Successful isolation and cultivation of Cylindrospermopsis raciborskii strains isolated from finished drinking water samples

This work presents the successful establishment of Cylindrospermosis raciborskii cultures isolated from water samples collected at the exit point of Water Treatment Plant (WTP). An intense bloom dominated by filamentous cyanobacteria (Aphanizomenon spp., Planktothrix spp., Cylindrospermosis raciborskii, Anabaena spp.) occurred in the summer of 2015 in Roxo reservoir (south Portugal). Several cyanotoxins (microcystins, saxitoxins, cylindrospermopsin) were detected in raw and treated water, although at levels below the corresponding regulatory and/or guideline values. Nevertheless, this bloom caused intense unpleasant odour and taste in the water supplied to the populations and cyanobacterial cells (up to 1000 cells.mL-1) were detected in finished water samples collected at the exit point of WTP. Treated water samples collected at the WTP and at the city water deposit were inoculated in Z8 culture medium and cyanobacterial growth was followed by optical microscopy. After 30 days, cyanobacterial growth was observed showing resistance to the treatment processes and maintenance of reprodution capacity. Interestingly, morphometric and molecular analysis revealed the presence of C. raciborskii. Three isolates of this species were obtained and none were cylindrospermopsin- or microcystins-producers, as confirmed by Enzyme Linked Immunosorbent Assays (ELISA) and by amplification of genes (PS, PKS, mcyA-cd, mcyAB, mcyB) involved in those cyanotoxin synthesis. However, the ELISA for saxitoxins was positive for the 3 isolates and confirmation of this toxin production is in progress. To our knowledge, this is the first report on the establishment of successful cultures of C. raciborskii that survived to conventional water treatment processes.N/

Treatment of water for human consumption: a case of cyanobacterial survival

No verão de 2015 ocorreu um episódio de mau odor/sabor na água de abastecimento proveniente da albufeira do Roxo. Este episódio foi atribuído à presença de um bloom cianobacteriano que incluía espécies produtoras de compostos voláteis (geosmina, MIB). De facto, foi detetada a presença de cianobactérias na água tratada, à saída da ETA, ainda que em densidades relativamente baixas. No entanto, desconhecia-se se estes organismos mantinham a viabilidade celular e a capacidade de se desenvolver na rede de distribuição, a jusante da ETA e, desta forma, contaminar a água para consumo humano. Assim, amostras de água colhidas à saída da ETA e no Reservatório de abastecimento público foram inoculadas em meio de cultura e o crescimento celular foi seguido por microscopia ótica. Após 30 dias, verificou-se o crescimento algal, o que demostra que algumas espécies resistiram ao tratamento na ETA e mantiveram a capacidade de se reproduzir. Curiosamente, uma dessas espécies foi o Cylindrospermopsis raciborskii, considerada até há pouco tempo uma espécie tropical. Irá proceder-se à caracterização molecular e toxigénica desta espécie. Atualmente está a decorrer um protocolo de colaboração entre o Instituto Nacional de Saúde Doutor Ricardo Jorge e a AgdA, com o objetivo de se estudar a distribuição de cianobactérias, cianotoxinas e compostos voláteis na albufeira do Roxo.In the summer of 2015 an unpleasant odour/taste occurred in the water for human consumption supplied from Roxo Reservoir. This episode was attributed to the presence of a cyanobacterial bloom composed by species producing volatile compounds (geosmin, MIB). In fact, cyanobacteria were detected in treated water, at the exit of Water Treatment Plant (WTP), although at relatively low densities. However, it was unknown if these organisms maintained their viability and the growth capacity in the distribution network and, as such, to contaminate the drinking water. Thus, treated water samples collected at the WTP and at the water deposit were inoculated in culture medium and cell growth was followed by optical microscopy. Algal growth was observed 30 days after culturing, which shows that some species resisted the treatment in the WTP and maintained the capacity to reproduce. Interestingly, one of those species was Cylindrospermopsis raciborskii, considered until recently a tropical species. Molecular and toxigenic characterization of this species will be carried out. A collaboration protocol between INSA I.P. and AgdA, is currently being carried out with the aim of studying the distribution of cyanobacteria, cyanotoxins and volatile compounds in the Roxo reservoir.Elsa Dias, bolsa da Fundação para a Ciência e Tecnologia (SFRH/BPD/77981/2011)info:eu-repo/semantics/acceptedVersio

Cyanotoxins: from poisoning to healing – A possible pathway?

