La multi-résistance aux drogues des bactéries est un problème majeur en clinique. L'un des mécanismes de résistance consiste à effluer les composés toxiques hors de la cellule grâce à des protéines de la membrane interne nommées pompes d'efflux. Ces protéines appartiennent à cinq familles (MFS, RND, MATE, SMR et ABC) et peuvent fonctionner en association avec deux autres types de protéines (protéine du périplasme et protéine de la membrane externe) pour former un canal. Dans le cadre d'une thématique de recherche basée sur l'étude des mécanismes de résistance auxdrogues de la bactérie pathogène Legionella pneumophila, une approche bioinformatique menée sur lesgénomes de trois souches séquencés (souches Lens, Paris et Philadelphia) a permis d'identifier des protéinespouvant participer à l'efflux. Notre but a été de vérifier l'implication de ces protéines dans la résistance auxdrogues et dans la virulence de Legionella en ciblant un ou plusieurs gènes codant pour des composants desystèmes d'efflux. Pour inactiver les gènes, nous avons choisi une stratégie de recombinaison homologue. Lesrecombinants ont été testés pour leur sensibilité à des composés toxiques afin de voir si les gènes ciblés jouentun rôle dans l'efflux d'E. coli. Un de ces mutants, le mutant MF201, altéré pour le gène codant pour une protéinehomologue à TolC chez E. coli s'est avéré être 2 à 16 fois plus sensible aux drogues testées comparé à lasouche sauvage. De plus, ce mutant présente un défaut important de virulence dans Acanthamoeba castellanii,Dictyostelium discoideum et les macrophages U937. Ce premier résultat implique que la protéine TolC-like deLegionella aurait un rôle clef dans la relation hôte pathogène et sous-tend un lien entre multi-résistance auxdrogues et virulence. Par ailleurs une étude de l'expression des gènes codant pour des pompes à efflux a étéinitiée afin de comprendre leur rôle au cours du cycle infectieux de Legionella.

L'étude s'intéresse à la bactérie Legionella pneumophila (Lpn) dans les biofilms des affluents du lac du Bourget. Elle confirme le caractère ubiquiste de cette bactérie, principalement sous sa forme non cultivable. Elle met aussi en évidence, le caractère local des facteurs naturels de régulation : seule la température de l'eau intervient dans la majorité des sites. Ce déterminisme local a aussi été mis en évidence avec l'étude du réseau hydrographique aixois qui reçoit à la fois des eaux thermales et celles de la rivière Le Tillet. Les perturbations thermique et chimique liées aux arrivées d'eau chaude sont détectables jusqu'à la partie aval du réseau. L'effet d'enrichissement des apports en Lpn qui leur correspondent reste localisé ce qui peut être la manifestation de processus naturels d'autoépuration. Nos résultats montrent également un comportement différent des organismes du groupe des Pseudomonas et des bactéries apparentées vis-à-vis des caractéristiques physico-chimiques. Des expériences effectuées en réacteurs biologiques ont montré qu'une augmentation de la vitesse ou de la température de l'eau est favorable à la présence de Lpn. Un modèle de transport de charges a été construit à partir duquel une gestion de l'utilisation du jet d'eau situé à proximité de la plage municipale d'Aix-les-Bains pourrait être faite pour tenir compte des périodes durant lesquelles des fragments de biofilms susceptibles de contenir des Lpn sont transportés en masse par le réseau aixois en direction de la plage. / The study concerns the occurrence of the bacteria Legionella pneumophila (Lpn) in biofilms from Lake Bourget tributaries. It confirms the ubiquity of the bacteria in flowing waters, but mainly as viable but non culturable state. It highlights that main environmental control factors are local, at the exception of water temperature which is involved in most studied sites. Local control of biofilms development has also been demonstrated in the study of the Aix-les-Bains hydrological network which receives hot springs water and water from the Tillet river. Thermal and chemical disturbances induced by hot waters entrances could still be detected at the farthest downstream part of the network. Biofilms enrichment in Lpn related to theses incomings kept localized possibly due to self-purification processes. Our results depict different answers of microorganisms like Pseudomonas and related bacteria towards physicochemical characteristics. Two experiments were performed based on biological reactors, showing that increases of water temperature or water flow, both enhance Lpn concentration. An hydrological transport model has been designed to serve the management of a waterspout sited by the Aix-les-Bains beach. It is written especially to address periods where biofilms transport holding high Lpn concentrations should be avoided.

Background: Community acquired pneumonia causes 5,9% of deaths in elderly South Africans. Mortality rates are increased in those in whom initiation of therapy with an appropriate agent has been delayed. Whereas Mycoplasma pneumoniae and Legionella pneumophila are sensitive to the macrolides or tetracycline, they do not respond to the currently recommended first-line agents for community acquired pneumonia, penicillin or a cephalosporin. It was therefore necessary to assess the prevalence of exposure to these 2 organisms in the elderly in order to determine whether a modification in the recommendations may be justified. Methods: Study population and survey: Subjects were residents of 4 old age homes in Cape Town who were older than 60 years and willing to participate. Written consent was obtained, a demographic and medical history questionnaire was completed, and a sample of blood was drawn. Laboratory methods: The indirect fluorescent antibody tests (Zeus Scientific Inc, New Jersey, USA) were used to detect the presence of antibodies to Mycoplasma pneumoniae and Legionella pneumophila. Results: The participation rate in this study was high, with 88,4% (677/766) taking part. Seropositivity for both of these organisms was low. There were 17 participants (2, 51 %) with antibodies to mycoplasma (IgG only in 8, IgM only in 1, and both IgG and IgM in the remaining 8). Titres were low with only 1 IgM titre of 16, and only 3 IgG titres of 64. Antibodies to Legionella were demonstrated in only 9 participants (1,33%). All these titres were 128 or above. Conclusions: It is concluded that first-line therapy for community acquired pneumonia should adhere to the current guidelines published by the South African Pulmonology Society. There is no indication for the routine use of agents active against Legionella or mycoplasma. Clearly, these antibiotics should be introduced if specific pointers to infection with one of these organisms are found. Because of the low seropositivity rate, the indirect fluorescent antibody test for these 2 agents has a high specificity in this population. This may be of use in making a diagnosis in an acute infection Further studies are required to elucidate the immunological response to these organisms in elderly persons. A further survey should be done to determine the seropositivity rate to these agents in community dwelling elderly.

Tesis por compendio / [EN] Legionella pneumophila is a strictly environmental and opportunistic pathogen that can cause severe pneumonia after inhalation of aerosols with enough bacterial load. Outbreaks and sporadic cases are usually localized in temperate environments, and the reservoirs are often water-related sources where biofilms are created. The existence of non-cultivable forms of the bacteria increases the risk for public health, as culture-based methods may miss them, thus complicating the environmental investigations of the sources. Genetic classification through the Sequence-Based Typing (SBT) technique allowed an increased discrimination among L. pneumophila strains compared to previous methods. SBT data can also be used for genetic variability and population structure studies, but a more exhaustive analysis can be performed using high-throughput genome sequencing strategies. This thesis describes the use of both SBT and genomic sequencing to evaluate and provide solutions to different public health needs in L. pneumophila epidemiology. We have focused in the Comunidad Valenciana (CV), the second region in Spain with the highest incidence of Legionellosis, with special interest in the city of Alcoy, where recurrent outbreaks have occurred since 1998. Firstly, SBT data were used to gain a deeper insight into the genetic variability and distribution of the most abundant Sequence Types (ST) in the CV area. We have shown that the level of variability in this region is comparable to that from other countries, revealing the existence of both locally and broadly extended profiles. Approximately half of the observed genetic diversity was found to result from geographical and temporal structure. Secondly, L. pneumophila detection from environmental sources remains a challenge for public health. A comparison between water and biofilm samples using a sensitive touchdown PCR (TD-PCR) strategy revealed that the use of biofilms increased by ten-fold the detection rate. This method allowed evaluating the hidden uncultivable L. pneumophila diversity in the locality of Alcoy and the real-time investigation of a Legionellosis outbreak affecting a hotel in Calpe (Southeast of Spain) in 2012. Thirdly, genomic sequencing was applied to a set of 69 strains isolated during 13 outbreaks occurred in Alcoy in the period 1999-2010, mainly the recurrent ST578. Higher intra-outbreak variability than expected was observed, pointing to the potential existence of multiple sources in this endemic area or high environmental diversity. Interestingly, above 98% of the genomic variability in this ST was found as being incorporated through recombination processes rather than through point mutations. Finally, a metagenomic analysis of environmental biofilms from Alcoy revealed a microbial community dominated by Proteobacteria, Cyanobacteria, Actinobacteria and Bacteroidetes. Despite the known endemism of Legionella in this area, the genus was only found in a relative abundance ranging 0.01-0.07%, which explains the low recovery from environmental sources. In summary, the results from this thesis can benefit public health efforts to control this pathogen in the environment, as we provide new insight into its molecular epidemiology, with immediate applications to surveillance and outbreak investigations. / [ES] Legionella pneumophila es un patógeno oportunista estrictamente ambiental capaz de causar neumonía debido a la inhalación de aerosoles con suficiente carga bacteriana. Los brotes y casos esporádicos suelen producirse en ambientes templados y los reservorios encontrarse en zonas con agua donde pueden crearse biopelículas microbianas. La existencia de formas no cultivables de la bacteria aumenta el riesgo para la salud pública, ya que los métodos estándar basados en cultivo microbiológico no pueden detectarlas, complicando las investigaciones ambientales. La clasificación genética basada en el método Sequence-Based Typing (SBT) permite un mayor poder de discriminación entre cepas de L. pneumophila en comparación con métodos previos. Los datos derivados del SBT pueden utilizarse para estudios de variabilidad genética y estructura poblacional. Sin embargo, puede llevarse a cabo un análisis más exhaustivo mediante técnicas de secuenciación genómica de alto rendimiento. Esta tesis describe la utilización tanto de SBT como de secuenciación genómica para evaluar e incluso proponer soluciones a diferentes necesidades en salud pública relacionadas con la epidemiología de L. pneumophila. Nos centramos en la Comunidad Valenciana (CV), la segunda región en España con mayor incidencia de Legionelosis, con especial interés en la localidad de Alcoy, donde ocurren brotes de forma recurrente. En primer lugar, utilizamos datos derivados de SBT para conocer mejor la variabilidad y la distribución de los perfiles genéticos (Sequence Types, ST) en el área de la CV. Mostramos que el nivel de variabilidad en sólo esta región es comparable a la de otros países, con perfiles extendidos local y globalmente. Aproximadamente la mitad de la diversidad genética observada se estima que procede de estructuración geográfica y temporal. En segundo lugar, la detección de L. pneumophila a partir de fuentes ambientales sigue suponiendo un reto para la salud pública. En esta tesis realizamos una comparación entre la detección mediante touchdown PCR (TD-PCR) a partir de muestras de agua y biopelículas microbianas y mostramos que estas últimas proporcionan un aumento de 10 veces en la tasa de detección de la bacteria. Este método permitió evaluar la diversidad no cultivable de L. pneumophila en la localidad de Alcoy y la investigación a tiempo real de un brote en un hotel en Calpe (Sudeste de España) en 2012. A continuación, aplicamos la secuenciación genómica a 69 cepas aisladas durante 13 brotes ocurridos en Alcoy en el período 1999-2010, principalmente el recurrente ST578. Se observó mayor variabilidad entre cepas de un mismo brote que la esperada, lo cual apunta a la existencia potencial de múltiples fuentes en este área, o alta diversidad ambiental. Además, se observó que más del 98% de la variabilidad genómica fue introducida por procesos de recombinación y no de mutación puntual. Finalmente, se realizó un análisis metagenómico de biopelículas ambientales recogidas en Alcoy. Se encontró que la comunidad está dominada por Proteobacteria, Cyanobacteria, Actinobacteria y Bacteroidetes. A pesar del conocido endemismo de Legionella en el área, este género sólo se encontró en una abundancia relativa entre 0.01-0.07%, lo cual explica su baja tasa de recuperación a partir de muestras ambientales. En resumen, los resultados de esta tesis pueden ser de utilidad para los programas de control de este patógeno llevados a cabo por las autoridades de salud pública, ya que proporcionan una nueva percepción de su epidemiología molecular, con aplicación inmediata a la vigilancia e investigación de brotes. / [CA] Legionella pneumophila és un patogen oportunista estrictament ambiental capaç d'ocasionar pneumònia degut a la inhalació d'aerosols amb la suficient carga bacteriana. Els brots i casos esporàdics solen ocórrer en ambients temperats, i els reservoris solen trobar-se en zones amb aigua on poden crear-se biopel·lícules microbianes. La existència de formes no cultivables del bacteri augmenten el risc per a la salut pública, ja que els mètodes estàndard basats en el cultiu microbiològic no poden detectar-les, complicant les investigacions ambientals. La classificació genètica basada en el mètode Sequence-Based Typing (SBT) permet un major poder de discriminació entre soques de L. pneumophila en comparació amb previs mètodes. Les dades derivades del SBT poden utilitzar-se per a estudis de variabilitat genètica i estructura poblacional, però un anàlisis més exhaustiu pot dur-se a terme a través de tècniques de seqüenciació genòmica d'alt rendiment. Esta tesis descriu la utilització tant del SBT com de la seqüenciació genòmica per a avaluar i proposar solucions a diferents necessitats en salut pública relacionades amb l'epidemiologia de L. pneumophila. Ens centrem en la Comunitat Valenciana (CV), la segona regió d'Espanya amb la major incidència de Legionel·losi, amb especial interès en la localitat d'Alcoi, on els brots ocorren de forma recurrent des de 1998. Primer, hem utilitzat dades derivades del SBT per a conèixer millor la variabilitat i la distribució dels perfils genètics (Sequence Types, ST) en l'àrea de la CV. Mostrem que el nivell de variabilitat en només aquesta regió és comparable a la d'altres països, amb perfils estesos tant de forma local com més amplia. Aproximadament la meitat de la diversitat genètica observada s'estima que procedeix d'estructuració geogràfica i temporal. Segon, la detecció de L. pneumophila a partir de fonts ambientals continua suposant un repte per a la salut pública. En aquesta tesis realitzem una comparació entre la detecció mitjançant touchdown PCR (TD-PCR) a partir de mostres d'aigua i biopel·lícules microbianes i mostrem que aquestes últimes proporcionen un augment de deu vegades en la tassa de detecció. A més, aquest mètode ens va permetre avaluar la diversitat no cultivable de L. pneumophila a la localitat d'Alcoi i la investigació a temps real d'un brot de Legionelosis que va afectar a un hotel en Calp (Sud-est d'Espanya) a l'any 2012. Tercer, vam aplicar la seqüenciació genòmica a 69 soques aïllades durant 13 brots ocorreguts a Alcoi en el període 1999-2010, principalment el recurrent ST578. Es va observar una major variabilitat entre soques d'un mateix brot de l'esperada, apuntant a l'existència potencial de múltiples fonts en aquesta àrea, considerada endèmica, o alta diversitat ambiental. A més, es va observar que més del 98% de la variabilitat genòmica havia sigut introduïda a partir de processos de recombinació i no de mutació puntual. Finalment, es va realitzar una anàlisi metagenòmica de biopel·lícules ambientals recollides a Alcoi. Varem trobar que la comunitat està dominada per Proteobacteria, Cyanobacteria, Actinobacteria i Bacteroidetes. A pesar del conegut endemisme de Legionella en l'àrea, aquest gènere només es va trobar en una abundància relativa entre 0.01-0.07%, el qual explica la seua baixa tassa de recuperació a partir de mostres ambientals. En resum, els resultats d'aquesta tesi poden ser d'utilitat per als programes de control d'aquest patogen duts a terme per les autoritats de salut pública, ja que proporcionen una nova percepció de la seua epidemiologia molecular, amb aplicació immediata a la vigilància i la investigació de brots. / Sánchez Busó, L. (2015). Genetic and genomic variability of Legionella pneumophila: applications to molecular epidemiology and public health [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52854 / TESIS / Premios Extraordinarios de tesis doctorales / Compendio

<p>The actin cytoskeleton is involved in many essential cellular events such as mitosis, cell migration, control of epithelial barrier function, and adherence of immune cells. Given the essential roles of the actin cytoskeleton, it is not surprising that it is a common target for bacterial virulence factors. <em>Legionella pneumophila</em>, the causative agent of Legionnaire’s pneumonia, establishes a replicative compartment using effectors secreted by its Dot/Icm secretion system. At least four Dot/Icm effectors, VipA, Ceg14, LegK2 and RavK have been shown to modulate the host actin cytoskeleton. Here, we identified RavJ (lpg0944) as an additional effector that interferes with the actin cytoskeleton in mammalian cells. We demonstrated that RavJ is a transglutaminase that functions to induce crosslink between actin and members of the Motin protein family, leading to inhibition of the binding between actin and ADF/cofilin. We also found that LegL1 (lpg0945) is a metaeffector of RavJ, which suppresses the transglutaminase activity of RavJ by blocking its enzymatic domain. </p>

Legionella pneumophila is a human pathogen commonly found in natural and artificial aquatic environments and can cause a condition called legionellosis. Monitoring for legionellae is therefore important for protecting public health and identifying its environmental sources is a way to prevent illness. This has resulted in development of several control strategies to identify these sources. One of these strategies is to construct a valid method to detect Legionella pneumophila and monitoring these methods is a way to ensure the method remain effective at tracing infection.  The current version of standardized method is called ISO 11731:2017 and supersedes its former version called ISO 11731:1998. The former version uses a combination of heat and acid solution treatment to reduce interfering microorganisms in water with high background, whereas the current version separates the treatment by subdividing the sample in three parts. One part is subjected to heat treatment, one with acid solution treatment and one remains untreated. Therefore, the aim of this study is to analyse how this difference in method strategy will affect detection of Legionella pneumophila between the current and its former version of ISO 11731. To do this, this study divided the experiment into two parts: experiment A was aimed at evaluating the validity of the method and experiment B was designed to study repeatability in terms of dispersion and performance data range. For experiment A: 14 samples were tested using both ISO 11731:2017 and 11731:1998 to see how the results differentiated. Six are natural samples and was appointed based on their previous results that showed positive for Legionella. Four samples were spiked with different serotypes of Legionella and the remaining four were spiked with both Legionella and Legionella-inhibited bacteria. For experiment B, three certified reference material with different concentration of Legionella pneumophila serotype 1 was tested in repeatability conditions with each sample producing ten replicates.  