Thèse de doctorat : Physique des aérosols : Paris 12 : 2005. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 246 réf.

Bibliography: leaves 246-304. / xi, 304, [43] leaves, [35] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes experiments aimed at characterising the potential virilant factors of Legionella longbeachae sg 1, an important human pathogen which is responsible for nearly half of all clinical cases of Legionella related pneumonia reported each year. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 2000

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 2000. / Bibliography: leaves 246-304.

Thesis (Ph. D.)--Auburn University. / Abstract. Includes bibliographical references (p. 129-151).

Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Computerdatei im Fernzugriff.

Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Computerdatei im Fernzugriff.

Würzburg, Universiẗat, Diss., 2000. / Dateien im PDF-Format.

Unsaturated fatty acids of known degree, position, and configuration of unsturation were esterified, and stereospecifically dihydroxylated at the double bond(s). cis-Hydroxylation was effected using Woodward's reagent (silver acetate/iodine/acetic acid), while trans-hydroxylation was effected using Fenton's reagent (hydrogen peroxide/ferrous sulfate/acetic acid). Diols derived from monounsaturated esters were recovered quantitatively, while tetraols derived from diunsaturates were lost, presumably during extensive washing. The stereospecifically dihydroxylated esters were analyzed by thin-layer chromatography on borate-impregnated silica gel plates, and by gas-liquid chromatography, on a nonpolar capillary column, as the trimethylsilyl, acetyl, n-butylboronyl, isopropylidene, and trifluoroacetyl derivatives. For each derivative, the erythro and threo diols were readily separable, and some resolution of positional isomers was observed. Thus, the cis/trans configuration, and in some instances, the position of unsaturation of the original monounsaturated fatty acid may be deduced. However, gas chromatography-mass spectrometry of appropriately derivatized diol esters is required for unambiguous determination of position of unsaturation in most cases. These reactions are simple, use readily available reagents, and require relatively little operator attention. Further, they do not require specialized apparatus, as do hydrogenation and ozonolysis, or potentially toxic chemicals, such as osmium tetroxide. This series of analyses was applied to the unsaturated non-hydroxylated fatty acids of Legionella species, which were shown to be monounsaturated and of the cis Δ9 family. Position of unsaturation was confirmed by gas chromatography-mass spectrometry.

Ineffective bacterial monitoring in water systems represents a danger to public health and can result in costly disease outbreaks. Of interest is Legionella pneumophila, a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. The Center for Disease Control stated that reported cases of Legionnaires’ disease have quadrupled since 2000 and ranks L. pneumophila as the number one cause of waterborne disease outbreaks in the United States. This threat is expected to increase given an aging population who are more susceptible to L. pneumophila infection and rising global temperatures that can promote L. pneumophila growth. Presently, Public Health agencies recommend bacterial culturing for the detection of L. pneumophila in environmental samples, however, this process can take up to ten days to complete. Consequently, there is a delay between sample collection and subsequent L. pneumophila detection, creating an opportunity for a Legionnaires’ disease outbreak to occur. There is a great need to develop a field-appropriate device that can provide early-stage detection of L. pneumophila in water as a means of mitigating Legionnaires’ disease outbreaks. We propose the use of DNAzymes for the development of such a device. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have successfully isolated an RNA-cleaving fluorescent DNAzyme (RFD) specific for the detection of L. pneumophila using in vitro selection. Thorough characterization of the DNAzyme has revealed key structural features influencing kinetics, specificity and sensitivity. In addition, the ability of the DNAzyme to function in cooling tower water, and conservation of the DNAzyme target across Legionella bacteria, has been investigated. In the future we plan to incorporate this RFD into a field-appropriate paper-based device which would play a key role in managing infectious diseases and preventing large-scale outbreaks. / Thesis / Master of Health Sciences (MSc) / Legionella pneumophila is a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. Numerous Legionnaires’ disease outbreaks have occurred, with the most common source of exposure to L. pneumophila coming from contaminated cooling towers. Presently, bacterial culturing is used to determine if a cooling tower is contaminated with L. pneumophila, however this process can take up to 10 days to complete. To address this delay, we plan to develop a rapid paper-based test for L. pneumophila detection in cooling tower water using DNAzymes. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have isolated a DNAzyme that can specifically detect L. pneumophila and characterized its properties. In the future we plan to incorporate this DNAzyme into a field-appropriate paper-based test which would play a key role in managing Legionnaires’ disease outbreaks.

Legionella is the causative agent of Legionnaires' disease which hospitalizes 8,000 to 18,000 people in the United States each year. The disease in transmitted through inhalation or aspiration of water containing the bacterium and can be acquired within the home. Studies have found that 0-37% of domestic water heaters contain Legionella, making household hot water systems a potential route of exposure. The objective of this research was to evaluate different methods for testing environmental samples for Legionella pneumophila and to analyze potable water conditions that affect survival of free living Legionella pneumophila in hot water tanks. Three heat pretreatment methods (50ºC for 30 minutes, 55ºC for 15 minutes, and 60ºC for 3 minutes) were not effective at recovering Legionella in this study. There was no statistically significant difference between the three acid pretreatment methods that were tested (pH 2.0 with a neutralizing solution, pH 2.2, and the CDC method). Six media (BCYE, DGVP, PCV, GPCV, CCVC, and GPVA) exhibited similar Legionella recovery, except for when high levels of non-Legionella organisms were present, in which case BCYE demonstrated lower recovery. When disinfectant was present, if sodium thiosulfate was not added before the disinfectant, Legionella recovery was lower. However, this result was not statistically significant for free chlorine until after 5 minutes. Pseudomonas aeruginosa (up to 67.5 cfu/ml) and pyocyanin (up to 9 mg/l) did not have an effect on Legionella recovery under the tested conditions. Environmental factors affecting survival of free living Legionella pneumophila in hot water tanks were also studied. After one day exposure in small-scale simulated water heaters at 55ºC, viable Legionella could not be recovered. At 44ºC, Legionellae were recovered after one day but only at very low levels after eight days. Between 23 and 37ºC, Legionella could survive longer than eight days. Copper (Cu²⁺) concentrations above 2160 ppb were found to be toxic to Legionella, but iron (Fe³⁺) between 1 and 2160 ppb did not affect survival. Above pH 11 survival was greatly reduced. No effect was observed between pH 5-10. When glass fiber filters were added to the reactors and they were seeded with tap water and sediment slurry, Legionellae were retained in 7 of 16 reactors for 327 days. The results of this work will assist in optimal identification of Legionella via microbial analysis of potable water samples, thereby assisting in prevention and diagnosis of factors contributing to Legionnaires' disease, especially in settings with high-risk patients (e.g. hospitals). Water systems studying Legionella amplification in domestic hot water systems can use simulated or real distribution system sampling to reproduce and study factors that prevent or reduce Legionella growth and persistence. / Master of Science