Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

Made available in DSpace on 2016-06-02T19:31:43Z (GMT). No. of bitstreams: 1 2506.pdf: 900946 bytes, checksum: 97bcd58db188bc414e74218995bbe7c1 (MD5) Previous issue date: 2009-05-27 / Financiadora de Estudos e Projetos / A hundred sixty samples of water were collected to assess the sanitary and ecoepidemiological conditions of collective swimming pools in São Carlos SP. The samples were collected between August/2007 and Abril/2008. The pools were characterized by application of a questionnaire, answered by people responsable for the water treatment. Physical and chemical analysis included: pH, temperature, alkalinity, free chlorine concentration and turbidity. All water samples were analyzed for heterotrophic plate counts by pour plate method. Membrane filter technique was used to isolate amoebae and other bacteria. Eleven pools were heated and nine were unheated. Half of the pools was indoors and the other half was outdoors. Seventeen pools were chlorinated and three pools had a combined disinfection, two using chlorine and UV and one using chlorine and ozone. Only 13 samples (8%) conformed within the desired standards for pH, turbidity and free chlorine concentration at the same time. Twelve samples (7.5 %) were unacceptable for heterotrophic bacteria and 63 (39 %) were unsatisfactory for the presence of total coliforms. Escherichia coli was detected in two samples. Moreover, 102 samples (64 %) were contaminated with at least one microorganism.The percentages of occurrence were: 14 % for Staphylococcus spp, 35 % for Pseudomonas spp, 33 % for Mycobacterium spp and 21 % for free-living amoebae. The amoebae identified were used to recover bacteria possibly present intracellularly . Of these 33 samples positive for protozoa, 18 (54 %) harbored Mycobacterium spp, 15 (45 %) harbored Pseudomonas spp and one (3 %) harbored Staphylococcus spp. Escherichia coli was not recovered from amoebae. The physical and chemical parameters most related to the occurrence of microorganisms in the samples studied were pH and chlorine. No significant relationship was identified between water temperature and occurrence of microorganisms. Overall, the inappropriate forms of treatment and maintenance of swimming pools studied might create conditions for the development of microorganisms, including those potentially pathogenic for human. / Com o intuito de avaliar as condições ecoepidemiológicas e sanitárias de piscinas coletivas da cidade de São Carlos SP, foram coletadas 160 amostras de água em 20 piscinas, entre Agosto/2007 e Abril/2008. As piscinas foram caracterizadas através de aplicação de questionário junto aos responsáveis pelo tratamento da água. Os parâmetros físico-químicos analisados foram: pH, temperatura, alcalinidade, concentração de cloro livre e turbidez. A análise microbiológica foi realizada através de contagem de bactérias heterotróficas (método de pour plate ) e de concentração em membrana filtrante para a pesquisa de coliformes totais, Escherichia coli, Staphylococcus spp, Pseudomonas spp, Mycobacterium spp e amebas de vida livre. Entre as piscinas analisadas, 11 eram aquecidas e nove não-aquecidas. Metade das piscinas localizava-se em ambiente interno e a outra metade em ambiente externo. Dezessete piscinas eram cloradas e três delas faziam tratamento de desinfecção combinado: duas utilizando cloro e UV e uma utilizando cloro e ozônio. Apenas 13 amostras (8 %) se apresentaram dentro dos padrões desejados para pH, turbidez e concentração de cloro livre ao mesmo tempo. Doze amostras (7,5 %) estavam fora do padrão sugerido para bactérias heterotróficas e 63 (39 %) foram consideradas insatisfatórias quanto à presença de coliformes totais. Escherichia coli foi detectada em duas amostras apenas. Em relação aos outros microrganismos pesquisados, 102 amostras (64 %) apresentaram-se com pelo menos um deles. As porcentagens de ocorrência foram: 14 % para Staphylococcus spp, 35 % para Pseudomonas spp, 33 % para Mycobacterium spp e 21 % para amebas de vida livre. As amebas detectadas foram utilizadas para recuperar bactérias possivelmente presentes em seu interior. Das 33 amostras positivas para esses protozoários, 18 (54 %) demonstraram hospedar Mycobacterium spp, 15 (45 %) abrigavam Pseudomonas spp e uma (3 %) apresentava Staphylococcus spp internalizados. Escherichia coli não foi recuperada a partir de células amebianas. Os parâmetros físico-químicos mais relacionados com a ocorrência de microrganismos nas amostras estudadas foram o pH e o cloro. Não foi identificada relação significativa entre a temperatura da água e a ocorrência de microrganismos. De forma geral, foi possível observar certo descuido quanto às formas adequadas de tratamento e manutenção das águas das piscinas estudadas, o que pode colocar em risco a saúde dos freqüentadores, ao criar condições para o desenvolvimento de microrganismos, inclusive para aqueles potencialmente patogênicos.

Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

Il est aujourd’hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l’environnement. Ainsi, et jusqu’à maintenant, toutes les espèces d’amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l’ensemble des études a porté sur un nombre restreint d’espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n’ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d’une souche d’amibe appartenant à l’espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l’existence d’une phagocytose interamibienne entre différentes espèces d’amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l’inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l’importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L’analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L’inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna / Free living amoeba is a known vector of L. pneumophila in the environment and it has been shown to favour bacterial growth. Until now, all studied amoeba species showed a capacity to support the growth of the bacterium responsible for the Legionnaire’s disorder. However, these studies were related to a restricted number of amoebic species, and their relative capacity to support the bacterial growth. We compared the capacity of various amoebic species to support the proliferation of various strains of L. pneumophila. These studies highlighted the particular properties of a strain belonging to the Willaertia magna species. This amoeba has, contrary to all the other species, the capacity to inhibit and decrease L. pneumophila proliferation. We also demonstrate the existence of an inter-amoebic phagocytosis between various species of amoebas, a phenomenon never studied to our knowledge. The consequences of this inter-amoebic phagocytosis on the growth and proliferation of L. pneumophila are also reported. Electron microscopy strongly suggests that the bacterium cannot inhibit the phagolysosomal fusion in W. magna contrary to permissive amoebic species. These observations suggest the importance of membrane phenomena in the capacity of the bacteria to infest their amoebic host. Compared analysis of the lipidic composition of various amoebic species shows in W. magna a high expression of phosphatidylcholine the major phospholipid. The inhibition of the PE N-methyltransferase biosynthesis pathway of this phospholipid results in a strong reduction of the amoebic growth, suggesting that this metabolic pathway plays an important role in the resistance capacity of W. magna to L.pneumophila.

In this thesis, the results of microfossil, biofacies, and geochemical analyses (µXRF) of the Upper Cretaceous (Turonian) Ferron – Notom delta, Utah, USA are reported and discussed. The Notom delta is the oldest of three clastic deltas in the Ferron Sandstone Member, Mancos Shale Formation. Foraminifera and testate amoebae were recovered from ninety-eight mudstone samples among five well-exposed outcrops (Caineville North, Steamboat, Blue Hills, Neilson Wash, and Coalmine Wash). Detailed observations showed foraminifera, and testate amoebae tests have undergone post-burial compression (flattening), dissolution, and transport/reworking; therefore, identification of these assemblages to their species level is difficult. The micropaleontological analysis of the Ferron-Notom delta consists of three studies. Morphogroup analysis was applied on foraminifera and testate amoebae, where a relationship between the test morphology and habitat was established. Morphogroup analysis resulted in four main morphogroups and eleven morphotypes and were assigned to life mode, environment, and feeding strategies. Three foraminifera morphogroups and one testate amoebae morphogroup were established indicate a range of environments, from shallow shelf to shallow shelf to lagoon/estuary environments. Biofacies analysis using the morphotypes was applied on three outcrops (Caineville North, Steamboat, and Blue Hills). We use the morphotypes to define the four main biofacies using cluster analysis, and biodiversity indices. Four biofacies showed marine and fluvial (freshwater) influences. Salinity and OM indices were derived from the relationship of foraminifera morphotypes (BiS, TrS, TS) and testate amoebae morphotypes (Ta-F, Ta-D, Ta-S) that follows lithofacies trends. Because of the under-representation of calcareous foraminifera (due to taphonomic and/or diagenetic factors), the biodiversity indices are treated herein as relative measures. Despite this taphonomic bias, the agglutinated foraminifera and testate amoebae morphogroups show trends with salinity both among the outcrops and stratigraphically within the outcrops. The Blue Hills outcrop represents the most landward and lowest salinity environment (tidally-influenced backwater), Steamboat is more coast proximal with a higher salinity of the delta front and fluvial estuarine environments, and the Caineville North outcrop represents the most coast proximal (fluvial/estuarine to deltaic/prodeltaic) with salinities ranging from low to medium. It appears that the landward transport and coastward of tests was a significant source of taphonomic bias. Nonetheless, the assemblages provide useful depositional information that correlates with previously documented lithofacies data. A salinity index based on a ratio of trochospiral taxa versus testate amoebae was found to provide a useful measure of coastal proximity that matches lithofacies trends. The µXRF analysis was conducted on twenty-nine mudstone samples from the Caineville North outcrop to examine elemental proxies for paleo-salinity (Sr/Ba), organic matter (K/S), redox (V/Ni), and sediment sources (Zr/Rb, Ti/Fe, Ti/Ca). Twenty-nine surface mudstone samples collected from four lithofacies representing prodelta, delta front, fluvial valley fill and shelf environments. Fourteen elements (Ti, Fe, Ca, Sr, Ba, Ni, Rb, Zr, S, V, Cu, Mn, Si, and K) were used to calculate proxy ratios to detect variations in terrigenous sediments, carbonate production, salinity, grain size, and fluvial inputs in the Caineville North outcrop. Elemental data showed good correspondence between the elemental data and the lithofacies and microfossil indices. Sr/Ba and Ca/Fe showed relationships with carbonate content and proximity to the shoreline. The paleo-salinity proxy (Sr/Ba) did not respond to salinity, but was more responsive to lithological change of carbonate content. The microfossil salinity index seems to be a more accurate paleo-salinity indicator. The proxy for fluvial input of sediment (Zr/Rb and Ti/Fe) agree well with the lithofacies trends, and Zr/Rb and Ti/Fe showed highest values within the fluvial valley fill facies, with higher variability of Ti/Fe compared to the other lithofacies (prodelta, delta front, and shelf), corresponding to response to the proximity of the depositional sites to a fluvial sediment source. The redox proxy V/Ni matched the previously derived microfossil OM index ("bolivind-type" taxa) showing a strong relationship between eutrophication and redox trends. The study aims to establish important baseline geochemical compositions of sediment sources to establish patterns and trends with sediment succession in the deeper basin (offshore; i.e. Mancos Shale). Potentially, these nearshore to fluvial trends will provide important geochemical data to assess changes in paleoclimate, and sea-level in offshore sediment successions. / Dissertation / Doctor of Philosophy (PhD)

<p>High density sampling (n=50) was conducted in Cootes Paradise, a shallow wetland on the western shoreline of Lake Ontario near the city of Hamilton. Cootes Paradise is an urban wetland that has been affected by pollutants and nutrients and invasive carp. Thecamoebian analyses paired with site specific environmental measurements (depth, sp. conductivity, temperature, DO and pH) and substrate characteristics (textural and organic content -LOI) show relationships (R² = 0.6) with depth (0-1m) and corresponding sp. conductivity (0.5 to 0.65 mS/cm) and temperature (26.5 to 30.5 °C). Q-mode cluster analysis recognized two biofacies. Biofacies 1 samples (n= 26) are found in the deeper areas (0.70 ± 0.27 m) and dominated by <em>C. tricuspis</em> 36 ± 8% (1 std), <em>L. vas</em> 18 ± 13% and <em>D. protaeiformis “claviformis”</em> 14 ± 6%. Mean water temperature is 28.0 ± 0.6 °C and conductivity at 0.56 ± 0.04 mS/cm. This assemblage has low species diversity (SDI=1.9 ± 0.3) which indicates a transitional environment. Biofacies 2 contains samples (n= 24) which are found in shallower areas (0.38 ± 0.15 m) and the assemblage is characterized by <em>C. constricta “aerophila”</em> 25 ± 8%, <em>C. tricuspis</em> 18 ± 5%, <em>Cyclopyxis sp.</em> 9 ± 6 % and <em>L. vas</em> 9 ± 4 %. The SDI for Biofacies 2 is 2.2 ± 0.2 and like Biofacies 1 shows a transitional environment. The average temperature is and 29.0 ± 1.0 °C with mean sp. conductivity also slightly higher than Biofacies 1 at 0.6 ± 0.04 mS/cm.</p> / Master of Science (MSc)

Dans un contexte de réchauffement climatique, la fonction puits de carbone des tourbières à sphaignes est susceptible d’être altérée en raison d’une modification des interactions sphaignes-microorganismes, responsables de l’accumulation de carbone. L’objectif de cette thèse a été (1) d’identifier les interactions chimiques entre les polyphénols des sphaignes et les communautés microbiennes des sphaignes et (2) d’évaluer l’impact du réchauffement climatique sur ces relations.Un dispositif expérimental (Open Top Chambers) simulant in situ une hausse modérée des températures (+ 1°C) a été installé sur la tourbière de Frasne (25). La hausse des températures a provoqué une modification du réseau alimentaire microbien avec l’augmentation de la biomasse des bactéries et une baisse importante (-70%) de la biomasse des prédateurs (amibes à thèque). Le réchauffement climatique a également induit une baisse de la production de polyphénols, diminuant ainsi leur effet inhibiteur sur les microorganismes. En parallèle à cette baisse, une hausse des activités enzymatiques phénoloxydases a également été enregistrée. Le réchauffement a ainsi modifié les relations polyphénols – phénoloxydases, deux éléments essentiels du cycle du carbone des tourbières.Les différents changements induits par le réchauffement climatique (polyphénols, phénoloxydases, réseau trophique microbien) ont aussi conduit à une modification des relations « sphaignes-microorganismes » via une accélération potentielle du recyclage des nutriments, ce qui pourrait influencer sur le long terme le fonctionnement de l’écosystème tourbière. / Carbon stocks of Sphagnum-peatlands are likely to be altered by climate warming due to important changes in “Sphagnum-microorganisms” relationships which are responsible for carbon sequestration. The objectives of the thesis are (1) to identify the chemical interactions between Sphagnum-polyphenols and Sphagnum associated microbial communities and (2) to understand the impact of warming on these relationships.An experimental design using Open-Top-Chambers (OTC) was used to simulate an increase of air temperatures (+1°C) on a peatland situated in Jura Mountains (Frasne, 25). Temperature increase induced a modification of the microbial food web with an increase of the biomass of bacteria and a strong decrease of the biomass of top-predators (-70%), i.e. testate amoebae. Warming also induced a decrease of Sphagnum-polyphenols, diminishing their inhibitory effect on microbial communities. In parallel, an increase of phenoloxidase enzymatic activities was recorded in OTC. Climate warming thus modified polyphenol-phenoloxidase interplay, two crucial components of carbon cycle in peatlands. Warming effects on the different components – i.e. polyphenols, phenoloxidases, microbial food web – induced a modification of Sphagnum-microbial communities interplay, favoring carbon and nutrient recycling, which could alter peatland functioning in long-term.

O presente trabalho explora os aspectos taxonomicos, ecologicos, morfologicos, biometricos e biogeograficos dos Arcellinida Kent, 1880 coletados no Parque Ecologico do Rio Tiete, Sao Paulo Brasil. Foram encontrados organismos pertencentes a cerca de 30 taxons nominais, no entanto, a revisao da literatura, novos dados morfologicos obtidos atraves do Microscopio Eletronico de Varredura e medidas biometricas realizadas com grande numero de individuos permitem afirmar que muitos destes taxons estao se referindo a mesma entidade na natureza. Logo, na presente pesquisa sao descritas, com detalhes de distribuicao geografica, morfologia ultra-estrutural, morfometria e ecologia, especies pertencentes a quatro familias e cinco generos: Difflugia corona Wallich, 1864; Difflugia gramen Penard, 1902; Difflugia lanceolata Penard, 1890; Difflugia claviformis Penard, 1899; Difflugia gigantea Chardez, 1967; Centropyxis aculeata (Ehrenberg, 1838); Netzelia wailesi (Ogden, 1980); Lesquereusia modesta Rhumbler, 1895; Lesquereusia mimetica Penard, 1911; Arcella hemisphaerica Perty, 1852; Arcella gibbosa Penard, 1890; Arcella discoides Ehrenberg, 1871 e Arcella brasiliensis Cunha, 1913. Sao discutidas inovacoes taxonomicas para que a comparacao de dados obtidos usando tecnicas atuais com aqueles reportados na literatura tradicional seja feita da maneira mais explicita possivel, de modo a delimitar melhor o conceito taxonomico de cada especie abordada. / The present survey explores the taxonomic, ecologic, morphologic, biometric and biogeographic aspects of the Arcellinida Kent, 1880 collected at the Ecological Park of the Tiete River, Sao Paolo Brazil. Around 30 nominal taxa were identified, however, a review of the literature and new morphologic data obtained via the Scanning Electron Microscope and biometric measures with a large number of individuals allow the inference that many of these taxa are referring to the same natural entity. Therefore, the present work describes species from four families and five genera, along with details about geographic distribution, ultra-structural morphology, morphometry and ecology: Difflugia corona Wallich, 1864; Difflugia gramen Penard, 1902; Difflugia lanceolata Penard, 1890; Difflugia claviformis Penard, 1899; Difflugia gigantea Chardez, 1967; Centropyxis aculeata (Ehrenberg, 1838); Netzelia wailesi (Ogden, 1980); Lesquereusia modesta Rhumbler, 1895; Lesquereusia mimetica Penard, 1911; Arcella hemisphaerica Perty, 1852; Arcella gibbosa Penard, 1890; Arcella discoides Ehrenberg, 1871 e Arcella brasiliensis Cunha, 1913. Taxonomic innovations are discussed in order to make comparison of recent data with those reported on traditional literature a more explicit practice, allowing a better understanding of each species taxonomic concept.

Legionella pneumophila ist ein fakultativ intrazellulär replizierendes Bakterium und der Erreger der Legionärskrankheit, einer schweren Pneumonie. Das Typ IVB Dot/Icm Proteinsekretionssystem und dessen Effektoren sind wesentlich an der Virulenz des Bakteriums beteiligt. Das Ziel der vorliegenden Arbeit war die Charakterisierung der Patatin-ähnlichen Proteine von L. pneumophila - insbesondere von PatA, das vom Dot/Icm Sekretionssystem in Wirtszellen eingeschleußt wird. Im Rahmen dieser Arbeit wurden folgende Ergebnisse erzielt: Die 11 Patatin-ähnlichen Proteine von L. pneumophila zeigen hauptsächlich Lysophospholipase A-Aktivität. PatA besitzt außerdem Phosphatidylglyzerol-spezifische Phospholipase A-Aktivität. Serin-72, welches in ein G-X-S-X-G Lipasemotiv eingebettet ist, ist für die Aktivität des Proteins essentiell. PatA ist nach Expression in A549 Epithelzellen in der Zytoplasmamembran oder einer damit eng assoziierten Struktur lokalisiert, die lipolytische Aktivität ist hierfür nicht entscheidend. Die Deletion einer C-terminalen Proteinregion führt zum Verlust der membranständigen Lokalisation. Virulenzattenuierte L. pneumophila Mutanten bilden unter Präsenz von Amöben - im Gegensatz zu Wildtypstämmen - eine Koloniemorphologie aus, die Scattermorphologie genannt wurde. Auf Basis der Scattermorphologie wurde eine Transposon-mutagenisierte Legionella Klonbank auf Wirtszellkolonisationsdefekte überprüft. Dabei wurden 119 kolonisationsdefekte Mutanten isoliert und 70 neue putative Wirtszellkolonisationsgene, darunter zwei Gene Patatin-ähnliche Proteine (patD, patF), identifiziert. patD befindet sich in einem Operon mit bdhA, dem Gen einer putativen 2-Hydroxybutyrat-Dehydrogenase. Das Operon spielt eine Rolle im Poly-Beta-Hydroxybutyrat (PHB) Stoffwechsel des Bakteriums und wird für die Replikation in Wirtszellen benötigt. Die Studie liefert die ersten experimentell fundierten Ergebnisse, die die Bedeutung des PHB-Metabolismus für die Virulenz des Bakteriums belegen. / Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal pneumonia. One mayor virulence determinant is the Dot/Icm Type IVB secretion system and its effector proteins. Aim of the present work was the characterization of patatin-like proteins of L. pneumophila, and in particular PatA, which is carried by the Dot/Icm secretion system into host cells. Within this work following results were obtained: The 11 patatin-like proteins of L. pneumophila possess majorly lysophospholipase A activity. L. pneumophila PatA additionally shows Phosphatidylglycerole-specific phospholipase A activity. Serin-72, which is embedded in an G-X-S-X-G lipase motiv is essential for the lipolytic activity of PatA. PatA locates to, or close to, the cytoplasmic membrane when expressed in A549 epithelial cells. The lipolytic activity of PatA is not required for membrane targeting and deletion of a C-terminal region abolishes proper targeting. Virulence attenuated L. pneumophila mutants, develop an easy recognizable phenotype during co-culture with A. castellanii on agar plates that was named „scatterphenotype“. On the basis of the scatterphenotype, a new assay was developed allowing screening of huge clone banks with respect to amoebae sensitivity, a marker for reduced virulence. Here, a collection of several thousand transposon mutagenized L. pneumophila clones was screened and a total of 119 amoebae sensitive mutants was isolated. Among those, 70 novel putative host cell colonization and virulence genes were identified including two members of the patatin-like protein family (patD, PatF). patD is cotranscribed with bdhA, therefore forming an operon. bdhA encodes a putative 3-hydroxybutyrate dehydrogenase. The operon is involved in the poly-beta-hydroxybutyrate (PHB) metabolism of L. pneumophila and is needed for replication in host cells. The study provides the first experimentally funded data showing the linkage of PHB metabolism and virulence of L. pneumophila.

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.