Integration of Indicator Alarm Signals for Ecosystem‐Based Fishery Management

We consider the problem of regulating the rate of harvesting a natural resource, taking account of the wider system represented by a set of ecological and economic indicators, given differing stakeholder priorities. This requires objective and transparent decision making to show how indicators impinge on the resulting regulation decision. We offer a new scheme for combining indicators, derived from assessing the suitability of lowering versus not lowering the harvest rate based on indicator values relative to their predefined reference levels. Using the practical example of fisheries management under an “ecosystem approach,” we demonstrate how different stakeholder views can be quantitatively represented by weighting sets applied to these comparisons. Using the scheme in an analysis of historical data from the Celtic Sea fisheries, we find great scope for negotiating agreement among disparate stakeholders.

Highly CO2 sensitive extruded fluorescent plastic indicator film based on HPTS

Highly-sensitive optical fluorescent extruded plastic films are reported for the detection of gaseous and dissolved CO2. The pH-sensitive fluorescent dye used is 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS, PTS-) coated on the surface of hydrophilic fumed silica and the base is tetrabutylammonium hydroxide (TBAH). The above components are used to create an HPTS pigment (i.e. HPTS/SiO2/TBAH) with a high CO2 sensitivity (%CO2(S=1/2) = 0.16%) and fast 50% response (t50↓) = 2 s and recovery (t50↑) = 5 s times. Highly CO2-sensitive plastic films are then fabricated, via the extrusion of the HPTS pigment powder in low-density polyethylene (LDPE). As with the HPTS-pigment, the luminescence intensity (at 515 nm) and absorbance (at 475 nm) of the HPTS plastic film decreases as the %CO2 in the ambient gas phase increases. The HPTS plastic film exhibits a high CO2 sensitivity, %CO2(S=1/2), of 0.29%, but a response time ˂2 min and recovery time ˂40 min, which is slower than that of the HPTS pigment. The HPTS plastic film is very stable under ambient conditions, (with a shelf life ˃ six month when stored in the dark but under otherwise ambient conditions). Moreover, the HPTS-film is stable in water, salt solution and even in acid (pH=2), and in each of these media it can be used to detect dissolved CO2.

An evaluation of the effectiveness of an algorithm intervention in reducing inappropriate faecal samples sent for Clostridium difficile testing.

Background: Clostridium difficile (C. difficile) is a leading cause of infectious diarrhoea in hospitals. Sending faecal samples for testing expedites diagnosis and appropriate treatment. Clinical suspicion of C. difficile based on patient history, signs and symptoms is the basis for sampling. Sending faecal samples from patients with diarrhoea ‘just in case’ the patient has C. difficile may be an indication of poor clinical management.

Aim: To evaluate the effectiveness of an intervention by an Infection Prevention and Control Team (IPCT) in reducing inappropriate faecal samples sent for C. difficile testing.

Method: An audit of numbers of faecal samples sent before and after a decision-making algorithm was introduced. The number of samples received in the laboratory was retrospectively counted for 12-week periods before and after an algorithm was introduced.
Findings: There was a statistically significant reduction in the mean number of faecal samples sent post the algorithm. Results were compared to a similar intervention carried out in 2009 in which the same message was delivered by a memorandum. In 2009 the memorandum had no effect on the overall number of weekly samples being sent.

Conclusion: An algorithm intervention had an effect on the number of faecal samples being sent for C. difficile testing and thus contributed to the effective use of the laboratory service.

Laterality as an indicator of emotional stress in ewes and lambs during a separation test

We assessed motor laterality in sheep to explore species-specific brain hemi-field dominance and how this could be affected by genetic or developmental factors. Further, we investigated whether directionality and strength of laterality could be linked to emotional stress in ewes and their lambs during partial separation. Forty-three ewes and their singleton lambs were scored on the (left/right) direction of turn in a y-maze to rejoin a conspecific (laterality test). Further, their behavioural response (i.e. time spent near the fence, vocalisations, and activity level) during forced separation by an open-mesh fence was assessed (separation test). Individual laterality was recorded for 44.2 % ewes (significant right bias) and 81.4 % lambs (equally biased to the left and the right). There was no significant association in side bias between dams and offspring. The Chi-squared test revealed a significant population bias for both groups (p < 0.05). Evolutionary adaptive strategies or stimuli-related visual laterality may provide explanation for this decision-making process. Absolute strength of laterality (irrespective of side) was high (Kolmogorov–Smirnov test, dams: D = 0.2; p < 0.001; lambs: D = 0.36, p < 0.0001). The Wilcoxon test showed that lateralised lambs and dams spent significantly more time near each other during separation than non-lateralised animals (p < 0.05), and that lateralised dams were also more active than non-lateralised ones. Arguably, the lateralised animals showed a greater attraction to their pair because they were more disturbed and thus required greater reassurance. The data show that measures of laterality offer a potential novel non-invasive indicator of separation stress.

Neuropeptides display antimicrobial activity against cariogenic and endodontic bacteria

Introduction: Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. It is therefore possible that the nervous system employs neuropeptides as antimicrobial agents by delivering them rapidly and precisely to innervated sites such as the dental pulp. Objectives: The aim of this study was to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), which we have previously shown to be present in dental pulp, displayed antimicrobial activity against the cariogenic bacterium Streptococcus mutans and the endodontic bacterium Enterococcus faecalis. Methods: Neuropeptides were purchased from Bachem and utilised in antibacterial assays using a previously described ultra sensitive radial diffusion method. Results: Antimicrobial activity was identified as clear zones around neuropeptide-containing wells. NPY was found to exhibit antimicrobial against both Streptococcus mutans and Enterococcus faecalis. SP and VIP were shown to exhibit antimicrobial activity against Streptococcus mutans only. The neuropeptides NKA and CGRP did not show antimicrobial activity against either micro-organism. Conclusion: This study is the first to describe an antimicrobial role for neuropeptides in pulp biology. The antimicrobial actions of neuropeptides contribute a novel aspect to pulpal defence against cariogenic and endodontic bacteria worthy of further investigation.

Lipopolysaccharide modification in Gram-negative bacteria during chronic infection

The Gram-negative bacterial lipopolysaccharide (LPS) is a major component of the outer membrane that plays a key role in host-pathogen interactions with the innate immune system. During infection, bacteria are exposed to a host environment that is typically dominated by inflammatory cells and soluble factors, including antibiotics, which provide cues about regulation of gene expression. Bacterial adaptive changes including modulation of LPS synthesis and structure are a conserved theme in infections, irrespective of the type or bacteria or the site of infection. In general, these changes result in immune system evasion, persisting inflammation, and increased antimicrobial resistance. Here, we review the modifications of LPS structure and biosynthetic pathways that occur upon adaptation of model opportunistic pathogens (Pseudomonas aeruginosa, Burkholderia cepacia complex bacteria, Helicobacter pylori and Salmonella enterica) to chronic infection in respiratory and gastrointestinal sites. We also discuss the molecular mechanisms of these variations and their role in the host-pathogen interaction.

Genotoxicity in humans and indicator species exposed to uranium wastes

Os riscos dos resíduos provenientes da extração do urânio estão associados ao seu conteúdo em metais e radionuclídeos, o que levanta preocupações às autoridades governamentais e à população em geral. As populações humanas e outras espécies animais que vivem em zonas de exploração de urânio poderão estar expostas à radiação através de resíduos e poeiras radioactivase também através de água e alimentos contaminados. A determinação dos riscos deste tipo de contaminantes é feita, principalmente, através da análise química de amostras ambientais, dando-se menos importância à determinação de efeitos biológicos. A determinação de efeitos biológicos causados pela exposição a poluentes, tem-se revelado muito importante para uma avaliação da qualidade ambiental, de modo a providenciar indicações acerca dos efeitos negativos nos seres vivos e também para complementar a informação dada pelas análises químicas de amostras ambientais. Este facto levou ao estabelecimento de biomarcadores, que consistem em respostas biológicas adversas que são específicas de uma exposição a toxinas ambientais, para serem usadas como ferramentas de avaliação da qualidade ambiental. Neste trabalho foram analisadas respostas a nível molecular e celular, em minhocas, ratinhos do campo e humanos, a fim de determinar o risco químico e radiológico dos resíduos provenientes da mina de urânio da Cunha Baixa. Este trabalho teve também como objectivo a clarificação das respostas subjacentes à exposição a metais e radionuclídeos, a fim de permitir o desenvolvimento de potenciais novos biomarcadores moleculares. Durante este trabalho foram efectuados ensaios com minhocas, em que estas foram expostas durante 56 dias a solo contaminado proveniente da mina de urânio da Cunha Baixa, em laboratório e in situ. Durante a exposição foram analisados vários parâmetros, como o crescimento, reprodução, bioacumulação de metais e radionuclídeos, histopatologia, danos no DNA, citotoxicidade e perfil de expressão genética. Para além disso, foram amostrados ratinhos do campo na mina de urânio da Cunha Baixa e numa área de referência para determinação de danos no DNA, níveis de expressão e mutações em genes supressores de tumores e também bioacumulação de metais. Por fim, foram recolhidas amostras de sangue em voluntários saudáveis pertencentes à população da aldeia da Cunha Baixa, para determinação de danos no DNA, imunofenotipagem e quantificação de metais no sangue. Os resultados revelaram que as minhocas assim como os ratinhos do campo foram negativamente afetados pela exposição aos resíduos mineiros em todos os níveis de organização biológica aqui analisados, o que faz destes organismos bons indicadores para a determinação do risco destas áreas contaminadas, evidenciando o potencial risco da exposição a estes contaminantes. Para além disso, o estudo feito à população da Cunha Baixa revelou danos no ADN e diminuição de populações importantes de células imunitárias (nomeadamente linfócitos T e NK), o que poderá resultar da exposição aos resíduos da mina, tornando as pessoas mais susceptíveis ao desenvolvimento de processos de carcinogénese. O presente estudo contribuiu significativamente para a caracterização dos riscos da exposição a resíduos provenientes de minas de urânio abandonadas, evidenciando os seus efeitos negativos a nível molecular e celular, que potencialmente poderão causar instabilidade genómica e aumentar o risco de desenvolvimento de doenças genéticas.

