Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-136).

<i>Pseudomonas aeruginosa</i> displays high levels of tolerance and resistance to many antimicrobial agents. Much of this tolerance is related to the nature of the Gram-negative cell envelope and in particular, the outer membrane. The outer membrane plays an important role in excluding harmful molecules from the cell, whilst being selectively permeable to other solutes via its implanted proteins (outer membrane proteins or OMPs). In order to exert their antibacterial action, antimicrobial agents must enter the cell and attain sufficiently high concentrations at their target site(s). The OMPs are highly sensitive to environmental changes and have a physiological ability to respond to such changes. It is thought that the altered cell envelope structure contributes to the accessibility of antimicrobial agents into the cell interior and resistance to such agents is related to over expression or loss of certain OMPs. Brozel and Cloete (1994) observed a gradual increase in tolerance to increasing concentrations of biocide upon exposure of <i>P. aeruginosa</i> to KathonTM, a commercial biocide containing 1.15% v/v 5-chloro-N-methylisothiazolone (CMIT) and 0.35% v/v N-methylisothiazolone (MIT). This adaptation was associated with the concurrent disappearance of a 35kDa OMP, designated T-OMP. Therefore, they concluded that the biocide entered the sensitive cells via the T-OMP and that the observed resistance was the result of the absence of this OMP. The aim of this investigation was to induce tolerance in cultures of <i>P. aeruginosa</i> PAOl towards the pure active forms of the three isothiazolone biocides 1,2-benzisothiazolone (BIT), MIT, CMIT and the thiol-interactive agent thiomersal (used as a positive control). An increase was observed in the minimum inhibitory concentrations (MIC) of all four biocides by at least 58% between the sensitive and resistant cultures. In some cases the percentage increase in MIC was in excess of 150%. However, when the tolerant cells were removed from the presence of the biocide, the MIC began to decrease, indicating a loss in tolerance. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) analysis of the OMP profiles from the tolerant-induced cultures illustrated the loss of T-OMP in all cases. Analysis of the sensitive and resistant cultures using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) indicated that the T-OMP disappeared in the tolerant cultures. However, these observations also suggested that other outer membrane alterations occur concurrently in T-OMP depleted tolerant cells. Investigations into the cross-resistance of the resistant cultures towards the other test biocides, indicated that resistance was, to some extent, transferable, once it had been developed towards one member of the biocide group. Following routine passaging of the resistant cultures on gradient plates two distinct colonial morphologies were observed, mucoid and non-mucoid. An increase in the cell surface hydrophobicity was noted between the mucoid and non-mucoid cultures, which indicated a loss or reduction in the B-band OPolysaccharide. However, there were no observable differences in the lipopolysaccharide banding patterns between the mucoid and non-mucoid cells. These observations suggested that other alterations were occurring in the tolerant cells upon exposure to biocide, over and above the simple disappearance of T-OMP. Therefore, it is suggested that the observed tolerant development in biocide exposed cells, was not solely due to the loss of T-OMP. Investigations into Gram-negative bacteria isolated from contaminated industrial samples preserved with isothiazolone compounds exhibited higher MICs towards the preservative biocides than would normally be expected in the species of bacteria isolated and identified. However, there were no observable alterations in their OMP profiles.

No description available.

Orientador: Wilter Ricardo Russiano Vicente / Banca: Maria Denise Lopes / Banca: Fabiana Ferreira de Souza / Resumo: Atualmente, estudos têm demonstrado a utilização da toxina botulínica do tipo A (TB-A) no tratamento de afecções prostáticas, como a hiperplasia prostática benigna. Ainda, sabe-se que alguns componentes bioquímicos do plasma seminal são relativamente específicos para a regulação da função espermática. O objetivo deste trabalho foi avaliar os possíveis efeitos deletérios ou benéficos da TB-A sobre a fertilidade de cães com HPB. Foram utilizados 18 cães machos, sem raça definida com sinais de HPB. Os animais foram divididos ao acaso em 3 grupos de 6 cães que receberam injeção intraprostática de solução salina de NaCl 0,9% (GC), solução contendo 250U (GI) e 500U de TB-A (GII). Quatro amostras foram colhidas antes da aplicação e 2, 4 e 8 semanas após o tratamento. Foi mensurado o pH e dosadas as concentrações de proteína total, cloretos totais, cálcio, potássio e sódio das amostras de plasma seminal. Ainda, foi realizada eletroforese SDS-Page utilizando géis de poliacrilamida nas concentrações de 12 e 18%. Os resultados foram avaliados por meio de análise de variância (ANOVA) e ao teste de Kruskall-Wallis (p<0,05). Não foram observadas diferenças significativas nos parâmetros bioquímicos avaliados. Outrossim, foram constatadas 31 bandas protéicas, com pesos moleculares variando de 106,2 a 3,9 kDa semelhantemente aos achados descritos na literatura. Destarte, pode-se concluir que os tratamentos com 250U e 500U de TB-A não alteraram os perfis bioquímico e protéico do plasma seminal de cães com HPB e, portanto podem ser considerados uma boa opção para cães destinados à reprodução ou envolvidos em programas de criopreservação de gametas. / Abstract: Recently, botulinum neurotoxin type A (BoNT-A) application in the lower urinary tract has been extended to prostate disorders such as benign prostatic hyperplasia (BPH). Nevertheless, sperm function is highly dependent on ionic and protein environment. The goal of the present study was to evaluate the effects of BoNTA treatment on seminal plasma biochemical and eletrophoretic profile in dogs with BPH. Eighteen mature male mongrel dogs with BPH, were use in this study. They were randomly divided in three groups, which received intraprostatic injection of 0,9% NaCl normal saline solution (GI), 250U(GII) and 500U (GIII) of BoNT-A. All animals were previously conditioned to semen collection and four semen samples were collected before treatment and at 2, 4 and 8 weeks after injection. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, total protein concentration (TP), total chlorides (TC), calcium (Ca), potassium (K), and sodium (Na). Additionally, one-dimensional sodium dodecyl sulfatepolyacrilamide gel eletrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigel. One way ANOVA and Kruskall-Wallis with Bonferoni as a post hoc test were used to comparison of means (P<0.05). Average pH, TP, TC, Ca, K did not vary significantly at any time point and among each studied group and maintained normal values for the specie. Similarly as described in the literature, the eletrophoresis analysis of the pooled eluded fractions, identified 31 bands, with molecular weights varying from 106.2 to 3.9 kDA, In conclusion, the treatment with 250U and 500U of BoNT-A did not modify the biochemical or protein profiles of the seminal plasma of dogs with BPH. / Mestre

Protein-carbohydrate recognition is an important target for inhibitor development. Improved inhibitor design requires a fundamental molecular basis of these interactions. This thesis describes the preliminary structural studies on three carbohydrate processing enzymes, UDP-galactopyranose mutase, alpha-D-glucose-1-phosphate thymidylyltransferase and TDP-glucose 4,6-dehydratase. These enzymes are found in important human pathogens such as Mycobacterium tuberculosis and Salmonella typhimurium. The major focus of the thesis has been on UDP-galactopyranose mutase, the enzyme responsible for catalysing synthesis of the thermodynamically unfavourable 5 membered ring form of galactose, UDP-galactofuranose from the thermodynamically favoured 6 membered ring form, UDP-galactopyranose. UDP-galactofuranose plays a key role in mycobacterial cell walls. This thesis also describes work with concanavalin A. This legume lectin is an invaluable model for the study of protein-carbohydrate interactions. Two concanavalin A complexes are discussed. Both structures clear up misunderstandings in the literature and provide an insight into designing enzyme inhibitors.

Cells of S. marina (N.C.M.B, 778) were disrupted using a Hughes press and a purified cell wall fraction obtained using a previously reported method for halococcal wall isolation. This procedure was monitored by examination of thin sections in the electron microscope and the final wall preparation was seen to be relatively free of cytoplasmic and membranous contaminants. However, treatment of the wall fraction with crude trypsin did appear to remove particulate surface components. The total ninhydrin-positive components detectable accounted, for only about 14% of the cell wall dry weight. The major amino acids present were glycine, alanine, glutamic acid and aspartic acid although very small amounts of others were detected. The amino sugar components included glucosamine and galactosamine although these only accounted for some 60% of the total amino sugars. The remainder was probably made up of one or more of four unidentified, acid-labile components detected on amino acid analysis and by paper chromatography. This is in accord with the finding of unusual, labile amino sugars in the cell walls of other halococcal species. Approximately 37% of the cell wall dry weight was made up of the neutral sugars, glucose, galactose and mannose which were present in eguimolar amounts. In addition, the wall was found to contain a negligible lipid (0.1% dry weight) and a high ash (9.2% dry weight) content. The poor recovery of organic material after analysis is almost certainly due to the lability of some of the more unusual (and in this work unidentified) components. Attempts to selectively solubilise the wall material with a view to identifying discrete polymers met with some success. In Particular, treatment with trichloroacetic acid (TCA) at 35&deg; extracted all of the glactosamine from the wall (in addition to other components) but none of the unknown component, X1. Further treatment with TCA at 60 extracted all of another unknown component, X1. These results suggest that some degree of resolution of different polymers constituting the wall may be possible and may have been achieved here. Treatment of S. marina with the antibiotics, D-cycloserine, novobiocin, bacitracin, penicillin G and vancomycin, known to affect cell wall biosynthesis in other bacteria, was carried out. Possible effects of the antibiotics were monitored by electron microscopy and turbidimetric estimation of bacterial growth. Only novobiocin and bacitracin had any effect on growth but this was marked; in both cases growth was prevented by addition of the antibiotic. The other three antibiotics all lost their antibiotic activity (against appropriate indicator organisms) when incubated over a period of a few hours in Dundas medium. It is suggested that this may be a significant consideration when explaining the antibiotic insensitivity of microorganisms, such as S. marina whose doubling times are of the same order of magnitude as that necessary for antibiotic inactivation. Thin sections of control and antibiotic-treated cells showed interesting ultrastructural features comparable with those seen in more conventional halophilic cocci. Some minor ultrastructural changes were seen in some of the anti-biotic-treated cells, the most notable being extensive plasmolysis in the case of novobiocin. However, none of the antibiotics tested appeared to cause cell lysis or osmotic fragility which may preclude their use as agents for the non-destructive removal of the cell wall.

