published_or_final_version / Biological Sciences / Master / Master of Philosophy

This study evaluated the stability of oral bacteria in healthy subjects and documented community shifts in smokers and oral/periodontal disease by employing PCR-RFLP, DGGE and sequence analysis of the 16S rDNA gene from metagenomes and plate-wash (cultured) bacteria of oral wash from 15 participants,. A stable core of bacterial DNA fingerprint was detected within and between subjects and did not change over time when analyzed in smokers and healthy non-smokers. Signature bands in smokers, non-smokers and periodontal disease subjects were evident suggesting the presence of potential indicators of health and poor oral health. Taxon diversity was higher in smokers including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and Pyrobaculum but highest in periodontal disease. The two techniques successfully aligned the subjects within appropriate categories (based on their oral microbial genetic patterns)confirming their diagnostic suitability. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

The environment is increasingly recognised as a key player in the emergence and mobilisation of antibiotic resistance, which negatively impacts human health, healthcare systems, and farming practices worldwide. Recent work has demonstrated concentrations of antibiotics in the natural environment may select for resistance in situ, but a scarcity of meaningful data has prevented rigorous environmental risk assessment of antibiotics. Without such data, mitigation strategies, such as improved antibiotic stewardship or environmental discharge limits, cannot be effectively designed or implemented. This thesis designed and developed two methods for determining effect concentrations of antibiotics in complex microbial communities, thereby generating a significant amount of data to address this knowledge gap. Minimal selective concentrations (MSCs) were determined in long term selection experiments for four classes of antibiotic at concentrations as low as 0.4 μg/L, which is below many measured environmental concentrations. Lowest observed effect concentrations were determined using a short term, growth based assay which were highly predictive of MSCs. A novel finding was significant selection for cefotaxime resistance occurred at a wide range of antibiotic concentrations, from 125 μg/L - 64 mg/L, which has important clinical implications. Determination of MSC in single species assays was also shown to be a poor predictor of MSC in a complex microbial community. Co-selection for antimicrobial resistance was demonstrated in selection experiments and through improved understanding of class 1 integron evolution, assessing selective effects on resistance gene acquisition using a novel PCR method and next-generation sequencing. In the final study, a novel resistance determinant (UDP-galactose 4-epimerase) conferring cross-resistance to biocides and antibiotics was discovered, providing a target for further study. These findings indicate selection and co-selection for antimicrobial resistance is likely to occur in the environment, and provides the means to rapidly generate further data to aid in the development of appropriate mitigation strategies.

The Tat protein export system transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli, the Tat system is composed of the TatA, TatB and TatC proteins. TatB and TatC assemble into a multimeric receptor complex that recognises and binds the substrate, before the TatA protomers cluster at the TatBC complex to facilitate substrate transport. A genetic screen was devised to explore the oligomeric state of TatC, reasoning that the isolation of dominant negative TatC variants that inactivate the Tat system in the presence of a functional copy of wild type TatC would provide strong evidence TatC is an obligate oligomer. Single dominant negative TatC substitutions were isolated that were located in the first and second periplasmic loops of TatC. These substitutions did not prevent TatC from interacting with TatB, TatA, itself or with a Tat substrate. Blue Native PAGE analysis showed that the TatC variants were unable to form the 440 kDa TatBC complex. Surprisingly, the substitutions did not prevent TatC:TatC self-interactions in the periplasmic regions, detected by disulphide cross-linking, but they did abolish a substrate-induced interaction at the fifth transmembrane helix of TatC. Fluorescence microscopy experiments revealed that the dominant negative TatC variants prevented the polymerisation of TatA-YFP in vivo. These results show that TatC possesses at least two interaction interfaces and imply that the periplasmic loops are critical for the transition between substrate binding and TatA polymerisation. Accessibility of single cysteine substitutions in TatC was probed by PEG-Mal labelling in intact cells. TatB was shown to be important for the proper insertion of TatC into the membrane. The absence of TatA led to accessibility changes in the vicinity of the fifth transmembrane domain of TatC, where both TatA and TatB are known to dock. This suggests that TatA and TatB may share an overlapping binding site.

In this thesis, the application of molecular microbiology methods to understand wastewater treatment bio-reactions is described. Two different wastewater treatment systems were chosen for the experimental work. Firstly; the activated sludge processes at two different facilities in Sweden (Västerås and Eskilstuna) were investigated and compared in a context where low temperatures can affect the efficiency of the nitrogen removal performance in terms of nitrification. Initially, fluorescence in situ hybridization (FISH) was utilised in order to quantify some of the species involved in ammonia and nitrite oxidation at Västerås, providing information on how the different communities react to decreasing temperatures. Then, the polymerase chain reaction (PCR), cloning-sequencing method was employed in order to study the composition of the ammonia oxidizing bacteria (AOB) community at the same two wastewater treatment plants (WWTPs). Secondly; the potential use of constructed wetlands for the treatment of winery wastewater was studied. High ethanol concentration artificial wastewater with and without inorganic nutrients (nitrates and phosphates) was fed in a set of pilot-scale constructed wetlands. Pollutant removal performance and enzyme activity tests were carried out. Additionally, the bacterial community structure was investigated by means of denaturing gradient gel electrophoresis (DGGE). In the first set of studies it was shown that the AOB population which plays a major role in nitrifying reactors presented a seasonal shift and a higher diversity at Västerås during winter time, while the nitrification performance maintained stable levels and the ammonia removal efficiency increased. Thus, the higher ammonia removal efficiency at Västerås could be related to the diversity of the AOB population composition. Lastly, when constructed wetlands were in focus, the differential effects of ethanol and nutrients over the chemical oxygen demand (COD) removal performance were proven. In fact, the addition of nutrients on one of the experimental wetlands increased the COD (ethanol) removal and supported the maintenance of a bacterial population similar to the control wetland (no ethanol added). In conclusion, both studies proved a strong relationship between process performance (pollution removal) and the dynamics of the bacterial communities involved.

The pathogen Vibrio cholerae uses cations as a primary currency of virulence and environmental persistence, using gradients of those cations to move, acquire nutrients, and control virulence gene expression. An understanding of the overlapping roles of bioenergetics and chemotaxis in the virulence and environmental survival of V. cholerae issues from a large body of prior work, but the interplay of each component is not yet clearly understood. To this end, the activity of the antiporters Vc-NhaP1, Vc-NhaA, and Vc-NhaB was assayed, as was the sodium transporting respiratory pump NQR, and environmental stimuli were paired with potential motilitylinked sensors. The Vc-NhaP1 antiporter was found to be a K⁺(Na⁺)/H⁺ antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-logarithmic phase, after 4-6 hours of culturing. Using a pH-sensitive GFP protein, cytosolic pH was shown to be dependent on K⁺ in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless E. coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K⁺ or Na⁺ ions for protons within a broad pH range (7.25 to 9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K⁺(Na⁺)/H⁺ antiporter of the CPA- 1 family that is required for bacterial pH homeostasis and growth in an acidic environment. Further, a model system comprised of a V. cholerae strain lacking both the nqr operon and the ORFs of Vc-nhaA or Vc-nhaB was generated and tested with and without lactate. These strains, along with the single mutants of nqr, Vc-nhaA, and Vc-nhaB, were assessed for aerobic growth as a function of media pH and cation concentration (Na⁺, Li⁺, or K⁺). Loss of Vc-NhaA and, to a lesser extent, Vc-NhaB, was better observed when NQR was absent but lactate was added to facilitate replenishment of the quinone pool. Loss of Vc-NhaA in this background inhibited growth most at basic pH under increasing Na⁺ and Li⁺ conditions, and loss of Vc- NhaB in this background inhibited was most severe in acidic conditions in the presence of 0-100 mM Na⁺ or Li⁺. We also observed the growth inhibition of Vc- NhaA in the absence of NQR and in the presence of lactate and 100-450 mM Li⁺, which has not been previously reported. These growth defects were restored upon expression of the cognate antiporter gene on an inducible expression vector. Lastly, potential chemotaxis stimuli were correlated with cognate methyl-accepting chemotaxis protein (MCP) receptors. The homology of MCP sensory domains among Vibrionaceae demonstrated a subset were unique to V. cholerae. Of these unique MCPs, transposon insertion in VC0098 significantly reduced chemotaxis swarm diameter towards Na⁺ and K⁺. Additionally, the MCP VCA0663 was shown, by transposon mutagenesis and complementation, to direct chemotaxis towards N-acetylglucosamine. Additional observations are described concerning the chemotaxis defects incurred by transposon mutagenesis of MCPs in vitro towards mucin, bile, or L-serine. MCP strains were also tested in vivo for 4 and 24 hours in the infant mouse model of infection. None of the observed chemotaxis defects showed complete loss of chemotaxis by transposon mutagenesis, in line with the hypothesis that the large number of MCPs encoded by V. cholerae result in redundant chemotaxis sensory functions. These findings add to the understanding of how bioenergetics and chemotaxis interact within V. cholerae, a foundation from which the bacterium can be understood and, eventually, controlled. / Graduation date: 2012

The intestinal microbiota is a massive and complex community, essential to the human host for good health and well-being. However, this population has been associated with gastrointestinal disease, and remains poorly understood. The aim of this study was to develop and validate DNA-based assays for the intestinal microbiota and to apply these methodologies to faecal samples collected from healthy volunteers and patients with gastrointestinal disease. Over 250 faecal samples were analysed using temporal temperature gradient gel electrophoresis (TTGE) and real time PCR. Validated assays had high sensitivity and reproducibility. Healthy individuals displayed a high level of temporal stability during short term studies (≤ 6 weeks) and long term studies (1-4years). Analysis of faecal samples provided by identical and fraternal twins demonstrated an influence of host genetics over the composition of the predominant bacteria in children. Two intervention studies, bowel lavage and the Atkins' diet, were carried out to monitor the impact of environmental change on the population's stability in healthy volunteers. Following bowel lavage, microbial populations rapidly recovered to control densities, however the stability of the population was disturbed. Introduction of the Atkins' diet, led to a significant change in the composition of the microbial population. A preliminary study of the intestinal microbiota in disease groups was undertaken. Significant differences were detected between inflammatory bowel disease groups and controls. Cluster analysis in these patients indicated a potential association between the composition of the predominant bacterial population and disease localisation. The studies reported here demonstrate that the faecal microbiota in healthy individuals is a highly stable population under the influence of both host genetics and environmental variables, however the population present in patients with inflammatory bowel disease exhibits differences compared to healthy controls.

Please read the abstract in the section 05summary, of this document / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

No description available.

BACKGROUND: Regulatory T cells (Tregs) are an essential subset of T cells that despite over 10 years of research have yet to be fully characterized. These suppressive immune cells, derived from the thymus, express high levels of interleukin-2 receptor alpha-chain (CD25). Tregs are needed to maintain self-tolerance and to control responses to non-self-antigen. The mechanism of Treg repression is unknown. The direct cell-to-cell contact through binding of cell surface molecules as well as secretion of suppressive cytokines has been shown to suppress the proliferation of Thl and Th2 cells against auto, allo and foreign antigens. The role of Tregs in regulating B cell response is also uncertain. The objective of this study is to determine how the removal of T regulatory cells can increase B cell responses in vitro. METHOD: This study focuses on the effect Tregs have on the generation of an antigen specific immune response in vitro. T cells with and without Tregs were co-cultured with monocyte derived dendrtic cells. The antigen specific activation was determined by analyzing cytokine production using intracellular cytokine staining, a flow based assay. Next, the effects of Tregs on both recall and naive B cell responses was analyzed using a co-culture of B cells, CD4 T cells with and without Tregs and monocyte-derived dendritic cells. Analysis of lymphoproliferation, activation, and antibody production was analyzed by using flow cytometry, Elispot and ELISA assays. RESULTS: An antigen specific response against gp120 was generated in naive T cell culture. Tregs were shown to inhibit antigen specific cytokine production in CD4 T cell culture to de novo antigens. The activation in the absence of Tregs was superior to the addition of exogenous factors of IL-2 and IL-7 with a third less non-specific background activation. When analyzed in a TT recall B cell assay, however, the removal of Tregs proved to have an inhibitory effect on antigen secreting cells detected by Elispot. This inhibition appeared at both a 1: 1 and 1 :4 T to B cell ratios though was slightly decreased at the 1 :4 ratio. The same was true for na1ve B cell assay showing a decrease in the generation ofMSPl-42 IgM antigen secreting cells. ELISA assays also confirmed the results showing a nearly 2.5 fold decrease in the amount of MSP 1-42 specific IgM Ab in the L TE cell culture supernatant. Conclusion: While the removal of T regulatory cells is beneficial for the activation of na1ve T cells, the removal of Tregs seems to be inhibitory to B cell activation in the LTE. This inhibitory effect maybe due to T cells becoming too stimulatory before culture with B cells. Studies involving a wider range of T to B cell ratios and culture times may be beneficial to determine if the depletion of Tregs would benefit this culture method.