Hydrological and photochemical influence on dynamics of estuarine bacteria

Os estuários são ecossistemas complexos, onde os processos físicos, químicos e biológicos estão intimamente ligados. A dinâmica bacteriana num estuário reflete a interação e a elevada variação temporal e espacial desses processos. Este trabalho teve como objetivo elucidar as interações entre os processos físicos, fotoquímicos e microbiológicos no sistema estuarino da Ria de Aveiro (Portugal). Para tal, foi realizada uma abordagem inicial no campo, durante a qual as comunidades bacterianas na coluna de água foram caracterizadas em termos de abundância e atividade ao longo de 2 anos. O estudo foi realizado em dois locais distintos, escolhidos por tipificarem as características marinhas e salobras do estuário. Estes locais possuem diferentes hidrodinâmicas, influências fluviais e, quantidade e composição de matéria orgânica. Numa perspectiva mecanicista, foram realizadas simulações laboratoriais no sentido de elucidar a resposta das bactérias à matéria orgânica foto-transformada. As comunidades bacterianas no estuário adaptam-se a diferentes regimes de água doce, desenvolvendo padrões de abundância e atividade distintos nas zonas marinha e salobra. Os elevados caudais dos rios induzem estratificação vertical na zona marinha, promovendo o fluxo de fitoplâncton do mar para o estuário, do bacterioplâncton do estuário para o mar, e estimulam a importação de bactérias aderentes a partículas na zona salobra. O transporte advectivo e os processos de ressuspensão contribuem para aumentar 3 vezes o número de bactérias aderentes a partículas durante os períodos de intensas descargas fluviais. Adicionalmente, a atividade bacteriana no estuário é controlada pela concentração de azoto inerente à variações de água doce. O fornecimento de azoto em associação com a fonte dos substratos bacterianos induzem alterações significativas na produtividade. O padrão de variação vertical de comunidades bacterianas foi distinto nas duas zonas do estuário. Na zona marinha, as bactérias na microcamada superficial (SML) apresentaram taxas de hidrólise mais elevadas, mas menores taxas de incorporação de monómeros e produção de biomassa que na água subjacente (UW), enquanto na zona salobra, as taxas de hidrólise e incorporação foram similares nos dois compartimentos, mas a produtividade foi significativamente mais elevada na SML. Apesar da abundância bacteriana ter sido semelhante na SML e UW, a fração de células aderentes a partículas foi significativamente maior na SML (2-3 vezes), em ambas as zonas do estuário. A integração dos resultados microbiológicos com as variáveis ambientais e hidrológicos mostraram que fortes correntes na zona marinha promovem a mistura vertical, inibindo o estabelecimento de uma comunidade bacteriana na SML distinta da UW. Em contraste, na zona de água salobra, a menor velocidades das correntes fornece as condições adequadas ao aumento da atividade bacteriana na SML. Características específicas do local, tais como a hidrodinâmica e as fontes e composição da matéria orgânica, conduzem também a diferentes graus de enriquecimento superficial de matéria orgânica e inorgânica, influenciando a sua transformação. Em geral, o ambiente da SML estuarina favorece a hidrólise de polímeros, mas inibe a utilização de monómeros, comparativamente com água subjacente. No entanto, as diferenças entre as duas comunidades tendem a atenuar-se com o aumento da atividade heterotrófica na zona salobra. A matéria orgânica dissolvida cromófora (CDOM) das duas zonas do estuário possui diferentes características espectrais, com maior aromaticidade e peso molecular médio (HMW) na zona de água salobra, em comparação com a zona marinha. Nesta zona, a abundância bacteriana correlacionou-se com a350 e a254, sugerindo uma contribuição indireta das bactéria para HMW CDOM. A irradiação do DOM resultou numa diminuição dos valores de a254 e a350, e, em um aumento do declive S275-295 e dos rácios E2:E3 (a250/a365) e SR. No entanto, a extensão de transformações foto-induzidas e as respostas microbianas são dependentes das características iniciais CDOM, inferidas a partir das suas propriedades ópticas. A dinâmica estuarina influencia claramente as atividades heterotróficas e a distribuição dos microorganismos na coluna de água. A entrada de água doce influencia a dinâmica e os principais reguladores das comunidades bacterianas no estuário. Os processos fotoquímicos e microbianos produzem alterações nas propriedades ópticas da CDOM e a combinação desses processos determina o resultado global e o destino da CDOM nos sistemas estuarinos com influência na produtividade nas áreas costeiras adjacente.