In conclusion, based on results assessed in this study ISO 11731:1998 was more suitable to analyse water with higher concentration of interfering microorganisms. By a combination of heat and acid solution treatment: it maximizes the reduction of interfering microorganisms which facilitates Legionella to cultivate on agar. ISO 11731:2017 was more efficient in recovering different serotypes of Legionella. Although, there were a significant increase in dispersion and performance data range results in ISO 11731:2017. This indicates that since there is an additional dilution step added in acid solution treatment: it increases the risk of human error and therefore a greater vulnerability to the method.

No description available.

As waterborne disease originating in potable water plumbing systems (such as Legionnaires' Disease and Nontuberculous Mycobacterial (NTM) infections) continue to increase, it is important to better understand the cause(s), responsible parties and interventions to prevent disease. This dissertation begins with a literature review characterizing the propensity of building (premise) plumbing to enhance or diminish opportunistic pathogen growth, including Legionella. It then holistically examines the problem at the field, bench and pilot scale by first discovering problems with lead and Legionella in Flint, MI, during an event popularly referred to as the Flint Water Crisis in 2014-2016. Four years were then spent simulating critical factors hypothesized to have triggered the Legionella outbreak in residences and in a large hospital in Flint. In parallel with that work, pilot scale rigs were operated for several years, to examine the important role of water heater system design and operation on energy efficiency, hot water delivery, and Legionella. The first chapter literature review is entitled "Critical Review of the Propensity of Premise Plumbing Pipe Materials to Enhance or Diminish the Growth of Legionella and Other Opportunistic Pathogens." It examines the complex environments found in premise systems, focusing primarily on the role of pipe materials. The effects of metallic (copper, iron) and plastic pipe materials on opportunistic pathogens and Legionella include their effect on nutrient availability, disinfectant levels, and the composition of the broader microbiome. Design, configuration, and operation are also examined in terms of their potential for influencing opportunistic pathogens. This chapter demonstrates that pipe materials have the potential to stimulate or inhibit pathogen growth, dependent on circumstance and water chemistry. This chapter will be submitted to the journal Pathogens. The field study in this work first predicted, discovered and then exposed problems with lead and Legionella in Flint, Michigan. A citizen science project that sampled Flint water in August 2015, demonstrated a city-wide problem with water lead exceeding the EPA limit of 15 µg/L after corrosion control was interrupted. Follow-up sampling events between August 2015 and August 2017 demonstrated that the switch back to the original water source and addition of enhanced corrosion control in October 2015, dramatically reduced lead, copper and iron levels flowing into consumer homes. Entitled "Evaluating Water Lead Levels During the Flint Water Crisis," this work was published in Environmental Science and Technology in 2018. After our Virginia Tech team's work helped expose a Legionnaires' disease outbreak that killed twelve people and sickened nearly one hundred individuals, the started to explore possible links between corrosion control, plumbing materials and disinfection that could help explain the trajectory of disease in Flint and elsewhere. Three separate experiments were performed using bench-scale simulated glass water heaters. Two of the studies attempted to simulate what occurred in Flint homes before, during and after the water crisis in relation to factors that either encouraged or discouraged Legionella growth, while the third examined the more benign Blacksburg tap water and a broader range of influential plumbing conditions. The first study entitled "Copper Pipe, Lack of Corrosion Control, and Uncontrolled pH Influenced the Trajectory of the Flint Legionnaires' Disease Outbreak," determined that the very low pH levels in summer 2015 and interruption of phosphate corrosion control, could cause explosive growth of Legionella in PEX plumbing held at warm temperature, without disinfectant and with constant mixing. Under the same conditions copper pipe had antimicrobial properties that markedly reduced Legionella in our experiments. This work has been submitted for review to Environmental Science and Technology. The second companion study conducted at a higher pH, without mixing and with trace chlorine, found 2.5 log10 lower levels of Legionella compared to the worst-case conditions in the aforementioned study, demonstrating the importance of mixing and traces of chlorine. Higher levels of disinfectant and the presence of copper pipe also enhanced control of Legionella. This manuscript is titled "Interactive Effects of Copper Pipe, Stagnation, Corrosion Control, and Disinfectant Residual Influenced Reduction of Legionella pneumophila during Simulations of the Flint Water Crisis," and it has been published in Pathogens. The third simulated glass water heater study examined the disinfection of opportunistic pathogens in the presence of six different premise plumbing materials or conditions in Blacksburg tap water. Generally speaking, all of the premise plumbing materials reduced disinfection of opportunistic pathogens compared to a control condition with glass surfaces. Chlorine decay was catalyzed by iron pipe, warmer temperature and the presence of organic matter, increasing the persistence of Legionella. Magnesium anodes in particular, encouraged much higher Legionella growth compared to all other materials. This work titled "Chlorine and Chloramine Disinfection of Legionella