[ENG] Cyanobacteria are photosynthetic microorganisms known for their multiple, and occasionally dual, ecologic roles. Cyanobacteria are major contributors to oxygen production on earth, but they often bloom in freshwater environments, depleting oxygen and inducing massive fish kills by anoxia. In addition, cyanobacteria are primary producers and the base of the food web in aquatic ecosystems, but they often “crowd out” other phytoplanktonic organisms by competing for nutrients. Cyanobacteria produce an array of beneficial, biologically active compounds, but some of their secondary metabolites are also known to be highly toxic to humans. Many cyanobacterial characteristics are still a mystery and raise many unsolved questions: Why do they bloom? How do they “communicate” with each other and “synchronise” to flourish? How do they colonise so many diverse habitats and are resistant to the most adverse of environments? Why do some strains produce toxins while others do not? Current research on cyanobacteria may provide answers to these “old” questions, but may also challenge us to consider new perspectives. In this paper, we will discuss the potential therapeutic application of cyanobacterial toxins, most of which are known as potent toxicants, but some of which have a non-negligible potential for drug discovery.[ES] Las cianobacterias son microorganismos fotosintéticos conocidos por sus múltiples y a veces opuestos, roles ecológicos: son importantes contribuyentes en la producción de oxígeno en la Tierra, pero a menudo tienen floraciones en ambientes de agua dulce y llegan a agotar el oxígeno del agua, en este caso inducen la muerte masiva de peces por anoxia; son productores primarios y la base de la cadena alimenticia en los ecosistemas acuáticos, pero a menudo impiden la presencia de otros organismos fitoplanctónicos con los que compiten por los nutrientes disponibles; producen una amplia diversidad de compuestos biológicamente activos explotables para aplicaciones útiles, pero algunos de sus metabolitos secundarios también son conocidos por ser altamente tóxicos para los seres humanos. Muchas características de las cianobacterias son todavía un misterio y plantean muchas preguntas sin resolver: ¿por qué tienen floraciones? ¿cómo se comunican entre sí y se sincronizan para producir floraciones? ¿cómo se las arreglan para colonizar hábitats tan diversos y resistir en los entornos más adversos? ¿por qué algunas cepas producen toxinas, mientras que otras no lo hacen? etc. La investigación actual sobre cianobacterias puede dar respuestas a esas preguntas antiguas, pero también puede desafiarnos para debatir nuevas perspectivas. En este artículo discutiremos la posible aplicación terapéutica de las cianotoxinas, más conocidas por ser tóxicos potentes aunque empiezan a mostrar tímidamente una potencialidad creciente en el descubrimiento y desarrollo de nuevos fármacos.Elsa Dias- Foundation for Science and Technology for the grant SFRH/BPD/77981/201

Mercury methylation versus demethylation: main processes involved

It is well known that mercury presents high toxicity, causing a great damage to the environment and living organisms; however, its properties depend on the mercury species present. Organomercury compounds, where methylmercury is included, cause more concern. Since 60-70’s, several methylation mechanisms are known. Generally, methylmercury can be formed naturally in the aquatic environment by two general pathways: chemical methylation (abiotic) and microbial (biotic) processes. At the same time, methylmercury can be also decomposed abiotically or by the action of several demethylating microbes, or demethylators, ranging from anaerobes to aerobes. Regarding the biotic methylmercury demethylation, two distinct vias - oxidative and reductive - might be used by those microorganisms, differing in the final products obtained. In relation to the reductive processes, two pathways might occur. The first one involves the mercury resistance operon (mer) whereas the second one involves sulphide ions; however, the former is considered to be the most common pathway. Regarding the mer operon, some bacteria only carry on a narrow-spectrum operon (merA), being only able to reduce inorganic mercury (Hg(II)) to elemental mercury (Hg0). On the other hand, others beyond this operon also carry on a broad-spectrum operon (merB). These microorganisms are able to decompose methylmercury to Hg0. Taking into account all of these processes, in the present work the most referred methylation and demethylation mechanisms found in aquatic environments are discussed, as well as the environmental factors that influence them. Factors related with the inorganic mercury/methylmercury availability and those that affect directly the activity of methylators and demethylators are also referred. Generally, the relationships encountered are complex and sometimes significant shifts on the microbial communities may be observed. These changes can alter the processes involving the mercury species, as well as the final products obtained. In conclusion, the abiotic factors and the type of microorganisms that are present in the environment, including their genetic patrimony, influence significantly the presence and the type of the mercury species. Furthermore, there are environmental factors, such as redox conditions, sulphides and organic matter that also affect the mercury dynamic and the equilibrium existents

The Kidney Vero-E6 Cell Line: A Suitable Model to Study the Toxicity of Microcystins

Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with contaminated water. This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant. The studies of MCs effects on cell lines often lead to contradictory results, given the fact that distinct MC toxicity endpoints (mainly cytotoxicity and genotoxicity) have been studied in diverse cell lines (and cell clones) under distinct exposure conditions (different doses-ranges, time of exposure, MCs variants, etc). In our work with Vero-E6 cells we tested MCLR (both pure toxin and from cyanobacterial extracts of M. aeruginosa) within a wide range of concentrations (1 nM- 200 μM), using several endpoints and methodologies (cytotoxicity, morphology, genotoxicity, protein expression). In this chapter we will summarize our results and discuss the utility of Vero-E6 cell line to evaluate the toxicological properties of MCLR

Topic Segmentation: How Much Can We Do by Counting Words and Sequences of Words

In this paper, we present an innovative topic segmentation system based on a new informative similarity measure that takes into account word co-occurrence in order to avoid the accessibility to existing linguistic resources such as electronic dictionaries or lexico-semantic databases such as thesauri or ontology. Topic segmentation is the task of breaking documents into topically coherent multi-paragraph subparts. Topic segmentation has extensively been used in information retrieval and text summarization. In particular, our architecture proposes a language-independent topic segmentation system that solves three main problems evidenced by previous research: systems based uniquely on lexical repetition that show reliability problems, systems based on lexical cohesion using existing linguistic resources that are usually available only for dominating languages and as a consequence do not apply to less favored languages and finally systems that need previously existing harvesting training data. For that purpose, we only use statistics on words and sequences of words based on a set of texts. This solution provides a flexible solution that may narrow the gap between dominating languages and less favored languages thus allowing equivalent access to information

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento, Farmácia (Toxicologia), 2009, Universidade de Lisboa, Faculdade de FarmáciaDisponível no documentoFundação para a Ciência e Tecnologia (SFRH/BD/10585/2002

A Importância do Brincar na Relação Inter-Pares

O presente Relatório Final surge da unidade curricular da Prática de Ensino Supervisionada. O estágio foi realizado em contexto de Creche, numa fundação do Instituto Secular das Cooperadoras da Família, em sala de creche, com crianças dos dois aos três anos. O seguinte estudo centra-se em perceber “Qual a importância do brincar na relação inter-pares”. O principal objetivo deste estudo foi perceber até que ponto a brincadeira e a relação entre os seus pares, contribui para o desenvolvimento da criança. A metodologia utilizada foi de carácter Qualitativo, sendo que as notas de campo foram o instrumento utilizado na recolha de dados, sendo uma observação direta e participante. Concluo que só poderá existir desenvolvimento, se for proporcionado à criança através de uma relação de afetos, respeito e confiança, experiências significativas e vivências felizes. E para que essas vivências sejam felizes e significativas, nada mais importante que estimular na criança, a brincadeira e a relação inter-pares, contribuindo assim para a construção da sua personalidade e identidade

Risk levels of toxic cyanobacteria in portuguese recreational freshwaters

Portuguese surface freshwaters are widely used as a source of drinking water as well as bathing water. Cyanobacterial blooming in these water resources are common and are often associated with cyanotoxin production. The Portuguese legislation for drinking water (Decreto-Lei nº306/2007) establishes the regulatory level of 1 μg/L for total microcystins in treated water. This parameter is determined when eutrophication of water is suspected and when the number of potentially toxic cyanobacteria exceeds 2000 cells/mL. Conversely, the Portuguese legislation concerning the quality of bathing water (Decreto-Lei nº 135/2009), that was transposed form the European Directive nº2006/7/CE, do not include any guideline for cyanobacterial cells nor microcystins concentrations. It only recommends that when the bathing water profile indicates a potential for cyanobacterial proliferation, appropriate monitoring shall be carried out to enable timely identification of health risks. When cyanobacterial proliferation is detected visually, it is the responsibility of the local health delegate to evaluate the associated risk. If any risk has been identified or presumed, health and environmental authorities should implement the adequate management measures to prevent exposure, including information to the public. According to national specificities, some European countries complemented the European Directive nº2006/7/CE and implemented their own guidance or regulations, based on cyanobacterial cell numbers, biovolumes, pigments and/or cyanotoxin concentrations (Ibelings et al., 2015; Chorus, 2012). Prior to establishing regulatory or guideline values, it will be fundamental to characterize Portuguese inland bathing waters concerning the frequency, density, specie composition and toxicity of cyanobacterial blooms. These data are available but not systematized at a national scale. In this work we present the results of the monitoring of cyanobacteria and microcystins in 8 freshwater reservoirs located in the centre of Portugal largely used for bathing and recreational activities. These results will contribute to identify the cyanobacterial blooms profile and to assess the risk level of toxic cyanobacteria in Portuguese recreational freshwaters.N/