Browsing by Subject "E. coli"
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Item A Geographical Approach to Tracking Escherichia coli and Nutrients in a Texas Coastal Plains Watershed(2011-02-22) Harclerode, CaraCarters Creek in Brazos County, Texas, like many surface water reaches in the Texas Gulf Coast region, has been identified for bacteria and nutrient impairment on the Texas Commission on Environmental Quality (TCEQ) 303(d) List. Carters Creek drains a rapidly urbanizing watershed and has been found to carry high concentrations of dissolved organic carbon (DOC), nitrate, phosphate and sodium. These constituents have a severe impact on the creek?s capacity for healthy aquatic life and increase the potential for eutrophication downstream. The creek has also had chronic high Escherichia coli counts, making the creek unsuitable for contact recreation according to the accepted standard for surface water quality, which is a geometric mean of 126 CFU per 100 ml. In this study, grab samples were taken twice monthly from fifteen sites on Carters Creek and its subcatchments from July 2007 to June 2008. The samples were analyzed for E. coli, DOC, total N, NO3-N, NH4-N, Na+, K+, Mg2+, Ca2+, F-, Cl-, Br-, NO2-, SO42- and PO4-3. Mean annual DOC concentrations varied from 24.8 mg/L in Carter at Boonville Road to 55.5 mg/L in Wolfpen Creek; sodium varied from 33 mg/L in Carter at Old Reliance Road to 200 mg/L, also in Wolfpen Creek. Burton 4, the subcatchment with the highest geometric mean for E. coli with 2547 CFU/100 mL, was also sampled with greater geographical intensity for E. coli and optical brightener fluorescence at 445 nm to identify any leaking sewer pipes, but no evidence of defective pipes was found. During both the spring season and annual high flow (storm events), E. coli counts were positively correlated with total urban land use, probably caused by storm runoff carrying residues from impervious surfaces into the stream. High flow E. coli also had a negative relationship with potassium and a positive relationship with calcium, possibly suggesting a bioflocculation effect. Sites downstream of wastewater treatment plants (WWTPs) showed higher nitrate, phosphate, sodium, potassium, chloride and fluoride than other urban subcatchments. Creeks with golf courses carried more phosphate, sodium and fluoride than subcatchments without golf courses or WWTPs.Item Adaptive Evolution for the Study of Complex Phenotypes in Microbial Systems(2013-07-24) Reyes Barrios, Luis HumbertoMicrobial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.Item Bacterial Source Tracking in Impaired Watersheds: Evaluation of Culture-Dependent and -Independent Methods for Increased Source Specificity and Improved Management(2013-05-02) Martin, Emily CBacterial contamination due to excessive levels of bacteria is a confounding problem and remediation of impaired watersheds relies on the detection of fecal indicator bacteria and then assessing the source of said bacteria. Bacterial source tracking (BST) is an approach for assessing potential sources of this contamination. The purpose of this study was to utilize both cultivation-independent and ?dependent methods to improve the ability to track sources of fecal contamination. First, E. coli community composition was assessed across three standard water quality assessments including USEPA Methods 1603 and 1604, and Colilert?, to determine their impact on BST library-based performance. Results indicate that the three assessed methods of enumeration and isolation may select for different populations of E. coli and standardized methods may be warranted if library-dependent BST is part of a research plan. Next, BST techniques were used to enumerate and characterize E. coli communities across various dairy manure management techniques used in the Leon River watershed in central Texas to determine effectiveness of BST efforts in tracking contamination from dairy manure. Results of this study indicated that manure and effluent management strategies which employed means to remove solids from the manure tended to decrease the levels of E. coli in the effluent. Some E. coli genotypes were found across the managerial treatments even though there were no clear seasonal trends or site groupings among the dataset. The vast majority of the isolates classified using the Texas E. coli BST library were correctly classified back to their major source class, thus increasing confidence in the methods currently being utilized to track dairy fecal contributions in this Central Texas watershed. Finally, deer bacterial fecal communities from south and central Texas were analyzed using 454-pyrosequencing to assess the potential for the development of a deer-specific BST marker. Microbial communities did not cluster by site or year suggesting that deer fecal communities in these Texas regions are stable over time and could be amenable to marker development.Item Bioremediation of the organophosphate methyl parathion using genetically engineered and native organisms(Texas A&M University, 2005-11-01) Diaz Casas, Adriana Z.Toxic waste disposal problems have become enormous due to the proliferation of xenobiotic compounds for use in agricultural, industrial and numerous other applications. Organophosphate (OP) pesticides are commonly used in agriculture and their toxicity is associated with inhibition of cholinesterase in the exposed organism. Some OPs have been shown to produce OP-induced delayed neuropathy (OPIDN). The overall goal of the work described in this thesis was to develop bacterial consortia to remediate hazardous substances at significantly higher rates than found with natural systems. Specifically, degradation of methyl parathion (MP) by hydrolysis with a genetically engineered Escherichia coli was investigated along with degradation of one of the resulting products, p-nitrophenol (PNP), by Sphingobium chlorophenolicum ATCC 53874. Simultaneous degradation of both MP and PNP was investigated using a consortium of a genetically engineered Escherichia coli and a native S. chlorophenolicum. Concentrations of MP and PNP were measured by high performance liquid chromatography (HPLC). Non-growing freely suspended recombinant OPH+ E. coli cells efficiently degraded MP without addition of nutrients for growth. Maximum reactor productivity was found with a biomass concentration of 25 g/L. Substrate inhibition did not occur up to 3 g MP/L. The simple Michaelis-Menten kinetic model for enzymatic reactions provided a good fit of the degradation data with Vm=11.45 ??mol/min??g-biomass and Km=2.73 g/L. B. cepacia failed to degrade PNP under the experimental conditions evaluated, so further studies were not conducted. Growing cultures of S. chlorophenolicum degraded PNP at concentrations up to 0.1 g/L without a lag phase in mineral salts glutamate medium. Parameters such as initial pH, growth medium and growth stage for addition of PNP were important degradation factors. The bacterium exhibited substantial growth in the degradation process. Hydroquinone (HQ) or nitrocatechol (NC) were not identified as products of PNP degradation. The recombinant OPH+ E. coli and S. chlorophenolicum consortium failed to degrade PNP when starting with higher concentrations of MP. The presence of organic solvent in the bacterial consortium degradation medium negatively affected the degradation of PNP. The genetically engineered organism efficiently degraded high concentrations of MP, but the resulting high concentration of intermediate product (PNP) inhibited growth of the native type organism. Biodegradation by consortia of genetically engineered non-growing and native-type organisms generally will be limited by the growing native-type organism.Item Challenges of Pathogen Control in Beef Cattle Production and Processing in South Texas(2013-01-04) Haneklaus, Ashley NThis multi-phase project was designed (1) to evaluate existing post-harvest process controls and intervention strategies used to reduce Escherichia coli O157:H7, (2) to evaluate the impacts of cattle source and environmental factors on Salmonella prevalence in bovine lymph nodes, and (3) to evaluate sanitary conditions of feedyards in South Texas. The ultimate goal of this project was to identify and implement measures that reduce E. coli O157:H7 in beef harvest facilities, and Salmonella prevalence in feedyards. To evaluate process control of E. coli O157:H7 throughout the beef harvest process, samples were collected from harvest floor processing areas at two commercial beef slaughter establishments, and enumerated for aerobic plate counts, E. coli/coliform, and Enterobacteriaceae. To survey existing Salmonella prevalence, bovine lymph nodes (n = 307) were collected from beef carcasses at a commercial beef processing plant. Lymph nodes were extracted from cattle sourced from seven feedyards. Salmonella prevalence in lymph nodes was found to be 0% in cattle sourced from only one of the seven yards. Lymph nodes from cattle sourced from the other feedyards yielded positive samples, with varying prevalence. Of the remaining six feedyards, one feedyard yielded 88.2% prevalence of Salmonella in bovine lymph nodes, which was significantly higher than all other feedyards (42.9, 40.0, 40.0, 24.0, and 4.0%). The prevalence of Salmonella in the feedlot environment was compared among three feedyards; one yard had 65.0% environmental prevalence of Salmonella, which was statistically higher than the other feedyards surveyed. Of the two remaining yards, one had 0% prevalence of Salmonella in fecal and soil samples, which was also the feedyard with 0% prevalence of Salmonella in lymph nodes. Findings include (1) the significance of effective sanitary dressing procedures and intervention strategies in a beef harvest environment, (2) that there is clear feedyard-to-feedyard variation with relation to Salmonella prevalence in bovine lymph nodes, and (3) that differences in environmental factors existed among feedyards although the reasons remain unclear.Item Characterization and evaluation of Escherichia coli biotype I strains for use as surrogates for enteric pathogens in validation of beef carcass interventions(2009-05-15) Cabrera-Diaz, ElisaAntimicrobial interventions implemented in slaughter establishments for the reduction of enteric pathogens on beef carcasses must be validated to demonstrate efficacy under commercial operation conditions. Validation studies can be conducted