Doctor of Philosophy / Department of Biology / Lynn Hancock / Enterococci are the third leading cause of nosocomial infections including urinary tract infections (UTI), surgical site infections (SSI) and blood stream infections. Enterococci are also found in the gastrointestinal tracts of humans, and other mammals. We elucidated the influence of the transcriptional regulator RpoN on enterococcal biofilm formation, virulence potential and cell wall architecture and proposed a potential involvement for carbohydrate metabolism in these processes. Biofilms are held together by matrix (BM) components such as extracellular DNA (eDNA) released by cell death from a sub-population of cells. The rpoN mutant (ΔrpoN) was resistant to autolysis as well as fratricide-mediated cell death and eDNA was not detected in planktonic as well as biofilm cultures. Unlike the parental strain V583, the ΔrpoN mutant formed proteinase K sensitive biofilms, suggesting that protein as well as eDNA serves as an important matrix component. The rabbit model of endocarditis was used to assess the effect of rpoN deletion on enterococcal virulence. Rabbits infected with ΔrpoN had reduced bacterial burden in heart, blood, liver, kidney and vegetation in comparison to the parental strain. The growth defect of ΔrpoN in physiologically relevant glucose levels (5 mM) partially explains the reduced bacterial burdens observed in the virulence study. Microarray analysis of ΔrpoN showed that 10% of the genome is differentially regulated by RpoN. Deletion of rpoN also protects Enterococcus faecalis from lysis in the absence of known modulators of cellular lytic events such as O-acetylation and D-alanylation. Of the four identified enhancer binding proteins in E. faecalis, MptR regulates the RpoN-dependent mannose/glucose uptake system (MptABCD) and the ΔmptR mutant phenocopied the ΔrpoN mutant in the eDNA release and growth assays. Because MptC and MptD have been shown to be the cellular receptors for class IIa and IIc bacteriocins, we are presently testing the hypothesis that these receptors may serve as a global receptor for bacteriocins. In conclusion, our data demonstrates that alterations in the metabolic state of the bacterium, as observed in the ΔrpoN mutant could be responsible for the switch in biofilm matrix composition, and this switch in turn likely influences the virulence potential of the bacterium.

Master of Science / Department of Biology / Helmut Hirt / Enterococcus faecalis is one of the most frequently encountered enterococcal isolates and accounts for about 80% of enterococcal infections. Treatment of enterococcal infections has become increasingly difficult as this organism has a high incidence of antibiotic resistance. Lipoteichoic acid (LTA) is an essential amphiphilic polymer on the surface of most Gram positive bacteria. While the molecule's exact role is not yet fully understood, a role in cell-cell contact during conjugation enabling the spread of extra chromosomal elements has been discussed. LTA also has implications in regulating autolysis, sequestering cations to the cell surface, adhesion, biofilm formation, antibiotic resistance, UV sensitivity, acid tolerance, and virulence. The gene ebsG was identified in a mutant of E. faecalis with major alterations in LTA structure and decreased ability to act as a recipient in conjugative mating. ebsG codes for a 119 kDa protein with only weak homology to other surface proteins of Gram positive bacteria. Transcriptional linkage analysis indicated ebsG and its downstream genes are organized in an operon. LTA analysis reveals a higher glycosyl content of the molecule in the mutant during stationary phase. Compared to wild type OG1RF, the mutant is more sensitive to nisin, shows higher autolysis activity during stationary phase, and is better able to serve as a recipient in plasmid transfer. Our data indicate ebsG and the members of the operon play a role in LTA structure and may act to degrade LTA.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Escherichia coli O157:H7 is responsible for over 70,000 cases of human illness every year in the United States. Most cases occur in children under the age of five, the elderly, or other immune-compromised people. A small percentage of these cases will develop a life threatening complication, hemolytic uremic syndrome. Cattle are the reservoir host for E. coli O157:H7 and serve as the main source of contamination of meat products and other food sources. The beef cattle industry is diverse with producers caring for as few as one to as many as thousands of cattle. The first objective of this research was to examine three major production systems (conventional, organic, and natural) in the U.S. and the published performance effects of the various technologies used in each system. The second objective was to determine if a newly licensed E. coli O157:H7 SRP® (SRP) vaccine administered to cows pre-partum could achieve successful passive transfer in their offspring. The third objective was to determine if colostrum obtained from SRP vaccinated heifers could protect against an oral challenge with an E. coli K99+ strain. The fourth objective was to examine the shedding characteristics, health, and performance effects of calves born to SRP-vaccinated cows that also receive SRP vaccination themselves. The technologies used in conventional beef cattle production resulted in significant improvements in health and performance of beef cattle. Vaccinating cows pre-partum with SRP resulted in passive transfer in calves consuming their colostrum. Calves that achieved successful passive transfer shed less E. coli K99+ and had improved fecal consistency compared to placebo. When calves were vaccinated with SRP at branding, weaning, and arrival to the feedyard there was no difference in fecal E. coli O157:H7 shedding on arrival to the feedyard or at harvest. Vaccinating calves with SRP had no effects on performance or health outcomes. Vaccinating cattle with SRP may provide protection against other pathogenic E. coli strains and warrants further investigation. The timing of vaccination appears to be an important consideration in order to ensure maximum vaccine efficacy.

