Avaliação metagenómica do hidrogenoma de águas minerais naturais (Região Norte de Portugal) e águas termais (Região da Galiza - Espanha)
Data
2018-10-04
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
Nos últimos anos, as águas termais e as águas minerais naturais têm vindo a ganhar especial atenção por parte da comunidade científica, nomeadamente, na prevenção e no tratamento de algumas doenças, mas também devido aos organismos microbianos que existem nestes habitats. Os microrganismos constituem uma fonte para a obtenção de novos compostos, tanto para a medicina como para a biotecnologia. Este trabalho teve por objetivo estudar, pela primeira vez, a diversidade taxonómica e funcional das comunidades microbianas existentes nas águas minerais naturais da Região Norte de Portugal e aumentar o conhecimento já existente da diversidade microbiana das águas termais da Região da Galiza. Para isso, recorreu-se à utilização das NGS (Next Generation Sequencing) em conjunto com a sequenciação Shotgun e a sequenciação do gene 16S rRNA, permitindo analisar o respetivo Hidrogenoma das águas em estudo. Foram colhidas, filtradas e analisadas quanto aos seus parâmetros físico-químicos duas réplicas de cada uma das cinco amostras de água. Posteriormente procedeu-se à extração do seu DNA metagenómico. As amostras de DNA resultantes da extração, foram sequenciadas com a plataforma Illumina MiSeq para a obtenção das sequências do gene 16S rRNA e com a plataforma Illumina HiSeq 4000 para a obtenção das sequências Shotgun. Após a obtenção das sequências foram utilizadas ferramentas bioinformáticas, nomeadamente as ferramentas FASTQC e MG-RAST server, permitindo assim obter o perfil taxonómico e funcional das sequências. Os principais iões responsáveis pelo agrupamento das águas provenientes da Região da Galiza foram, os compostos de enxofre (SO3 -2 e S-2 ) e o CO3 -2 no caso das amostras ES_PR e ES_TI e o pH no caso da amostra ES_BUR. Já relativamente às águas provenientes da Região Norte de Portugal, os iões (Mg, Ca e Sr) agruparam a água de PT_SA num grupo diferente da água PT_CH. Esta última foi agrupada de forma distinta graças aos compostos azotados (NO3 - e NH4 + ), C, RS, Na e Rb. A sequenciação do gene 16S rRNA resultou aproximadamente num total de 804 158 reads com um comprimento médio de 426pb. Na sequenciação Shotgun, utilizando apenas duas das amostras em estudo, conseguiram obter-se 94 973 018 reads, com um comprimento médio de 150pb e utilizando apenas duas das amostras em estudo. A sequenciação do gene 16S rRNA mostrou uma diversidade elevada de microrganismos em todas as águas analisadas, contudo, existe uma clara predominância bacteriana dos filos Proteobacteria (nas amostras de água PT_CH, ES_BUR e PT_SA), seguido do filo Firmicutes (nas águas ES_PR e ES_TI), Deinococcus-Thermus (nas águas PT_CH e ES_BUR), Aquificae (nas águas ES_BUR e PT_CH) e Nitrospira (nas águas PT_CH, ES_TI, ES_PR e ES_BUR). As técnicas utilizadas neste trabalho, assim como os resultados obtidos, podem num futuro próximo, permitir um melhor entendimento sobre a composição das comunidades microbianas e de que forma esses microrganismos interferem na constituição físico-química das águas termais e minerais naturais, muitas vezes associada a propriedades medicinais. Futuramente, o hidrogenoma poderá vir a ser utilizado como uma ferramenta auxiliar na prática de hidrologia médica.
In recent years, thermal waters and natural mineral waters have been gaining the special attention of the scientific community, namely in the prevention and treatment of some diseases, but also because of the microbial organisms that exist in these habitats. Micro-organisms are a source of new compounds for both medicine and biotechnology. The aim of this work was to, for the first time, study the taxonomic and functional diversity of the microbial communities present in the natural mineral waters of the northern region of Portugal and to increase the existing knowledge of the microbial diversity of the thermal waters of the region of Galicia. For this, we used Next Generation Sequencing (NGS) together with Shotgun sequencing and 16S rRNA gene sequencing, which made it possible to analyse the respective Hydrogenome of the waters under study. Two replicas of each of the five water samples were collected, filtered and analysed in relation to their physico-chemical parameters, after which their metagenomic DNA was subsequently extracted. The DNA samples which resulted from the extraction were sequenced with the Illumina MiSeq platform to obtain the 16S rRNA gene sequences and with the Illumina HiSeq 4000 platform to obtain the Shotgun sequences. After obtaining the sequences, bioinformatics tools were used, namely the FASTQC and MG-RAST server tools, thus allowing the taxonomic and functional profile of the sequences to be obtained. The main ions responsible for the grouping of the waters deriving from the region of Galicia were, sulfur compounds (SO3 -2 and S-2 ) and CO3 -2 in the case of the ES_PR and ES_TI samples and pH in the case of the ES_BUR sample. With regard to the waters deriving from the northern region of Portugal, the ions (Mg, Ca and Sr) grouped the PT_SA water into a group that was different to the PT_CH water. The latter was grouped differently thanks to the nitrogen compounds (NO3 - and NH4 + ), C, RS, Na and Rb. The sequencing of the 16S rRNA gene resulted in a total of approximately 804,158 reads with an average length of 426pb. In the Shotgun sequencing, 94,973,018 reads with an average length of 150 bp were obtained, using only two of the samples under study. The sequencing of the 16S rRNA gene showed a high diversity of microorganisms in all analyzed waters, however, there is a clear bacterial predominance of Proteobacteria phylum (in the PT_CH, ES_BUR and PT_SA water samples), followed by the phylum Firmicutes (in the ES_PR and ES_TI waters), Deinococcus-Thermus (in the PT_CH and ES_BUR waters), Aquificae (in the ES_BUR and PT_CH waters) and Nitrospira (in the PT_CH, ES_TI, ES_PR and ES_BUR waters). The techniques used in this work, as well as the results obtained, may, in the near future, allow for a better understanding of the composition of the microbial communities and how these microorganisms interfere in the physicochemical constitution of thermal and natural mineral waters often associated with medicinal properties. In the future, the hydrogenome may be used as an auxiliary tool in the practice of medical hydrology.
In recent years, thermal waters and natural mineral waters have been gaining the special attention of the scientific community, namely in the prevention and treatment of some diseases, but also because of the microbial organisms that exist in these habitats. Micro-organisms are a source of new compounds for both medicine and biotechnology. The aim of this work was to, for the first time, study the taxonomic and functional diversity of the microbial communities present in the natural mineral waters of the northern region of Portugal and to increase the existing knowledge of the microbial diversity of the thermal waters of the region of Galicia. For this, we used Next Generation Sequencing (NGS) together with Shotgun sequencing and 16S rRNA gene sequencing, which made it possible to analyse the respective Hydrogenome of the waters under study. Two replicas of each of the five water samples were collected, filtered and analysed in relation to their physico-chemical parameters, after which their metagenomic DNA was subsequently extracted. The DNA samples which resulted from the extraction were sequenced with the Illumina MiSeq platform to obtain the 16S rRNA gene sequences and with the Illumina HiSeq 4000 platform to obtain the Shotgun sequences. After obtaining the sequences, bioinformatics tools were used, namely the FASTQC and MG-RAST server tools, thus allowing the taxonomic and functional profile of the sequences to be obtained. The main ions responsible for the grouping of the waters deriving from the region of Galicia were, sulfur compounds (SO3 -2 and S-2 ) and CO3 -2 in the case of the ES_PR and ES_TI samples and pH in the case of the ES_BUR sample. With regard to the waters deriving from the northern region of Portugal, the ions (Mg, Ca and Sr) grouped the PT_SA water into a group that was different to the PT_CH water. The latter was grouped differently thanks to the nitrogen compounds (NO3 - and NH4 + ), C, RS, Na and Rb. The sequencing of the 16S rRNA gene resulted in a total of approximately 804,158 reads with an average length of 426pb. In the Shotgun sequencing, 94,973,018 reads with an average length of 150 bp were obtained, using only two of the samples under study. The sequencing of the 16S rRNA gene showed a high diversity of microorganisms in all analyzed waters, however, there is a clear bacterial predominance of Proteobacteria phylum (in the PT_CH, ES_BUR and PT_SA water samples), followed by the phylum Firmicutes (in the ES_PR and ES_TI waters), Deinococcus-Thermus (in the PT_CH and ES_BUR waters), Aquificae (in the ES_BUR and PT_CH waters) and Nitrospira (in the PT_CH, ES_TI, ES_PR and ES_BUR waters). The techniques used in this work, as well as the results obtained, may, in the near future, allow for a better understanding of the composition of the microbial communities and how these microorganisms interfere in the physicochemical constitution of thermal and natural mineral waters often associated with medicinal properties. In the future, the hydrogenome may be used as an auxiliary tool in the practice of medical hydrology.
Descrição
Palavras-chave
Hidrogenoma , Águas minerais naturais , Águas Termais , 16S rRNA , Shotgun , Illumina , NGS , Metagenómica