Oil hydrocarbon-bacteria interactions in coastal environments

The ability of microorganisms to use oil hydrocarbons as a source of carbon and energy is crucial for environmental oil detoxification. However, there is still a lack of knowledge on fundamental aspects of this process on specific habitats and under different climate scenarios. In the first phase of this work, the culturable fraction of the oil hydrocarbon (OH) degrading bacteria from the sea surface microlayer (SML) of the estuarine system Ria de Aveiro was characterized. In the second phase, the impact of oil contamination on the active bacterial community was studied under climate change scenarios. Pseudomonas emerged as the prevailing genera among OH degrading bacteria in the SML. Moreover, culture-independent methods revealed that the relative abundance and diversity of Gammaproteobacteria, in which Pseudomonas is included, varies along an estuarine gradient of contamination. In order to access the impact of oil contamination on microbial communities under climate change scenarios, an experimental life support system for microcosm experiments (ELLS) was developed and validated for simulation of climate change effects on microbial communities. With the ELSS it is possible to simulate, in controlled conditions, fundamental parameters of the dynamics of coastal and estuarine systems while maintaining community structure in terms of the abundance of the most relevant members of the indigenous bacterial community. A microcosm experiment in which the independent and combined impact of ultraviolet radiation, ocean acidification and oil contamination on microbial communities was conducted. The impact on bacterial communities was accessed with a 16S RNA (cDNA) based barcode pyrosequencing approach. There was a drastic decrease of Desulfobacterales relative abundance after oil contamination under the reduced pH value estimated for 2100, when compared to present values. Since members of this order are known OH degraders, such a significant decrease may have consequences on OH detoxification of contaminated environments under the pH levels of the ocean expected for the future. Metagenome predictions based on the 16S RNA database indicated that several degradation pathways of OH could be affected under oil contamination and reduced water pH. Taken together, the results from this work bring new information on the dynamics of OH degrading bacteria in coastal and estuarine environments under present and future climate scenarios.

Photodynamic inactivation of bacteria by cationic porphyrins : their cellular targets and potential environmental applications

Photodynamic inactivation (PDI) is defined as the process of cell destruction by oxidative stress resulting from the interaction between light and a photosensitizer (PS), in the presence of molecular oxygen. PDI of bacteria has been extensively studied in recent years, proving to be a promising alternative to conventional antimicrobial agents for the treatment of superficial and localized infections. Moreover, the applicability of PDI goes far beyond the clinical field, as its potential use in water disinfection, using PS immobilized on solid supports, is currently under study. The aim of the first part of this work was to study the oxidative modifications in phospholipids, nucleic acids and proteins of Escherichia coli and Staphylococcus warneri, subjected to photodynamic treatment with cationic porphyrins. The aims of the second part of the work were to study the efficiency of PDI in aquaculture water and the influence of different physicalchemical parameters in this process, using the Gram-negative bioluminescent bacterium Vibrio fischeri, and to evaluate the possibility of recycling cationic PS immobilized on magnetic nanoparticles. To study the oxidative changes in membrane phospholipids, a lipidomic approach has been used, combining chromatographic techniques and mass spectrometry. The FOX2 assay was used to determine the concentration of lipid hydroperoxides generated after treatment. The oxidative modifications in the proteins were analyzed by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Changes in the intracellular nucleic acids were analyzed by agarose gel electrophoresis and the concentration of doublestranded DNA was determined by fluorimetry. The oxidative changes of bacterial PDI at the molecular level were analyzed by infrared spectroscopy. In laboratory tests, bacteria (108 CFU mL-1) were irradiated with white light (4.0 mW cm-2) after incubation with the PS (Tri-Py+-Me-PF or Tetra-Py+-Me) at concentrations of 0.5 and 5.0 μM for S. warneri and E. coli, respectively. Bacteria were irradiated with different light doses (up to 9.6 J cm-2 for S. warneri and up to 64.8 J cm-2 for E. coli) and the changes were evaluated throughout the irradiation time. In the study of phospholipids, only the porphyrin Tri-Py+-Me-PF and a light dose of 64.8 J cm-2 were tested. The efficiency of PDI in aquaculture has been evaluated in two different conditions: in buffer solution, varying temperature, pH, salinity and oxygen concentration, and in aquaculture water samples, to reproduce the conditions of PDI in situ. The kinetics of the process was determined in realtime during the experiments by measuring the bioluminescence of V. fischeri (107 CFU mL-1, corresponding to a level of bioluminescence of 105 relative light units). A concentration of 5.0 μM of Tri-Py+-Me-PF was used in the experiments with buffer solution, and 10 to 50 μM in the experiments with aquaculture water. Artificial white light (4.0 mW cm-2) and solar irradiation (40 mW cm-2) were used as light sources.

Bio-removal of toxic metals by metal resistant anaerobic bacteria: molecular characterization and performance studies

Tese de dout., Ciências Biotecnológicas (Biotecnologia Ambiental), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.