Legionella pneumophila est une bactérie opportuniste qui émerge de l'environnement après multiplication dans des amibes et peut infecter accidentellement les macrophages alvéolaires humains, provoquant une pneumonie sévère, la légionellose. La capacité de L. pneumophila à survivre dans ses cellules hôtes est strictement dépendante du système de sécrétion de type 4 Dot/Icm, qui sécrète un large répertoire d'effecteurs dans le cytosol de l'hôte. Identifier la contribution individuelle de chaque protéine bactérienne sécrétée par le système Dot/Icm, dans le cycle infectieux de L. pneumophila reste un enjeu majeur pour comprendre les bases moléculaires de la virulence des légionelles. Mes travaux de thèse participent à cet objectif en caractérisant la voie cellulaire ciblée par la protéine kinase LegK2. Des tests d'interaction et de phosphorylation ont identifié le complexe nucléateur d'actine ARP2/3 comme cible de LegK2. Suite à l'adressage de LegK2 à la surface de la vacuole après sa translocation dans le cytosol de l'hôte, l'interaction LegK2-ARP2/3 inhibe la polymérisation d'actine sur le phagosome. Cette inhibition permet à Legionella de diminuer le trafic des endosomes tardifs et/ou des lysosomes vers le phagosome et favorise ainsi l'évasion du phagosome à la voie de dégradation endocytique. L'interaction LegK2-ARP2/3 met en évidence un mécanisme original de virulence dans lequel le remodelage local du cytosquelette d'actine de la cellule hôte permet à la bactérie de manipuler le trafic vésiculaire pour échapper aux défenses de l'hôte / Legionella pneumophila is an opportunistic bacterium that emerges from the environment after multiplication in protozoans and can accidentally infect human alveolar macrophages leading to a severe pneumonia, the legionellosis. The L. pneumophila ability to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Deciphering the individual contribution of each bacterial protein translocated by the Dot/Icm system in the L. pneumophila infectious cycle remains a major challenge to understand the molecular basis of Legionella virulence. My works contribute to this objective by characterizing the cellular pathway targeted by the protein kinase LegK2. Interaction and phosphorylation assays identified the actin nucleator ARP2/3 complex as the target of LegK2. Following the LegK2 addressing to the vacuole surface after its translocation into host cytosol, LegK2- ARP2/3 interplay inhibits the actin polymerization on the phagosome. This inhibition allows Legionella to decrease the late endosome/lysosome trafficking towards the phagosome and promotes the phagosome evasion from endocytic degradation pathway. LegK2-ARP2/3 interplay highlights an original mechanism of virulence wherein the local actin cytoskeleton remodeling of host cell allows bacteria to hijack the vesicles trafficking in order to escape host-cell defenses

Please read the "Background" (p v) in the section 00front of this document Copyright 2002, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Bartie, C 2002, The life and career of the South African dramatric soprano Marita Napier, DPhil thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-11142007-125718 / > / Thesis (DPhil (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Legionella pneumophila is an intracellular pathogen and the etiological agent of Legionnaires’ disease, a severe atypical pneumonia. This bacterium is ubiquitous to freshwater ecosystems where it spreads in the planktonic form but is primarily found associated with protozoa. Protozoa serve as a niche for its replication because the extracellular environment often does not offer sufficient nutrients to support the growth of this bacterium. L. pneumophila is an opportunistic pathogen in humans and utilizes an arsenal of virulence factors to colonize hosts and cause Legionnaires’ disease. The transition between extracellular and intracellular milieus triggers a series of metabolic, morphological and genetic changes that define two developmental stages in this bacterium: replicative and transmissive. Relatively high concentration of nutrients triggers the replicative stage of growth, where the bacterium has the appearance of a thin, elongated rod without the presence of flagella. In addition, is characterized by active metabolism and expression of genes required for productive replication. In contrast, once nutrient levels are relatively low, L. pneumophila switches to its transmissive form. In this form, the bacterium activates a genetic program that includes the expression of many traits associated with the transmissive stage, such as coccoid cell shape, motility, pigmentation and important virulence factors. These multifaceted changes in gene expression leading the differentiation from replicative to the transmissive form, are controlled by two-component regulatory systems. Specifically, the LetAS two-component system plays a key role in the regulation of cell morphology and in the production of the pigment pyomelanin. Here we report the identification of a LetAS-regulated protein, Lpg0586 (designated as Larp1), capable of inducing changes in cell morphology and pigment production. We found that Larp1 expression was accompanied by accumulation of the RecA protein, but evaluation of recA deletion mutants indicated that RecA is not involved in cell morphology changes in L. pneumophila. The specific reason as to why RecA accumulates upon Larp1 expression remains to be elucidated. However, we show that upon synthetic HGA treatment, L. pneumophila cultures display cell elongation and increased RecA levels. Lastly, Larp1 expression restored pyomelanin production in an un-pigmented mutant and increased the transcription of important genes involved in the pyomelanin production pathway. Based on these findings, Larp1 is the first LetAS-regulated protein reported to be involved in pyomelanin production.

