The overarching aim of this thesis was to describe the ecology of the assemblages of free-living nematodes in the sediments of nearshore marine and estuarine waters on the microtidal lower west coast of Australia. The thesis also provides descriptions of ten previously undescribed species as well as develops and tests a habitat classification scheme for the Swan River Estuary using these biotic assemblages. The first section of my thesis has determined the ways in which the characteristics of the nematode assemblages in nearshore marine waters along the microtidal lower west coast of Australia are related to habitat type, time of year and shore-perpendicular zones. Three habitat types, which had previously been identified on the basis of a suite of enduring environmental characteristics, could be broadly described as highly sheltered from wave activity and containing dense seagrass (habitat type 1), moderately sheltered from wave activity and with sparse seagrass (habitat type 2) and relatively exposed to wave activity and with no seagrass (habitat type 6). Sampling in five consecutive seasons yielded > 15 000 nematodes, representing 75 species. The number of species and densities in habitat type 1, and particularly those in its subtidal zone, were far greater than those in the other two habitat types. Both of these biotic variables underwent marked seasonal changes, declining to low levels during winter. The compositions of the assemblages differed significantly among the three habitat types, with the differences between habitat types 1 and 6 being particularly marked. Paracomesoma sipho, Dichromadora sp., Marylynnia annae and Pomponema sp., which, on the basis of their buccal cavity morphology, are assumed to feed primarily on benthic diatoms, were particularly abundant at the most sheltered habitat type, whereas Gonionchus australis, Theristus sp. and Bathylaimus australis, which are assumed to be deposit feeders, were relatively abundant at the most highly exposed habitat type. The compositions of the assemblages differed among seasons and were most discrete in spring, due to marked increases in the densities of certain species. However, differences in the compositions in the different zones of each habitat type were relatively small, presumably reflecting the influence of the small tidal regime of this region. The second component of this thesis has determined the ways in which the density, number of species, species composition and trophic structure of free-living nematode assemblages in the subtidal waters of a large microtidal estuary change spatially and temporally, and has explored whether those four biotic characteristics are related to certain environmental factors. Based on data derived from samples collected seasonally at 12 sites throughout the estuary, the densities and number of species of nematodes decreased progressively with distance from estuary mouth, to reach a minimum at sites where salinities were most variable, and then increased slightly in the uppermost part of the estuary where salinities were least. Densities were also generally greatest in spring, due largely to increases in the abundance of epistrate-grazing species at the time when the amount of primary food (microphytobenthos) peaked. The spatial distribution of the composition of the nematode assemblages was closely correlated with salinity and, to a lesser extent, grain-size composition and amount of particulate organic material in the sediment (%POM). Although species composition changed sequentially along the estuary, the change was particularly pronounced between sites above and below the area where salinities started to decline markedly and become more variable and %POM increased markedly. This reflected, in particular, far greater abundances of Spirinia parasitifera at the six downstream sites, and of Theristus sp. 1 at the six sites further upstream. Species composition underwent pronounced seasonal cyclical changes at all sites, presumably reflecting interspecific differences in the timing of peak reproduction and thus of recruitment. The trophic structure of the nematode assemblages changed both spatially and temporally in relation to the relative abundance of different food sources. Thus, for example, non-selective deposit feeders, such as Theristus sp. 1, dominated samples in the upper estuary, where %POM was by far the greatest, and was rare or absent at downstream sites. Conversely, epistrate grazers, such as species of the Chromadoridae, were most abundant at downstream sites in spring, when the density of the microphytobenthos reached its maximum. The data for the nematode assemblages in nearshore subtidal marine sediments of the lower west coast of Australia were compared with those in nearshore subtidal sediments in the upper and lower regions of the Swan River Estuary. The densities and average species richness in cores from the marine environment were much lower than in cores from both estuarine regions. However, the total number of species