine success or extinction.Item Characterization of ferredoxin-dependent enzymes from plants and cyanobacteria(Texas Tech University, 1988-08) Sung, Jeou-derNot availableItem Culturability and dynamics of bacterial communities along the Pine Canyon Watershed of Big Bend National Park, TX(2007-12) Clark, Jeb; Zak, John; Francisco, Michael J. D. S.; Jeter, Randall M.Soil bacterial communities are known to play a significant role in ecosystems as they are responsible for, among other things, nutrient cycling, and plant and rhizosphere interactions. Those bacterial communities which persist in arid environments have gained special attention over the past several years as life in these environments is often believed to be limited due to a lack of available water. Since soil bacteria play such a significant role in the environment, understanding how seasonal changes in soil moisture and temperature affect bacterial community structure and function is critical for understanding how soil bacteria contribute to the functioning of arid ecosystems. However, the study of soil bacterial communities has been limited due to difficulties experienced in culturing organisms from the environment. Furthermore, PCR-based, culture-independent techniques have become increasingly popular amongst researchers, but these techniques which impose a bias limiting observations to only the most DNA-dominant bacterial species, species which are not believed to play a large functional role in the ecosystem. Hence, a large and possibly important proportion of the bacterial population could be commonly overlooked. A year-long, two-part research project on the environmental effects on soil bacterial communities in three sites along the Pine Canyon Watershed of Big Bend National Park began in August 2005. The first part examined the DNA-dominant bacterial community structure using PCR and Denaturing Gradient Gel Electrophoresis along with Fatty Acid Methyl Ester analysis. These techniques revealed a stable DNA-dominant bacterial community structure throughout the summer and winter seasons, and no significant changes in bacterial structure were recorded during a large precipitation event during August 2006. The second study utilized a culture-based approach to examine the functional bacterial communities along the three sites. The culture-based approach revealed that two different assemblages of soil bacteria exist, a culturable-dominant and a culturable-non-dominant. The culturable-dominant assemblage remained relatively stable throughout the seasonal changes and during the precipitation event of August 2006 and consisted largely of bacterial species of the Arthrobacter and Burkholderia genus. However, the culturable-non-dominant assemblage responded to seasonal changes by either increasing or decreasing in population density and increased in density during the precipitation event. The responses observed in the culturable-non-dominant assemblage may provide an explanation towards the difficulty many researchers experience in culturing organisms from the environment. Furthermore, culture-dependant techniques proved to be more sensitive in detecting changes in the bacterial community structure than culture-independent based techniques. Also, to aide in the collection of data for the second study, computer software was developed using the C# computer language and has been made available at: http://www.biol.ttu.edu/faculty/jzak/dgge_soft.Item Dynamics of marine pelagic bacterial communities on the Texas-Louisiana shelf(2009-05-15) Anitsakis, Erin ColleenMicrobial community interactions within many ecosystems are still relatively unknown. Investigating links between environmental dynamics and shifting pelagic bacterial community structures on the Texas-Louisiana shelf, Eubacterial community profiles of Operational Taxonomic Units (OTUs) were generated using Automated Ribosomal Intergenic Spacer Analysis (ARISA) of the 16S rDNA and 23S rDNA intergenic spacer region. This ITS region is highly variable in both length and sequence. Community diversity was assessed by the comparison of ARISA-generated community fingerprints of samples collected from four distinct regions along the Texas-Louisiana shelf in a cross-shelf pattern on 10m, 20m, and 40m isobaths. Incubations of samples with a thymidine analog, 5-Bromodeoxyuridine (BrdU), allowed for the isolation and analysis of the actively growing subset within the total bacterial population. Community composition was determined through the construction of clone libraries for sequencing and putative phyla affiliation of community 16 rRNA genes. Hydrographic data were also collected for analysis of shifts in microbial community diversity correlated with a variety of influential environmental factors. ARISA profiles of Eubacterial species richness suggest strong distinction between the two communities found within Zones A and C along the Texas-Louisiana Shelf. Further analysis of salinity gradients originating from the two main fluvial sources, the Mississippi and the Atchafalaya Rivers, identified possible sources of variation between the individual communities. Whereas composition of these communities remains discrete between regions, the active subset of the population becomes more similar across the shelf through the summer. Possibly due to undersampling of hypoxic sites, no relationship could be determined between hypoxia formation and the Eubacterial community dynamics. Several OTUs within the communities were identifiable as ? - and ? - Proteobacteria, Actinobacteria, Synechococcus, Prochlorococcus, and Cytophaga/Flavobacterium/Bacteroides. Through validation studies of 5-Bromodeoxyuridine field sampling, this study indicates the power of BrdU incorporation and ARISA analysis to study a dynamic environmental system and explore the factors that determine the structure of the pelagic community on the Texas-Louisiana Shelf.Item Ecological mechanisms underlying soil microbial responses to climate change(2013-12) Waring, Bonnie Grace; Hawkes, Christine V.Soil microbes influence the global carbon cycle via their role in the decomposition and formation of soil organic matter. Thus, rates of ecosystem processes such as primary production, soil respiration, and pedogenesis are sensitive to changes in the aggregate functional traits of the entire microbial community. To predict the magnitude and direction of microbial feedbacks on climate change, it is necessary to identify the physiological, ecological, and evolutionary mechanisms that underlie microbes’ responses to altered temperature and rainfall. Therefore, I examined microbial community composition and function in relation to manipulations of resource availability and precipitation in two contrasting ecosystems: a tropical rainforest at La Selva Biological Station, Costa Rica, and a semi-arid grassland in central Texas. I conducted a leaf litter decomposition experiment at La Selva to identify the physiological constraints on microbial allocation to extracellular enzymes, which degrade organic matter. I found strong evidence that microbial enzyme production is decoupled from foliar stoichiometry, consistent with weak links between leaf litter nutrients and decomposition rates at the pan-tropical scale. Next, to examine whether ecological trade-offs within microbial communities may drive shifts in carbon cycling at local spatial scales, I quantified changes in soil fungal and bacterial community composition in response to an in situ precipitation exclusion experiment I established at La Selva. Although drought-induced shifts in community structure were small, large increases in biomass-specific respiration rates were observed under dry conditions. These findings suggest that physiological adjustments to drought may constitute an important feedback on climate change in wet tropical forests. Finally, I focused on microbial community responses to climate change within a meta-community framework, using a reciprocal transplant experiment to investigate how dispersal shapes bacterial community structure along a natural rainfall gradient in central Texas. I found that soils from the wet end of the precipitation gradient exhibited more plastic functional responses to altered water availability. However, soil bacterial community composition was resistant to changes in rainfall and dispersal, preventing functional acclimatization to precipitation regime. Together, the results of these experiments emphasize the potential for physiological plasticity or microevolutionary shifts within microbial populations to drive ecosystem carbon cycling under climate change.Item Effects of Wet and Dry Weather Events on Bacteria (Enterococci) and the Public Health Threat from the Re-Suspension of Sediment Sequestered Enterococci(2014-06-04) Bare, Ryan MichaelApproximately 66% of Texas surface waters are impaired with bacteria from fecal waste, including several tributaries and segments within the Galveston Bay system. This study was conducted in the waters of the Marina Del Sol marina on Clear Lake in Kemah, Galveston County, Texas, USA. A series of hypothesis were tested; 1) rainfall and subsequent runoff from stormwater is the primary cause of elevated Enterococcus levels in the waters in Marina Del Sol, 2) hotspots of Enterococcus will be present in the waters in Marina Del Sol and 3) the concentration of Enterococcus will increase from the marina entrance to the rear of the marina. Sampling was conducted at 10 stations between 0800 and 1100 every Monday, Thursday, and Saturday over five weeks in June and July 2013. Enterococci concentrations were quantified using the IDEXX Enterolert method for detection and enumeration estimation, Fluorogenic Substrate Enterococcus Test, Multi-well procedure and three-day rainfall accumulation prior to sampling was recorded from NOAA?s Climate Data Online. Eleven dry weather and four wet weather events occurred during the sampling period with the largest rainfall accumulation at 1.39 inches. The geometric means of wet versus dry weather samples were not significantly different (Mann Whitney). Two hotspots were found yielding geometric means of 42.98 and 41.25 MPN, which exceed the U.S. EPA primary contact recreation limit of 35 MPN. Additionally, the EPA single sample maximums (104 CFU/100 mL) were exceeded at nine out of ten sampling stations at least once, including a spike of 1,445 MPN and 1,198 MPN. A low to high gradient of Enterococci, from the entrance to the back portion of the marina, was evident. The results from the initial summer study indicated that the stormwater retention pond to the west of the marina could be a possible source of Enterococci. The fourth hypothesis, states that sediments are a source of elevated Enterococcus concentrations in the water at the Marina Del Sol marina, was tested during a follow up study. Sediment and water samples were collected on the 13th of November, 2013 between the hours of 0900 and 1400. Six stations in the stormwater retention pond were sampled. In addition, three of the original sampling stations in the marina were sampled. A stormwater outfall was found to be a concentrated source of Enterococci into the retention pond (12,098 MPN/100 mL). Data from these two studies indicate that there are numerous sources that contribute to the concentrations of Enterococci in the marina. A gateway effect is occurring between the increasingly built environment of the Galveston Bay marinas and the natural environment.Item Evaluation of In-House Windrow Composting as a Poultry Litter Treatment Prior to Land Application(2013-08-05) Winkler, ScottThe land application of poultry litter as a fertilizer is a common practice due to the low cost and high availability of poultry litter in some regions. However, land application can create concerns related to runoff water quality and odor. An experiment was conducted to determine the effectiveness of in-house windrow composting (IWC) of poultry litter prior to land application in terms of bacteria, odors and nutrients compared to untreated (fresh) litter. In the second part of the research, the objective was to quantify the number and distribution within poultry houses of selected water quality indicator bacteria in litter. Comparison of fresh and IWC litter showed that Escherichia coli (E. coli) was present in very low concentrations on day 1 in fresh litter (20 cfu/g) and IWC litter (55 cfu/g), but the levels were undetectable in both litter types on day 9 in Trial 1. In Trial 2, E. coli levels were undetectable in IWC litter before and after the IWC process. Similarly, fresh litter had undetectable E. coli levels on day 1, but 185 cfu/g on day 10. Additionally, nutrient analysis and moisture content results showed no significant differences between fresh and IWC litter. To evaluate odor differences between fresh and IWC litter, volatile gases were collected onto sorbent tubes and into Tedlar bags from wind tunnel flux chambers placed directly on litter piles prior to land application. The concentrations of 13 compounds commonly associated with animal manure were then determined by GC/MS. Analysis of volatile gas samples resulted in significant changes of various individual odorants, while olfactometry analysis of Tedlar bag air samples resulted in reduced detection threshold values for IWC litter compared to the fresh litter. These results indicate the possible mitigating effects IWC may have on odors associated with litter. In the survey of bacterial distribution within poultry houses, litter counts varied greatly within house sections and between farms. Regression analysis revealed that bacterial counts and litter moisture content are significantly related, thus explaining much of the variation in litter bacterial counts within a house. These results indicate that IWC could be a useful best management practice to reduce E. coli levels and odor associated with poultry litter prior to land application, but factors such as moisture content, initial bacteria concentrations, and windrow size all affect the level of bacteria and odor reduction.Item Evolution of microbial populations with spatial and environmental structure(2010-05) Miller, Eric Louis; Meyers, Lauren Ancel; Bennett, Philip C.; Bull, James J.; Hawkes, Christine V.; Hillis, David M.Rarely are natural conditions constant, but generally biologists study microbes in artificially constant environments in the laboratory. I relaxed these assumptions of constant environments through time and space as I investigated how microbial populations evolve. First, I examined how bacteriophage evolved in the presence of permissive and nonpermissive hosts. I found that bacteriophage evolved discrimina- tion in mixed environments as well as in one of two environments with homogeneous, permissive hosts. This showed the asymmetry of host-shifting in viruses as well as the possibility of large, and somewhat unpredictable, pleiotropic effects. Secondly, I reconstructed ancestral environmental conditions for soil bacteria groups using phy- logenetics and environmental variables of extant species’ habitats. These generaliza- tions suggested characteristic phenotypes for several phylogenetic groups, including uncultured Acidobacteria. Lastly, I collected genetic sequences and global collection information for 65 bacteria genera across the domain. In examining the relation- ship between genetic distance, environmental conditions, and geography, I observed positive relationships specifically between genetic distance and geography or genetic distance and environmental conditions for bacteria from land sites but not from wa- ter sites. Phylogenic classifications or phenotypes of the genera could not predict these correlations. In all of these projects, variations in the environment created evolutionary signals that hinted at past environments of microbial populations.Item Fiber Optic Micro-endoscopy for Detection of Bacteria in Early Stages of Infection(2012-02-14) Mufti, Nooman SadatMycobacterium tuberculosis, the bacterium that causes tuberculosis, has an incubation period ranging from a few months to several years following infection via inhalation into the lungs. Whole body fluorescence scanners are used to image and monitor the growth of fluorescent protein expressing strains of M. tuberculosis in the lungs of animal models. Accurate quantitative analysis of bacterial growth during the early stages of infection inside lungs remains elusive, due to tissue absorption and scattering of photons emitted by the low numbers of bacteria deep in tissue. Fiber optic micro-endoscopy is uniquely suited to provide a novel solution to this problem by delivering light excitation directly to and collecting fluorescence from the infection site located in the lungs of an animal model, thereby enabling detection of fluorescent bacteria during the early stages of infection. In this thesis, I present a contact probe fiber bundle fluorescence micro-endoscope with a range of LED based excitation wavelengths, 4 ?m resolution, a 750 ?m field of view, and a 1 mm outer diameter. This system has detected tdTomato and GFP expressing Bacillus Calmette-Gu?rin (BCG) bacteria in vitro. Additionally, images of bacterial regions of infection obtained in mice subcutaneously infected with tdTomato expressing bacteria at concentrations ranging from 106 to 101 Colony Forming Units (CFU) and intra-tracheally infected mice at 106 CFU demonstrate the micro-endoscope?s capability to detect and resolve regions of bacterial infection in vivo. By relaying the bacterial fluorescence image from the infection site to an external detector, we are able to increase the sensitivity to early stages of infection.Item From chaos to harmony : public participation and environmental policy(2011-12) Dulay, Marcel; Eaton, David J.; Rodriguez, Victoria E.; Spelman, William G.; Browning, Larry D.; Maxwell, Madeline M.Water quality issues in the Leon River watershed in Texas exemplify the challenges water resource managers and the public face in the ongoing effort to improve water quality in our nation’s water bodies. Some pollutant sources are difficult to regulate and likely managed through non-regulatory means, such as voluntary action. The Leon River challenge is how to go beyond regulations to address the concerns of citizens and produce options they want to develop and implement voluntarily that address a common good. This dissertation argues that voluntary measures work only if those who must take action support the action, otherwise conflict can occur. Thus, it is critical to learn what people are willing to do to promote the public good (e.g., swimmable streams). This can be achieved through an effective public process. Public participation processes may have barriers that impede success, such as inadequate access, intimidation, competing interests, limited accountability, and scientific mistrust. This dissertation developed process enhancements to overcome these barriers based on documented public participation principles. This research tested whether specific enhancements can improve the quality of a public process and achieve desired process outcomes. This dissertation reports on quasi-experiments with stakeholders making actual environmental decisions. The findings suggest that these enhancements are capable of reducing conflict and reducing the time to produce environmental policy. Five process enhancements (representation, film, narratives, deliberative decision-making, and decision support) were put into operation to provide options for government agencies and stakeholders to consider when undertaking public participation processes. The lack of access can be avoided by giving stakeholders voice with representation through different types of meetings levels (e.g., focus groups and town hall meetings). Films, when captured, edited, and shown to others, can remove the mechanisms typically associated with the intimidation perceived by speakers during discussions. Narratives were used to collect information about stakeholders to develop a deeper understanding of the diversity of interests affected by a policy, avoiding