Three Legionella-like organisms isolated from patients with pneumonia are shown to belong to the species Legionella sainthelensi by DNA hybridization studies and to a new serogroup, serogroup 2, by serological studies (ATCC 49322). L. sainthelensi serogroup 2 and L. santicrusis are indistinguishable bu slide agglutination, but are separable on the basis of their cell wall fatty acid profiles. © 1990.

Diese Arbeit befasst sich mit der Untersuchung des Metabolismus sowie der Reaktivität verschiedener Wirk- und Arzneistoffe mittels flüssigchromatographischer und massen-spektrometrischer Methoden, sie gliedert sich dabei in vier Projekte. Zur Bestimmung des Metabolitenprofils wurde ein passendes In-vitro-Inkubationssystem mit Cytochrom-P-450-Systemen entwickelt. So wurden der Metabolismus und die Pharmakokinetik der Mip-Inhibitoren SF110, SF235 und SF354 gegen Legionellen, sowie neuer antitrypanosomaler Verbindungen MB209, MB343 und MB444 und von Daptomycin bestimmt. Darüber hinaus wurde die antibakterielle Aktivität des Daptomycins gegenüber einem unbekannten Staphylokokkus-Stammes S. sciuri ermittelt. Außerdem wurden Reaktivitätsuntersuchungen neu synthetisierter Inhibitoren gegen Tuberkulose und S. aureus durchgeführt. Die untersuchten Mip-Inhibitoren lieferten ein Metabolitenprofil, welches durch Ester- und Amidhydrolysen sowie Hydroxylierungen geprägt wurde. Die Verbindung SF110 schien dabei bereits eine gewisse Instabilität der Esterbindung aufzuweisen, da auch im Blindwert entsprechende Spaltprodukte identifiziert werden konnten. Die Hauptmetabolite von SF235 und SF354 bildeten sich durch unterschiedliche Hydrolysen, da die Spaltung des Moleküls von den jeweiligen Substituenten abhängig ist. Innerhalb dieser Substanzklasse dominiert die mikrosomale Enzymkatalyse, da der größte metabolische Umsatz sowie die meisten Metabolite mittels mikrosomaler Fraktion des Menschen bzw. der Maus gefunden wurden. Die Klasse der Mip-Inhibitoren wird somit vor allem durch Cytochrom-P-450-Enzyme umgesetzt, wobei die Hydrophilie durch Einführung polarer OH-Gruppen der Moleküle erhöht wird. Die Hydroxylierung scheint dabei positionsspezifisch, bedingt durch sterische Hinderungen oder dirigierende Einflüsse, abzulaufen. Stabilitätsvergleiche zwischen SF110, SF235 und SF354 zeigten, dass die Einführung einer Amidbindung anstelle der korrespondierenden Esterbindung die Substanzklasse maßgeblich metabolisch stabilisiert. Im Rahmen des murinen In-vivo-Metabolismus wurde beobachtet, dass SF235 einem deutlich stärkeren Metabolismus unterlag als SF354 und sich der Metabolismus vor allem innerhalb der ersten 30 min vollzog. Demgegenüber zeigten die In-vitro-Ergebnisse gegenteilige Ergebnisse, bei denen SF354 die am stärksten metabolisierte Substanz war. Diese widersprüchlichen Ergebnisse deuten darauf hin, dass In-vitro-Modelle nur als Anhaltspunkt verwendet werden sollten, um mögliche Trends abzuleiten. Metabolismusstudien der Chinolonamide, die gegen die afrikanische Schlafkrankheit wirken sollen, veranschaulichten, dass die größte enzymatische Umsetzung aller drei getesteten Verbindungen mittels cytosolischer Fraktion erfolgte. Die Enzymreaktionen werden vermutlich durch ALDH bzw. MAO dominiert und nicht durch CYP bzw. FMO. Die gebildeten Metabolite in den verschiedenen Fraktionen unterlagen (ω-1)-Oxidationen, N-Desalkylierungen, Amidhydrolysen und aromatischen Hydroxylierungen. Auffallend war, dass eine Hydroxylierung am aromatischen Benzylring nur erfolgen konnte, sofern der Benzylaromat keinen Fluorsubstitutenten trug, da dieser desaktivierend wirkte. Die aromatische Hydroxylierung am Chinolonamid erfolgte dagegen bei allen drei Substanzen. Es wurde somit lediglich eine Hydroxylierung am Benzylring von MB343 festgestellt. Die enzymatische Aktivität aller Substanzen folgte einer Reaktionskinetik 1. Ordnung. Die unterschiedlichen Stabilitäten der Substanzen zeigten einen deutlichen Trend: MB209 wurde, da es die instabilste Verbindung darstellt, im größten Maße umgesetzt, gefolgt von den stabileren Derivaten MB343 und MB444. Die Untersuchung der enzymatischen Aktivitäten zeigte, dass die drei Substanzen, verglichen mit der Leitstruktur GHQ168, eine um den Faktor zehn geringere Aktivität aufwiesen [19]. Aufgrund der eingeführten Fluoratome weisen die Substanzen somit eine wesentlich höhere Stabilität auf. Diese Ergebnisse wurden durch die Untersuchung der Halbwertszeit bestätigt, bei der MB444 den höchsten Wert besaß. Weiterhin ist die Position des Fluorsubstituenten am Chinolongerüst ausschlaggebend für die metabolische Stabilität, wobei MB444 aufgrund des para-Fluorsubstituenten am Chinolonamid die stabilste Verbindung darstellt. Durch Inkubation von Daptomycin mit unterschiedlichen S. sciuri-Isolaten wurde ein möglicher Inaktivierungsmechanismus beobachtet, bei dem das Antibiotikum durch Spaltung des cyclischen Aminosäureringes, durch Deacylierung des Fettsäureschwanzes, einer Kombination beider Mechanismen oder durch eine Spaltung des heteroaromatischen Ringsystems von Tryptophan inaktiviert wurde. Die Proteasen des Daptomycin-resistenten S. sciuri-Isolats TS92 führten zu einem Daptomycinabbau von 35 %, unabhängig von der eingesetzten Menge des Arzneistoffes. Das Ausmaß des Abbaus scheint darüber hinaus vom eingesetzten Inkubationsmedium abhängig zu sein, da die Proteasen voraussichtlich auf ein bestimmtes Nährmedium angewiesen sind. Der sensitive S. sciuri-Stamm TS93 lieferte die höchste Abbaurate an Daptomycin mit 55 % und widerlegt damit die Vermutung, dass Daptomycin die geringste antibakterielle Aktivität gegenüber diesem S. sciuri-Stamm aufweist. Im In-vitro-Metabolismus zeigte Daptomycin insgesamt eine sehr geringe Umsetzungsmenge mit maximal 5 % nach 4 h und einer geringen Metabolitenbildung. Hier wurde nur ein Metabolit gefunden, welcher auch mittels S. sciuri-Inkubation identifiziert wurde. Dieser Mechanismus könnte somit auf anderem Wege verlaufen. Die Reaktivitätsstudien der kovalenten Inhibitoren der FadA5-Thiolase gegen Tuberkulose zeigten, dass nur die Verbindungen C1 und C4 eine Reaktivität gegenüber der Aminosäure Cystein93 im aktiven Zentrum besaßen, die somit für den gewünschten Einsatzzweck geeignet sein könnten. Weiterhin wurde bei den kovalenten Inhibitoren der Enoyl-ACP-Reduktase mit dem Enzym FabI, welches im aktiven Zentrum ein Tyrosin besitzt, keine Reaktion festgestellt, da keine Addukte identifiziert wurden. Dies ist vermutlich auf die Unlöslichkeit im verwendeten TRIS-Puffer zurückzuführen. / This work deals with the investigation of the metabolism as well as the reactivity of different drug candidates as well as active pharmaceutical substances by means of liquid chromatographic and mass spectrometric methods. It is divided into four projects. In order to determine the metabolite profile, a suitable in-vitro incubation system using cytochrome P-450-systems was developed. Thus, the metabolism and pharmacokinetics of the Mip inhibitors SF110, SF235, and SF354 against Legionella, as well as of new antitrypanosomal compounds MB209, MB343, and MB444 and of daptomycin were determined. In addition, the antibacterial activity of daptomycin against an unknown Staphylococcus strain S. sciuri was determined. In addition, reactivity studies of newly synthesized inhibitors against tuberculosis and S. aureus were performed. The Mip inhibitors investigated showed a metabolite profile being characterized by ester and amide hydrolysis as well as hydroxylation. The ester moiety of compound SF110 seemed to be unstable, as metabolites could be also identified in the negative control. The major metabolites of SF235 and SF354 were formed by different hydrolyses, whereby the cleavage mechanism of the molecule is dependent on the respective substituents. The substance class is dominated by microsomal enzyme catalysis, as the highest metabolic turnover and, eventually, the most metabolites were found using a microsomal fraction of the human and mouse. The class of Mip inhibitors is thus represented mainly by cleavage due to cytochrome P-450 enzymes, wherein the hydrophilicity of the substrate is increased by introducing polar hydroxyl groups. Hydroxylation seems to be site specific due to steric hindrance or directing influences. Comparing the stability of SF110, SF235, and SF354 revealed that introducing an amide bond instead of an ester bond significantly stabilizes the substance class metabolically. In murine in vivo metabolism results, SF235 was found to be metabolized more significantly than SF354 within the first 30 min of incubation. In contrast, the in vitro results showed the opposite. SF354 was the most metabolized substance. The contradictory results suggest that in vitro models should only be used as an indicator to derive possible trends. Metabolism studies of quinolonamides active against African sleeping sickness, demonstrated that the highest enzymatic conversion of all three tested compounds was caused by the cytosol fraction. The enzyme reactions are probably catalyzed by ALDH or MAO and not by CYP or FMO, respectively. The formed metabolites found in various fractions were subject to (ω-1)-oxidations, N-dealkylations, amide hydrolyses, and hydroxylations. It was observed that hydroxylation could only take place on the aromatic benzyl ring if it did not carry any fluorine substituents having a deactivating effect. The aromatic hydroxylation of the quinolonamide, however, was carried out in all three substances. Thus, only hydroxylation on the benzyl ring of MB343 was observed. The enzymatic activity of all substances followed a first-order kinetic. The different stabilities of the substances had a clear trend: MB209 showed the highest enzymatic activity as it represents the most unstable compound, followed by MB343 and MB444. The enzymatic activities of the three substances were ten times lower compared to the enzymatic activity of lead structure GHQ168 [19], which exhibits a much higher stability due to the fluorine atoms. These results were confirmed by a half-life study in which MB444 was the most stable compound. The position of the fluorine substituent on the quinolone determines the metabolic stability, making MB444 the most stable compound because it carries a p-fluorine substituent on the quinolonamide. When incubating Daptomycin with different S. sciuri isolates, a possible inactivation mechanism of the antibiotic agent was observed in which the cyclic amino acid ring was opened, the fatty acid tail deacylated, or a combination of both mechanisms as well as the heteroaromatic ring system of tryptophan was cleaved. The proteases of the daptomycin-resistant S. sciuri isolate TS92 led to a daptomycin degradation of 35%, regardless of the initial concentration used. The degradation also seems to depend on the incubation medium since the proteases probably rely on a specific nutrient medium. The sensitive S. sciuri strain TS93 showed the highest degradation rate of daptomycin with 55 % and thus refutes the assumption that it has the smallest antibacterial sensitivity against daptomycin. Overall, DAP showed a very low in vitro metabolism with a conversion rate of maximum 5% after 4 h and a low metabolic rate. Here, only one metabolite could be found which was also identified by means of S. sciuri incubation. Thus, this mechanism could proceed in a different way. The reactivity studies of the covalent inhibitors of the thiolase of the FadA5 enzyme against tuberculosis showed that only compounds C1 and C4 targeted cysteine93, thus being suitable for the desired purpose. Furthermore, no reaction was observed for in the covalent inhibitors of the enoyl-ACP reductase with the enzyme FabI, which carries a tyrosine in the active site, since no adducts were identified. This is probably due to the insolubility in the TRIS buffer.

Bakterien Legionella pneumophila orsakar sjukdomen Legionella vilket har ökat i världen de senaste 20 åren och har troligtvis ett ännu större mörkertal. Den frodas i vattenrör mellan temperaturen 25–50 grader och vid installation av varmvattenrör i byggnader måste det tas hänsyn i riskzoner. Ett speciellt riskområde är varmvattenavstick som skapas för att kunna dra vidare vattenrör i framtiden. Där utsätts vattnet för större risk för stillastående och sjunkande temperaturprofil beroende på längd. Med hjälp av COMSOL Multiphysics® Modelling Software har ett teoretiskt ramverk för dimensionering av varmvattenavstick i byggnader skapats. Resultatet visar på hur stor strömningshastighet i vattenflödet, isoleringstjocklek och luftrörslängd har påverkan på hur långt avsticket kan vara utan att understiga kritisk temperatur. Resultatet kan användas som beslutsstöd givet att liknande avstickmodell och flödesprofil används samt om strömningshastighet i röret inte är längre än det i rapporten. / The bacterium Legionella pneumophila causes the disease Legionella which has seen an increase in the world over the last 20 years and likely has an even greater number of unrecorded cases. It thrives in water pipes between the temperature of 25-50 degrees and when installing hot water pipes in buildings certain factors must be taken into account. A special risk area is hot water branches that are created to be able to extend water pipes in the future. There, the water is at greater risk of stagnant and decreasing temperature profile depending on its length. With the help of COMSOL Multiphysics® Modelling Software, a theoretical framework for the dimensioning of hot water distances in buildings has been created. The result shows how the velocity in the water flow, the insulation thickness and the length of the air pipe have a great influence on how long the branch can be without falling below the critical temperature. The results can be used as decision support if one has a similar branch model and flow profile and also given a that a lower flow rate is not used.

Legionella pneumophila is a pathogen that can proliferate in premise (i.e., building) plumbing and, when aerosolized during water use, infect the lungs of exposed individuals and cause a deadly form of pneumonia known as Legionnaires' disease. Given that it is one of the primary sources of tap-water associated disease throughout much of the world, this organism has been the subject of intense research, ranging from aiming to understand key aspects of its physiology that allow it to proliferate in premise plumbing, to the specific virulence factors that make it so infectious to humans. The work presented here starts with a comprehensive review of published studies related to the L. pneumophila proteome, i.e., the set of expressed proteins associated with a given strain under a given set of environmental conditions, showing how the field has progressed in parallel to improvements in mass spectrometry technologies and how proteomics can be used as a tool to understand this unique and important organism. Copper is a natural antimicrobial that can be present in drinking water due to passive release from copper pipes or intentionally dosed (e.g., copper-silver ionization systems) for microbial control. However, some L. pneumophila strains have recently been found to exhibit copper resistance, an adaptive process that is not fully understood at the physiological level. Chapter Two describes the copper survivability of three outbreak-associated strains of L. pneumophila and examines the copper-induced proteome of QC1, a strain found to display high resistance to copper. Pairwise comparisons of the proteomes of copper-resistant and copper sensitive strains indicated that L. pneumophila QC1 adapts to copper exposure via the induction of redox and metal homeostasis proteins, while concomitantly inducing motility and pathogenesis related proteins, suggestive that copper induces a search for a host protozoan strain for protection. In 2014 and 2015, Flint, Michigan experienced the largest per capita community-wide Legionnaires' Disease outbreak in US history. The outbreak was associated with a change in the source of the municipal drinking water supply from Detroit water, which was sourced from the Great Lakes and subject to appropriate corrosion control, to the Flint River, which was not appropriately controlled for corrosivity. The underlying drivers of this outbreak have been debated and include: elevated iron in the water serving as a nutrient for L. pneumophila, diminished chlorine in the water due to reactions with iron, reduced copper in the water due to shifts in pH influencing release from copper pipes, and shifts in potentially key components of the microbial community. In Chapter Three of this dissertation, we employ controlled microcosm studies to establish a fundamental understanding of interactive effects of pipe material and water of varying iron bioavailability (ferric chloride, ferrous chloride and ferric pyrophosphate) on the microbial community and its relationship with L. pneumophila numbers. The combination of copper pipes and Flint River water decreased the diversity of the microbial community to a larger degree than copper pipes with Detroit water, implying greater copper bioavailability in the former condition. Several Order were found to be significantly associated with high or low numbers of culturable L. pneumophila recovered from the