spp., L. pneumophila, and Acanthamoeba Under Warm Water Premise Plumbing Conditions," has been submitted to Microorganisms. Results of a six-year pilot study titled "Elucidating the Role of Water Heater System Configuration in Energy Efficiency, Consumer Comfort and Legionella Proliferation," examined different types of residential-sized water heater systems with plastic pipes including: a standard tank system with water stagnant between uses, a recirculating tank system with flowing water between uses, and an on-demand system which only heated water and had flow during use. Considering the volume of water in each tank between 38 and 47 ° C as a measure of Legionella growth risk, with a heater setpoint at 48 °C (118 °F) the recirculating system had 90% of its volume at risk daily compared to only 24% of the standard system volume. The on-demand system used a minimum of 10% less energy than the standard tank, and 50% less energy than the recirculating tank, and had one tenth of the volume at risk of growing Legionella than either tank system. In fact, it was only by contriving a system to keep distal lines artificially warmed to above room temperature, that Legionella growth could occur in the on-demand system, whereas it rose to 107 L. pneumophila MPN per liter in a normally operating recirculating system. On the other hand, the on-demand heaters were repeatedly subject to mechanical malfunction during the study, and had difficulty delivering water at the desired temperature and flow rates versus traditional tank systems. This manuscript will be submitted to Water Research. / Doctor of Philosophy / Recent water crises in Flint, Michigan and Legionnaires' Disease outbreaks in Flint, New York City, and Quincy, Illinois have demonstrated the need to better understand the cause(s), responsible parties, and interventions required to prevent waterborne diseases. As waterborne disease originating in building plumbing systems (premise plumbing), such as Legionnaires' Disease and Nontuberculous Mycobacterial infections, continue to increase each year, the burden on healthcare systems and impact on public health also grows. In this dissertation, a literature review, a field study of water in Flint, small-scale laboratory studies, and residential-sized water heater systems were examined to study interactions between water chemistry, premise plumbing, and disease-causing opportunistic pathogens (OPs) with a focus on Legionella, the OP which causes Legionnaires' Disease. The first chapter literature review is entitled "Critical Review of the Propensity of Premise Plumbing Pipe Materials to Enhance or Diminish the Growth of Legionella and Other Opportunistic Pathogens." It examines the complex environments found in premise systems, and the important role of pipe material selection. The effects of metallic (copper, iron) and plastic pipe materials on opportunistic pathogens and Legionella include their impact on nutrient availability, disinfectant levels, and the composition of the broader microbiome. Design, configuration, and operation of plumbing systems are also examined in terms of their potential for influencing growth of opportunistic pathogens. This chapter demonstrates that pipe materials have the potential to stimulate or inhibit pathogen growth, dependent on circumstance and water chemistry. The field study in this work exposed problems with lead and Legionella in Flint, Michigan, during an event known in the popular press as the Flint Water Crisis 2014-2016. In August 2015, a citizen science sampling campaign demonstrated a citywide lead in water problem. After a federal emergency was declared, follow-up sampling events between August 2015 and August 2017 demonstrated that the switch back to the original water source, enhanced disinfection, and corrosion control under federal direction had reduced lead levels to half of the EPA limit. Additionally, the pipe material installed between each home and the water main (service lines) affected levels of lead and copper in water, with the lowest lead concentrations measured in homes with copper service lines. After our teams' work in Flint helped expose a Legionnaires' disease outbreak that killed twelve people and sickened nearly one hundred other individuals, we started to explore possible links between corrosion control, plumbing materials, and disinfection that could affect the trajectory of disease in Flint and elsewhere. Three follow-up studies, using small glass bottles to simulate water heaters, provided more specific comparisons between water conditions in premise plumbing and OP occurrence. Two studies expanded on phosphate corrosion control, chlorine (disinfectant) decay, and pH-related research questions, which arose during the Flint water crisis field study. The first determined that properly treated drinking water and some mixing could inhibit Legionella growth in copper pipes. The second study found that without mixing, copper could be antimicrobial and reduce Legionella growth even if the water chemistry was slightly altered with higher pH. The third simulated water heater study examined the reduction of OPs using a chlorine or chlorine plus ammonia disinfectant to reduce Legionella in the presence of six different plumbing conditions. The reduction of Legionella with chlorine was inhibited when carbon was increased and in the presence of a magnesium anode rod, a necessary water heater component. A six-year study using a residential-sized water heater system holistically examined three different types of water heater systems with plastic pipes: one using a standard water heater tank with water stagnant between uses, one using a water heater tank connected to a recirculating pump to provide constantly flowing water, and one tankless (on-demand) heater which only heated water and provided flow during use. Using temperature as an indicator of risk for Legionella growth, the recirculating system at a temperature setpoint of 48 °C (118 °F) would be at high risk for Legionella growth (water volume at 38-47 °C or 100-117 °F) in 90% of the tank volume each day, whereas the standard system would only be at high risk in 24% of the tank each day. The on-demand system provided the safest alternative for hot water distribution with virtually undetectable levels of Legionella risk when the pipes were kept at room temperature as per normal operation. The on-demand system also used at least 50% less energy than the recirculating system and 10% less energy than the standard system; however, we were not successful in finding a reliable on-demand system that could also provide hot water at the desired temperature and flow rate.

The opportunistic pathogen Legionella is the leading cause of reported waterborne disease outbreaks in the United States. Legionella can thrive under the warm, stagnant, low-disinfectant conditions characteristic of premise (i.e., building) plumbing systems, making it challenging to identify effective interventions for its control. Copper (Cu) is a promising antimicrobial that can be dosed directly to water via copper-silver ionization systems or released naturally via corrosion of Cu pipes to help control growth of Legionella and other pathogens. However, prior research has shown that Cu does not always reliably control Legionella and sometimes seems to even stimulate its growth. A deeper understanding of the mechanistic effects of Cu on Legionella, at both pure-culture and real-world scales, is critical in order to inform effective controls for Legionella. The overarching objective of the research embodied by this dissertation was aimed at elucidating the chemical and microbial interactions in premise plumbing that govern efficacy of Cu for Legionella control through a series of complementary bench-, pilot-, and field-scale studies. A critical review and synthesis of the literature identified important knowledge gaps in relation to antimicrobial effects of Cu. In particular, changes in the pH, phosphate corrosion control, and rising levels of natural organic matter (NOM) in distributed water are predicted to be important controlling factors. The type of sacrificial anode rod material employed in water heaters was also identified as an underappreciated factor, which directly affects pH, evolution of hydrogen gas as a microbial nutrient, and release of metals (such as aluminum) that bind copper. Microbiological factors: including growth phase of Legionella (e.g., exponential or stationary), strain-specific Cu tolerance, background microbiome composition, and the possibility that viable but non-culturable (VBNC) Legionella might still cause human disease, were also identified as major confounding factors. These knowledge gaps are addressed from various dimensions across each chapter of the dissertation. The effects of pH, orthophosphate corrosion inhibitor concentration, and NOM were examined in bench-scale pure culture experiments over a range of conditions relevant to drinking water. Cupric ions and antimicrobial effects were drastically reduced at pH >7.5, especially in the presence of phosphate, which precipitates copper, or NOM, which complexes the Cu in a form that is less bioavailable. Chick-Watson disinfection models indicated that soluble Cu was the most robust correlate with observed Cu antimicrobial effects across a range of tested waters. This new knowledge suggests that measuring soluble rather than total Cu would be much more informative to guide practitioners in dosing. The research also demonstrated that changes in pH or orthophosphate that have been made to control corrosion over the last few decades, have significantly altered Cu chemistry in buildings, undermining antimicrobial capacity and increasing likelihood of Legionella growth. Pilot-scale experiments confirmed that soluble Cu is an effective indicator of Cu antimicrobial capacity, even in more complex environments represented by realistic hot water plumbing systems. In particular, dosing of orthophosphate, which is widely added by drinking water utilities to control corrosion, directly reduces soluble copper and overall antimicrobial capacity. In some cases, Cu added together with orthophosphate apparently promoted the growth of Legionella, providing an example of at least one circumstance where Cu addition can induce interactive effects that elevate Legionella compared to a control system with trace Cu. It was also demonstrated for the first time that different water heater sacrificial anode types are subject to different corrosion processes, which indirectly influence Cu antimicrobial capacity. Specifically, aluminum ions released from aluminum anode corrosion at 1 mg/L can form an Al(OH)3 gel, which can remove >80% of the soluble Cu from water and reduce Cu antimicrobial effects by >2-log at pH=7. Corrosion from magnesium anodes was found to dramatically increase the pH from 6.8 to >8, which correspondingly reduces Cu antimicrobial capacity. Cu deposition further increased the anode corrosion rate and promoted evolution of hydrogen gas, which is a potent electron donor that stimulates autotrophic microbial growth especially with a magnesium anode. Electric powered anodes did not release metals or alter pH and thus did not diminish Cu antimicrobial capacity. Still, across the pilot-scale experiments, even very high levels of Cu (>1.2 mg/L) at low pH (<7) failed to fully eradicate culturable Legionella. The much lower than expected antimicrobial efficacy of Cu in the pilot-scale hot water plumbing systems was found to be partially explained by the properties of the strain that colonized the systems. Based on fitting the data to a Chick-Watson disinfection model, the outbreak-associated strain that was inoculated into the systems was estimated to be 7 times more tolerant to Cu compared to the common lab strain applied in the bench-scale tests. Further, exponential growth phase L. pneumophila were found to be 2.5 times more susceptible to Cu relative to early stationary phase cultures. It is important to also recognize that, in the pilot-scale systems, drinking water biofilms and the amoeba hosts that colonize them can further shield Legionella from the antimicrobial effects of Cu. Application of shotgun metagenomic sequencing offered the opportunity to more deeply examine the response of Legionella and other pathogens to Cu dosed to the pilot-scale hot water systems in the context of the broader microbiome. It was found that metagenomic analysis provided a sensitive indication of the bioavailability of Cu to the broader microbial community inhabiting the hot water systems, further confirming that the outbreak-associated strain of Legionella that colonized the rigs was relatively tolerant of Cu. Functional gene analysis provided further insight into the mechanistic action of Cu, suggesting multi-modal action of both membrane damage and interruption of nucleic acid replication. The metagenomic analysis further revealed that protozoan host numbers tended to increase in the pilot-scale systems with time, and this could also increase the potential for Legionella proliferation with time. Additional pure culture studies aiming to further assess the mechanistic action of Cu provided strong evidence that Cu can induce a VBNC state for Legionella. This is a concern, given that other studies have indicated that VBNC Legionella are still capable of causing legionellosis. However, VBNC cells are not detected by conventional culturing. Multiple lines of evidence supported the conclusion that Cu induced a VBNC state for Legionella, including membrane integrity, enzyme activity, ATP generation, and Amoebae resuscitation assays applied to two different strains of L. pneumophila. After exposure to Cu, up to a 5-log (99.999%) reduction in culturable Legionella was observed, whereas corresponding reductions in the various viability measures were only by <1-log (90%). In other words, conventional culturing may miss up to 99.99% of the Legionella that is still capable of causing disease. To our knowledge, this is the first study that has assessed the potential for Cu-induced VBNC Legionella. Additional research is needed to further quantify the contribution of VBNC status to challenges in effective Cu-based control of Legionella in premise plumbing. This research further examines, for the first time, the proteomic response of Legionella to Cu, comparing both presumably VBNC and culturable cells. Functional annotation of proteins that were differentially produced by the cells in response to Cu addition revealed that VBNC L. pneumophila modulated its proteome to favor cell membrane- and motility-related proteins, while reducing production of other proteins related to primary metabolism compared to culturable cells. These observations are consistent with the metagenomic-based observations and support the hypothesis that Cu inactivates cells by damaging the cell membrane. The findings also confirmed reduced general cell metabolism that is characteristic of a VBNC state. This dissertation highlights the important and complex effects of Cu on Legionella growth in potable water systems as modified by water chemistry, water heater anode type, characteristics of the surrounding microbiome, and Legionella strain characteristics and growth status. The findings raise important questions about how to measure disinfectant efficacy and provide fundamental new knowledge that can help to better optimize the application of Cu as an antimicrobial to drinking water systems and better protect public health. / Doctor of Philosophy / The opportunistic pathogen Legionella is the leading cause of reportable waterborne disease outbreaks in the United States. Legionella is capable of growing in drinking water plumbing systems in homes, hospitals, hotels, and other buildings. Legionella is spread by inhaling tiny droplets of water that are suspended in the air when using the water, for example when showering, resulting in a severe and deadly form of pneumonia called Legionnaires' Disease. Copper is a promising antimicrobial that can be dosed directly into a building's water system by installing a copper-silver ionization system. There is also interest in understanding whether copper released naturally from copper pipes could help control Legionella. However, prior research indicates that copper sometimes inhibits, sometimes has no effect, and sometimes even seems to stimulate Legionella growth. The purpose of this dissertation was to better understand how the chemical properties of the drinking water, such as pH, presence of corrosion inhibitors that are commonly added to the water by utilities, and natural organic matter impact the ability of copper to kill Legionella. Impacts of the design of the drinking water system were also examined, for example, the material used in the anodes of water heaters to prevent corrosive damage to other system components was hypothesized to change the water chemistry in such a way that could also interfere with copper disinfection. Finally, the effect of the strain of Legionella, its growth phase (exponential or stationary), and culturability status (culturable versus viable but non-culturable) was also examined. Experiments were conducted over a wide range of conditions, from bench-scale pure culture experiments of a few days to full-scale plumbing systems over a period of 3.5 years. The complementary approaches maximize the strength of scientific conclusions about approaches that can more effectively control Legionella. Several discoveries were made as a result of this research that can help to improve the use of copper for controlling Legionella in drinking water systems. In particular, it was found that it is best to keep the pH less than 7.5, because above pH 7.5 copper reacts with orthophosphate corrosion inhibitor or natural organic matter in the water in a manner that makes it less potent to microbes. Through disinfection modeling it was found that soluble copper was the best predictor of the ability to kill Legionella. Therefore, it is recommended from this research that practitioners should monitor soluble copper instead of total copper for the purpose of assessing Legionella control. From the pilot-scale experiments, it was further found that the type of anode installed in the water heater can affect the ability of copper to kill Legionella. Magnesium anodes performed the worst, likely because they raised the pH above the recommended level of 7.5. Aluminum anodes were also a problem because aluminum ions released form an aluminum hydroxide gel that can remove more than 80% of the soluble copper from water. Electric powered anodes did not reduce copper antimicrobial effects by raising pH or forming a gel, but they are much less commonly used. A surprising finding throughout this study was that very high levels of copper (>1.2 mg/L) were required to measurably reduce Legionella in the pilot-scale systems. In the pure culture experiments, it was found that the outbreak-associated strain from Quincy, IL, that was inoculated into the system was highly copper tolerant. This demonstrated that the strain of Legionella that colonizes a particular drinking water system could be the reason why copper is sometimes less effective. Pure culture experiments also found that stationary phase Legionella are more difficult to kill than exponential phase Legionella, which could explain some discrepancies among lab studies reported in the literature. A particularly noteworthy discovery of this research was that copper can make it appear as if Legionella have been killed, because the traditional culture media indicate that there is no growth on the Petri dish; however, they are in fact still alive and capable of causing human disease. This is referred to as a "viable but non-culturable (VBNC)" state. The VBNC state of Legionella was confirmed using an array of techniques (membrane integrity, enzyme activity, ATP generation, and amoebae resuscitation) for two strains of L. pneumophila. We also examined how VBNC Legionella cellular functions were impacted by copper using whole cell proteome, i.e., analysis of all of the proteins extracted from Legionella. Copper induced VBNC Legionella modulated its proteome to favor cell membrane and motility related proteins, and reduced others related to primary metabolism compared with culturable cells. These results were consistent with those obtained via shotgun metagenomic analysis of the microbial community DNA in the pilot-scale water systems. Given the potential for VBNC organisms to prevail in systems disinfected with copper, it is recommended to supplement culture-based monitoring with molecular-based monitoring, e.g., with quantitative polymerase chain reaction. This dissertation highlights the important and complex effects of copper on Legionella growth in potable water systems. The findings help to inform guidance on how to improve the antimicrobial effect of copper, through adjusting the water chemistry, selecting appropriate water heater anodes, and optimizing the overall hot water system design. The dissertation also helps to inform improved strategies for monitoring the efficacy of copper for killing Legionella in real-world systems. Overall, the findings can help to improve policy and practice aimed at reducing the incidence of Legionnaires' Disease and protecting public health.

Legionella pneumophila est un pathogène intracellulaire, agent de la Légionellose, et dont le réservoir dans l’environnement est constitué d’amibes aquatiques comme Acanthamoeba castellani. Mes objectifs de thèse étaient l’identification de mécanismes moléculaires contrôlant la virulence et la multi-résistance chez L. pneumophila, et en particulier l’exploration du rôle des protéines « GGDEF/EAL ». Les domaines GGDEF et EAL sont retrouvés dans des enzymes permettant respectivement de synthétiser (diguanylate cyclase, DGC) ou dégrader (phosphodiestérase, PDE) le di-GMP cyclique, un second messager spécifique des bactéries, qui participe au contrôle de fonctions clés comme la virulence ou la mobilité. L. pneumophila Lens possède 22 gènes codant des protéines GGDEF/EAL, et dont la plupart sont exprimés lorsqu’elle est dans sa phase virulente. L’activité enzymatique des 22 protéines « GGDEF/EAL » a été analysée in vitro : sur 10 protéines purifiées, 6 sont des DGC, dont 2 présentes une double activité DGC/PDE. L’inactivation de 5 gènes des 22 gènes et la surexpression de 2 autres entrainent une baisse de la virulence vis-à-vis d’A. castellanii. De plus, nous avons montré que l’activité DGC d’au moins 2 de ces protéines est requise lors du cycle infectieux. Enfin, nous avons décrit un système à deux composants original comprenant l’histidine kinase (HK) Lpl0330, capable de s’autophosphoryler sur un nouveau domaine HisKA, retrouvé dans 64 autres HK potentielles, et Lpl0329, le premier régulateur de réponse à double activité enzymatique caractérisé, dont la phosphorylation conduit à moduler le taux de di‐GMPc en favorisant une de ses deux activités (Levet-Paulo et al., 2011). / Legionella pneumophila is an intracellular pathogen found in aquatic environments where it replicates in protozoan hosts. My objectives were to identify molecular mechanisms that control virulence and multidrug resistance in L. pneumophila, and especially to explore the role of proteins “GGDEF/EAL” in virulence. GGDEF and EAL domains are found in enzymes able to synthesize (diguanylate cyclase, DGC) or degrade (phosphodiesterase, PDE) c-di-GMP, respectively. C-di-GMP is a bacterial second messenger which plays a key role in regulating main functions including motility, and virulence. L. pneumophila Lens contains 22 genes encoding GGDEF/EAL proteins and most of them are expressed simultaneously with genes encoding virulence factors. The enzymatic activities of the 22 GGDEF/EAL proteins of L. pneumophila Lens were assayed in vitro. Among the 10 proteins purified, 6 showed a DGC activity and 2 contained both activities. The role of the GGDEF/EAL proteins of L. pneumophila Lens on virulence was investigated. Inactivation of 5 genes and overexpression of 2 other genes led to a significant decrease in virulence. Moreover, DGC activity of at least two of these proteins is required for bacterial virulence. Finally, an original two-component system was identified comprising Lpl0330, a histidine kinase able to autophosphorylate on a new HisKA domain, and Lpl 0329, a protein with dual in vitro DGC/PDE activity. Phosphorylation of Lpl0329 led to a decrease in its DGC activity only, giving the first example of a bifunctional enzyme which modulates synthesis and turnover of c-di-GMP in response to phosphorylation (Levet-Paulo et al., 2011).