Social behaviour involving drug resistance: the role of initial density, initial frequency and population structure in shaping the effect of antibiotic resistance as a public good.

Bacteria sometimes cooperate with co-inhabiting cells. Pathogenic bacteria, for example, often produce and excrete virulence factors, eventually benefitting both producer and non-producer cells. The role of social interactions involving antibiotic resistance, however, has been more elusive. Enzymes that inactivate ß-lactam antibiotics such as ampicillin or penicillin (ß-lactamases) are good candidates as public goods. Nonetheless, it has been claimed that bacteria harbouring plasmids of natural origin coding for ß-lactamase almost do not protect sensitive bacteria. This does not fit with the fact that ampicillin-sensitive bacteria can be isolated from subjects undergoing ampicillin treatment. We hypothesised that there are two non-exclusive explanations for the discrepancy between previous works: (1) the range of values of demographic conditions (such as initial strain frequency, initial total cell density or habitat structure) has not been broad enough to include most scenarios, or (2) there are interactions between some of these factors. We performed experiments with Escherichia coli bacterial cells to measure the degree of protection of sensitive cells when co-cultured with cells harbouring RP4, R16a or the R1 plasmids, all of natural origin and coding for ß-lactamases, and in presence of ampicillin. In these co-cultures, performed in structured and non-structured environments, both the initial total cell density and the initial frequency of sensitive cells spanned four orders of magnitude. We found protection of sensitive cells in 63% of tested conditions. All factors (plasmid, structure, frequency and density) significantly affect levels of protection. Moreover, all factors interact, with interactions revealing large or very large effect sizes.

Effects of ivermectin on Danio rerio: a multiple endpoint approach: behaviour, weight and subcellular markers.

Ivermectin (IVM) is a broad acting antihelmintic used in various veterinary pharmaceuticals. It has been shown that IVM enters the aquatic compartment and adversely affects organisms including fish. This study is based on the hypothesis that long term exposure to IVM affects fish and thus, the main objective was to assess the chronic effects of 0.25 and 25 µg IVM/L to zebrafish using multiple endpoints representative of several levels of biological organization: weight, behaviour (swimming and feeding) and subcellular markers including biomarkers for oestrogenicity (vitellogenin-VTG), oxidative stress (catalase-CAT and glutathione-S-transferase-GST) and neurotransmission (cholinesterase-ChE). Concentrations as low as 0.25 µg IVM/L disrupted the swimming behaviour, causing fish to spend more time at the bottom of aquaria. Such reduction of the swimming performance affected the feeding ability which is likely responsible for the weight loss. The effects on weight were gender differentiated, being more pronounced in males (0.25 µg IVM/L) than in females (25 µg IVM/L). Fish exposed to 25 µg/L exhibited darker coloration and mild curvature of the spine. No effects on VTG and AChE were observed, but a reduction on CAT and GST levels was observed in fish exposed to 25 µg IVM/L, although these alterations probably only reflect the general condition of the fish which was significantly compromised at this concentration. Despite that predicted environmental concentrations of IVM are below 0.25 µg/L, the behavioural effects may be translated into important ecological impacts, e.g. at predator-prey interactions where fish competitive advantage can be decreased. Future work should address the link between behaviour disruption and population fitness. The current study was based on a one experiment and multiple endpoint (anchored) approach, allowing the results to be integrated and linked towards a mechanistic understanding.

Impacts of aqueous extracts of wildfire ashes on aquatic life-stages of Xenopus laevis: Influence of plant coverage.

Wildfires have emerged as a global ecological concern due to their wide-ranging off-site effects. One particular consequence is the adverse impact on aquatic environments, as wildfires are acknowledged as a significant source of aquatic contamination through ash runoffs containing toxic compounds. Yet, amphibian response to this source of contamination remains largely undocumented. This study assessed how ash runoffs from Eucalyptus sp. and Pinus sp. affect early aquatic life-stages of Xenopus laevis. Embryos and tadpoles were exposed, respectively, for 96 h and 14 days to serial concentrations (26.9% - 100%) of aqueous extracts of ashes (AEAs; 10 gL-1) composed of eucalypt (ELS) and pine (PLS) ashes. Mortality and development were monitored, and biometric data (snout-to-vent, tail and total length, and weight) measured. Sub-individual endpoints regarding oxidative stress (catalase-CAT; total glutathione-TG; lipid peroxidation-TBARS), neurotoxicity (acetylcholinesterase-AChE), transformation metabolism (glutathione-S-transferase-GST) and energetic metabolism (carbohydrate, lipid and protein content and O2 consumption), were also measured. The two AEAs induced no significant lethal effects on embryos or tadpoles. However, in general, AEAs caused a developmental delay in both life stages. Effects of AEAs on biometric endpoint were only reported for tadpoles, which showed a decreased body length (snout-to-vent, tail and total) and weight (embryos were not weighed), with PLS exerting higher effect than ELS. As for the sub-individual endpoints, embryos showed mostly no alterations on the activity of the monitored parameters, except for PLS, which reduced embryos' carbohydrate content (at ≥59.2%) and increased O2 consumption (at ≥35.0%). Regarding tadpoles, AEA exposure decreased the activity of CAT and GST (at ≥26.0%) and decreased carbohydrate (at ≥26.0%) and lipid (at ≥45.5%), whereas oxygen consumption increased (at ≥26.0%) only on PLS. Overall, the tested AEAs differentially affected amphibians across life-stages, indicating that plant coverage might affect ash toxicity.

New Enclosure for in vivo Medical Imaging of Zebrafish With Vital Signs Monitoring.

Lately, the use of zebrafish has gained increased interest in the scientific community as an animal model in preclinical research. However, there is a lack of in vivo imaging tools that ensure animal welfare during acquisition procedures. The use of functional imaging techniques, like Positron Emission Tomography (PET), in zebrafish is limited since it requires the animal to be alive, representing a higher instrumentation complexity when compared to morphological imaging systems. In the present work, a new zebrafish enclosure was developed to acquire in vivo images while monitoring the animal's welfare through its heartbeat. The temperature, dissolved oxygen, and pH range in a closed aquatic environment were tested to ensure that the conditions stay suitable for animal welfare during image acquisitions. The developed system, based on an enclosure with a bed and heartbeat sensors, was tested under controlled conditions in anesthetized fishes. Since the anesthetized zebrafish do not affect the water quality over time, there is no need to incorporate water circulation for the expected time of PET exams (about 30 min). The range of values obtained for the zebrafish heart rate was 88-127 bpm. The developed system has shown promising results regarding the zebrafish's heart rate while keeping the fish still during the long imaging exams. The zebrafish enclosure ensures the animal's well-being during the acquisition of in vivo images in different modalities (PET, Computer Tomography, Magnetic Resonance Imaging), contributing substantially to the preclinical research.

ORMOPLEXEs for gene therapy: In vitro and in vivo assays.

Gene therapy stays on the cutting edge of biomedical research, being the design of the optimal gene delivery vector one of the key requests. Silica-based nanoparticles (NPs) have emerged as promising non-viral gene delivery vector, due to their high biocompatibility, nontoxicity, non-immunogenicity, biodegradability and enormous bioconjugation versatility. In this work a sol-gel methodology for the synthesis of amino-functionalized silica NPs (NH2-ORMOSIL NPs) was optimized, and NPs were characterized by TEM and FTIR. In a first step NH2-ORMOSIL NPs were bioconjugated with a plasmid DNA, pVAX1-GFP, assembling an ORMOPLEXE, confirmed by agarose gel electrophoresis. In a second step, in vitro studies have been performed with cultured CHO cells, where ORMOPLEXEs transfection was proved by CLSM. In vivo transfection efficiency and bio-distribution were performed in Zebrafish (Danio rerio) embryos, assessed by FM. Finally, NPs ecotoxicity was studied in zebrafish embryos by following the mortality and developmental endpoints.

Biochemical and metabolic effects of a short-term exposure to nanoparticles of titanium silicate in tadpoles of Pelophylax perezi (Seoane).

This study aimed to evaluate sublethal effects of a short-term exposure (96 h) to titanium silicate nanoparticles (TiSiO(4)-NP) on Pelophylax perezi tadpoles. Tadpoles were exposed to five concentrations of TiSiO(4)-NP (8.2, 10.2, 12.8, 16 and 20 mg/L) plus a control. Effect criteria were: mortality, cholinesterases, glutathione S-transferases, lactate dehydrogenase, and catalase activities, and alanine and lactate contents. Light scattering was used for physical characterization of TiSiO(4)-NP suspensions, revealing a high aggregation state of the NP, consistent with low z-potential values (<30 mV). Mortality among TiSiO(4)-NP treatments was <11%. Significant differences relatively to the control were observed at the biochemical level (for CAT and LDH) and in lactate and alanine contents, which may end-up in increased oxidative stress. Overall, some of the monitored endpoints suggest metabolic alterations in TiSiO(4)-NP exposed tadpoles, highlighting the potential of TiSiO(4)-NP long-term effects on these organisms.