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

A Method For Producing Airborne Pollen Source Inventories: An Example of Ambrosia (Ragweed) on the Pannonian Plain

We present here a simple methodology for calculating species inventories for allergenic pollen that can be used by atmospheric transport models. Ragweed (Ambrosia) species distribution or infection level on the Pannonian Plain has been used as an example of how the methodology can be used. The Pannonian Plain is one of the three main regions in Europe recognized as being polluted by Ambrosia. The methodology relies on spatial variations in annual Ambrosia pollen counts, knowledge on ragweed ecology and detailed land cover information. The results of this analysis showed that some of the highest mean annual ragweed pollen concentrations were witnessed around Kecskemét in central Hungary and Novi Sad in northern Serbia. These areas are also the areas with the highest density of Ambrosia habitats. The resulting inventory can be entered into atmospheric transport models in combination with other components such as a phenological model and a model for daily pollen release, in order to simulate the movement of ragweed pollen from the Pannonian Plain. The methodology is likely to be generally applicable for creating inventories of species distribution of allergenic plants. The main requirement is availability of: detailed land cover information; pollen indexes; a list of the most important habitats; and a region of interest that is mainly influenced by local sources.

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.

Airborne Olive Pollen Counts are not Representative of 1 Exposure to the Major Olive Allergen Ole e 1

Pollen is routinely monitored, but it is unknown whether pollen counts represent allergen exposure. We therefore simultaneously determined olive pollen and Ole e 1 in ambient air in C"ordoba, Spain, and "Evora, Portugal, using Hirst-type traps for pollen and high-volume cascade impactors for allergen. Pollen from different days released 12-fold different amounts of Ole e 1 per pollen (both locations P < 0.001). Average allergen release from pollen (pollen potency) was much higher in C"ordoba (3.9 pg Ole e 1/pollen) than in "Evora (0.8 pg Ole e 1/pollen, P = 0.004). Indeed, yearly olive pollen counts in C"ordoba were 2.4 times higher than in "Evora, but Ole e 1 concentrations were 7.6 times higher. When modeling the origin of the pollen, >40% of Ole e 1 exposure in "Evora was explained by high-potency pollen originating from the south of Spain. Thus, olive pollen can vary substantially in allergen release, even though they are morphologically identical.

Back-trajectories Show Export of Airborne Fungal Spores (Ganoderma sp.) From Forests to Agricultural and Urban Areas in England

We propose here the hypothesis that all of United Kingdom (UK) is likely to be affected by Ganoderma sp. spores, an important plant pathogen. We suggest that the main sources of this pathogen, which acts as a bioaerosol, are the widely scattered woodlands in the country, although remote sources must not be neglected. The hypothesis is based on related studies on bioaerosols and supported by new observations from a non-forest site and model calculations to support our hypothesis. Hourly concentrations of Ganoderma sp. spores were measured from 2006 to 2010 using a 7-day volumetric spore trap at the city of Worcester. The concentrations peak during the night and early in the morning. This suggests that the main spore sources are located a few hours away with respect to air masses transport and reach urban areas thanks to air masses transport. The back-trajectory analysis was applied to determine the location of Ganoderma sp. spore sources. The analysis of back-trajectories demonstrated that 78% of the air masses reached Worcester from a 180° arc direction from the East to West. Three episodes were selected for detailed investigation and they revealed that during the episodes air masses always passed main UK woodlands before the arrival in Worcester, independently of their origin, but the long distance transport under certain conditions might be possible. Our studies suggest that the sources of UK Ganoderma sp. spores are mainly to be found in UK. Hence our studies suggest that research and mitigation strategies in UK should give their main attention to national sources, without neglecting the contribution from long distance transport.