microcosms. Most notably, the Order Pseudomonadales was significantly associated to the reactors with low culturable L. pneumophila. This order contains Pseudomonas species known to inhibit the growth of L. pneumophila. The findings reported in this dissertation can be used to develop more informed management practices for drinking water systems to reduce the risk of Legionnaires' Disease outbreaks associated with premise plumbing. Specifically, 1) copper might be inducing a more pathogenic form of copper resistant L. pneumophila, 2) the use of corrosive control in municipal water systems goes beyond the influence on lead and copper pipes, but also on the microbial community, which in part influences L. pneumophila, and 3) there are organisms, such as Pseudomonadales species, associated with environments with low culturable L. pneumophila which might be introduced to the drinking water systems as probiotics. / Doctor of Philosophy / Legionella pneumophila is a microbe found in drinking water plumbing systems. This organism causes Legionnaires' Disease, a severe form of pneumonia that particularly affects immunocompromised individuals. Due to its health and economic impact, there are worldwide efforts to understand the biology of this organism, from the conditions that allows it to grow in the drinking water plumbing, to the specific components that allows it to infect humans. In this dissertation, we first review the published studies related to the L. pneumophila proteome, a powerful tool used to functionally describe biological organisms. This first chapter showed how proteomics can be used to understand this unique and important organism. In the next chapter we studied how copper metals may influence the proteome of L. pneumophila. Copper pipes have been extensively used to control the growth of microorganisms in drinking water systems, however some studies have reported that copper may promote the growth of L. pneumophila. In this chapter, we showed that a copper resistant strain of L. pneumophila adapts to copper exposure by inducing motility and pathogenesis related proteins, suggesting that it might be more infectious. In the last chapter of this dissertation, we investigated the combined effect of pipe material and water chemistry, on the microbial community and its relationship with L. pneumophila. The combination of copper pipes and a more corrosive water decreased the diversity to a larger degree, in comparison to the other evaluated conditions. Several organisms were also identified to be significantly associated with the high or low culturable L. pneumophila. This is of particular interest because they might be used as potential probiotics to control the growth of L. pneumophila. The findings reported in this dissertation can help to better understand the significance of water chemistry and pipe material, particularly copper pipes, for the purpose of reducing risk of Legionnaires' Disease outbreaks associated with drinking water systems.

Understanding and limiting public health threats resulting from exposure to opportunistic pathogens (OPs) in domestic water (i.e., hot/cold water for human use) will be one of the grand challenges for water safety in the 21st century. This dissertation anticipates some of the complexities in balancing stakeholder goals and developing building standards to limit OP growth, and advances scientific understanding of OP survival and proliferation in domestic plumbing systems. In a cross-sectional survey of water- and energy-efficient buildings, domestic water age ranged from 8 days to 6 months and resulted in pH and temperature fluctuations, rapid disinfectant residual decay up to 144 times faster than municipal water delivered to the buildings, and elevated levels of OP gene markers. This motivates future work to determine how to maintain high quality and safe water while preserving the sustainability goals of these cutting-edge buildings. Head-to-head pilot-scale experiments examining OP growth in recirculating hot water systems revealed that elevated temperature had an overarching inhibitory effect on L. pneumophila growth where temperatures were maintained. However, control was undermined in distal branches, especially when density-driven convective mixing gradients maintained ideal growth temperatures and delivered nutrients to the otherwise stagnant branches. These results resolve discrepancies reported in the literature regarding the effects of flow, and identify important system design and operational conditions that facilitate OP growth. Advancements were also made in understanding how corrosion can trigger OP growth. In Flint, MI, corrosive Flint River water damaged iron pipes, releasing iron nutrients, consuming chlorine residual, and supporting high levels of L. pneumophila in large building systems. This likely triggered two unprecedented clusters of Legionnaire's disease. In pilot-scale systems, copper released from copper pipes, but not dosed as soluble cupric, triggered release of >1,100 times more H2 into the water due to deposition corrosion. The organic carbon fixed by autotrophic hydrogen oxidation has the potential to facilitate OP growth, but more work is needed to understand the limits of this mechanism. Finally, well-controlled laboratory experiments confirmed past reports from field surveys that the use of chloramines trigger a trade-off between controlling Legionella and allowing non-tuberculous Mycobacteria to persist. / Ph. D.

Water conservation efforts have led to a decrease of flow rates in buildings, increasing water retention time (WRT) and sometimes opportunistic pathogens (OPs) growth. A novel experiment with replicated distal pipes operated at commonly used flow rates was designed to evaluate the effects of water age, flush frequency, flow rate, pipe diameter, water temperature, disinfectant residual presence, and microbial regrowth in hot and cold pipes. In cold water, total bacterial regrowth was a function of water age, plateauing after approximately 6 days at cell counts 20 times higher than influent water with minimal disinfectants. In warm (40 °C) water, most regrowth occurred in the heater tank, reducing the relative growth in the pipes. When cold water with ~1 mg/L chloramine was present, cold-water total bacteria regrowth plateaued after about 2 days WRT with cell counts 14 times higher than influent water, but regrowth still occurred in the heater tank. With 1 mg/L chloramine and elevated heater temperature (60 °C), regrowth in the tank was suppressed and cell counts in the pipes increased 82 times above cold-water influent levels at 7.5 days WRT. Legionella spp. and Mycobacterium spp. demonstrated opposite responses to flow rate with chloramine minimization. The highest levels of Legionella spp. (1.7 log higher than influent) were present when flow velocity was >2 feet per second (fps), but the highest levels of Mycobacterium spp. (1.5 log higher than influent) were observed at the lowest flow velocity (0.33 fps). This study highlights the tradeoffs between water conservation and water quality. / Master of Science / Regulations that decrease flow rates of faucets and showers have driven water conservation in buildings, increasing the time water sits in pipes and tanks (i.e., water retention time or WRT) elevating the likelihood of harmful bacterial growth. A novel faucet rig was designed to carry out a comprehensive experiment revealing the combined effects of WRT, flush frequency, flow rate, pipe diameter, water temperature, and disinfectant residual presence on water quality at the tap. In water without disinfectant, growth in cold water pipes increased with WRT, but in hot water the growth of bacteria occurred mostly in the warm water tank at 40 °C, which is a temperature known to leave a system vulnerable to bacterial growth. Cold pipes with a disinfectant residual saw a decrease in bacterial regrowth in comparison to cold pipes without disinfectant. However, if there was a disinfectant residual and an elevated water heater temperature set point in the tank, regrowth occurred when water was in the pipes at room temperature and there were lower disinfectant residuals. Potentially harmful bacteria, like Legionella spp. and Mycobacterium spp., which cause Legionnaires' disease and nontuberculous Mycobacteria (NTM) infections, grew more readily at higher flow rates, whereas others grew less readily, but all harmful bacteria were reduced by lowering WRT to less than ≈ 2 days and maintaining the water at 60 °C with a disinfectant. This study has important implications for regulations requiring minimum disinfectant levels to buildings, faucet flow rates regulations, and design and operation of building plumbing systems.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010 / La fréquentation des spas peut entraîner des infections par les bactéries Legionella (pneumonies sévères) et Pseudomonas aeruginosa (lésions cutanées). Peu de données sont toutefois disponibles sur la contamination microbiologique de ces bassins. Des paramètres microbiologiques et physicochimiques de l'eau de 95 spas du Québec ont été mesurés durant l'été 2008. Les gestionnaires ont aussi répondu à un questionnaire sur leur entretien. Une proportion de 2% des spas était contaminée par Escherichia' coli, 41% par Pseudomonas aeruginosa et 22% par Legionella. Dans 25% des spas, des bactéries ont été détectées en concentrations préoccupantes. Les paramètres physicochimiques s'écartaient fréquemment des valeurs recommandées. Seulement quatre gestionnaires étaient formés (4%). Une bonne filtration, un nettoyage fréquent du spa et une désinfection adéquate limitaient efficacement la contamination microbiologique. Des efforts devraient être faits pour adapter les réglementations aux spas qui abritent un écosystème différent des piscines. Une meilleure formation des gestionnaires est également nécessaire.