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Nowadays, microplastics (MPs) and adsorbed pollutants are considered a global thread to marine ecosystems. This study describes the effects of pollutants and MPs ingestion on fish brains through the assessment of oxidative stress biomarkers and monoaminergic neurotransmitters using gilthead seabream (Sparus aurata) as fish model. Juveniles were experimentally exposed to three different dietary treatments for 90 days: Control treatment (C) consisted of standard feed; Virgin treatment (V) contained feed enriched with 10% of MPs; and Exposed treatment (E) consisted of feed with 10% of MPs that were exposed to seawater in an anthropogenically impacted area for 2 months in order to enrich the plastic with the pollutants within the water column. Sampling was made at the start of the experiment (T0), at the end of the dietary treatments (T90) and after a posterior detoxification period of 30 days (T120). Results evidenced that a MPs and pollutants enriched diet increases the activity of some of the oxidative stress biomarkers (e.g. CAT and GST), and it was shown for the first time alterations on dopaminergic and serotonergic system activity on seabream brains, indicating potential neurofunctional effects associated to MPs and pollutants ingestion. In addition, results showed a tendency to recover enzymatic and brain monoaminergic neurotransmitter levels after a 30-day detoxification period. In conclusion, MPs and pollutants exposure for 90 days induced oxidative stress and changes on monoaminergic activity in the brain of S. aurata.

Environmental enrichment is considered as a recommended tool to guarantee or improve the welfare of captive fish. This study demonstrates for the first time that structural environmental enrichment enhances cognition, exploratory behaviour and brain physiological functions of gilthead seabream (Sparus aurata). Seabream was reared in groups (n = 15) during 60 days under two different treatments: enriched tanks with plant-fibre ropes (EE) or bare/non-enriched tanks (NE). Fish were then exposed to a purpose-built maze for 1 h every second day in four trials. Analysis of video recordings showed that seabream under EE conditions presented higher overall exploratory behaviour, spatial orientation and learning capability compared to seabream from NE conditions. Results from brain monoamines analyses may suggest increased recent dopaminergic activity in telencephalon, known to be involved in learning processes; and increased serotonergic activity in cerebellum, involved in the coordination of balance, movements and orientation. In addition, EE-reared fish showed increased antioxidant activity in whole brain, with no apparent oxidative damage. Structural EE seemed to induce an hormetic response on juvenile seabream, improving their welfare status during captivity. Application of this kind of physical structure might be feasible at fish farms as a passive and noninvasive tool to improve welfare of intensively cultured seabream.

Current production systems of finfish aquaculture, and in particular intensive farming systems, can cause welfare problems leading not only to poor condition of the fish but also to a decrease in product quality. Adding structural environmental enrichment (EE) to bare rearing environments may improve the welfare of certain cultured fish. In this study we experimentally demonstrate the positive effects of adding structural EE on rearing environments of juvenile seabream (Sparus aurata). Fish maintained for 35 days with EE showed less aggression and interactions with the net pen, and lower erosion of pectoral and caudal fins, compared to fish kept in bare conditions (non-enriched, NE). In addition, EE modified the horizontal distribution of fish in the experimental cage, increasing the use of the inner areas. Non-significant effects of EE were observed on fish body condition and growth, and on brain monoamines levels and mortality. Nevertheless, this work highlights the potential use of structural EE to improve welfare of juvenile seabream, which might be feasible to apply at larger-commercial scale.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here., Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Sociedad Española de Neumología y Cirugía Torácica, European Commission, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Non-typeable Haemophilus infl uenzae (NTHi) is an opportunist pathogen well adapted to the human upper respiratory tract and responsible for many respiratory diseases. In the human airway, NTHi is exposed to pollutants, such as alkylating agents, that damage its DNA. In this study, we examined the signifi cance of genes involved in the repair of DNA alkylation damage in NTHi virulence. Two knockout mutants, tagI and mfd, encoding N3methyladenine-DNA glycosylase I and the key protein involved in transcription-coupled repair, respectively, were constructed and their virulence in a BALB/c mice model was examined. This work shows that N3-methyladenine-DNA glycosylase I is constitutively expressed in NTHi and that it is relevant for its virulence. [Int Microbiol 2014; 17(3):159-164]Keywords: Haemophilus infl uenzae · alkylating agents · virulence · genes tagI and mfd

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K.pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K.pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K.pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K.pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K.pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis. Prevailing belief states that the human pathogens Klebsiella pneumoniae is an extracellular pathogen. However, in this work, we demonstrate that K. pneumoniae co-opts the maturation of the phagosome manipulating a PI3K-AKT-RAB14 signalling cascade. Here, we also demonstrate that by preventing the activation of this cascade, the macrophages eliminate intracellular Klebsiella. © 2015 John Wiley

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae. ©2013, American Society for Microbiology.

[EN]Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful

[Background] Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction., [Results] The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence., [Conclusion] Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.