Identification of Potential Sources of Airborne Olea Pollen in the Southwest Iberian Peninsula

This study aims to determine the potential origin of Olea pollen recorded in Badajoz in the Southwest of the Iberian Peninsula during 2009–2011. This was achieved using a combination of daily average and diurnal (hourly) airborne Olea pollen counts recorded at Badajoz (south-western Spain) and Évora (south-eastern Portugal), an inventory of olive groves in the studied area and air mass trajectory calculations computed using the HYSPLIT model. Examining olive pollen episodes at Badajoz that had distinctly different diurnal cycles in olive pollen in relation to the mean, allowed us to identify three different scenarios where olive pollen can be transported to the city from either distant or nearby sources during conditions with slow air mass movements. Back trajectory analysis showed that olive pollen can be transported to Badajoz from the West on prevailing winds, either directly or on slow moving air masses, and from high densities of olive groves situated to the Southeast (e.g. Andalucía). Regional scale transport of olive pollen can result in increased nighttime concentrations of this important aeroallergen. This could be particularly important in Mediterranean countries where people can be outdoors during this time due to climate and lifestyle. Such studies that examine sources and the atmospheric transport of pollen are valuable for allergy sufferers and health care professionals because the information can be incorporated into forecasts, the outputs of which are used for avoiding exposure to aeroallergens and planning medication. The results of studies of this nature can also be used for examining gene flow in this important agricultural crop.

Variation of the Group 5 Grass Pollen Allergen Content of Airborne Pollen in Relation to Geographical Location and Time in Season

Allergies to grass pollen are the number one cause of outdoor hay fever. The human immune system reacts with symptoms to allergens from pollen. Objective: We investigated the natural variability in release of the major group 5 allergen from grass pollen across Europe. Methods: Airborne pollen and allergens were simultaneously collected daily with a volumetric spore trap and a high-volume cascade impactor at 10 sites across Europe for 3 consecutive years. Group 5 allergen was determined with a Phl p 5 specific ELISA in two fractions of ambient air: Particulate Matter (PM) >10μm and 10μm>PM>2.5μm. Mediator release by ambient air was determined in FcεR1-humanized basophils. Origin of pollen was modeled and condensed to pollen potency maps. Results: On average grass pollen released 2.3 pg Phl p 5/pollen. Allergen release per pollen (potency) varied substantially, ranging from 0 to 9 pg Phl p 5/pollen (5 to 95% percentile). The main variation was locally day-to-day. Average potency maps across Europe varied between years. Mediator release from basophilic granulocytes correlated better with allergen/m3 (r2=0.80, p<0.001) than with pollen/m3 (r2=0.61, p<0.001). In addition, pollen released different amounts of allergen in the nonpollen bearing fraction of ambient air depending on humidity. Conclusion: Across Europe, the same amount of pollen released substantially different amounts of group 5 grass pollen allergen. This variation in allergen release is on top of variations in pollen counts. Molecular aerobiology, i.e. determining allergen in ambient air, may be a valuable addition to pollen counting.

Potential Sources of Airborne Alternaria spp. spores in South-west Spain

Fungi belonging to the genus of Alternaria are recognised as being significant plant pathogens, and Alternaria allergens are one of themost important causes of respiratory allergic diseases in Europe. This study aims to provide a detailed and original analysis of Alternaria transport dynamics in Badajoz, SW Spain. This was achieved by examining daily mean and hourly observations of airborne Alternaria spores recorded during days with high airborne concentrations of Alternaria spores (N100 s m−3) from 2009 to 2011, as well as four inventory maps of major Alternaria habitats, the overall synoptic weather situation and analysis of air mass transport using Hybrid Single Particle Lagrangian Integrated Trajectory model and geographic information systems. Land use calculated within a radius of 100 km from Badajoz shows that crops and grasslands are potentially the most important local sources of airborne Alternaria spores recorded at the site. The results of back trajectory analysis showthat, during the examined four episodes, the two main directions where Alternaria source areas were located were: (1) SW–W; and (2) NW–NE. Regional scale and long distance transport could therefore supplement the airborne catch recorded at Badajoz with Alternaria conidia originating from sources such as crops and orchards situated in other parts of the Iberian Peninsula.

Risk of Exposure to Airborne Ambrosia Pollen from Local and Distant Sources in Europe – an Example from Denmark

Background. Ambrosia artemisiifolia L. is a noxious invasive alien species in Europe. It is an important aeroallergen and millions of people are exposed to its pollen. Objective. The main aim of this study is to show that atmospheric concentrations of Ambrosia pollen recorded in Denmark can be derived from local or more distant sources. Methods. This was achieved by using a combination of pollen measurements, air mass trajectory calculations using the HYPLIT model and mapping all known Ambrosia locations in Denmark and relating them to land cover types. Results. The annual pollen index recorded in Copenhagen during a 15-year period varied from a few pollen grains to more than 100. Since 2005, small quantities of Ambrosia pollen has been observed in the air every year. We have demonstrated, through a combination of Lagrangian back-trajectory calculations and atmospheric pollen measurements, that pollen arrived in Denmark via long-distance transport from centres of Ambrosia infection, such as the Pannonian Plain and Ukraine. Combining observations with results from a local scale dispersion model show that it is possible that Ambrosia pollen could be derived from local sources identified within Denmark. Conclusions. The high allergenic capacity of Ambrosia pollen means that only small amounts of pollen are relevant for allergy sufferers, and just a few plants will be sufficient to produce enough pollen to affect pollen allergy sufferers within a short distance from the source. It is necessary to adopt control measures to restrict Ambrosia numbers. Recommendations for the removal of all Ambrosia plants can effectively reduce the amount of local pollen, as long as the population of Ambrosia plants is small.

The Long Distance Transport of Airborne Ambrosia Pollen to the UK and the Netherlands from Central and South Europe

Background: The invasive alien species Ambrosia artemisiifolia (common or short ragweed) is increasing its range in Europe. In the UK and the Netherlands airborne concentrations of Ambrosia pollen are usually low. However, more than 30 Ambrosia pollen grains per cubic metre of air (above the level capable to trigger allergic symptoms) were recorded in Leicester (UK) and Leiden (NL) on 4 and 5 September 2014. Objective: The aims of this study were to determine whether the highly allergenic Ambrosia pollen recorded during the episode could be the result of long distance transport, to identify the potential sources of these pollen grains and describe the conditions that facilitated this possible long distance transport. Methods: Airborne Ambrosia pollen data were collected at 10 sites in Europe. Back trajectory and atmospheric dispersion calculations were performed using HYSPLIT_4. Results: Back trajectories calculated at Leicester and Leiden show that higher altitude air masses (1500m) originated from source areas on the Pannonian Plain and Ukraine. During the episode, air masses veered to the west and passed over the Rhône Valley. Dispersion calculations showed that the atmospheric conditions were suitable for Ambrosia pollen released from the Pannonian Plain and the Rhône Valley to reach the higher levels and enter the air stream moving to Northwest Europe where they were deposited at ground level and recorded by monitoring sites. Conclusions: The study indicates that the Ambrosia pollen grains recorded during the episode in Leicester and Leiden were probably not produced by local sources, but transported long distances from potential source regions in East Europe, i.e. the Pannonian Plain and Ukraine, as well as the Rhône Valley in France.

In vitro transference and molecular characterization of bla TEM genes in bacteria isolated from Portuguese ready-to-eat foods

The principal aim of this study was to investigate the possibility of transference to Escherichia coli of β-lactam resistance genes found in bacteria isolated from ready-to-eat (RTE) Portuguese traditional food. From previous screenings, 128 β-lactam resistant isolates (from different types of cheese and of delicatessen meats), largely from the Enterobacteriaceae family were selected and 31.3% of them proved to transfer resistance determinants in transconjugation assays. Multiplex PCR in donor and transconjugant isolates did not detect bla CTX, bla SHV and bla OXY, but bla TEM was present in 85% of them, while two new TEMs (TEM-179 and TEM-180) were identified in two isolates. The sequencing of these amplicons showed identity between donor and transconjugant genes indicating in vitro plasmid DNA transfer. These results suggest that if there is an exchange of genes in natural conditions, the consumption of RTE foods, particularly with high levels of Enterobacteriaceae, can contribute to the spread of antibiotic resistance.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas

Biophysical Chemistry 110 (2004) 83–92