Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells

An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NPbacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NPcell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, e.g., via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, e.g., by SEM, for evidence of NPbacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NPbacteria interactions.financial support from the following sources: grant SFRH/BPD/47693/2008 from the Portuguese Foundation for Science and Technology (FCT); FCT Strategic Project PEst-OE/EQB/LA0023/2013; project “BioHealth Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2−O Novo Norte), QREN, FEDER; project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, ref. FCOMP-01-0124-FEDER- 027462

Towards a simple global-standard bioassay for a key ecosystem process: organic-matter decomposition using cotton strips

Cotton-strip bioassays are increasingly used to assess ecosystem integrity because they provide a standardized measure of organic-matter decomposition – a fundamental ecosystem process. However, several different cotton- strip assays are routinely used, complicating the interpretation of results across studies, and hindering broader synthesis. Here, we compare the decay rates and assemblages of bacteria and fungi colonizing the three most commonly used cotton materials: Artist’s canvas, Calico cloth, and Empa fabric. Cotton strips from each material type were incubated in 10 streams that span a wide range of physicochemical properties across five ecoregions. Additionally, to evaluate responses to environmental stress without potentially confounding biogeographical effects, we deployed identical bioassays in five streams across an acidification gradient within a single ecoregion. Across all streams decomposition rates (as tensile strength loss [TSL]) differed among the three cotton ma- terials; Calico cloth decomposed fastest (time to 50% TSL [T50]=16.7d), followed by the Empa fabric (T50 = 18.3 d) and then Artist’s canvas (T50 = 21.4 d). Despite these differences, rates of TSL of the three cotton materials responded consistently to variation in environmental conditions; TSL of each fabric increased with stream temperature, dissolved-nutrient concentrations and acid-neutralizing capacity, although Artist’s canvas and Calico cloth were more sensitive than Empa fabric. Microbial communities were similar among the mate- rials, and values of community structure (e.g., phylotype richness and diversity) were comparable to those reported for decaying leaves in streams from the same region, the major natural basal carbon resource in forested-stream ecosystems. We present linear calibrations among pairs of assays so that past and future studies can be expressed in a “common currency” (e.g., Artist’s-fabric equivalents) ‘past and future studies’ repeated two times in the sentence. Lastly, given its relatively low within-site variability, and the large number of streams where it has been used (> 700 across the globe), we recommend Artist’s fabric for future work. These results show that cotton provides an effective and realistic standardized substrate for studying heterotrophic microbial assemblages, and acts as a reasonable proxy for more chemically complex forms of detritus. These findings add to growing evidence that cotton-strip bioassays are simple, effective and easily standardized indicators of het- erotrophic microbial activity and the ecosystem processes that result

Calcium–magnesium liming of acidified forested catchments: Effects on humus morphology and functioning

International audienc

Evaluation multi-échelles de la toxicité des nanoparticules sur les bactéries

International audienc

Analysis of factors affecting arsenic speciation in neutral contaminated mine drainage

As2014 : proceedings of the 5th International Congress on Arsenic in the Environment, May 11-16, 2014, Buenos AiresInternational audienc

Impact of CeO2 nanoparticles on the functions of freshwater ecosystems: A microcosm study

International audienceWe investigated the impact of CeO2 nanoparticles (NPs) with different sizes, shapes and coatings on the function of a freshwater experimental ecosystem. We hypothesized that the different types of NPs would have different effects on the organisms involved in leaf litter decomposition and could differentially affect this process. Experiments were conducted in microcosm under environmentally relevant conditions with low CeO2 NP concentrations (1 mg L-1). Leaf litter decomposition, leaf-associated fungal biomass, bacterial community diversity and toxicity on Chironomus riparius larvae were studied. A decrease in teratogenicity (mouthpart deformities) in chironomid larvae was observed with citrate-coated spherical NPs, suggesting a hormesis effect. In contrast, exposure to non-coated, spherical NPs led to increased teratogenicity in chironomids, changes in bacterial community diversity and decreased leaf litter decomposition. Large, non-coated plates induced changes in bacterial assemblages, whereas no effect on fungal biomass was observed. These results are discussed and several hypotheses are presented to explain the results. Leaf litter decomposition is a marker that is frequently used to assess freshwater ecosystems' health. Extending its use to nano-ecotoxicology enables the study of NP impact on the function of ecosystems. This study shows that leaf litter decomposition and mouthpart deformities in chironomid larvae are sensitive, congruent markers of the environmental impact of CeO2 NPs under these experimental conditions. © The Royal Society of Chemistry 2016

Bactericidal effect of titanium dioxide nanoparticles: an overview of the toxicity mechanism

International audienceBactericidal effects of NP-TiO2 under dark condition are still the subject of debate. As an example, some authors found that NP-TiO2 have no effect on the laboratory model strain E. coli while others found for this strain EC50 values differing from 10 magnitude orders. Such contradictory results may be explained by multiple causal factors, including differences in the intrinsic characteristics of NP-TiO2 and bacterial cells used in these studies, exposure conditions for toxicity assessment and even the method used to assess the toxicity, the latter being often inappropriate for nanoparticle toxicity assessment. Increasing knowledge about exposition parameters which trigger NP toxicity and associated molecular mechanisms would contribute to properly evaluate and predict NP effects/fate in the environment.In our study, analysis of the interactions between nanoparticles (NPs) and bacteria highlighted the paramount role played by interfacial electrostatic interactions (NP-cell vs. NP-NP interactions) in determining the extent of NP toxicity and the importance of physico-chemical parameters such as pH and ionic strength in controlling these interactions. In condition of interaction, we showed that nanoparticles causes E.coli membrane depolarization and loss of membrane integrity leading to higher cell permeability. A transcriptomic analysis highlighted that deregulated genes are involved in the response to osmotic stress, metabolism of various cell envelope components and the uptake/metabolism of other endogenous and exogenous compounds. In addition, a significant number of deregulated genes encode proteins localized in the membrane and periplasmic space. All in all, results indicate that the primary effect of NP-TiO2 is initiated at the cell envelope level (membrane depolarization, loss of integrity) triggering an osmotic stress response in bacteria. These results are supported by the observed massive leakage of intracellular K+/Mg

Multibiomarker assessment of titanium nanoparticles (nTiO2) sublethal effects on the freshwater bivalve Dreissena polymorpha and the amphipod Gammarus roeseli: influence of shape and crystal structure

International audienc

Evaluation des effets du dioxyde de cerium nanoparticulaire (nCeO2) sur les invertebres dulçaquicoles Dreissena polymorpha et Gammarus roeseli a l’aide d’une batterie de biomarqueurs

International audienc

Multibiomarker assessment of cerium and titanium dioxide nanoparticles (nCeO2 and nTiO2) sublethal effects on freshwater invertebrates

International audienc