Master of Science / Food Science Institute -- Animal Science & Industry / Daniel Y.C. Fung / In February 2008, the FDA released a draft Compliance Policy Guide (CPG) on Listeria monocytogenes and proposed that ready-to-eat (RTE) foods that do not support the growth of L. monocytogenes may contain up to 100 CFU/g of this pathogen. Frozen foods such as ice cream fall in that category since they are consumed in the frozen state. However, other frozen foods, such as vegetables and seafood that are thawed and served at salad and food bars, may support the growth of Listeria and would not be allowed to contain 100 CFU/g according to the draft CPG. In the current study, growth curves were generated for L. monocytogenes inoculated onto four thawed frozen foods - corn, green peas, crabmeat, and shrimp - stored at 4, 8, 12, and 20ºC. Growth parameters, lag phase duration (LPD), and exponential growth rate (EGR) were determined using a two-phase linear growth model and the Square Root Model. The results demonstrated that L. monocytogenes has a very short LPD on these thawed frozen foods during refrigerated storage and that there would be several orders of magnitude of growth (i.e., more than 1.7 log increase at 4 ºC) of the organism before the product is found to be organoleptically unacceptable. Although it would not be possible to take advantage of any extended lag phase duration caused by freeze injury to the organism, frozen foods containing less than 100 CFU/g of L. monocytogenes that are thawed, or thawed and cooked, and then consumed immediately, should not represent a public health hazard.

Master of Science / Department of Grain Science and Industry / Bhadriraju Subramanyam / Organic, hard red winter wheat of 11% moisture was tempered with distilled water to moisture levels of 16 and 18% and held for 8, 16, and 24 h. At each moisture and holding time wheat was unexposed (control) or exposed to infrared radiation for 1, 1.5, and 2 min using a bench-top flameless catalytic infrared emitter. The mean external grain temperatures for 16% mc wheat measured with thermocouples during infrared exposure of 1, 1.5, and 2 min ranged from 77.4-83.1, 93.7-101.2, and 91.2-98.3°C, respectively; corresponding mean internal temperatures were 67.3-76.4, 80.0-85.6, and 81.3-93.2°C. Minor differences in kernel moisture, hardness, and weight were observed among treatments. Tempered wheat after infrared exposure among treatments lost 1.5-2% moisture. Infrared exposure of wheat reduced initial bacterial loads (6.7×10[superscript]4 CFU/g) by 98.7% and fungal loads (4.3×10[superscript]3 CFU/g) by 97.8% when compared with those on untreated wheat. Wheat tempered to 18% and exposed for 2 min to infrared radiation lost 2% moisture, and this wheat when milled had a yield of 73.5%. The color of flour from infrared- exposed wheat was slightly dark (color change, ΔE = 0.31) when compared with untreated flour. Differential scanning calorimetry showed that flours from infrared exposed wheat had lower enthalpy (3.0 J/g) than those unexposed to infrared (3.3 J/g). These flours were adversely affected because they had longer mixing times (7-15 min) at all infrared exposures due to the presence of insoluble polymeric proteins (up to 60%). Microbial loads in flour from wheat tempered to 18% and exposed for 1-2 min had 0.6-2.4 log reduction compared to flour from untreated wheat. Wheat tempered to 18% moisture with electrolyzed-oxidizing (EO) water reduced bacterial and fungal loads up to 66%. EO water tempered wheat exposed for 1, 1.5, and 2 min to infrared radiation showed microbial reductions of 99.5% when compared with control wheat. Infrared treatment of tempered wheat cannot be recommended as it adversely affected flour functionality. The use of EO water for tempering as opposed to potable water that is generally used in mills slightly enhances microbial safety of hard red winter wheat.

Doctor of Philosophy / Division of Biology / Ari M. Jumpponen / Microorganisms are critically important for establishing and maintaining ecosystem properties and processes that fuel and sustain higher-trophic levels. Despite the universal importance of microbes, we know relatively little about the rules and processes that dictate how microbial communities establish and assemble. Largely, we rely on assumptions that microbial community establishment follow similar trajectories as plants, but on a smaller scale. However, these assumptions have been rarely validated and when validation has been attempted, the plant-based theoretical models apply poorly to microbial communities. Here, I utilized genomics-inspired tools to interrogate microbial communities at levels near community saturation to elucidate the rules and patterns of microbial community assembly. I relied on a community filtering model as a framework: potential members of the microbial community are filtered through environmental and/or biotic filters that control which taxa can establish, persist, and coexist. Additionally, I addressed whether two different microbial groups (fungi and bacteria) share similar assembly patterns. Similar dispersal capabilities and mechanisms are thought to result in similar community assembly rules for fungi and bacteria. I queried fungal and bacterial communities along a deglaciated primary successional chronosequence to determine microbial successional dynamics and to determine if fungal and bacterial assemblies are similar or follow trajectories similar to plants. These experiments demonstrate that not only do microbial community assembly dynamics not follow plant-based models of succession, but also that fungal and bacterial community assembly dynamics are distinct. We can no longer assume that because fungi and bacteria share small propagule sizes they follow similar trends. Further, additional studies targeting biotic filters (here, snow algae) suggest strong controls during community assembly, possibly because of fungal predation of the algae or because of fungal utilization of algal exudates. Finally, I examined various technical aspects of sequence-based ecological investigations. These studies aimed to improve microbial community data reliability and analyses.

Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / Halogenated organic compounds have had widespread and massive applications in industry, agriculture, and private households, for example, as degreasing solvents, flame retardants and in polymer production. They are released to the environment through both anthropogenic and natural sources. The most common chlorinated solvents present as contaminants include tetrachloroethylene (PCE, perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and persistence in the environment, threatening human and environmental health. This contribution provides insight on PCE degradation at laboratory and field scale at a former dry cleaning site in Manhattan, KS. Biostimulation experiments included combinations and concentrations of the following nutrients: soy oil methyl esters (SOME), yeast extract (YE), glucose, lactate, methanol and cheese whey. Bioaugmentation studies used KB-1 bacterial consortium (commercially available culture containing Dehalococcoides). This culture is known to complete the degradation of PCE to a safe end product, ethene. Concentrations of PCE and its degradation intermediates were monitored in the gas phase of the microcosm vials. Biostimulation of the natural ground water and soil microflora did not completely degrade PCE as cis-DCE (c-DCE) accumulated in the sample. Bioaugmented microcosms containing YE and SOME created reducing conditions for KB-1 culture, resulting in ~ 90% dechlorination of PCE to methane and c-DCE. Cheese whey microcosms containing 0.05% cheese whey inhibited the KB-1 culture. This inhibition was due to a drop of pH that inhibited the culture activity. Lower concentrations of cheese whey (e.g. 0.01% to 0.025%) reduced PCE and generated methane in KB-1 augmented microcosms. Based on microcosm results, a pilot bioremediation field study was conducted for a dry cleaning site contaminated with PCE. Ground water flow threatened public water wells located 1.5 miles from the source. Concentrations of PCE in the aquifer was 15 mg/L above the maximum contaminant level of 5 µg/L. Tracer studies with potassium bromide (KBr) were conducted before, during and after the bioremediation study. Nutrient solutions prepared with YE, SOME, lactate and glucose were used for biostimulation and preconditioning of ground water prior to KB-1 injection. Nutrients were provided twice during the pilot study to supplement microbial growth and cheese whey was used. During biostimulation no degradation beyond DCE was evident. The addition of KB-1 reduced PCE and DCE concentrations in the monitoring wells of the pilot study area. Total chlorinated ethene concentrations did not reach background levels 2 years after the last nutrient addition. Tracer results showed that microbial growth decreased ground water velocity during the study, but returned to normal conditions 1 year after the last nutrient addition. In this study we were able to show that native microbial population was not able to degrade PCE to final end products. Therefore, it was necessary to introduce KB-1 culture a long with nutrients to support complete reductive dechlorination of PCE.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / The enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections.