MSc (Microbiology) / Department of Microbiology / See the attached abstract below

CAPES; CNPq; Fundação Araucária / A alta demanda por água de qualidade e livre de contaminações, em um contexto de elevado crescimento populacional e econômico, é uma questão que preocupa a sociedade. Porém, a atividade humana, poluindo alterando o equilíbrio e a dinâmica dos recursos naturais produz impacto direto sobre as bacias hidrográficas de onde se capta a maior parte dessa água. A detecção de bactérias do grupo coliforme, em especial a Escherichia coli, é amplamente utilizada como parâmetro microbiológico da qualidade da água, seja na avaliação de sua potabilidade ou na verificação da eficácia do tratamento. Assim, propõem-se uma metodologia que, fazendo uso da bioluminescência ultrafraca, ou emissão de fótons ultrafraca (ultra-weak photon emission – UWPE) proveniente destas bactérias, possibilita detectar a presença destes microrganismos em amostras de água, de forma mais rápida que nos métodos atuais (que podem levar até 48 horas), além de dispensar testes bioquímicos e ser operacionalmente mais simples. O método consiste em detectar padrões de UWPE proveniente de amostras de água previamente incubadas em meio nutritivo EC, com e sem tratamento, utilizando para tal, uma instrumentação especialmente projetada, baseada em dispositivo fotomultiplicador capaz de efetuar a contagem dos fótons emitidos da amostra colocada no interior de uma câmara escura. Assim, por meio dos padrões temporais da UWPE com duração de 24 horas, foi possível discriminar as amostras de água tratada das não-tratadas (contaminadas), comparando-se os padrões obtidos destas amostras, com aqueles obtidos a partir de amostras de controle de uma cepa padrão de Escherichia coli ATCC 8739 incubadas em meio EC. O padrão bem definido da UWPE da amostra de controle serviu, portanto, de referência para avaliar a presença de bactérias do grupo coliforme em amostras sem tratamento. Dos resultados dos ensaios com cepa padrão de Escherichia coli, água tratada e não tratada foi possível concluir que o método proposto utilizando a avaliação dos perfis temporais de UWPE pode ser usado como um método alternativo de avaliação da qualidade sanitária da água com as vantagens supracitadas. / The high demand for quality water, free of contamination, in a high population growth context and economic is an issue that concerns the society. However, human activity polluting is changing the balance and dynamics of natural resources has a direct impact on the watershed, where it is captures most of this water. The detection of coliform group, in especially Escherichia coli, is widely used as microbiological parameter for water quality, as well as a tool to evaluate its potability or verification of the efficacy of treatment. Thus, proposed a methodology that makes use of the ultraweak bioluminescence, or ultra-weak photon emission (UWPE) from these bacteria, it’s possible to detect the presence of these microorganisms in water samples, faster than current methods (which can take up to 48 hours), besides dispensing biochemical tests and being operationally simpler. The method consists in detecting UWPE patterns from water samples previously incubated in EC nutrient broth, with and without treatment, using instrumentation specifically designed with a photomultiplier device capable of counting photons emitted from samples inside a dark chamber. Thus, through UWPE temporal patterns with a duration of 24 hours, it was possible to discriminate the treated water samples from the untreated water samples (contaminated), comparing the patterns obtained from these samples with those obtained from control samples of a standard strain of Escherichia coli ATCC 8739 incubated in EC nutrient broth. The well-defined pattern of the control sample served, therefore, as a reference to evaluate the presence of bacteria in the coliform group in untreated water samples. From the results of the standard strain of Escherichia coli, treated and untreated water, it was possible to conclude that the proposed method using the UWPE temporal profile evaluation can be used as an alternative method of evaluating the sanitary quality of water with the aforementioned advantages.

The purpose of our study was to describe the dynamics of E. coli in the rivers of the Seine watershed. This watershed is characterized by a poor microbiological water quality due to an important population density, industrial activities and intensive agriculture. The main objectives of our study were to obtain field microbiological data describing the system and to understand the underlying mechanisms in order to build a model able to describe and predict the fate of the fecal bacteria at the scale of the whole watershed. This model was built in order to help management of microbiological water quality. <p>To be able to describe and modelize the fate of fecal bacteria at the scale of such a large and complex system, it was first necessary to identify and quantify the sources of contamination and to be able to describe the processes affecting these bacteria once discharged to the rivers. <p>The quantification of E. coli in the natural aquatic systems through the traditional culture-based techniques has been strongly questioned during the last years because they have a long response delay (24 to 48 h) and they do not take into account the viable but not culturable bacteria (VBNC) which nevertheless could represent a health thread. For this reason we developed in this study two alternative methods: the direct (without passing through a cultivation step) measurement of the â-D-glucuronidase activity and a fluorescent in situ hybridization with an E. coli specific probe coupled with a viability test. The first has the main advangate to give a very rapid evaluation of the E. coli content of the water while the second one was able to enumerate VBNC E. coli. <p>The study of the sources of contamination of the rivers revealed the predominance, in this anthropogenicised watershed, of the point sources (effluents from wastewater tretament plants). Diffuse sources soil leaching and surface runoff) may nevertheless be locally important and have also be quantified. The influence of the land use on these diffuse sources has been quantified demonstrating that soil leaching and surface runoff of grazed areas was mainly responsible of fecal contamination in rural areas.<p>The attachment of E .coli to particles in the river waters was studied as it can influence the fate of this kind of bacteria in the aquatic systems. A linear regression was found between the percentage of particle-associated E. coli and the SM content of the water. Settling velocities of particle-associated E. coli were determined. The mortality rate of E. coli was analysed following various perspectives: the river order, the attachment of E. coli to particles, the importance of the cultivability loss in the disappearance of these bacteria and the importance of protozoan grazing. The results confirmed the importance of grazing and showed that the abondance of free living E. coli bacteria decreased two times more rapidly than particle-associated E. coli. In the Seine river, we showed that the net loss of culturable E. coli was 1.5 time higher than the net loss of viable E. coli. Finally, no relationship was found between mortality rate and river order. <p>These results have been used to build two models CF-SENEQUE and CF-SiAM-3D which describe E. coli dynamics respectively for the whole Seine river watershed and for the Seine for the estuary. Comparisons model calcultaions with field data of fecal coliforms abundances showed that these models correctly simulated the longitudinal distribution of fecal colifoms in the main rivers of the Seine watershed and in the estuary. These models were used to test the impact on the microbiological water quality of various scenarii of wastewater management .<p> / Doctorat en sciences, Spécialisation biologie animale / info:eu-repo/semantics/nonPublished

MESHWR / Department of Hydrology and Water Resources / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF