Berlin, Humboldt-Univ., Diss., 2002.

No presente trabalho faz-se uma revisão da literatura sobre pneumonia por Legionella pneumophila e se comparam estes dados com a série da autora, que se compõe de 10 casos esporádicos desta pneumonia, adquiridos na comunidade, ocorridos no perÍodo entre outubro de 1983 e maio de 1989. Todos os pacientes desta série eram do sexo masculino e de cor branca, com idade variando entre 36 e 71 anos. Os sintomas mais freqüentes foram febre alta, calafrios, cefaléia, tosse seca e mialgias. A hipótese diagnóstica baseou-se nos dados clínicos, radiológicos e laboratoriais. Em todos os casos o critério de comprovaçao diagnóstica foi a imunofluorescência indireta para Legionella. Salienta-se a importância do reconhecimento desta doença, que ainda apresenta um baixo Índice de suspeição em nosso meio. Procura-se tanto ressaltar os principais achados clínicos e radiológicos como também contribuir com orientações diagnósticas e terapêuticas. ApÓs a análise dos dados obtidos no trabalho, a autora concluiu que: 1. Os achados da série nao diferem daqueles descritos na literatura. 2. A casuística é restrita para o traçado de um perfil da doença no Rio Grande do Sul. 3. Quadros pneumônicos com má resposta clÍnica à penicilina ou derivados, associados a lesões radiolÓgicas com rápida mutabilidade, devem chamar a atenção para este diagnóstico. 4. A infreqüência deste diagnóstico em nosso meio deve-se ao baixo Índice de suspeição. Logo, a divulgação de informações sobre a doença pode resultar em substancial acréscimo ao registro de casos. / In the present work a review is made on the 1iterature about pneumonia by Legione11a pneumophi1a and the data are compared with the series presenteà by the author, composed of 10 sporadic cases of this pneumonia, acquired in the community, October, 1983 and May, 1989. between A11 the patients of this group were ma1e, caucasian, age varying from 36 to 71. The most frequent symptoms were high fever, chills, headache, dry cough and myalgia. The diagnostic hypothesis was baseà on laboratory, radiological and clinicai data. For all the cases, the criterion for diagnostic comprovation was indirect immunofluorescence for Legionella. The importance of the recognition of this disease s emphasized for it still shows a very low 1evel of suspicion in Rio Grande do Sul. The author looks for to ernphazise the main clinicai and radiological findings as well as contributes with diagnostics and therapeutical orientations. After the analysis of the data obtained in the present work the author concludes that: 1. The findings of this series does no differ from those described in the 1iterature. 2. The casuistic is too restricted to draw a profile of the disease in Rio Grande do Sul. 3. Pneumonias with bad clinicai response to penicillin or its derivatives, associated with radiologic lesions with rapid mutability shall call the attention for this diagnosis. 4. The low frequency of this diagnosis in our country is due to the low index of suspicion. Thus, the divulgation of information about the disease could result in substantial increase in the record of new cases.

abstract: Granular activated carbon (GAC) filters are final polishing step in the drinking water treatment systems for removal of dissolved organic carbon fractions. Generally filters are colonized by bacterial communities and their activity reduces biodegradable solutes allowing partial regeneration of GAC's adsorptive capacity. When the bacteria pass into the filtrate due to increased growth, microbiological quality of drinking water is compromised and regrowth in the distribution system occurs. Bacteria attached to carbon particles as biofilms or in conjugation with other bacteria were observed to be highly resistant to post filtration microbial mitigation techniques. Some of these bacteria were identified as pathogenic. This study focuses on one such pathogen Legionella pneumophila which is resistant to environmental stressors and treatment conditions. It is also responsible for Legionnaires' disease outbreak through drinking water thus attracting attention of regulatory agencies. The work assessed the attachment and colonization of Legionella and heterotrophic bacteria in lab scale GAC media column filters. Quantification of Legionella and HPC in the influent, effluent, column's biofilms and on the GAC particles was performed over time using fluorescent microscopy and culture based techniques. The results indicated gradual increase in the colonization of the GAC particles with HPC bacteria. Initially high number of Legionella cells were detected in the column effluent and were not detected on GAC suggesting low attachment of the cells to the particles potentially due to lack of any previous biofilms. With the initial colonization of the filter media by other bacteria the number of Legionella cells on the GAC particles and biofilms also increased. Presence of Legionella was confirmed in all the samples collected from the columns spiked with Legionella. Significant increase in the Legionella was observed in column's inner surface biofilm (0.25 logs up to 0.52 logs) and on GAC particles (0.42 logs up to 0.63 logs) after 2 months. Legionella and HPC attached to column's biofilm were higher than that on GAC particles indicating the strong association with biofilms. The bacterial concentration slowly increased in the effluent. This may be due to column's wall effect decreasing filter efficiency, possible exhaustion of GAC capacity over time and potential bacterial growth. / Dissertation/Thesis / Masters Thesis Civil and Environmental Engineering 2014

No presente trabalho faz-se uma revisão da literatura sobre pneumonia por Legionella pneumophila e se comparam estes dados com a série da autora, que se compõe de 10 casos esporádicos desta pneumonia, adquiridos na comunidade, ocorridos no perÍodo entre outubro de 1983 e maio de 1989. Todos os pacientes desta série eram do sexo masculino e de cor branca, com idade variando entre 36 e 71 anos. Os sintomas mais freqüentes foram febre alta, calafrios, cefaléia, tosse seca e mialgias. A hipótese diagnóstica baseou-se nos dados clínicos, radiológicos e laboratoriais. Em todos os casos o critério de comprovaçao diagnóstica foi a imunofluorescência indireta para Legionella. Salienta-se a importância do reconhecimento desta doença, que ainda apresenta um baixo Índice de suspeição em nosso meio. Procura-se tanto ressaltar os principais achados clínicos e radiológicos como também contribuir com orientações diagnósticas e terapêuticas. ApÓs a análise dos dados obtidos no trabalho, a autora concluiu que: 1. Os achados da série nao diferem daqueles descritos na literatura. 2. A casuística é restrita para o traçado de um perfil da doença no Rio Grande do Sul. 3. Quadros pneumônicos com má resposta clÍnica à penicilina ou derivados, associados a lesões radiolÓgicas com rápida mutabilidade, devem chamar a atenção para este diagnóstico. 4. A infreqüência deste diagnóstico em nosso meio deve-se ao baixo Índice de suspeição. Logo, a divulgação de informações sobre a doença pode resultar em substancial acréscimo ao registro de casos. / In the present work a review is made on the 1iterature about pneumonia by Legione11a pneumophi1a and the data are compared with the series presenteà by the author, composed of 10 sporadic cases of this pneumonia, acquired in the community, October, 1983 and May, 1989. between A11 the patients of this group were ma1e, caucasian, age varying from 36 to 71. The most frequent symptoms were high fever, chills, headache, dry cough and myalgia. The diagnostic hypothesis was baseà on laboratory, radiological and clinicai data. For all the cases, the criterion for diagnostic comprovation was indirect immunofluorescence for Legionella. The importance of the recognition of this disease s emphasized for it still shows a very low 1evel of suspicion in Rio Grande do Sul. The author looks for to ernphazise the main clinicai and radiological findings as well as contributes with diagnostics and therapeutical orientations. After the analysis of the data obtained in the present work the author concludes that: 1. The findings of this series does no differ from those described in the 1iterature. 2. The casuistic is too restricted to draw a profile of the disease in Rio Grande do Sul. 3. Pneumonias with bad clinicai response to penicillin or its derivatives, associated with radiologic lesions with rapid mutability shall call the attention for this diagnosis. 4. The low frequency of this diagnosis in our country is due to the low index of suspicion. Thus, the divulgation of information about the disease could result in substantial increase in the record of new cases.

Please read the abstract on p 79-80 of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

<p>The modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitin-interacting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen <i>Legionella pneumophila</i>: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of host-pathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides <i>Legionella</i>.</p>

Legionella pneumophila (Lp) is a gram-negative, intracellular parasite. These bacteria evade the host response with the help of a Type IVb secretion system (T4bSS), composed of Defective in organelle trafficking (Dot) and Intracellular multiplication (Icm) proteins. This secretion system delivers over 300 effectors into the host, and a large number of these molecules are dependent on IcmS and IcmW. These effectors are essential for the bacterium’s survival in the host. This work and previous studies have shown that IcmS and IcmW interact to stabilize each other and the C-terminal "tail" of DotL (residues 655-783), a coupling protein in the T4bSS, binds to the IcmSW complex to further increase its stability. All three components are α-helical, making the complex amenable to structural studies by X-ray crystallography and cryo-electron microscopy. Three maps of the IcmSW-DotL-tail complex (~42 kDa) were generated from cryo-EM images recorded with a Volta phase plate and K2 Summit direct electron detector at 500-1000 nm under-defocus. The final maps were processed with RELION-2 and resolved to 5.5-6.5 Å resolution using 57k, 60k and 80k particles, respectively. Concurrent with work in this thesis, a crystal structure of IcmSW-DotL(656-783) was solved by Dr. Byung-Ha Oh’s group at KAIST. This structure was used as a comparative model for our cryo-EM 3D reconstructions which were determined to evaluate size limits imposed on single particle methods with current technology and to provide snapshots of the complex in solution. Comparisons between the crystal structure and cryo-EM maps show that the overall structure is similar in solution, but there is significant flexibility within each subunit with a repositioning of some α-helices and surface loops. Flexibility in the absence of a central subunit (LvgA), and a low number of good particles may have limited the final resolution. Although the current maps were determined at α-helical resolution, this work provides a road map for solving near atomic structures at or near the size of IcmSW-DotL-tail. This structural technology will provide a means to probe the solution structure and function of biological machines in a large range of sizes and conformations.

The attachment and/or penetration of animal cells by two strains of Legionella pneumophila was studied in three vertebrae cell lines in vitro . The study focused on (1) differences in attachment and penetration between the two bacterial strains (an environmental isolate, Johannesburg-2, and a clinical isolate, Chicago-8) and between the cell lines (Hep-2, WI-38 and a murine line); (2) effects of L. pneumophila on cell morphology and growth; and (3) the effects of pyruvate and six sugars or sugar derivatives (D-mannose, D-Galactose, D-Glucose, L-glucose, D-fructose, and 2-deoxy-D-glucose).

Two species of cyanobacteria (Fischeralla sp. 29161 and Phormidium autumnale) and one species of green algae (Fritschiella tuberosa) were found to promote survival of Legionella pneumophila in mineral salts medium cocultures. During the early stage of incubation Fischerella sp. supported growth of Legionella pneumophila even though the bacteria would not grow in the algae-free basal medium.

The effect of copper on Legionella pneumophila in potable water plumbing systems is dependent on its dose and water chemistry. For instance, prior research demonstrated that the presence of aluminum hydroxide from anode rods in water heaters can bind copper and render high doses non-toxic. On this basis it was also hypothesized that iron hydroxide would have similar effects and that lower levels of copper may act as a nutrient encouraging Legionella growth. Here we conducted complementary experiments at bench, microcosm and pilot-scale to evaluate the effect of copper speciation and dose on L. pneumophila. At bench-scale, the addition of 5 mg/L as Fe iron hydroxide to a solution with 1 mg/L copper decreased soluble copper from > 90% down to < 20% at pH 6.5-7. The reduction in soluble copper caused ~3-logs higher L. pneumophila culturability when iron was added with copper when compared to a condition with copper alone. In a 9-month microcosm test using simulated glass water heaters with PEX pipe, a complete range of copper doses (0, 4, 30, 250 and 2000 g/L) were tested in triplicate. Over the first phase of research covered herein, the L. pneumophila levels were low at the four lowest doses of copper, and non-detectable at the highest dose. Moreover, total cell counts were highest at 250 g/L copper, lowest at 2000 g/L copper, and in between these extremes at the lower copper doses. This ongoing experiment will continue for months after this thesis was complete. Pilot-scale experiments were conducted with anode rods removed from tank water heaters, to examine the effects of unprotected corrosion of the steel on iron release and Legionella pneumophila levels in systems with 1) added copper (WH-Cu), 2) copper and phosphate corrosion inhibitor (WH-Cu+PO4-3), 3) phosphate corrosion inhibitor alone (WH-PO4-3) and 4) a control with neither copper nor inhibitor (WH-Control). While there were slight differences in iron between the conditions, the iron concentration in the water of the tanks did not dramatically increase compared to when the powered anodes were still installed and reducing corrosion. Because the iron level was usually < 0.1 mg/L, the released iron dose was not high enough to reduce soluble copper. In fact, oddly, soluble copper increased by 37-183%, mostly likely because the installed anode rods were suppressing copper release. Consequently, with the anode rods removed, the dose of 2000 g/L copper still had a strong antimicrobial effect. The levels of L. pneumophila increased in the order WH-Cu (2.6-logs CFU/L) < WH-PO4-3 (5.1-logs CFU/L) ≈ WH-Control (5.1-logs CFU/L). The addition of phosphate precipitated some of the added copper, such that the condition with copper and phosphate [WH-Cu+PO4-3 (4.2-logs CFU/L)] had L. pneumophila in between the condition with copper alone and that with no added copper. When the copper dose in the pilot rig was reduced to 1000 g/L, Legionella increased somewhat in the system with added copper compared to the control, and L. pneumophila increased in the water heaters in the order WH-Cu (3.4-logs CFU/L) < WH-Cu+PO4-3 (4.3-logs CFU/L) < WH-PO4-3 (4.9-logs CFU/L) ≈ WH-Control (5.0-logs CFU/L). Overall, an antimicrobial effect of copper was maintained in the water heaters even after the anodes were removed and iron in the water increased slightly. If iron corrosion and release to water were much higher without the anode rods than observed in this study, it would be predicted that the higher iron would have reduced the copper antimicrobial effect. / Master of Science / The effect of copper on Legionella pneumophila in potable water plumbing systems depends on its dose and water chemistry. For instance, prior research demonstrated that the presence of aluminum hydroxide from anode rods in water heaters can bind copper and render high doses non-toxic. It was also hypothesized that iron hydroxide would have similar effects and that lower levels of copper may act as a nutrient encouraging Legionella growth. Here we conducted complementary experiments at bench, microcosm and pilot-scale to evaluate the effect of copper's chemistry and dose on L. pneumophila. At bench-scale, the addition of a high level of iron to a solution with copper decreased the amount of copper available to Legionella from > 90% down to < 20% at pH 6.5-7. The reduction in bioavailable copper caused ~3-logs higher L. pneumophila when iron was added with copper when compared to a condition with copper alone. In a 9-month microcosm test using simulated glass water heaters with PEX pipe, a complete range of copper doses (0, 4, 30, 250 and 2000 g/L) were tested. The L. pneumophila levels were low and there were no significant differences between the five doses in the beginning phases of the experiment reported herein. However, total cell counts were highest at 250 g/L copper, lowest at 2000 g/L copper, and in between these extremes at the lower copper doses. This suggests that 250 g/L may be optimal in encouraging overall microbial growth. Pilot-scale experiments were conducted with anode rods removed from water heaters to examine the effects of unprotected corrosion of the steel tank on iron release and Legionella pneumophila levels. The four water heaters had added copper (WH-Cu), copper and phosphate corrosion inhibitor (WH-Cu+PO4-3), phosphate corrosion inhibitor alone (WH-PO4-3) and neither copper nor inhibitor (WH-Control). The iron concentration in the water of the tanks did not dramatically increase compared to when the powered anodes were used to reduce corrosion. Nevertheless, there were still slight differences in iron concentration between the replicate water heaters. When 2000 g/L copper was dosed, WH-Cu and WH-Control had statistically higher iron levels than WH-PO4-3, consistent with copper increasing corrosion of the unprotected tank. However, because the iron level was usually < 0.1 mg/L, the released iron dose was not high enough to reduce bioavailable copper. In fact, bioavailable copper went up when the anode rod was removed, most likely because the anode rods were protecting brass plumbing from corrosion. With the anode rods removed a dose of 2000 g/L copper had a strong antimicrobial effect. The levels of L. pneumophila increased in the order WH-Cu < WH-PO4-3 ≈ WH-Control. The addition of phosphate reduced some of the bioavailable copper, such that the condition with copper and phosphate (WH-Cu+PO4-3) had L. pneumophila in between the condition with copper alone and that with no added copper. When the copper dose in the water heaters was reduced to 1000 g/L, Legionella increased somewhat in the system with added copper compared to the control, and L. pneumophila increased in the order WH-Cu < WH-Cu+PO4-3 < WH-PO4-3 ≈ WH-Control. Overall, an antimicrobial effect of copper was maintained in water heaters even after the anodes were removed and even though iron in the water increased slightly. If iron corrosion and release to water was much higher than occurred in this study, it would be expected that the iron could have reduced the copper antimicrobial effect.