gridlock from positional bargaining. Deliberative decision-making (no voting) can assure stakeholders have real and equitable decision-making power, with scenarios collaboratively developed that address the common good. Application of a decision support system (DSS) as an overlay to a scientific model can provide stakeholders direct access to science so they can develop scenarios, evaluate alternatives, and choose solutions.Item A GIS-based estimation of steady-state non-point source bacteria pollution in the Lower Rio Grande below Falcón Reservoir(2012-05) Lynch, Robin Shaw; Eaton, David J.; McKinney, Daene C.This report estimates the steady-state, non-point source bacteria pollution along the international river system of the Lower Rio Grande / Río Bravo between Falcón Reservoir and the Gulf of Mexico. The results from this report may be used by environmental agencies in the United States and México in order to develop a steady-state water quality model of the bacterial load in this river system. This report creates a GIS-based estimation of the steady-state, non-point source pollution from sources such as failing septic tanks, untreated sewage, grazing animals, and wildlife in the watershed. This report also provides recommendations for environmental agencies when developing the water quality model. The results and methodology developed for this report may be used as part of the Lower Rio Grande / Río Bravo Watershed Initiative, a binational pilot project to develop a plan to restore and protect the quality of the Rio Grande/Río Bravo.Item Growth Kinetics of Wildlife E. coli Isolates in Soil and Water(2012-07-16) Gallagher, MeghanBacteria are the major cause of surface water contamination in the United States. US Environmental Protection Agency (USEPA) uses the Total Maximum Daily Load (TMDL) process to regulate the E. coli loads from fecal sources in a watershed. Different point and non-point sources can contribute to the fecal contamination of a waterbody including municipal and on-site wastewater treatment plants, livestock, birds, and wildlife. Unfortunately, wildlife sources in many rural watersheds are poorly characterized. E. coli is also known to persist in waterbodies when no known fecal sources are present. In this study, E. coli from wildlife fecal material was enumerated. It was found that E. coli concentrations varied with the season the fecal samples were collected. When studying the fate of E. coli under different environmental factors, no growth was observed in soil at 4% moisture content and in water at 10 degrees C. The highest E. coli growth was recorded in water at 30 degrees C. It can be seen from these results that there was variation in the fate of E. coli under different environmental conditions. The fate of E. coli in the environment is a complex process and is influenced by many factors and their interactions, making it difficult to predict. The findings from this study along with additional studies can be used to improve the accuracy of model predictions to estimate the E. coli loads in watersheds.Item Immunogenicity and Effects on Fecal Microbiome of an Electron-Beam Inactivated Rhodococcus equi Vaccine in Neonatal Foals(2014-05-03) Bordin, Angela IlhaRhodococcus equi is a bacterium commonly isolated from soil that primarily causes pneumonia in foals and immunocompromised adult horses. Many vaccines were designed and tested to protect foals from developing pneumonia; however, to date, there is no vaccine that will protect foals from intrabronchial challenge with R. equi, except live, virulent R. equi. To evaluate electron-beam (e-beam) irradiation as a method of inactivation of R. equi, 2 concentrations (Concentration 1, 1 ? 10^(8) colony-forming units/ml [CFU/ml] or Concentration 2, 1 ? 10^(9) CFU/ml) of R. equi were submitted to a range of e-beam radiation doses, ranging from 0 to 7 kGy. All microorganisms of Concentrations 1 and 2 were adequately inactivated by 4 and 5 kGy, respectively, and the bacterial cell wall remained intact, whereas heat-inactivated samples indicated a compromised cell wall. Both concentrations were tested for immunogenicity and effects on fecal microbiome in neonatal foals. Mucosal and serum antibody responses were studied, as well as cell-mediated immune responses. Enteral administration of e-beam inactivated R. equi increased IFN-? production and generated naso-pharyngeal R. equi-specific IgA in newborn foals. The inactivated vaccine appeared safe and immunogenic in neonatal foals in the presence of maternal antibody. No impact of treatment on fecal microbiome composition or diversity was observed among vaccinated foals; however, marked and significant differences in microbial communities and diversity were observed between foals at 32 days of age relative to 2 days of age regardless of treatment. In conclusion, electron-beam irradiation is an appropriate method for inactivation of R. equi, and e-beam irradiated R. equi vaccine is immunogenic in neonatal foals. Also, age-related changes in immune responses