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Estudo da actividade antimicrobiana de isolados provenientes de ambientes vulcânicos da Ilha Terceira - Açores - contra microrganismos indicadores relacionados com ambientes alimentares

Tecnologias Alimentares e Saúde

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Dissertação de Mestrado em Tecnologia e Segurança Alimentar

Orientador: Professora Doutora Maria de Lurdes Nunes Enes Dapkevicius
Co-Orientador: Post-Doc Cristina Riquelme Gabriel
Post-Doc Airidas Dapkevicius


Márcia Catarina Mendes Coelho

Resumo

In the recent decades, many bacteria among which important food borne pathogens, have acquired resistance to the antibiotics that are presently available, raising the need to search for novel compounds with antimicrobial activity. Extreme environments are home to unique microbial communities that can be exploited as sources of antimicrobial compounds. Due to their volcanic origin, the Azores possess countless terrestrial extreme environments, such as lava tubes and sulfur vents, whose biotechnological potential is yet to be exploited.
The present work aimed at the morphophysiological characterization of isolates from biofilms in lava tubes – Terra Mole and Branca Opala - and from sulfur vents – Furnas do Enxofre, screening these isolates for their antimicrobial activity against food-related indicator microorganisms; isolating and preliminarily characterizing the anti-microbially active fractions of the most active bacteria.
Twenty-seven isolates from two lava tubes and three isolates from sulfur vents located in Terceira Island (Azores, Portugal) were studied. All isolates were characterized from the morphophysiological point of view (colony morphology, Gram reaction, catalase, oxidase, nitrate reduction and motility). Antimicrobial activity of the 30 isolates was initially studied against 8 indicator microorganisms (Proteus sp., Salmonella Typhimurium ATCC 14028, E.coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococus aureus ATCC 9144, Staphylococus aureus ATCC 29523, Listeria innocua ATCC 33090, Listeria monocytogenes ATCC 7466) by the cross-streak assay. Four representative
isolates with antimicrobial activity (GBO31C, GTM1B2, GBO69B and P2-1) were selected for studying the effect of cultivation conditions on their growth parameters (ODmax, μmax, e T.est.) and on their antimicrobial activity, evaluated by the well-assay. Identification of the isolates GBO31C, GTM1B3 and GBO69B was attempted by sequencing the genes that code for rRNA from the 16S subunit. The effect of sample preparation (whole culture vs. cell-free supernatant) on the antimicrobial activity of isolate P2-1 was evaluated by the well-assay and disk assay methods. Finally, preliminary characterization of the antimicrobial activity of cell-free supernatants from P2-1 was carried out by testing the effect of heat,
enzymes, and ammonium sulfate precipitation.
The 30 isolates under study displayed considerable phenotypic diversity, with 7 of the 16 possible morphological groups represented. Nitrate reduction and motility were frequent. Seventyseven percent of the studied isolates inhibited at least on of the 8 indicators tested. Concerning the sampling locations, all isolates from sulfur vents were inhibitory against the tested pathogens. In lava tubes, the frequency of antibacterial isolates was higher in the yellow and pink mats when compared with white and tan biofilms. Tests carried out in two isolates belonging to the Proteobacteria (GBO31C and GTM1B2) and one Enterococcus isolate (GBO69B) showed that cultivation conditions (temperature and culture media) influenced their antimicrobial activity. It also observed that optimal conditions for antibiosis differ from the optimal conditions for growth in the tested organisms. Comparison of the
results of the evaluation of antimicrobial activity of GBO31C, GTM1B2 and P2-1 by the cross-streak and well-assay methods indicated that the biofilm lifestyle enhanced antibiosis in these isolates. The same did not occur for isolate GBO69B. Isolate P2-1 (G+Ox+Cat-n) displayed broad-spectrum antimicrobial activity. In this isolate, removing the cells from the spent culture, heat treatment at 100°C, extraction with n-butanol and addition of proteinase K, α-chemotrypsin, trypsin and α-amylase did not affect antimicrobial activity, whereas thermal treatment at 121°C, precipitation with ammonium sulfate, treatment with neutrase, extraction with n-hexane and ethyl acetate eliminated the antibiosis effect.
This indicates that mid-polarity proteic compound(s) contribute to the antimicrobial activity of P2-1.
Our data show that bacterial isolates from Azorean (Terceira Island) extreme environments hold potential as a source of novel antibiotics and other antimicrobial compounds of industrial interest.

Segunda, 12 Maro, 2012

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Márcia Catarina Mendes Coelho
Aluno de Doutoramento

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