Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.

Consumer exposure to opportunistic pathogens in potable water systems poses a significant challenge to public health as manifested by numerous cases of pneumonia, non-tuberculosis lung disease, and keratitis eye infections. Water utilities have extensive understanding in control of heterotrophic and coliform bacteria re-growth in water distribution systems via disinfection, control of assimilable organic carbon (AOC), and biologically degradable organic carbon (BDOC). However, much little is known about the effect of AOC on the proliferation of heterotrophic bacteria and pathogens within premise plumbing. This thesis is the first systematic examination of opportunistic pathogen persistence and amplification in simulated glass water heaters (SGWH) as a function of influent organic matter concentration. The role of plumbing conditions that may internally generate AOC is critically examined as part of this evaluation. Strong correlations were often observed between influent organic matter and heterotrophic bacteria in effluent of SGWH as indicated by 16S rRNA gene abundance (average R2 value of 0.889 and 0.971 for heterotrophic organisms and 16S rRNA respectively). The correlation was strongest if water turnover was more frequent (every 48-72 hours) and decreased markedly when water changes were less frequent (stagnation up to 7 days). No simple correlations were identified between the concentration of pathogenic bacteria (L. pneumophila, M. avium, A. polyphaga, and H. vermiformis) and AOC, although correlations were observed between M. avium and TOC over a limited range (and only for a subset of experiments). Indeed, there was little evidence that Legionella and Acanthamoeba proliferated under any of the conditions tested in this work. Parallel experiments were conducted to examine the extent to which factors present in premise plumbing (e.g. sacrificial magnesium anode rods, cross-linked polyethylene, nitrifying bacteria, and iron) could influence water chemistry and influence growth of bacteria or specified pathogens. Although these factors could strongly influence pH, dissolved oxygen concentrations, and levels of organic matter (e.g. iron, magnesium, nitrifying), there was no major impact on effluent concentrations of either heterotrophic bacteria or premise plumbing pathogens under the conditions investigated. While additional research is needed to confirm these findings, at present, there is no evidence of correlations between organic matter and pathogen concentrations from SGWH under conditions tested. Substantial effort was also invested in attempting to identify SGWH and oligotrophic nutrient conditions that would consistently support L. pneumophila and A. polyphaga amplification. A review of the literature indicates no prior examples of large scale amplification of these microorganisms at nutrient levels commonly found in synthesized potable water. It is likely that a complex combination of abiotic and biotic factors (i.e. micronutrients, necrotrophic growth, ambient water temperature, disinfectant type and dose, plumbing materials, water usage patterns), which are not yet fully understood, control the amplification and viability of these pathogenic organisms in premise plumbing systems. / Master of Science

Legionella pneumophila, l’agent responsable de la légionellose est transmissible à l’Homme par les aérosols environnementaux et infecte les macrophages pulmonaires. Après l’exposition à différents stress L. pneumophila est capable de d’entrer dans un état Viable Non Cultivable (VBNC) qui semble être une stratégie de survie. L’objectif de nos travaux était d’étudier l’état VBNC de différentes souches de L. pneumophila après des traitements thermique et chimique et d’évaluer le pouvoir infectieux des formes VBNC envers les macrophages et les cellules épithéliales alvéolaires. Nous avons étudié les profils physiologiques de L. pneumophila de trois souches différentes. Les résultats montrent que pour chaque souche 3 populations peuvent être identifiées, les légionelles viables cultivables, les VBNC et les bactéries mortes. Lorsque soumises aux stress, chaque souche possède un profil physiologique propre et la présence ou non de bactéries VBNC était dépendante du traitement appliqué et de la souche utilisée. La deuxième partie fut relative à l’étude des traitements thermiques de 70°C pendant 30 min et des chocs au dioxyde de chlore de 4, 6 et 7 mg/L pendant 60 min à température ambiante sur ces VBNC. Aucune légionelle VBNC n’est capable de se développer au sein des cellules et aucune croissance sur milieu BCYE n’a été observée après co-culture. La suite de notre étude a été d’étudier le comportement, envers les macrophages, de L. pneumophila revivifiées après culture sur amibes. Les résultats montrent que les légionelles VBNC induites par choc thermique et revivifiées par co-culture sur Acanthamoeba polyphaga sont capables d’infecter de nouveau les macrophages. En conclusion, ces résultats suggèrent que: (i) les formes VBNC de L. pneumophila ne sont pas spontanément infectieuses pour les macrophages et les cellules épithéliales alvéolaires in vitro et (ii) elles peuvent devenir pathogènes pour les cellules humaines après revivification préalable sur A. polyphaga / Legionella pneumophila, the causative agent of legionellosis is transmitted to human through aerosols from environmental sources and invades lung’s macrophages. It also can replicate within various protozoan species in environmental reservoirs. Following exposures to various stresses, L. pneumophila enters a Viable Non Cultivable state (VBNC) which is likely to be a survival strategy. The objective of our work was to study the VBNC forms of several strains of L. pneumophila serogroup 1 obtained after thermal and chemical treatments and to evaluate the infectivity of these VBNC forms against macrophages and alveolar epithelial cells. First we studied the physiological patterns of the three different strains (Philadelphia GFP 008, 044 clinical and environmental RNN). For all strains we observed the presence of VBNC bacteria in the native (non stressed) state. The results show that for each strain, three populations of Legionella can be identified: viable and culturable, VBNC and dead cells. Once submitted to the various stresses, we observed that each strain had its own physiological pattern and the presence (or not) of VBNC bacteria was dependent on the applied treatment and the strain used. The second part was related to the study of the pathogenicity of these VBNC forms against macrophages or epithelial cells. The study focused on heat shock treatment at 70°C for 30 min and chlorine dioxide treatment at 4, 6 and 7 mg/L for 60 min at room temperature. The results show that no Legionella VBNC forms were able to grow within the cells and no growth on BCYE medium was observed after co-culture. Then we investigated the behavior of L. pneumophila resuscitated after culture on ameba within macrophages. The results shows that Legionella VBNC induced by heat shock treatment and resuscitated by Acanthamoeba polyphaga co-culture are able to infect macrophages. In conclusion, these results suggest that: (i) the VBNC forms of L. pneumophila are not infectious for macrophages and alveolar epithelial cells in vitro and; (ii) they can be pathogenic for human cells after revivification by an amoeba (A. polyphaga)

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