Sónia Mendo

Molecular Characterization of a New Class 3 Integron in Klebsiella pneumoniae

ABSTRACT Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum β-lactamase was encoded by blaGES-1, previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the blaGES-1 gene cassette was located on a new class 3 integron. The integron was 2,863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The blaGES-1 gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between blaOXA-10-type and aac(6′)-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the blaGES-1 gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50=145.7 mg/L) than adults (96 h-LC50=39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.

Congeneric Lantibiotics from Ribosomal In Vivo Peptide Synthesis with Noncanonical Amino Acids

Expanded repetoire: Synthetic amino acids translated into propeptides dramatically increase the chemical diversity of the two-component lantibiotic lichenicidin. This opens new routes towards novel and unique peptide antibiotic sequences, which could display features important for medical applications.

Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides

Eisenia andrei was exposed, for 56 days, to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure, to assess metals bioaccumulation, coelomocytes DNA integrity and cytotoxicity. Radionuclides bioaccumulation and growth were also determined at 0 h, 14 and 56 days of exposure. Results have shown the bioaccumulation of metals and radionuclides, as well as, growth reduction, DNA damages and cytotoxicity in earthworms exposed to contaminated soil. The usefulness of the comet assay and flow cytometry, to evaluate the toxicity of contaminants such as metals and radionuclides in earthworms are herein reported. We also demonstrated that DNA strand breakage and immune cells frequency are important endpoints to be employed in the earthworm reproduction assay, for the evaluation of soil geno and cytotoxicity, as part of the risk assessment of contaminated areas. This is the first study that integrates DNA damage and cytotoxicity evaluation, growth and bioaccumulation of metals and radionuclides in a sub lethal assay, for earthworms exposed to soil contaminated with metals and radionuclides.

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain

A Bacillus licheniformis strain, I89, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 °C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 °C and 100 °C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus.

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this ‘nonmodel’ species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator–activated receptor (PPAR) pathways. Application of rosiglitazone, a well‐known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT‐induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR‐responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.

The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems.

Evaluation of the sensitivity of genotoxicity and cytotoxicity endpoints in earthworms exposed in situ to uranium mining wastes

Earthworms were exposed for 56 days to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The exposure occurred in situ: the containers with contaminated soil were placed near the mine pit; the containers with reference soil were placed in a reference site. For the assessment of metals bioaccumulation, DNA damages, cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation, organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure. For the assessment of radionuclides bioaccumulation, animals were sampled after 0, 14 and 56 days of exposure. As for growth, organisms were sampled after 0, 14, 28 and 56 days of exposure. The reproduction assay was performed according to the OECD (2004) guideline. DNA damages were assessed by comet assay and flow cytometry was used to determine cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation. Results have shown a myriad of effects in the organisms exposed to the contaminated soil, namely: the inhibition of reproduction, growth reduction, DNA damages, cytotoxicity, changes in eleocytes fluorescence intensity, coelomocytes proliferation and bioaccumulation of metals and radionuclides. Our results showed that the evaluation of genotoxicity and cytotoxicity endpoints, along with other parameters at an individual level in standard reproduction assays conducted in situ, are important to improve the risk assessment process of areas contaminated with uranium and other radioactive mining wastes.

Histopathological changes in the earthworm Eisenia andrei associated with the exposure to metals and radionuclides

Earthworms were exposed for 56 d to a contaminated soil, from an abandoned uranium mine, and to the natural reference soil LUFA 2.2. Histological changes in earthworms body wall (epidermis, circular and longitudinal muscles) and gastrointestinal tract (chloragogenous tissue and intestinal epithelium) were assessed, after 0, 14 and 56 d of exposure. Results have shown alterations in all the studied tissues after 14 d of exposure (except for the intestinal epithelium), yet more severe effects were registered after 56 d of exposure. Herein we report histopathological alterations as a good biomarker for the evaluation of soil quality. We also demonstrate that morphological changes in the body wall and gastrointestinal tract, are important endpoints that could be added to earthworms standardized tests, for the evaluation of soil toxicity, as part of the risk assessment of contaminated areas.

Toxicity of tributyltin (TBT) to terrestrial organisms and its species sensitivity distribution

The contamination of the terrestrial environment by disposal of tributyltin (TBT) by contaminated harbour sediments, sewage sludge and/or biocide products has been raising concerns and it may pose a risk to soil invertebrates and plants. This study aimed to improve the amount and quality of data for TBT toxicity in soils in order to assess the ecological risk of TBT to the terrestrial ecosystems. For this, bioassays were performed with the species Porcellionides pruinosus, Folsomia candida, Brassica rapa and Triticum aestivum to evaluate the toxic effects of TBT (as chloride) on these species. Additionally, this study contributed to increase the amount of data concerning TBT toxicity on soil dwelling organisms. The results showed a dose-response relationship between TBT concentration and the increase of toxicity in all species tested. These results were collated with results from literature to construct species sensitivity distributions (SSDs) and to calculate the hazardous concentration at 5% (HC₅) for all data, for each type of soil and TBT formulation used. The HC₅ value for TBT in soil was 2.06 mg TBT/kg soil dw. Little information is available concerning the concentrations of TBT in soils. In addition the predicted no-effect concentration (PNEC) value was determined to be 30 μg/kg soil. Only one study was found referring to TBT contaminated soils, and where TBT concentrations were lower than 0.024 μg TBT/kg for the wetland soil. Therefore it can be concluded that the real TBT concentrations determined represent low risk for environmental effects. In conclusion, the construction of SSDs and the calculation of HC5 using all the data available showed to be a more suitable method rather than the construction of several SSDs for each soil and TBT types. Further investigations concerning TBT concentrations and toxicity on soil organisms need to be performed to increase data and improve risk calculations.