Identificación de Brucella melitensis como Ochrobactrum anthropi mediante MALDI-TOF MS / Identification of Brucella melitensis as Ochrobactrum anthropi by MALDI-TOF MS

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Hidden drivers of low-dose pharmaceutical pollutant mixtures revealed by the novel GSA-QHTS screening method.

The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture effect assessment of main pharmaceutical pollutants to date. An ecological scaling-up experiment confirmed that this subset of pollutants also affects typical freshwater microbial community assemblages. Contrary to our expectations and challenging established scientific opinion, the bioactivity of the mixtures was not predicted by the null mixture models, and the main drivers that were identified by GSA-QHTS were overlooked by the current effect assessment scheme. Our results suggest that current chemical effect assessment methods overlook a substantial number of ecologically dangerous chemical pollutants and introduce a new operational framework for their systematic identification.

Electrospun cellulose acetate composites containing supported metal nanoparticles for antifungal membranes.

Electrospun cellulose acetate composites containing silver and copper nanoparticles supported in sepiolite and mesoporous silica were prepared and tested as fungistatic membranes against the fungus Aspergillus niger. The nanoparticles were in the 3-50nm range for sepiolite supported materials and limited by the size of mesopores (5-8nm) in the case of mesoporous silica. Sepiolite and silica were well dispersed within the fibers, with larger aggregates in the micrometer range, and allowed a controlled release of metals to create a fungistatic environment. The effect was assessed using digital image analysis to evaluate fungal growth rate and fluorescence readings using a viability stain. The results showed that silver and copper nanomaterials significantly impaired the growth of fungi when the spores were incubated either in direct contact with particles or included in cellulose acetate composite membranes. The fungistatic effect took place on germinating spores before hyphae growth conidiophore formation. After 24h the cultures were separated from fungistatic materials and showed growth impairment only due to the prior exposure. Growth reduction was important for all the particles and membranes with respect to non-exposed controls. The effect of copper and silver loaded materials was not significantly different from each other with average reductions around 70% for bare particles and 50% for membranes. Copper on sepiolite was particularly efficient with a decrease of metabolic activity of up to 80% with respect to controls. Copper materials induced rapid maturation and conidiation with fungi splitting in sets of subcolonies. Metal-loaded nanomaterials acted as reservoirs for the controlled release of metals. The amount of silver or copper released daily by composite membranes represented roughly 1% of their total load of metals. Supported nanomaterials encapsulated in nanofibers allow formulating active membranes with high antifungal performance at the same time minimizing the risk of nanoparticle release into the environment.

Effect of PFOA/PFOS pre-exposure on the toxicity of the herbicides 2,4-D, Atrazine, Diuron and Paraquat to a model aquatic photosynthetic microorganism.

Pre-exposure to the perfluorinated compounds (PFCs) perfluorooctano sulphonate (PFOS) or perfluorooctanoic acid (PFOA) on the toxicity of four herbicides of different types and modes of action towards the self-luminescent recombinant cyanobacterium Anabaena CPB4337 was evaluated. The rationale of the approach is that both PFOS and PFOA as surfactants are known to modify cell membrane properties and pre-exposure to them might alter herbicide toxicity towards the cyanobacterium. Anabaena CPB4337 was pre-exposed during 72h to PFOS or PFOA at a concentration below their no observed effect concentration (NOEC). After pre-exposure, cells were exposed to increasing concentrations of 2,4-D Atrazine, Diuron and Paraquat and the toxicity was compared to that of non-pre-exposed ones. The data clearly showed that PFCs pre-treatment significantly altered the toxicity of the tested herbicides. However the effects resulting from PFOA and PFOS pre-exposure were not homogeneous for all the herbicides. In general PFOA pre-exposure resulted in increased herbicide toxicity except for atrazine, while PFOS pre-exposure resulted in increased toxicity for paraquat and diuron, and reduced toxicity for atrazine with no significant effect on 2,4-D toxicity. The strongest modifying effect was found for paraquat whose toxicity doubled with PFOA pre-exposure. Further analysis of membrane properties by flow cytometry revealed that both PFOA and PFOS were able to modify membrane integrity and membrane potential of Anabaena CPB4337 at the concentrations used in the pre-exposure experiments. These results reveal relevant indirect effects of PFCs pollution with eco-toxicological implications.

First evidences of PAMAM dendrimer internalization in microorganisms of environmental relevance: A linkage with toxicity and oxidative stress.

This article reports novel results on the toxic mechanisms of action of amine- and hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers toward microorganisms of environmental relevance, namely a cyanobacterium of the genus Anabaena and the green alga Chlamydomonas reinhardtii. We used PAMAM ethylenediamine core dendrimers from generations G2 to G4, which displayed a positive charge, measured as ζ-potential, in culture media. All amine-terminated and most remarkably the G4 hydroxyl-terminated dendrimer inhibited the growth of both microorganisms. The effect on the growth of the green alga was significantly higher than that on the cyanobacterium. With concentrations expressed in terms of molarity, there was a clear relationship between dendrimer generation and toxicity, with higher toxicity for higher generation. Hormesis was observed for hydroxyl-terminated dendrimers at low concentrations. The cationic dendrimers and G4-OH significantly increased the formation of reactive oxygen species (ROS) in both organisms. ROS formation was not related with the chloroplast or photosynthetic membranes and photosystem II photochemistry was unaffected. Cell damage resulted in cytoplasm disorganization and cell deformities and was associated to an increase in ROS formation and lipid peroxidation in mitochondria in the green alga; cell wall and membrane disruption with apparent loss of cytoplasmic contents was found in the cyanobacterium. It was determined for the first time that cationic PAMAM dendrimers were quickly and largely internalized by both organisms. These results warn against the generalization of the use of dendrimers, which may pose significant risk for the environment and particularly for primary producers which are determinant for the health of natural ecosystems.

A colloidal singularity reveals the crucial role of colloidal stability for nanomaterials in-vitro toxicity testing: nZVI-microalgae colloidal system as a case study.

Aggregation raises attention in Nanotoxicology due to its methodological implications. Aggregation is a physical symptom of a more general physicochemical condition of colloidal particles, namely, colloidal stability. Colloidal stability is a global indicator of the tendency of a system to reduce its net surface energy, which may be achieved by homo-aggregation or hetero-aggregation, including location at bio-interfaces. However, the role of colloidal stability as a driver of ENM bioactivity has received little consideration thus far. In the present work, which focuses on the toxicity of nanoscaled Fe° nanoparticles (nZVI) towards a model microalga, we demonstrate that colloidal stability is a fundamental driver of ENM bioactivity, comprehensively accounting for otherwise inexplicable differential biological effects. The present work throws light on basic aspects of Nanotoxicology, and reveals a key factor which may reconcile contradictory results on the influence of aggregation in bioactivity of ENMs.

Toxicity of five antibiotics and their mixtures towards photosynthetic aquatic organisms: implications for environmental risk assessment.

The individual and combined toxicities of amoxicillin, erythromycin, levofloxacin, norfloxacin and tetracycline have been examined in two organisms representative of the aquatic environment, the cyanobacterium Anabaena CPB4337 as a target organism and the green alga Pseudokirchneriella subcapitata as a non-target organism. The cyanobacterium was more sensitive than the green alga to the toxic effect of antibiotics. Erythromycin was highly toxic for both organisms; tetracycline was more toxic to the green algae whereas the quinolones levofloxacin and norfloxacin were more toxic to the cyanobacterium than to the green alga. Amoxicillin also displayed toxicity to the cyanobacterium but showed no toxicity to the green alga. The toxicological interactions of antibiotics in the whole range of effect levels either in binary or multicomponent mixtures were analyzed using the Combination Index (CI) method. In most cases, synergism clearly predominated both for the green alga and the cyanobacterium. The CI method was compared with the classical models of additivity Concentration Addition (CA) and Independent Action (IA) finding that CI could accurately predict deviations from additivity. Risk assessment was performed by calculating the ratio between Measured Environmental Concentration (MEC) and the Predicted No Effect Concentration (PNEC). A MEC/PNEC ratio higher than 1 was found for the binary erythromycin and tetracycline mixture in wastewater effluents, a combination which showed a strong synergism at low effect levels in both organisms. From the tested antibiotic mixtures, it can be concluded that certain specific combinations may pose a potential ecological risk for aquatic ecosystems with the present environmentally measured concentrations.

An insight into the mechanisms of nanoceria toxicity in aquatic photosynthetic organisms.

The effect of nanoceria on two aquatic photosynthetic organisms of ecological relevance, a green alga and a cyanobacterium, is reported. The main bioenergetic process of these organisms, photosynthesis, was studied by measuring both oxygen evolution and chlorophyll a fluorescence emission parameters. Nanoceria significantly inhibited photosynthesis in the cyanobacterium in the entire range of concentrations tested (0.01-100 mg/L), while a dual effect of nanoceria was found in the green alga with slight stimulation at low concentrations and strong inhibition at the highest concentrations tested. Chlorophyll a fluorescence experiments indicated that nanoceria had a significant impact on the primary photochemical processes of photosystem II. The primary cause of the observed photosynthetic inhibition by nanoceria is an excessive level of ROS formation; the results indicated a strong generation of reactive oxygen species (ROS) which caused oxidative damage, as evidenced by lipid peroxidation in both photosynthetic organisms. It is proposed that nanoceria can increase the production of hydrogen peroxide (a normal ROS by-product of light-driven photosynthesis) in both the green alga and the cyanobacterium; through an oxidative reaction, these ROS cause lipid peroxidation, compromising membrane integrity and also seriously impairing photosynthetic performance, eventually leading to cell death.

Chemical and toxicological evolution of the antibiotic sulfamethoxazole under ozone treatment in water solution.

This work studied the elimination paths of the sulfonamide antibiotic sulfamethoxazole by ozonation in fast kinetic regime. The ozonation runs were performed in conditions favouring either the direct attack of the ozone molecule or the indirect attack by ozone-generated radical species with initial concentration of 0.150 mM. When doses of ozone were transferred to the liquid phase 0.2mM, in no case did sulfamethoxazole remain in solution. Two main transformation pathways were found involving the preferential attack of molecular ozone or radical pathway and leading to the formation of six intermediates, which were identified by LC-ESI-QTOF-MS. Both routes took place simultaneously in the different conditions tested, leading to a hydroxylation reaction of the benzene ring, oxidation of the amino group on the benzene ring, oxidation of the methyl group and the double bond in the isoxazole ring and S-N bond cleavage. The most abundant reaction intermediate was that resulting from S-N bond cleavage. The toxicity of partially ozonated samples for Daphnia magna and Pseudokirchneriella subcapitata revealed the formation of toxic by-products during the early stages of reaction and the persistence of considerable toxicity after the total depletion of sulfamethoxazole.

Ecotoxicity assessment of lipid regulators in water and biologically treated wastewater using three aquatic organisms.

BACKGROUND, AIM, AND SCOPE: The high consumption of blood lipid regulators is leading to frequent reports of the occurrence of fibrates in natural streams and wastewater effluents. This paper describes a study undertaken to evaluate the acute toxicity of bezafibrate, clofibric acid, gemfibrozil, and fenofibric acid, a metabolite of fenofibrate whose ecotoxicity has not been previously reported. MATERIALS AND METHODS: The bioassays used were based on Vibrio fischeri, Daphnia magna, and Anabaena CPB4337 tests. Anabaena CPB4337 is a novel bioassay based on Anabaena sp. PCC 7120 strain CPB4337 bearing in the chromosome a Tn5 derivative with luxCDABE from the luminescent terrestrial bacterium Photorhabdus luminescens. RESULTS: The higher toxicity corresponded to fenofibric acid, with EC(50) as low as 1.72 mg/l for V. fischeri. Gemfibrozil was also toxic for Anabaena sp. with EC(50) of 4.42 mg/l. The study reports the results from toxicity tests using fortified real wastewater samples taken from the effluent of a wastewater treatment plant. The wastewater itself was found to be very toxic to Anabaena CPB4337 (84% of bioluminescence inhibition) whereas it did not have any negative effect on D. magna or V. fischeri. On the contrary, V. fischeri luminescence exhibited a stimulatory effect in wastewater. DISCUSSION: Except for fenofibric acid, the Anabaena bioassay was more sensitive than the D. magna and V. fischeri bioassays to bezafibrate, clofibric acid, and gemfibrozil. For the three toxicity tests, fortification resulted in lower measured toxicity for the four compounds, probably indicating a reduced bioavailability due to the interaction with other chemicals in the wastewater or with particulate matter. The observed decrease in toxicity associated to the use of a wastewater matrix was higher for the more hydrophobic compounds reaching one order of magnitude for bezafibrate and gemfibrozil. CONCLUSIONS: The Anabaena CPB4337 bioassay revealed a certain risk associated with the three less toxic compounds tested. Based on V. fischeri and D. magna bioassays, bezafibrate and gemfibrozil would have been considered non-toxic and harmful, respectively. The use of EC(50) data measured in wastewater increases the risk estimation. RECOMMENDATIONS AND PERSPECTIVES: Cyanobacteria, as primary producers with a key role in the carbon and nitrogen cycles, are a substantial component of the microbial food webs. Any detrimental effect on this group may have a negative impact in nutrient availability to organisms of higher trophic levels and should be considered in ecotoxicity assessment tests.