found in the marine environment was much greater than in the estuary. The compositions of the nematode assemblages were more variable in marine than estuarine sediments. The assemblages from the two estuarine regions were far more similar to each other than to those from the marine region at a species level, and also, but to a lesser extent, at the generic and family levels. While the trophic compositions of the nematode assemblages in the upper estuarine region was dominated by non-selective deposit feeding species and those of the lower estuarine region were dominated by epistrate grazing species during spring and non-selective deposit feeding species in other seasons, the dominant functional feeding groups varied among the sites representing the marine region. That variability presumably reflects differences in the relative contributions of the different potential food sources. Surprisingly, the trophic composition in the upper estuarine region, i.e. comprising predominantly non-selective deposit feeders, was similar to that at the very different environment of the most exposed marine site. The dominance of this feeding group at the marine site is assumed to be attributable to the fact that the only food source of any note is POM and, even then, it occurs in only small amounts. Taxonomic descriptions have been produced for ten new species of nematodes found during the ecological studies of the free-living aquatic nematofaunas of south-western Australia. These species were chosen because they were members of families for which the other species had been described and, in a number of cases, were important for distinguishing between the compositions of a priori groups. They comprised four species of Axonolaimidae, representing the genera Ascolaimus, Parascolaimus, Odontophora and Parodontophora, and six species of Desmodoridae, representing single species of Bolbonema, Eubostrichus, Catanema and Leptonemella and two species of Onyx. As a complement to the nematological study of the Swan River Estuary, a novel habitat classification system was developed and then applied in this environment. This system was based on enduring environmental characteristics and employed the relatively new multivariate statistical routines SIMPROF and LINKTREE. The applicability of habitat types produced by this classification system to biotic assemblages was tested using the data for the estuarine nematode assemblages described above. The results demonstrate that the compositions of the assemblages differed significantly among each of the habitat types defined by the classification system. While there were also significant differences between the compositions of the nematofaunas at sites belonging to the same habitat type, the extent of these differences were generally less than those between habitat types. A significant and strong correlation was also found between the spatial pattern exhibited by the environmental characteristics used to define habitat types, and that of the nematofauna.

Objectives. This study aimed to provide data on water intake from food and beverages of free-living adults in their natural environment, and investigate how this is affected by physiological, psychological, social and environmental factors. A further aim was to monitor the hydration status of free-living adults and relate this data to water intake. Methods The consumption of all food and beverages was recorded in a diary over three consecutive days by 80 healthy adults (40 males, 40 females), aged 18 to 65 years, who were instructed to continue their normal eating and drinking habits and lifestyles throughout the study. The data was analysed to determine total daily water intake and how this was affected by time of day, day of the week, presence of others, location of consumption, age and subjects mood. A further 20 healthy subjects (10 males, 10 females) repeated these procedures but also had blood samples taken for monitoring of blood indices (haemoglobin concentration, haematocrit, sodium concentration, and potassium concentration) and provided urine samples for the measurement of urine indices (volume, colour, specific gravity, osmolality and sodium, potassium, chloride, and creatinine concentrations). Results The total volume of water consumed by the 80 subjects was 2229  882 ml/day (mean  standard deviation). Females tended to consume more water than males (2402  827 ml/day vs 2056  911 ml/day, P = 0.079). Similar volumes were consumed by the additional 20 subjects in the hydration status study. Females appeared euhydrated; males appeared mild dehydrated, having a higher morning and 24-hour urine specific gravity and osmolality than females (P < 0.05, respectively). Conclusions The mean daily total volume of water consumed by females in this study was greater than the adequate intake value set by the European Food Safety Authority; for males it was lower. Females were euhydrated during the study but males tended to be mild dehydrated, reflecting their comparative water intakes.

Le génome de Mycobacterium tuberculosis, l’agent causal de la tuberculose, code pour une protéine ayant la capacité de se fixer sur la cellulose (Rv1987), une cellulase potentielle (Rv1090), et une cellulase pleinement active (Rv0062). Cette observation est surprenante, car la cellulose est un composant majeur des parois des cellules végétales, tandis que M. tuberculosis est un pathogène humain sans contact connu avec des plantes. Nous avons émis l’hypothèse que ces protéines pourraient jouer un rôle dans les interactions entre les mycobactéries du complexe M. tuberculosis avec les kystes d’amibes libres, dont la paroi contient également de la cellulose. Dans notre travail de thèse, nous avons cherché par une analyse in silico la présence de ces trois gènes chez toutes les bactéries ayant un génome complètement séquencé présentes dans la base de données CAZy (accessible en ligne à l’adresse www.cazy.org). Cette étude a montré que seulement 2,5% des bactéries codent pour les trois gènes simultanément. Parmi ces bacteries, nous avons ensuite confirmé expérimentalement par PCR et séquençage la présence des gènes Rv0062, Rv1090 et Rv1987 chez les mycobactéries du complexe M. tuberculosis. Nous avons ensuite vérifié la transcription de ces trois gènes chez la souche de référence M. tuberculosis H37Rv, puis produit dans Escherichia coli des protéines de fusion Rv1090 et Rv1987 et montré qu'elles étaient capables d'hydrolyser la cellulose (Rv1090) et de s’y fixer (Rv1987). De plus, nous avons mis en place un model expérimental d’interaction entre les mycobactéries du complexe M. tuberculosis et les amibes libres dans le but de comprendre le rôle des gènes Rv0062, Rv1090 et Rv1987. Dans un premier temps nous avons montré que M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii ainsi que Mycobacterium avium utilisé ici comme un controle positif étaient capables de survivre dans le cytoplasme des amibes libres telles que Acanthamoeba polyphaga. Ensuite, nous avons montré que M. tuberculosis et M. bovis mais pas M. canettii étaient capables de survivre à l’intérieur des kystes d’amibes. Enfin nous avons montré que M. tuberculosis, M. bovis et M. canettii étaient capables de survivre dans le sol pendant au moins 6 mois. Les données établies dans cette thèse soutiennent le rôle des cellulases dans la survie environnementale des mycobactéries du complexe M. tuberculosis, et ouvrent la voie à l’étude de cette phase méconnue dans le cycle de ces organismes / The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a protein with the ability to bind to cellulose (Rv1987), one potential cellulase (Rv1090), and one fully active cellulase (Rv0062). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. We hypothesized that these genes could play a role in the interactions between M. tuberculosis complex organisms and amoebal cysts, whose wall contains cellulose.In our thesis work, we have searched by in silico analysis for the presence of these three genes in all bacteria with complete sequenced genomes present in the CAZy database (available online at www.cazy. org). This study showed that only 2.5% of bacteria encode the three genes simultaneously. Among these bacteria we have confirmed experimentally by PCR and sequencing the presence of Rv0062, Rv1090 and Rv1987 in the M. tuberculosis complex organisms. We have checked the transcript of the three genes in the reference strain M. tuberculosis H37Rv and we subsequently produced Rv1090 and Rv1987 fusion proteins in Escherichia coli and demonstrated that they were indeed able to hydrolyze (Rv1090) and to bind (Rv1987) cellulose. In addition, we have developed an experimental model of interaction between M. tuberculosis organisms and the free-living amoebae in order to understand the role of Rv0062, Rv1090 and Rv1987 genes. Initially we have shown that M. tuberculosis, Mycobacterium bovis, Mycobacterium canettii and Mycobacterium avium used here as a positive control were able to survive in the cytoplasm of the free-living amoeba such as Acanthamoeba polyphaga. We have further shown that M. tuberculosis and M. bovis but not M. canettii were able to survive within the amoebal cysts. Finally we have shown that M. tuberculosis, M. bovis and M. canettii were able to survive in soil for at least 6 months. The data obtained in this thesis support the role of cellulase in the survival of M. tuberculosis complex organisms in the environment and pave the way for the study of this unknown phase in the cycle of these organisms.

Os nematoides causadores de doenças em cana-de-açúcar ocasionam graves problemas no rendimento desta cultura. Os gêneros mais agressivos são Meloidogyne e Pratylenchus que formam galhas e necroses nas raízes, respectivamente. A retirada da palha de cana-de-açúcar do campo para produção de etanol de segunda geração pode resultar em mudanças no comportamento das comunidades de nematoides. Assim o objetivo deste trabalho foi quantificar a abundância de nematoides em diferentes quantidades de palha presentes na superfície do solo. O experimento foi conduzido em Capivari - SP na unidade Bom Retiro (BR) e em Valparaíso - SP na unidade Univalem (UV). O tipo de solo de cada área experimental era, respectivamente, Latossolo Vermelho distrófico típico (textura média na camada superficial) e Argissolo Vermelho-Amarelo distrófico típico (textura arenosa na camada superficial). O delineamento foi o de blocos ao acaso com seis tratamentos e quatro repetições, compostos por quantidades de palha em Mg ha-1 de massa de matéria seca, sendo em BR (0; 4,2; 8,7; 15,1; 18,9; 23,3), e em UV (0; 3,9; 10,2; 12,5; 16,4; 13,2). As amostragens de solo e raiz coletadas na profundidade de 0-20 cm na linha e entrelinha da cultura foram realizadas em BR em maio e em UV em junho, ambas em 2015. Pratylenchus zeae teve maior dominância nas áreas, seguido de nematoides de vida livre, e Helicotylenchus sp. e Meloidogyne javanica apenas foi observada na área de solo arenoso e em médias densidades. O manejo de remoção da palha de cana-de-açúcar não promoveu mudanças significativas nas abundâncias das comunidades contidas no solo, tanto de textura média quanto arenosa. Por outro lado, houve interferência das quantidades de palha na infecção pelos nematoides nas raízes, no solo de textura média 4,2 Mg ha-1 de palha diminuiu a comunidade de nematoides e no solo de textura arenosa a maior comunidade de nematoides foi em 13,2 Mg ha-1 de palha. Todavia, múltiplos fatores podem ter afetado esses resultados, não apenas o manejo de remoção da palha. São necessários mais estudos em longo prazo para se afirmar o real efeito da retirada de palha do campo / The nematodes that cause diseases in sugarcane represent serious yield problems. The most aggressive genera are Meloidogyne and Pratylenchus that form gall and necrosis in the roots, respectively. Sugarcane straw removal from the field for second generation ethanol production may change the behavior of nematode communities. Thus the objective of this study was to quantify nematode abundance in different quantities of sugarcane straw left on soil surface. The experiment was conducted in Capivari - SP at Bom Retiro (BR) mill and Valparaíso - SP at Univalem (UV) mill. The soil type of each experimental area was, respectively, Oxisol (medium texture in the surface layer) and Alfisol (sandy texture in the surface layer). The experimental design was a randomized block with six treatments and four replicates, composed of quantities of straw in Mg ha-1 of dry matter mass, where in BR (0; 4,2; 8,7; 15,1; 18,9; 23,3), and in UV (0; 3,9; 10,2; 12,5; 16,4; 13,2). Soil and root samples were collected at 0-20 cm depths in the row and inter-row position of the sugarcane crop in BR in May and UV in June, both in 2015. Pratylenchus zeae had greater dominance in both evaluated areas, followed by free-living nematodes, and Helicotylenchus sp., Meloidogyne javanica was only observed in the sandy soil area and in medium densities. Due to the high densities it would be necessary to control nematodes in the areas. In terms of communities abundance in the soil there was no difference in relation to the treatments in the two areas. However, there was interference of the straw in the nematodes infection in the roots, and this result cannot be inferred by the straw deposition. Additional long-term studies are needed to understanding the effect of straw removal from the field on nematode community

Amebas de vida livre (AVL) são distribuídas mundialmente no solo e na água. Um número pequeno delas é considerado importante para a saúde dos seres humanos: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris e Sappinia diploidea. Algumas das infecções são oportunistas, ocorrendo em indivíduos imunocomprometidos, enquanto outras são não oportunistas. Amostras de água foram coletadas de banheira de hidromassagens e piscinas térmicas na cidade de Porto Alegre, RS, Brasil, com o objetivo de determinar a presença de Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e na amplificação por PCR com oligonucleotídeos gênero-específico. De 72 amostras analisadas, 20 (27,77%) foram positivas para amebas de vida livre, e identificadas morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 11 possuíam características compatíveis com o grupo morfológico II e 9 com o grupo III. Entre os isolados, 11(55%) foram considerados potencialmente patogênicos a partir de testes de osmotolerância e termotolerância. Somente 9 isolados quando submetidos à Reação da PCR, confirmaram pertencer ao gênero Acanthamoeba. A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição nos grupos genotípicos T3 (11,1%), T5 (11,1%), T4 (33,3%) e T15 (44,4%).Os resultados obtidos com este confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana nos ambientes de banheiras de hidromassagem e piscinas térmicas. / Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts, while others are non opportunistic. Water samples were collecyed from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Eschererichia coli. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. From 72 samples analyzed, 20 (27,77%) were positive for free-living amoebae, and the isolates were morphologically identified as belonging to the genus Acanthamoeba. Out of these, 11 presented morphological characteristics compatible with group II, and 9 with group III. Among the isolates, 11 (55%) were considered potentially pathogenic according to osmotolerance and temperature assays. The isolates when submitted to PCR reaction only 9 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compare to the sequences in the GenBank, showed that genotype distribution in group T3 (11,1%), T5 (11,1%), T4 (33,3%) and T15 (44,4%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health.

Les hydrocarbures aromatiques polycycliques (HAPs) sont des polluants organiques persistants (POP) émis essentiellement par les activités humaines suite à la combustion incomplète de la matière organique (industrie, chauffage, trafic routier...). En raison de leur faible taux de dégradation, de leur toxicité et de leur bioaccumulation, les HAPs font l'objet de plusieurs études d'écotoxicologie. La présente thèse, entreprise dans ce contexte par le biais d'une étude microcosmique se propose d'évaluer l'impact des HAPs sur la microflore et la méiofaune et d'appliquer différentes techniques de bioremédiation (biostimulation, bioaugmentation et combinaison des deux techniques) dans le but de dégrader les HAPs. Nos résultats ont montré que les HAPs sont toxiques aussi bien pour la microflore que pour la méiofaune de la lagune de Bizerte. Sur la microflore, l'effet toxique de ces contaminants s'est manifesté par l'inhibition de l'activité bactérienne à l'interface eau–sédiment et par la modification profonde de la structure des communautés bactériennes. En ce qui concerne la méiofaune, les HAPs ont entrainé une altération de la structure des communautés nématologiques. En effet, nous avons pu caractériser des espèces indicatrices de pollution par les HAPs. Ainsi, l'espèce Spirinia parasitifera dont la densité s'accroît dans tous les microcosmes contaminés par les HAPs paraît être une espèce “opportuniste” à la pollution par les HAPs. Cependant, Oncholaimus campylocercoïdes, fortement dominante dans tous les microcosmes témoins, a diminué de densité dans tous les microcosmes contaminés et a été considérée comme HAP-sensible. En analysant la structure des communautés microbiennes et méiofaunistiques, nous avons observé que la méiofaune joue un rôle structurant dans le maintien d'une communauté microbienne peu sensible aux effets des HAPs. Ce rôle structurant a été moins prononcé dans le cas d'ajout des sels nutritifs par la technique de biostimulation ; technique qui a présenté des effets non significatifs vis-à-vis des communautés nématologiques et a semblé plus efficace dans la minéralisation des HAPs et par conséquent à la réduction de leur effet toxique sur les organismes benthiques. Ces résultats suggèrent que la bioremédiation serait une alternative prometteuse à la dégradation des HAPs. / Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POP) emitted mainly by human activities due to the incomplete combustion of organic matter (industry, heating, traffic ...). Because of their low rate of degradation, their toxicity and their bioaccumulation, PAHs are main of concern in ecological studies. In this context, the objectives of this thesis were to assess the impact of PAHs on the benthic microflora and meiofauna and apply different techniques of bioremediation (biostimulation, bioaugmentation and combination of both treatments) in order to degrade PAHs, using experimental approach with microcosms.Our results showed that PAHs are toxic for microflora but also for meiofauna of Bizerta lagoon. The toxic effects of these contaminants were demonstrated by the inhibition of bacterial activity in the sediment/water interface and by profound changes in the structure of bacterial communities. PAHs provoked significant changes on meiofaunal community with the selection of nematode species that could be proposed as bioindicators of PAH pollution. Thus, Spirinia parasitifera which significantly (p<0.05) increased in PAH contaminated microcosms, suggesting that it is an "opportunistic» species to PAH pollution. In contrast, Oncholaimus campylocercoïdes, strongly dominant in control microcosms, decreased in PAH contaminated microcosms and seemed to be a ‘‘PAH-sensitive'' species.By analyzing the structure of microbial and meiofaunal community, we observed that the structural role of meiofauna on bacteria community structure was still evident even under PAH contamination despite the toxic effects on meiofauna. However, this structural role of meiofauna disappeared when nutrients were added to the sediment resulting in an almost complete removal of PAHs. Biostimulation seemed to be the most effective bioremediation strategy in the reduction of PAH toxic effects on benthic organisms. Overall, these results suggest that bioremediation using nutrient addition is a promising alternative technique for the degradation of PAHs in coastal polluted environments.

The purpose of this dissertation was to determine if the New Lifestyles NL-2000 (NL) and the Digi-Walker SW-200 (DW), waist-mounted devices, yield similar daily step counts as compared to the StepWatch 3 (SW), an ankle-mounted device, worn by adults and children in the free-living environment. For the first study, fifty-six adults (32.7 + 14.5 y) wore the devices for seven consecutive days. There were 20 normal weight, 18 overweight, and 18 obese participants. The NL and DW undercounted (pedometer error) similarly in the normal weight and overweight groups (-15.4% to -18.2%, respectively). However, the DW undercounted more than the NL in the obese group (-32.8% vs -23.9%, respectively). Stepwise regression revealed that both the NL and DW had more error (undercounted more) as a greater percentage steps were accumulated while walking slowly. The DW also had more error with greater BMI. Use of the DW in an obese population will result in twice the error as compared to a normal weight population and thus the DW should not be used to determine relationships between walking volume and adiposity For the second study, 74 children (13 ± 1.1 y) wore the same devices during one weekday. There were 33 normal weight, 21 overweight, and 20 obese participants. The error was determined for the NL and DW, and the values were similar in the normal weight and overweight groups (-10.8% to -15.4%, respectively). The DW undercounted more than the NL in the obese group (-27.3% vs -8.4%, respectively). The NL was very consistent regardless of BMI category, recording 89.1% (-10.8% error), 89.1% (-10.9% error), and 91.6% (-8.4% error) for the normal weight, overweight, and obese participants, respectively. Stepwise regression revealed that the DW undercounted more in participants with a high weight. Using the DW in obese children of this age group will result in significantly more undercounting when compared to normal weight children. The DW should not be used to determine relationships between walking volume and adiposity in this population. The NL undercounted by ~10%, regardless of BMI category.

Free-living amebae are ubiquitous in the environment and occasionally invade and parasitize host tissues causing illness in humans. Despite possibly frequent exposure to these organisms, infection is rare and why some people, healthy or not, end up with illness and others do not is still unclear. Human infections are rare; when illness does occur, it is often fatal. Only two papers have examined data from the literature and cases reported to the Centers for Disease Control and Prevention, and both were published over twenty years ago. The purpose of this study is to better document the epidemiology of Balamuthia and non-keratitis Acanthamoeba, give insight into trends of these infections over time, and contribute to the scientific and medical community by producing the only comprehensive review of all Balamuthia and non-keratitis Acanthamoeba cases in the United States from 1955 through 2009. This study also examines cases that have survived in an attempt to determine if there is evidence for the effectiveness of a particular treatment regimen. Only a small number of patients have survived these infections, so any evidence for a successful course of treatment could be crucial for future cases.

Amebas de vida livre (AVL) do gênero Acanthamoeba estão amplamente distribuídas no ambiente e podem tornar-se amebas patogênicas ao homem. O objetivo deste trabalho foi isolar em de água de torneira amebas de vida livre do gênero Acanthamoeba, identificá-las e classificá-las. Um total de 132 amostras de água de torneira foi coletado de escolas estaduais e municipais entre os meses de março a novembro de 2009. As amostras passaram pelo processo de filtração e as membranas foram semeadas em ágar não-nutriente 1,5% coberto por uma suspensão de E. coli inativadas pelo calor. Todas as amostras positivas para AVL foram submetidas à clonagem celular e identificadas como pertencentes ao gênero Acanthamoeba, através da morfologia dos cistos e trofozoitos e pela PCR utilizando oligonucleotídeos gênero-específicos que amplificam a região ASA.S1 do gene 18S rDNA. Ensaios fisiológicos de termo e osmotolerância foram utilizados para avaliar a patogenicidade dos isolados. Vinte sete isolados foram positivos para AVL e 10 foram identificados como pertencentes ao gênero Acanthamoeba tanto pelas características morfológicas quanto pela análise molecular. Destes, nove isolados apresentaram características do grupo II e um do grupo III, segundo Pussard e Pons (1977). A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição no grupo genotípicos T2 (40%), T2/T6 (40%), T6 (10%) e T4 (10%). Nos ensaios de termotolerância e osmotolerância 50% dos isolados obtiveram um baixo potencial patogênico. Os resultados indicaram a presença do gênero Acanthamoeba em água tratada no estado do RS, revelando sua importância epidemiológica e a necessidade de mais estudos para determinar sua distribuição no ambiente e seu potencial patogênico. / Free-living amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can become human pathogenic amoebae. The aim of this study was to isolate from tap water in free-living amoebae of Acanthamoeba, identify them and then classify them. A total of 132 samples of tap water was collected from state and municipal schools between march and november 2009. The samples passed through the filtration process and the membranes were seeded in non-nutrient 1.5% covered by a suspension of E. coli heatinactivated. All samples of AVL were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites by PCR using primers and genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Tests of physiological thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. Twenty seven isolates of AVL and 10 were identified as belonging to the genus Acanthamoeba through the morphological and molecular analysis. Nine of the isolates showed characteristics of group II and one isolate showed characteristics of group III, according Pussard and Pons (1977). The sequencing analysis by comparing the sequences submitted to GenBank, showed that genotype distribution in group T2 (40%), T2/T6 (40%), T6 (10%) and T4 (10%). In tests of thermotolerance and osmotolerance 50% of isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in the RS, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Foram coletadas amostras de água de piscinas térmicas e não térmicas na cidade de Porto Alegre, RS, Brasil entre os meses de maio de 2006 e março de 2007, com o objetivo de determinar a presença do gênero Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e trofozoítos e na amplificação por PCR com oligonucleotídeos gênero-específico. O potencial patogênico foi avaliado usando testes de osmotolerância e termotolerância. Das 65 amostras analisadas, 13 (20%) foram positivas para amebas de vida livre e identificados morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 9 possuíam características compatíveis com o grupo morfológico II e 4 com o grupo III. Todos os isolados identificados morfologicamente quando submetidos à Reação de PCR, confirmaram pertencer ao gênero Acanthamoeba e 38% (5/13) dos isolados foram considerados potencialmente patogênicos a partir dos testes de osmotolerância e termotolerância. Neste estudo, o método molecular de RAPD ("Random Amplified Polymorphic-DNA") foi utilizado para investigar a relação genética entre os 13 isolados de piscinas e dois isolados de referência da ATCC. De 10 oligonucleotídeos decaméricos testados, quatro foram selecionados por gerarem produtos de amplificação passíveis de análise. A similaridade entre os isolados foi calculada utilizando-se o coeficiente de Jaccard e o dendrograma construído pelo método da média das distâncias entre grupos ("Average Linkage"). Quatro grupos distintos (G1-G4) de isolados foram formados de acordo com a similaridade genética entre eles. Sugeriu-se que os isolados do G1 por agruparem-se aos isolados de referência de A. castellanii (ATCC 30010 e 50492) possam pertencer a esta espécie. Os dados fenotípicos, tais como, morfologia e testes de tolerância foram relacionados aos dados genotípicos de RAPD e permitiram a caracterização dos isolados. Os resultados deste primeiro estudo de isolamento e caracterização de Acanthamoeba de água de piscinas na cidade de Porto Alegre-RS, Brasil confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana. / Water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, RS, Brazil between May 2006 and March 2007, to determine the presence of Acanthamoeba in the water of swimming pools as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Escherichia coli. The identification of the isolates was based on the trophozoites and cysts morphology and on the amplification through PCR with genus-specific oligonucleotides. The potential pathogenic was assessed by osmotolerance and temperature tolerance assays. From the 65 samples analyzed, 13 (20%) were positive for free-living amoebae, and the isolates morphologically identified as belonging to the genus Acanthamoeba. Out of these, 9 presented characteristics compatible with morphological group II, and 4 with group III. All the morphologically identified isolates, when submitted to PCR, were confirmed as belonging to the genus Acanthamoeba, and 38% (5/13) of the isolates were considered potentially pathogenic according to osmotolerance and temperature tolerance assays. In this study, the molecular RAPD method (Random Amplified Polymorphic-DNA) was used to investigate the genetic relationship among 13 isolates from swimming pools and two strains from the ATCC reference. From the ten decameric oligonucleotides tested, four were selected for generating products of amplification possible to be analyzed. The similarity between isolates was calculated using the Jaccard coefficient and the dendrogram constructed by using the method of the average distances between groups ("Average Linkage"). Four distinct groups (G1-G4) of isolates were separated according to genetic similarity between them. It was suggested that the isolates from G1 once group up with the reference isolates of A. castellanii may belong to this species. The phenotypic data such as morphology and tolerance tests were related to RAPD genotypic data and led to the characterization of isolates. The results of this study about isolation and characterization of Acanthamoeba in swimming pools water at Porto Alegre, RS, Brazil confirm the presence of potentially pathogenic isolates which can present risks to human health.