An unusual form of copper tube corrosion, occurring in two hospitals, was investigated during two site surveys and due to the presence of characteristic perforations became known as pepper-pot pitting corrosion. The corrosion was found to occur predominantly in soft water areas but mainly in hot water systems maintained below 50'C. When monitoring the hot water system at a particular site the water temperature was found to decrease overnight with a corresponding decrease in the dissolved oxygen concentration and assimilable organic carbon. Copious amounts of biofilrn were recovered from the pipe surfaces thus it was hypothesised that metabolically active and respiring biofilm bacteria contributed to the creation of aggressive corrosive conditions at the copper tube surface. At control sites where this type of corrosion was not reported, the water temperature was found to be maintained above 50'C with reduced biofouling. A laboratory model, using filter-sterilised potable water as the sole carbon source, was developed to investigate the conditions under which corrosion and biofouling was occurring. Biofilm development was demonstrated up to 55'C. At 60"C biofouling was very much reduced; however, a decrease in the number of bacteria recovered from the planktonic phase was only observed at 65'C. Planktonic bacteria were found to be dominated by pseudomonads while the biofilm was dominated by other Gram negative bacteria. Control measures that would slow down or prevent corrosion were studied. Pasteurisation (60'C) was found to prevent biofouling as well as controlling re-established biofilms but was less effective against consortia that had been previously exposed to this temperature. For the removal and control of biofilm, sulphamic acid was more effective than citric acid which allowed re-growth to occur within 14 days. This unusual copper tube corrosion has resulted in increased use of alternative plumbing tube materials and therefore colonisation of copper and competitive plastic materials were investigated in the model system. Plastic materials were shown to encourage growth of Legionella pneumophila at 40'C whereas copper suppressed the growth of this water-borne pathogen. Results obtained in this investigation suggest that plastic plumbing systems pose a potential health risk by providing a means for transmission of pathogens such as L. pneumophila. The association of biofilms with pepper pot pitting led to new ideas about mechanisms of microbially induced corrosion of copper tubing. A number of techniques including SEM, SCLM and light microscopy were used to demonstrate the heterogeneity and metabolic activity of biofilms produced in the laboratory model and on pipe surfaces. Mosaic microcolonies, themselves are responsible for the initiation of differential concentration sites that are aggravated by exo-polysaccharides, metabolic activity and particulate matter in the aquatic environment. It is the localised distribution of initiated sites that could be responsible for the formation of multi-loci corrosion cells that are driven by an electrochemical potential forming the type of corrosion described as pepper-pot pitting.

No description available.

Bacterial abundances and production, and the size distribution of oxygen metabolism and chlorophyll a concentration were followed through two seasonal cycles in the Menai Strait (North Wales, U. K. ) and during austral summer in the Southern Ocean. In the Menai Strait, spring blooms were characterised by a diatom to Phaeocystis succession. In both the Menai Strait and the Southern Ocean, meso- and microphytoplankton dominated phytoplankton production and biomass during diatom blooms. Nanophytoplankton predominated when production and biomass were low, i. e. during the summer in the Menai Strait, in waters near the Polar Front, and in some samples from the Weddell Sea. In both ecosystems substantial respiration resided in the bacterial (< 0.8 gm) size-fraction. Consequently during the Menai Strait temporal study, phasing of respiration in relation to photosynthesis was strongly influenced by bacterial metabolism and abundance changes. The respiration maximum occurred 1-2 weeks after the Phaeocystis abundance maximum. An explanation for this temporal lag was sought by considering the time scales of flow of organic material between the phytoplankton and the bacteria. The observations were consistent with routes via a slowly cycling pool, such as polymeric organic material. This pool would function as a reservoir and result in microheterotrophic respiration persisting after the decline of photosynthesis, causing a positive to negative temporal sequence in net community production. There was no evidence for differences in any measure of microbial biomass between the Southern Ocean and the Menai Strait. General relationships could be derived for both ecosystems: (a) the biomass quotient (< 20 µm phytoplankton / unfractionated phytoplankton) generally increased sharply as unfractionated phytoplankton biomass decreased, (b) bacterial biomass generally increased as phytoplankton biomass increased, (c) the biomass quotient of bacteria to unfractionated phytoplankton increased sharply as unfractionated phytoplankton biomass decreased. Different relationships were derived for the oxygen fluxes in terms of phytoplankton biomass for the Southern Ocean and Menai Strait observations. In these relationships, the oxygen fluxes were generally relatively (relative to the explanatory variable: phytoplankton biomass) higher in the Menai Strait. In contrast, a single relationship for DCR in terms of GCP was fitted for both data sets. This difference is consistent with a temperature effect on the oxygen fluxes, with GCP and DCR similarly suppressed at lower temperatures.

No description available.