using surrogates which are nonpathogenic organisms that respond to a particular treatment in a manner equivalent to a target pathogen. The purpose of this study was to identify surrogates for enteric pathogens to validate antimicrobial interventions on beef carcasses. The growth, attachment, resistance properties as well as the response to interventions on beef carcasses of nonpathogenic fluorescent protein-marked E. coli strains were evaluated and compared to E. coli O157:H7 and Salmonella strains. Growth curves were performed in tryptic soy broth at 37?C and it was demonstrated that in general, growth parameters were not different among surrogates and target pathogens. Thermal resistance was compared in phosphate buffered saline (PBS) at 55, 60 and 65?C; D-values of surrogates were not different or were higher than those of target pathogens. The acid resistance of surrogates was not different to that of E. coli O157:H7 in PBS acidified with lactic acid at pH 2.5, 3.0 and 3.5. Some Salmonella serotypes were found to be less acid resistant than the surrogates. Survival of surrogates after storage at low temperatures (4?C and -18?C) was not different or was longer than survival of E. coli O157:H7 and Salmonella. Additionally, the cell surface hydrophobicity and attachment to beef carcasses surfaces was not different among surrogates and pathogens. Antimicrobial interventions were applied on carcass surfaces under laboratory controlled conditions. After application of hot water washes, D-values were not different among surrogates and pathogens, while no differences were observed in log reductions (CFU/cm2) among surrogates and pathogens when 2% L-lactic acid sprays at 25 and 55?C were applied, regardless of the temperature and volume of the acid solution. The response of surrogates to water washes and lactic acid sprays on beef carcasses was also evaluated in commercial slaughter facilities. Reductions of surrogates were not different to those of aerobic plate count, coliforms and E. coli. However, the surrogates showed less variation and provided more consistent results than traditional indicators.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 Design and evolution of synthetic biological systems(2006-08) Tabor, Jeffrey Jay; Ellington, Andrew D.The study of biology has undergone a fundamental change due to advancements in genetic engineering, DNA synthesis and DNA sequencing technologies. As opposed to the traditional dissective mentality of discovering genes via genetics, describing genetic behaviors through biochemistry, and then drawing diagrams of functional networks, researchers now have the potential (albeit limited) to construct novel biological molecules, networks, and even whole organisms with user-defined specifications. We have engineered novel catalytic DNAs (deoxyribozymes) with the ability to 'read' an input DNA sequence and then 'write' (by ligation) a separate DNA sequence which can in turn be detected sensitively. In addition, the deoxyribozymes can read unnatural (synthetic) nucleotides and write natural sequence information. Such simple nanomachines could find use in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of difficult to detect (short) nucleic acids such as microRNAs. As an extension of in vitro biological engineering efforts, we aimed to construct novel signal transduction systems in vivo. To this end, we used directed evolution to generate a catalytic RNA (ribozyme) capable of creating genetic memory in E. coli. In the end we evolved an RNA which satisfied the conditions of our genetic screen. Rather than maintaining genetic memory, however, the RNA increased relative cellular gene expression by minimizing the translational burden it imposed on the host cell. Interestingly, detailed mutational analysis of the evolved RNA led us to new studies on the relationship between ribosome availability and stochasticity in cellular gene expression, an effect that had frequently been alluded to in the literature, yet never examined. We have also taken a more canonical approach to the forward engineering of biological systems with unnatural behaviors. To this end, we designed a protein-based synthetic genetic circuit that allows a community of E. coli to function as biological film, capable of capturing and recapitulating a projected light pattern at high resolution (theoretically 100 mexapixels). The ability to control bacterial gene expression at high resolution could be used to ‘print’ complex bio-materials or deconvolute signaling pathways through precise spatial and temporal control of regulatory states.Item Development and application of the spatially explicit load enrichment calculation tool (select) to determine potential E. coli loads in watersheds(2009-05-15) Riebschleager, Kendra JeanAccording to the USEPA National Section 303(d) List Fact Sheet, bacterial pathogens are the leading cause of water quality impairments in Texas. The automated Spatially Explicit Load Enrichment Calculation Tool (SELECT) uses spatially variable factors such as land use, soil condition, and distance to streams to characterize pathogen sources across a watershed. The results support development of Total Maximum Daily Loads (TMDLs) where bacterial contamination is of concern. SELECT calculates potential E. coli loads by distributing the contributing source populations across suitable habitats, applying a fecal production rate, and then aggregating the potential load to the subwatersheds. SELECT provides a Graphical User Interface (GUI), developed in Visual Basic for Applications (VBA) within ArcGIS 9.X, where project parameters can be adjusted for various pollutant loading scenarios. A new approach for characterizing E. coli loads resulting from on-site wastewater treatment systems (OWTSs) was incorporated into the SELECT methodology. The pollutant connectivity factor (PCF) module was created to identify areas potentially contributing E. coli loads to waterbodies during runoff events by weighting the influence of potential loading, runoff potential, and travel distance. Simulation results indicate livestock and wildlife are potentially contributing large amounts of E. coli in the Lake Granbury Watershed in areas where these contributing sources are not currently monitored for E. coli. The bacterial water quality violations near Lake Granbury are most likely the result of malfunctioning OWTSs and pet waste in the runoff. The automated SELECT was verified by characterizing the potential E. coli loading in the Plum Creek Watershed and comparing to results from a prior study (Teague, 2007). The E. coli potential load for the watershed was lower than the previous study due to major differences in assumptions. Comparing the average ranked PCF estimated by physical properties of the watershed with the statistical clustering of watershed characteristics provided similar groupings. SELECT supports the need to evaluate each contributing source separately to effectively allocate site specific best management practices (BMPs). This approach can be used as a screening step for determining areas where detailed investigation is merited. SELECT in conjunction with PCF and clustering analysis can assist decision makers develop Watershed Protection Plans (WPPs) and determine TMDLs.Item Engineering and investigation of protease fine specificity(2010-12) Li, Haixin; Georgiou, George; Iverson, Brent; Hoffman, David; Robertus, Jon; Ren, PengyuThe Escherichia coli (E. coli) outer membrane protease OmpT is an endopeptidase of the omptin family in gram negative bacteria. OmpT cleave preferentially between two consecutive basic residues, especially Arg-Arg, and it has been classified as an aspartyl protease based on its crystal structure although biochemical confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001). Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT by employing novel strategies for the isolation of enzyme variants that cleave desired substrates from large combinatorial libraries screened by flow cytometry. However, the engineering of proteases with altered specificity beyond the P1 and P1’ residues of the substrate have not been demonstrated. By applying high throughput screening of large libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2 subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone viii PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold change in the selectivity for the P2 position in peptide substrates. Specifically, this enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type OmpT. Molecular modeling was then employed to provide insights on how mutations in OmpT mediated this change in P2 specificity. A long term goal of protease engineering is to generate highly specific enzyme variants that can be used for the irreversible inactivation of disease targets. The anaphylatoxin C3a is a key mediator in inflammation and has been implicated with multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could be therapeutically relevant. Using high throughput screening and directed evolution we successful isolated C3a cleaving enzyme variants and have characterized them biochemically. Finally as part of this dissertation we have employed high throughput screening methods to dissect the substrate specificity of members of the kallikrein family of mammalian proteases which are implicated in a number of physiological and disease functions. The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast and ovarian cancer tissues and has been shown to cleave peptides derived from human ix myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking simulations of consensus peptide substrates was used to infer possible identities of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor (GluR) and synphilin.Item Engineering of de novo pathways for biosynthesis of glutathione analogues in Escherichia coli(2010-05) Veeravalli, Karthik; Georgiou, George; Molineux, Ian J.; Appling, Dean R.; Mullins, Charles B.; Maynard, JenniferThe low molecular weight (L.M.W.) thiol redox couple formed by γ-L-glutamyl-L-cysteinyl glycine, also called glutathione (reduced and oxidized), is present in most eukaryotes and a few species of bacteria. Glutathione plays a role in numerous cellular processes by providing a means of shuttling electrons to different enzymatic systems. As a result, thiol-dependent redox metabolic processes are highly coupled. Due to tight coupling of redox reactions, it is difficult to understand how changes in the concentration of glutathione would affect a specific glutathione-dependent process. Interestingly, only a small subset of bacteria encode the canonical enzyme for the biosynthesis of glutathione, namely γ-glutamyl cysteine synthetase (gshA gene product). The mechanisms by which glutathione-dependent processes are carried out in bacteria which do not have the genes for biosynthesis of glutathione or other L.M.W. thiols is not well understood. A genetic selection to restore a glutathione-dependent phenotype in E. coli, lacking the gene involved in first step of glutathione biosynthesis (gshA), was used to address how bacteria lacking gshA might substitute for glutathione. Genetic and biochemical analyses of the E. coli mutants isolated in the selection revealed a de novo pathway for biosynthesis of γ-glutamyl cysteine, the product formed normally by GshA. Additionally we found that the unnatural analogue of glutathione, γ-glutamyl homocysteine could also be formed by this pathway. Bioinformatic analysis suggested that bacteria lacking gshA may use these de novo pathways for biosynthesis of γ-glutamyl cysteine or γ-glutamyl homocysteine, which could serve as potential substitutes for glutathione. The engineering of de novo biosynthetic pathways for γ-glutamyl cysteine and γ-glutamyl homocysteine provided us a strategy for engineering a pathway for biosynthesis of another unnatural analogue of glutathione, β-aspartyl cysteine. Both γ-glutamyl homocysteine and β-aspartyl cysteine could potentially be used as orthologus redox couples in E. coli operating in parallel to glutathione to shuttle electrons to specific pathways which may thus be decoupled from glutathione availability. Glutathione-dependent enzymes that can use orthologous redox couples instead are biochemically isolated from network of other redox reactions in the cell and could be used to direct metabolic fluxes to specific pathways with high efficiencies. Towards this end, we show that glutathione transferase, a glutathione-dependent enzyme, can be engineered to use analogous thiols like γ-glutamyl cysteine as cofactors.Item Estimation of E. coli Concentrations from Failing On-Site Wastewater Treatment Facilities (OWTS) Using GIS(2014-08-12) Virani, Afreen ShirazFailing Onsite Wastewater Treatment Systems (OWTSs) have been identified as a significant threat to water quality, discharging significant amounts of inadequately treated sewage effluents. When developing a Watershed Protection Plan (WPP), OWTS has often been difficult to assess due to technological, institutional and economic constraints. In Texas, contamination from bacterial pathogens is the primary source in water quality concern. According to the 2012 Texas Water Quality Inventory, the Dickinson Bayou watershed is listed as ?impaired?, due to bacteria. Since the bacterial levels in this watershed are not meeting the State?s recreation standards, actions are needed to improve the water quality. Poorly designed and maintained OWTS, along with inappropriate site characterization are major contributors of the bacteria in this watershed. The majority of the OWTS located in Dickinson Bayou are located in poorly drained soils increasing the likelihood of contaminated runoff into the surface waters. A prediction tool was developed using Geographic Information System (GIS) to assess failing OWTS and the potential E. coli contamination to surface waters. This tool will help identify different parameters affecting E. coli concentration in streams, which include: rainfall conditions, spatial connections of OWTS to stream network, age of the OWTS, and the failure rate of the OWTS. A spatially-explicit algorithm was developed to estimate E. coli concentrations in watersheds resulting from failing OWTS, and implemented using ArcGIS 10. Spatial analysis of accumulated E. coli concentrations in streams was made possible by GIS. The algorithm was automated using python programming language, ArcPy, to simulate E. coli concentrations in surface waters in a coastal Texas watershed for different rainfall conditions. This automated tool simulated potential E. coli loads and concentrations from failing OWTS across the Dickinson Bayou watershed in Texas. The tool was validated using observed runoff data in the Dickinson Bayou watershed. The highest potential E. coli loads were identified and the areas of concern were highlighted to more effectively apply Best Management Practices (BMPs). Results concluded that precipitation played a significant role in routing the E. coli loads to streams in the watershed. The potential E. coli concentration in streams decreased with increasing rainfall amount. Also, the simulation results showed the number of household size and the number of OWTS plays a major role in E. coli contribution in the watershed. The age of the OWTS and the hydrologic connectivity of those failing systems should be considered while simulating the E. coli concentrations in the stream. Regulators, planners, and watershed managers to make timely management decisions can use results from this automated tool.Item Estimation of E. coli Concentrations from Non Point Sources Using GIS(2012-10-19) Mckee, KynaWhen developing a Watershed Protection Plan (WPP) or a Total Maximum Daily Load (TMDL), it is often difficult to accurately assess the pollutant load for a watershed because not enough water quality monitoring data are available. According to the Texas Commission on Environmental Quality (TCEQ), there are 274 bacteria impairments in Texas water bodies out of 386 impaired water bodies. Bacteria water quality data are often more sparse than other types of water quality data, which hinders the development of WPPs or TMDLs. The Spatially Explicit Load Enrichment Calculation Tool (SELECT) was used to develop watershed protection plans for four rural watersheds in Texas that are impaired due to E. coli bacteria. SELECT is an automated Geographical Information System (GIS) tool that can assess pathogen loads in watersheds using spatial factors such as land use, population density, and soil type. WPPs were developed for four rural Texas watersheds: Buck Creek, Lampasas River, five sub watersheds of the Little Brazos River, and Geronimo Creek. A spatial watershed model was developed to simulate bacteria concentrations in streams resulting from non point sources using SELECT combined with a simple rainfall-runoff model and applied to the Geronimo Creek watershed. The watershed model applies a rainfall-driven loading function to the potential E. coli loads calculated by the output of SELECT. The simulated runoff volumes and E. coli concentrations from the model were compared to actual monthly E. coli data collected at two sampling sites near the outlet of a subwatershed. The results show how SELECT methodology was applied to each watershed and adapted based on stakeholder concerns and data availability. The highest potential contributors were identified and areas of concern were highlighted to more effectively apply best management practices (BMPs). The runoff volumes were predicted with very good agreement (E = 0.95, RSR = 0.21 to 0.22) for both sampling sites. The predicted E. coli concentrations did not agree with measured concentrations for both sites using eight different methods. The results indicate that the model does not include significant factors contributing to the transport of E. coli bacteria but can be modified to include these factors.Item Evaluation of Methods to Assess and Reduce Bacterial Contamination of Surface Water from Grazing Lands(2012-10-19) Wagner, KevinExcessive bacterial levels are a major water quality concern. Better methods are needed to quantify the proportion of bacterial loading contributed by various sources, and best management practices are needed to restore water quality. This study assessed the ability of alternative water supplies and grazing management to reduce E. coli loading from cattle and evaluated the ability of quantitative polymerase chain reaction analysis of total and bovine-associated Bacteroides markers (AllBac and BoBac, respectively) to determine the percentage of bovine-associated fecal contamination. Runoff from seven small watersheds, representing ungrazed, properly stocked, and overstocked conditions, was analyzed for E. coli, AllBac, and BoBac to assess grazing management impacts on E. coli runoff and the effectiveness of Bacteroides markers. To determine the effectiveness of alternative water, instream E. coli levels and cattle movement were evaluated before and after alternative water was provided. The study found that when alternative off-stream water was provided, the amount of time cattle spent in the creek was reduced 43 percent and the direct deposition of E. coli into Clear Fork of Plum Creek was estimated to be reduced from 1.11E 07 to 6.34E 06 colony forming units per animal unit per day. Observed pre- and post-treatment E. coli loads suggested similar reductions; however, this study could not conclusively attribute observed E. coli loading reductions to providing alternative water because of the lack of statistical significance of these observations, possibly due to decreased streamflow during Year 2 (due to drought) and a corresponding increase in E. coli levels. The study found that rotational stocking, if timed appropriately, was very effective at reducing E. coli runoff. The impact of grazing timing in relation to runoff events was more significant than the impact of grazing management (i.e. ungrazed properly stocked or overstocked) or stocking rate. When runoff occurred more than two weeks following grazing, E. coli levels in runoff were decreased more than 88 percent. Finally, data suggest that AllBac and BoBac markers are good indicators of recent fecal contamination from cattle. However, although elevated BoBac/AllBac ratios generally aligned well with cattle presence, this ratio appeared to underestimate the percentage of bovine-associated fecal contamination.Item Genomics analysis on the responses of E. coli cells to varying environmental conditions(2016-05) Yan, Xiwei; Wilke, C. (Claus); Lin, LizhenThe natural living environments of E. coli cells are diverse, varying from mammalian gastrointestinal tracts and soil. Each environment might require distinct metabolic pathways and transporter systems, and long-term evolution has established elaborate regulatory system for E. coli cells to quickly adapt to the changing conditions. Sensing outside stresses and then adopting a different phenotype enable them to take advantage of any possible nutrients and defend against hostile environment. A lot of regulatory mechanisms have been identified by genetic, biochemical and molecular biology methods, and our study aim to build a systematic view on the response of the whole genome to four different environmental conditions. We used statistical tests including Pearson’s tests and Spearman’s tests and multiple testing adjustments to identify feature genes that are induced or repressed significantly across treatment levels. The feature genes identified were partially supported by previous literatures, and some of the novel genes not found in any previous studies may infer a potential research blind spot. Additionally, we compared the correlation tests to the implementation of machine learning algorithms, and discussed the advantage and drawbacks of each method.Item In-plant Validation of Two Antimicrobial Agents Applied During the Production of Tenderized and/or Enhanced Beef Products(2013-08-28) Nelson, KaylaNumerous outbreaks of foodborne illness have been attributed to non-intact beef (e.g., tenderized, marinated, and enhanced) products contaminated with Escherichia coli O157:H7. Organic acids are commonly utilized in the beef industry as antimicrobial interventions, which must be validated to eliminate or reduce E. coli O157:H7 to an undetectable level. Rifampicin-resistant Biotype I E. coli O157:H7 surrogate microorganisms (ATCC BAA-1427, BAA-1428, and BAA-1430) were applied as a cocktail (7.8 log10 CFU/ml) to three beef products (boneless strip loins, top sirloin butts, and bottom sirloin flaps) prior to treatment with an antimicrobial intervention (2.5% Beefxide or 2.9% lactic acid). Products were then subjected to a single or multiple pass tenderization and/or marination process. Beefxide and lactic acid treatments resulted in statistically significant log reductions of the microorganisms (P < 0.05) on the surfaces for all three products. Surrogate microorganisms were recovered from interior samples of all three products after mechanical tenderization. Additionally, surrogate concentrations recovered from flap surface and internal samples taken post-tumbling and marination were statistically similar (P < 0.05). These data indicate that tenderization and marination processes can transfer microorganisms into the interior of whole-muscle cuts, and suggest Beefxide and lactic acid may be similar in their efficacy as an antimicrobial applied as an intervention in the production of non-intact beef products.Item Influence of autoinducer 2 (ai-2) and ai-2-like inhibitors generated from ground beef on escherichia coli o157:h7 protein expression(2009-05-15) Soni, Kamleshkumar A.Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.Item Inter-kingdom Recognition of Norepinephrine by E. Coli : Identification of the Receptors Involved in Chemotaxis(2012-10-19) Kim, Dae NyunThere are approximately 10^14 bacteria belonging to nearly 1000 different species in the human gastrointestinal (GI) tract that co-exist with host cells. Within the GI tract, signaling molecules secreted by both eukaryotic and prokaryotic cells are abundant. Recent studies have shown that both bacteria and human cells recognize and respond to the signals from each other, presumably to gain a competitive advantage. The cross-recognition of signals is known as Inter-kingdom (IK) signaling and this phenomenon is considered to be important in the onset of infections in the GI tract. Of the eukaryotic signaling molecules present in the GI tract, the neuroendocrine hormone norepinephrine (NE) is considered to be important in the context of infections as NE is produced at very high concentration in the intestine under post traumatic stress, is known to increase bacterial virulence and infection, and has also been shown to be a potent chemoattractant for GI tract pathogens such as enterohemorrhagic E. coli (EHEC). The focus of this study is on elucidating the mechanisms underlying the recognition and chemotaxis of bacteria towards NE. While chemotaxis has been typically investigated in the context of bacteria moving towards a metabolizable source (e.g., amino acids), chemotaxis is potentially important in the onset of infections in the human GI tract. In this study we use a microfluidic plug assay to investigate the receptor and mechanism utilized by a model bacterium Escherichia coli in its chemotactic response to NE. A series mutant of E. coli RP437 strains of knockouts for four MCP-encoding genes was used in this study. The results from the microfluidic plug assay were then confirmed quantitatively by capillary assay. We have shown that Tsr receptor is necessary for chemotaxis of NE for E. coli RP437, and attraction of E. coli towards NE may require an additional receptor. Results from the priming experiments suggest that exposure to NE may result in the de novo expression of co-receptor(s) that are crucial to chemotaxis towards NE. The requirement for high cell density also suggests the possibility that NE per se may not be an attractant for E. coli, but could be a precursor that is modified into a chemoattractant by cells. These results are expected to further our understanding of bacterial chemotaxis and its role in bacterial colonization and infection of the human GI tract.Item Methods for controlling Escherichia coli O157:H7 and Salmonella surrogates during the production of non-intact beef products(2012-10-15) Ulbrich, CarsonThis study evaluated methods for controlling Escherichia coli O157:H7 and Salmonella non-pathogenic bacterial surrogates during the production of marinated non-intact beef products. Hot (~30 degrees C) boneless, beef strip loins (n = 54, Institutional Meat Purchase Specification 180) were inoculated with one of two levels (approximately 5.8 and 1.9 log10 CFU/cm2, hereafter referred to as high- and low-inoculated, respectively) of non-pathogenic, rifampicin-resistant E. coli organisms used to simulate harvest floor contamination. The inoculated beef strip loins were chilled at 2 degrees C for 24 h, and then vacuum packaged and aged for 7 to 24 days at 2 degrees C. The beef strip loins were subjected to one of five treatments or control (no treatment). Spray treatments were: 2.5% L-lactic acid, 5.0% L-lactic acid, 1,050 ppm acidified sodium chlorite, 205 ppm peroxyacetic acid, and tap water. Lactic acid treatments were applied at ~53 degrees C, whereas the other sprays were applied at room temperature (~25 degrees C). Treated and control pieces were tumble marinated using a commercial marinade. Sample counts were collected throughout the experiment to track reductions in inoculated microorganisms as impacted by antimicrobial treatment and processing. For the high-inoculated strip loins, the 5.0% L-lactic acid treatment was most effective (P < 0.05) across treatments and control at reducing surrogate organisms on meat surfaces before marination, producing a 2.6 log10 CFU/cm2 reduction. The water treatment accounted for the least (P < 0.05) reductions across treatments and control of surrogate organisms on the meat surface before marination. Peroxyacetic acid produced the greatest reduction of surface surrogate organisms in the finished, marinated product. The water treatment resulted in greater internalization of surrogate microorganisms when compared to the control. Furthermore, certain less effective antimicrobial sprays such as water may facilitate internalization of surface bacteria, more so than non-treated subprimals. It is important that producers of non-intact beef products focus on using effective antimicrobial sprays that maximize reductions and minimize internalization of surface bacteria into the finished product.Item Microbial Indicators in Restaurant Salads: Correlation Between Salad Type, Restaurant Ownership Format, and Customer Business Volumes(2013-12-12) Prince, David WarrenFoodborne illness outbreaks associated with fresh produce have increased over the past decade. Food workers employed at full-service restaurants are found to perform risky food practices more often than food workers employed in other segments of the foodservice industry. The goal of this study was to determine if differences in restaurant ownership format, business volume, and salad type influenced the level of indicator organisms present in restaurant salads. Overall levels of heterotrophic bacteria, total coliforms, Escherichia coli, Enterococcus, male-specific coliphages, and somatic coliphages were determined by aerobic plate counts (APC), Colilert?, Enterolert?, and U.S. EPA (Method 1601 and 1602), respectively. Molecular methods including automated rep-PCR DiversiLab? system and reverse transcriptase-PCR were used for the DNA fingerprinting of E. coli and the genotyping of male-specific coliphages, respectively. All of the above mentioned indicator organisms were present in the restaurant salads. Comparisons between restaurant types found that levels of APC, Enterococcus, and male-specific coliphages were significantly higher in locally owned restaurants and levels of total coliforms and somatic coliphages were significantly higher in corporate restaurants. The levels for all indicator organisms were significantly higher in specialty salads compared to leafy greens salads. Comparisons between business volumes suggested that indicator organism counts were higher during low customer traffic sampling periods. These results suggested that there were differences in safe food handling practices between locally owned and corporate restaurants. Staffing and labor issues as a result of low customer traffic and the need for additional handling and preparation of specialty salads seemed to increase the risk of cross-contamination issues for fresh produce. DNA fingerprinting for E. coli revealed that the same organism was found at multiple restaurants. These results indicate that there was a common source of contamination somewhere between field production and distribution. Genotyping results for male-specific coliphages found that some of the produce had been exposed to human and animal sources of contamination. Overall, the monitoring for indicator organisms in restaurant salads found that there is still a need for improved education-based programs in the area of safe food handling practice associated with fresh produce for food workers in restaurants.