and the fecal microbial population occurred in healthy foals vaccinated enterally with e-beam inactivated R. equi. Mucosal vaccination does not result in major changes of the fecal microbiome in foals.Item Insight into a unique carbon resource partitioning mechanism in Aggregatibacter actinomycetemcomitans(2010-08) Brown, Stacie Anne, 1979-; Whiteley, Marvin; Meyer, Richard; Walker, James; McLean, RobertAggregatibacter actinomycetemcomitans is a Gram negative bacterium found exclusively in the mammalian oral cavity where it resides in the gingival crevice, the space between the tooth and gum tissue. Though it has historically been considered a common commensal organism, it is now appreciated that A. actinomycetemcomitans is an opportunistic pathogen associated with the diseases periodontitis and endocarditis. To cause infection, A. actinomycetemcomitans must interact and compete with neighboring bacteria for space and nutrients, though little is known about the physiology it employs within the gingival crevice. Using A. actinomycetemcomitans grown in a chemically defined medium containing carbon sources found in vivo, I use transcriptome analyses and growth studies to show that A. actinomycetemcomitans preferentially utilizes lactate over the phosphotransferase system (PTS) sugars glucose and fructose. Additionally, the presence of lactate or pyruvate inhibits the transport and metabolism of these sugars in a post-transcriptionally controlled process I have termed PTS substrate exclusion. Since lactate is an energetically inferior carbon source, PTS substrate exclusion appears to be a carbon resource partitioning mechanism that allows A. actinomycetemcomitans to avoid competition for energetically favorable sugars with other species, and I propose a model to describe this phenomenon. To begin to understand the mechanism of PTS substrate exclusion, I examine the first step of the proposed model by purifying and characterizing the L-lactate dehydrogenase (LctD) from A. actinomycetemcomitans. I demonstrate that, unlike other studied lactate dehydrogenases, the LctD from A. actinomycetemcomitans does not exhibit feedback inhibition in the presence of physiologically relevant concentrations of pyruvate, which supports my hypothesis that elevated intracellular pyruvate levels inhibit the PTS. The results of my studies provide insight into a new regulatory mechanism governing carbon utilization in this bacterium.Item Paper analytical devices for rapid, quantitative electrochemical detection of DNA and bacteria(2016-12) Brenes, Nicholas James; Crooks, Richard (Richard McConnell)In this thesis, two paper analytical devices (PADs) are described as proof of concept devices for point-of-care applications. The first PAD, termed the Esensor, was developed for quantitative detection of oligonucleotides. The detection component of the Esensor was based on DNA stem-loop probe hybridization with signal stranded DNA followed by transduction of an electrochemical signal via target-induced conformational switching. The electrochemical signal was produced by a redox label attached to the DNA stem-loop probe. The Esensor had a limit of detection of 30 nM for DNA, and the device-to-device reproducibility was better than 10%. Furthermore, the Esensor had a shelf life of at least 4 weeks and required only 20 µL of sample. The Esensor work presented in this thesis was published in Analytical Chemistry where the detection of DNA and thrombin was described.1 The Esensor work was completed in collaboration with Dr. Cunningham who is the primary author on the publication. This thesis focuses only on the detection of DNA by the Esensor, as I have made significant contributions to this portion of the work. The second PAD covered in this thesis was developed for the detection of whole-cell bacteria. The operation of the device involved a sandwich capture assay. Bacterial specificity was achieved using antibody-functionalized magnetic microbeads and silver nanoparticle (AgNP) labels. The AgNP labels allowed for electrochemical detection via anodic stripping voltammetry. In this sensor, there were two inherent forms of signal amplification: (1) magnetic concentration of microbeads complexed with bacteria at the working electrode surface and (2) electrochemical concentration of Ag+ ions at the working electrode surface. This PAD was nearly 100% specific for Escherichia coli (E. coli) in the presence of two additional bacterial species. The on-chip assay time was <4 min, the device fabrication was cost effective at $0.36 USD/device, and the limit of detection was 1.3 x 107 cells/mL. This device, termed the oSlipB, was similar to a PAD employed to detect DNA and proteins as previously reported;2–5 however, this new application of bacterial detection further exemplifies the versatility of this paper device.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 The Ecology of Aerobic, Heterotrophic Bacteria of Playa Lakes and Microcosms(Texas Tech University, 1971-05) Porter, Lucy B HillNot Available.