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- Modulatory effect of Gracilaria gracilis on European seabass gut microbiota community and its functionalityPublication . Gonçalves, Ana Teresa; Simões, Marco; Costa, Cátia; Passos, Ricardo; Baptista, TeresaSeaweeds are an important source of nutrients and bioactive compounds and have a high potential as health boosters in aquaculture. This study evaluated the effect of dietary inclusion of Gracilaria gracilis biomass or its extract on the European seabass (Dicentrarchus labrax) gut microbial community. Juvenile fish were fed a commercial-like diet with 2.5% or 5% seaweed biomass or 0.35% seaweed extract for 47 days. The gut microbiome was assessed by 16S rRNA amplicon sequencing, and its diversity was not altered by the seaweed supplementation. However, a reduction in Proteobacteria abundance was observed. Random forest analysis highlighted the genera Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae. SparCC correlation network analysis suggested several mutualistic and other antagonistic relationships that could be related to the predicted altered functions. These pathways were mainly related to the metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae. This study shows the beneficial potential of Gracilaria as a functional ingredient through the modulation of the complex microbial network towards fish health improvement.
- The involvement of SnRK1 complex in Gibberellin signalling pathway – a molecular approachPublication . Costa, Cátia Filipa Gaspar Veríssimo; Rodrigues, Américo do PatrocínioPrevê-se que a população mundial aumente, aproximadamente 34% em relação a 2009, até 2050. Para lidar com este aumento, é necessário aumentar o rendimento da agricultura. Um aumento no rendimento da agricultura requer plantas com a capacidade de resistir mais às condições de stress. Como elas são organismos sésseis, elas devem suportar e superar o stress biótico e abiótico. Frequentemente, a presença de stresses resulta na privação de energia, que é promovida pela redução da fotossíntese e pode levar à morte celular. A Sucrose non-fermenting1 protein kinase 1 (SnRK1) é responsável pelo restabelecimento da homeostasia energética e ao mesmo tempo intervém na regulação de processos de crescimento e desenvolvimento vegetais. Nesses mecanismos de regulação têm particular importância as fito-hormonas, tais como Ácido Abscísico (ABA) e as Giberelinas (GAs). As GAs são hormonas relacionadas com processos de desenvolvimento e de crescimento em plantas, envolvendo o alongamento das células, de que resulta um alongamento do hipocótilo e do tronco. As proteínas DELLA atuam como reguladores negativos dos efeitos de GA, na via de sinalização. Anteriormente, a interação entre as subunidades SnRK1 e as proteínas DELLA foi observada através de um ensaio de interação de proteínas, o Yeast Two-Hybrid. Neste estudo, a interação do complexo SnRK1 e das proteínas DELLA foi confirmada através de um teste de interação in vitro, o pull-down, o que sugere que as proteínas DELLA são substratos da SnRK1. Os resultados obtidos no presente trabalho mostram igualmente que a sobre expressão de SnRK1.1 origina um fenótipo de insensibilidade a GA. Globalmente, estes resultados sugerem que SnRK1 tem um papel importante na via de sinalização das giberelinas atuando como um regulador negativo.
- Detection of mcr-1 Gene in undefined Vibrio species isolated from clamsPublication . Valdez, Christian; Costa, Cátia; Simões, Marco; Carvalho, Carla C. C. R. De; Baptista, Teresa; Campos, MariaThe increase of antimicrobial resistant strains is leading to an emerging threat to public health. Pathogenic Vibrio are responsible for human and animal illness. The Enterobacteriaceae family includes microorganisms that affect humans, causing several infections. One of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filterfeeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In the present study, Vibrionaceae and coliforms microorganisms were isolated from clams. A microbial susceptibility test was performed using the disk diffusion method. From 43 presumptive Vibrio spp. and 17 coliforms, three Vibrio spp. with MICs to colistin > 512 mg L−1 were found. From the 23 antimicrobial resistance genes investigated, only the three isolates that showed phenotypic resistance to colistin contained the mcr‐1 gene. Genotypic analysis for virulence genes in EB07V indicated chiA gene presence. The results from the plasmid cure and transformation showed that the resistance is chromosomally mediated. Biochemical analysis and MLSA, on the basis of four protein‐coding gene sequences (recA, rpoB, groEL and dnaJ), grouped the isolates into the genus Vibrio but distinguished them as different from any known Vibrio spp.
- Real-time PCR protocol for detection and quantification of three pathogenic members of the Vibrionaceae familyPublication . Costa, Cátia; Ferreira, Guilherme D.; Simões, Marco; Silva, Joana L.; Campos, Maria J.Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using a new primer pair targeting the groEL gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL-1 in the case of V. alginolyticus and L. anguillara, and 48 CFU mL-1 for V. harveyi. Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with V. harveyi emphasize the importance of adjusting the DNA control’s standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR.