Unveiling the capacity of bioaugmentation application, in comparison with biochar and rhamnolipid for TPHs degradation in aged hydrocarbons polluted soil.

Persistent, aged hydrocarbons in soil hinder remediation, posing a significant environmental threat. While bioremediation offers an environmentally friendly and cost-effective approach, its efficacy for complex contaminants relies on enhancing pollutant bioavailability. This study explores the potential of immobilized bacterial consortia combined with biochar and rhamnolipids to accelerate bioremediation of aged total petroleum hydrocarbon (TPH)-contaminated soil. Previous research indicates that biochar and biosurfactants can increase bioremediation rates, while mixed consortia offer sequential degradation and higher hydrocarbon mineralization. The present investigation aimed to assess whether combining these strategies could further enhance degradation in aged, complex soil matrices. The bioaugmentation (BA) with bacterial consortium increased the TPHs degradation in aged soil (over 20% compared to natural attenuation - NA). However, co-application of BA with biochar and rhamnolipid higher did not show a statistically prominent synergistic effect. While biochar application facilitated the maintenance of hydrocarbon degrading bacterial consortium in soil, the present study did not identify a direct influence in TPHs degradation. The biochar application in contaminated soil contributed to TPHs adsorption. Rhamnolipid alone slightly increased the TPHs biodegradation with NA, while the combined bioaugmentation treatment with rhamnolipid and biochar increased the degradation between 27.5 and 29.8%. These findings encourage further exploration of combining bioaugmentation with amendment, like biochar and rhamnolipid, for remediating diverse environmental matrices contaminated with complex and aged hydrocarbons.

Assessment of Physico-Chemical and Toxicological Properties of Commercial 2D Boron Nitride Nanopowder and Nanoplatelets.

Boron nitride (BN) nanomaterials have been increasingly explored for potential applications in chemistry and biology fields (e.g., biomedical, pharmaceutical, and energy industries) due to their unique physico-chemical properties. However, their safe utilization requires a profound knowledge on their potential toxicological and environmental impact. To date, BN nanoparticles have been considered to have a high biocompatibility degree, but in some cases, contradictory results on their potential toxicity have been reported. Therefore, in the present study, we assessed two commercial 2D BN samples, namely BN-nanopowder (BN-PW) and BN-nanoplatelet (BN-PL), with the objective to identify whether distinct physico-chemical features may have an influence on the biological responses of exposed cellular models. Morphological, structural, and composition analyses showed that the most remarkable difference between both commercial samples was the diameter of their disk-like shape, which was of 200-300 nm for BN-PL and 100-150 nm for BN-PW. Their potential toxicity was investigated using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus Saccharomycescerevisiae, as human and environmental eukaryotic models respectively, employing in vitro assays. In both cases, cellular viability assays and reactive oxygen species (ROS) determinations where performed. The impact of the selected nanomaterials in the viability of both unicellular models was very low, with only a slight reduction of S. cerevisiae colony forming units being observed after a long exposure period (24 h) to high concentrations (800 mg/L) of both nanomaterials. Similarly, BN-PW and BN-PL showed a low capacity to induce the formation of reactive oxygen species in the studied conditions. Even at the highest concentration and exposure times, no major cytotoxicity indicators were observed in human cells and yeast. The results obtained in the present study provide novel insights into the safety of 2D BN nanomaterials, indicating no significant differences in the toxicological potential of similar commercial products with a distinct lateral size, which showed to be safe products in the concentrations and exposure conditions tested.

Fate assessment of commercial 2D MoS2 aqueous dispersions at physicochemical and toxicological level.

The physicochemical properties and the toxicological potential of commercially available MoS2 nanoparticles with different lateral size and degradation stage were studied in the present research work. To achieve this, the structure and stoichiometry of fresh and old aqueous suspensions of micro-MoS2 and nano-MoS2 was analyzed by Raman, while x-ray photoelectron spectroscopy allowed to identify more quantitatively the nature of the formed oxidized species. A, the toxicological impact of the nanomaterials under analysis was studied using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus S. cerevisiae as biological models. Cell viability assays and reactive oxygen species (ROS) determinations demonstrated different toxicity levels depending on the cellular model used and in function of the degradation state of the selected commercial nanoproducts. Both MoS2 nanoparticle types induced sublethal damage on the A549 cells though the increase of intracellular ROS levels, while comparable concentrations reduced the viability of yeast cells. In addition, the old MoS2 nanoparticles suspensions exhibited a higher toxicity for both human and yeast cells than the fresh ones. Our findings demonstrate that the fate assessment of nanomaterials is a critical aspect to increase the understanding on their characteristics and on their potential impact on biological systems along their life cycle.

Interaction Analysis of Commercial Graphene Oxide Nanoparticles with Unicellular Systems and Biomolecules.

The ability of commercial monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) to interact with different unicellular systems and biomolecules was studied by analyzing the response of human alveolar carcinoma epithelial cells, the yeast Saccharomyces cerevisiae and the bacteria Vibrio fischeri to the presence of different nanoparticle concentrations, and by studying the binding affinity of different microbial enzymes, like the α-l-rhamnosidase enzyme RhaB1 from the bacteria Lactobacillus plantarum and the AbG ß-d-glucosidase from Agrobacterium sp. (strain ATCC 21400). An analysis of cytotoxicity on human epithelial cell line A549, S. cerevisiae (colony forming units, ROS induction, genotoxicity) and V. fischeri (luminescence inhibition) cells determined the potential of both nanoparticle types to damage the selected unicellular systems. Also, the protein binding affinity of the graphene derivatives at different oxidation levels was analyzed. The reported results highlight the variability that can exist in terms of toxicological potential and binding affinity depending on the target organism or protein and the selected nanomaterial.

Molecular mechanisms involved in the response to desiccation stress and persistence in Acinetobacter baumannii.

Desiccation tolerance contributes to the maintenance of bacterial populations in hospital settings and may partly explain its propensity to cause outbreaks. Identification and relative quantitation of proteins involved in bacterial desiccation tolerance was made using label-free quantitation and iTRAQ labeling. Under desiccating conditions, the population of the Acinetobacter baumannii clinical strain AbH12O-A2 decreased in the first week, and thereafter, a stable population of 0.5% of the original population was maintained. Using label-free quantitation and iTRAQ labeling, 727 and 765 proteins, respectively, were detected; 584 of them by both methods. Proteins overexpressed under desiccation included membrane and periplasmic proteins. Proteins associated with antimicrobial resistance, efflux pumps, and quorum quenching were overexpressed in the samples subjected to desiccation stress. Electron microscopy revealed clear morphological differences between desiccated and control bacteria. We conclude that A. baumannii is able to survive long periods of desiccation through the presence of cells in a dormant state, via mechanisms affecting control of cell cycling, DNA coiling, transcriptional and translational regulation, protein stabilization, antimicrobial resistance, and toxin synthesis, and that a few surviving cells embedded in a biofilm matrix are able to resume growth and restore the original population in appropriate environmental conditions following a "bust-and-boom" strategy.

The Acinetobacter baumannii Omp33-36 porin is a virulence factor that induces apoptosis and modulates autophagy in human cells.

Acinetobacter baumannii is an extracellular opportunistic human pathogen that is becoming increasingly problematic in hospitals. In the present study, we demonstrate that the A. baumannii Omp 33- to 36-kDa protein (Omp33-36) is a porin that acts as a channel for the passage of water. The protein is found on the cell surface and is released along with other porins in the outer membrane vesicles (OMVs). In immune and connective cell tissue, this protein induced apoptosis by activation of caspases and modulation of autophagy, with the consequent accumulation of p62/SQSTM1 (sequestosome 1) and LC3B-II (confirmed by use of autophagy inhibitors). Blockage of autophagy enables the bacterium to persist intracellularly (inside autophagosomes), with the subsequent development of cytotoxicity. Finally, we used macrophages and a mouse model of systemic infection to confirm that Omp33-36 is a virulence factor in A. baumannii. Overall, the study findings show that Omp33-36 plays an important role in the pathogenesis of A. baumannii infections.

High-Performance Aramids with Intrinsic Bactericide Activity.

Aramids, renowned for their high-performance attributes, find applications in critical fields such as protective equipment, aerospace components, and industrial filters. However, challenges arise in scenarios in which frequent washing is impractical, leading to bacterial proliferation, especially in textiles. This study outlines a straightforward and scalable method for preparing aramid-coated textiles and films endowed with inherent bactericidal activity, achieved by reacting parent aramids with vanillin. The functionalization of the aramids with bactericide moieties not only preserved the high-performance characteristics of commercial aramids but also improved their crucial mechanical properties. Tensile tests revealed an increase in Young's modulus, up to 50% compared to commercial m-aramid, accompanied by thermal performance comparable to commercial m-aramids. The evaluation of these coated textiles as bactericidal materials demonstrated robust effectiveness with A parameters (antibacterial activity) of 4.31 for S. aureus and 3.44 for K. pneumoniae. Reusability tests (washing the textiles in harsh conditions) underscored that the bactericide-coated textiles maintain their performance over at least 5 cycles. Regarding practical applications, tests performed with reconstructed human epidermis affirmed the nonirritating nature of these materials to the skin. The distinctive qualities of these metal-free intrinsic bactericidal aramids position them as ideal candidates for scenarios demanding a synergy of high performance and bactericidal properties. Applications such as first responders' textiles or filters stand to benefit significantly from these advanced materials.

Bioaugmentation and vermicompost facilitated the hydrocarbon bioremediation: scaling up from lab to field for petroleum-contaminated soils.

The biodegradation of total petroleum hydrocarbon (TPH) in soil is very challenging due to the complex recalcitrant nature of hydrocarbon, hydrophobicity, indigenous microbial adaptation and competition, and harsh environmental conditions. This work further confirmed that limited natural attenuation of petroleum hydrocarbons (TPHs) (15% removal) necessitates efficient bioremediation strategies. Hence, a scaling-up experiment for testing and optimizing the use of biopiles for bioremediation of TPH polluted soils was conducted with three 500-kg pilots of polluted soil, and respective treatments were implemented: including control soil (CT), bioaugmentation and vermicompost treatment (BAVC), and a combined application of BAVC along with bioelectrochemical snorkels (BESBAVC), all maintained at 40% field capacity. This study identified that at pilot scale level, a successful application of BAVC treatment can achieve 90.3% TPH removal after 90 days. BAVC's effectiveness stemmed from synergistic mechanisms. Introduced microbial consortia were capable of TPH degradation, while vermicompost provided essential nutrients, enhanced aeration, and, potentially, acted as a biosorbent. Hence, it can be concluded that the combined application of BAVC significantly enhances TPH removal compared to natural attenuation. While the combined application of a bioelectrochemical snorkel (BES) with BAVC also showed a significant TPH removal, it did not differ statistically from the individual application of BAVC, under applied conditions. Further research is needed to optimize BES integration with BAVC for broader applicability. This study demonstrates BAVC as a scalable and mechanistically sound approach for TPH bioremediation in soil.

Phytostabilization of metal(loid)s by ten emergent macrophytes following a 90-day exposure to industrially contaminated groundwater.

Better understanding of macrophyte tolerance under long exposure times in real environmental matrices is crucial for phytoremediation and phytoattenuation strategies for aquatic systems. The metal(loid) attenuation ability of 10 emergent macrophyte species (Carex riparia, Cyperus longus, Cyperus rotundus, Iris pseudacorus, Juncus effusus, Lythrum salicaria, Menta aquatica, Phragmites australis, Scirpus holoschoenus, and Typha angustifolia) was investigated using real groundwater from an industrial site, over a 90-day exposure period. A "phytobial" treatment was included, with 3 plant growth-promoting rhizobacterial strains. Plants exposed to the polluted water generally showed similar or reduced aerial biomass compared to the controls, except for C. riparia. This species, along with M. aquatica, exhibited improved biomass after bioaugmentation. Phytoremediation mechanisms accounted for more than 60% of As, Cd, Cu, Ni, and Pb removal, whilst abiotic mechanisms contributed to ∼80% removal of Fe and Zn. Concentrations of metal(loid)s in the roots were generally between 10-100 times higher than in the aerial parts. The macrophytes in this work can be considered "underground attenuators", more appropriate for rhizostabilization strategies, especially L. salicaria, M. aquatica, S. holoschoenus, and T. angustifolia. For I. pseudacorus, C. longus, and C. riparia; harvesting the aerial parts could be a complementary phytoextraction approach to further remove Pb and Zn. Of all the plants, S. holoschoenus showed the best balance between biomass production and uptake of multiple metal(loid)s. Results also suggest that multiple phytostrategies may be possible for the same plant depending on the final remedial aim. Phytobial approaches need to be further assessed for each macrophyte species.

Macrophyte assisted phytoremediation and toxicological profiling of metal(loid)s polluted water is influenced by hydraulic retention time.

The present study reports findings related to the treatment of polluted groundwater using macrophyte-assisted phytoremediation. The potential of three macrophyte species (Phragmites australis, Scirpus holoschoenus, and Typha angustifolia) to tolerate exposure to multi-metal(loid) polluted groundwater was first evaluated in mesocosms for 7- and 14-day batch testing. In the 7-day batch test, the polluted water was completely replaced and renewed after 7 days, while for 14 days exposure, the same polluted water, added in the first week, was maintained. The initial biochemical screening results of macrophytes indicated that the selected plants were more tolerant to the provided conditions with 14 days of exposure. Based on these findings, the plants were exposed to HRT regimes of 15 and 30 days. The results showed that P. australis and S. holoschoenus performed better than T. angustifolia, in terms of metal(loid) accumulation and removal, biomass production, and toxicity reduction. In addition, the translocation and compartmentalization of metal(loid)s were dose-dependent. At the 30-day loading rate (higher HRT), below-ground phytostabilization was greater than phytoaccumulation, whereas at the 15-day loading rate (lower HRT), below- and above-ground phytoaccumulation was the dominant metal(loid) removal mechanism. However, higher levels of toxicity were noted in the water at the 15-day loading rate. Overall, this study provides valuable insights for macrophyte-assisted phytoremediation of polluted (ground)water streams that can help to improve the design and implementation of phytoremediation systems.

Comparative toxicological analysis of two pristine carbon nanomaterials (graphene oxide and aminated graphene oxide) and their corresponding degraded forms using human in vitro models.

Despite the wide application of graphene-based materials, the information of the toxicity associated to some specific derivatives such as aminated graphene oxide is scarce. Likewise, most of these studies analyse the pristine materials, while the available data regarding the harmful effects of degraded forms is very limited. In this work, the toxicity of graphene oxide (GO), aminated graphene oxide (GO-NH2), and their respective degraded forms (dGO and dGO-NH2) obtained after being submitted to high-intensity sonication was evaluated applying in vitro assays in different models of human exposure. Viability and ROS assays were performed on A549 and HT29 cells, while their skin irritation potential was tested on a reconstructed human epidermis model. The obtained results showed that GO-NH2 and dGO-NH2 substantially decrease cell viability in the lung and gastrointestinal models, being this reduction slightly higher in the cells exposed to the degraded forms. In contrast, this parameter was not affected by GO and dGO which, conversely, showed the ability to induce higher levels of ROS than the pristine and degraded aminated forms. Furthermore, none of the materials is skin irritant. Altogether, these results provide new insights about the potential harmful effects of the selected graphene-based nanomaterials in comparison with their degraded counterparts.

On the Tunability of Toxicity for Viologen-Derivatives as Anolyte for Neutral Aqueous Organic Redox Flow Batteries.

Viologen-derivatives are the most widely used redox organic molecules for neutral pH negative electrolyte of redox flow batteries. However, the long-established toxicity of the herbicide methyl-viologen raises concern for deployment of viologen-derivatives at large scale in flow batteries. Herein, we demonstrate the radically different cytotoxicity and toxicology of a series of viologen-derivatives in in vitro assays using model organisms representative of human and environmental exposure, namely human lung carcinoma epithelial cell line (A549) and the yeast Saccharomyces cerevisiae. The results show that safe viologen derivatives can be molecularly engineered, representing a promising family of negolyte materials for neutral redox flow batteries.

Toxicological assessment of pristine and degraded forms of graphene functionalized with MnOx nanoparticles using human in vitro models representing different exposure routes.

The development of novel advanced nanomaterials (NMs) with outstanding characteristics for their use in distinct applications needs to be accompanied by the generation of knowledge on their potential toxicological impact, in particular, that derived from different occupational risk exposure routes, such as inhalation, ingestion, and skin contact. The harmful effects of novel graphene-metal oxide composites on human health are not well understood, many toxicological properties have not been investigated yet. The present study has evaluated several toxicological effects associated with graphene decorated with manganese oxide nanoparticles (GNA15), in a comparative assessment with those induced by simple graphene (G2), on human models representing inhalation (A549 cell line), ingestion (HT29 cell line) and dermal routes (3D reconstructed skin). Pristine and degraded forms of these NMs were included in the study, showing to have different physicochemical and toxicological properties. The degraded version of GNA15 (GNA15d) and G2 (G2d) exhibited clear structural differences with their pristine counterparts, as well as a higher release of metal ions. The viability of respiratory and gastrointestinal models was reduced in a dose-dependent manner in the presence of both GNA15 and G2 pristine and degraded forms. Besides this, all NMs induced the production of reactive oxygen species (ROS) in both models. However, the degraded forms showed to induce a higher cytotoxicity effect. In addition, we found that none of the materials produced irritant effects on 3D reconstructed skin when present in aqueous suspensions. These results provide novel insights into the potentially harmful effects of novel multicomponent NMs in a comprehensive manner. Furthermore, the integrity of the NMs can play a role in their toxicity, which can vary depending on their composition and the exposure route.

Extracellular proteome of a highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii.

The study of the extracellular proteomes of pathogenic bacteria is essential for gaining insights into the mechanisms of pathogenesis and for the identification of virulence factors. Through the use of different proteomic approaches, namely Nano-LC and 2DE combined with MALDI-TOF/TOF, we have characterized the extracellular proteome of a highly invasive, multidrug-resistant strain of A. baumannii (clone AbH12O-A2). This study focused on two main protein fractions of the extracellular proteome: proteins that are exported by outer membrane vesicles (OMVs) and freely soluble extracellular proteins (FSEPs) present in the culture medium of A. baumannii. Herein, a total of 179 nonredundant proteins were identified in the OMV protein fraction and a total of 148 nonredundant proteins were identified in FSEP fraction. Of the OMV proteins, 39 were associated with pathogenesis and virulence, including proteins associated with attachment to host cells (e.g., CsuE, CsuB, CsuA/B) and specialized secretion systems for delivery of virulence factors (e.g., P. pilus assembly and FilF), whereas the FSEP fraction possesses extracellular enzymes with degradative activity, such as alkaline metalloprotease. Furthermore, among the FSEP we have detected at least 18 proteins with a known role in oxidative stress response (e.g., catalase, thioredoxin, oxidoreductase, superoxide dismutase). Further assays demonstrated that in the presence of FSEPs, bacterial cells withstand much higher concentrations of H2O2 showing higher survival rate (approximately 2.5 fold) against macrophages. In this study we have identified an unprecedented number of novel extracellular proteins of A. baumannii and we provide insight into their potential role in relevant processes such as oxidative stress response and defense against macrophage attack.

Toxicological assessment of nanocrystalline metal alloys with potential applications in the aeronautical field.

The development of new candidate alloys with outstanding characteristics for their use in the aeronautical field is one of the main priorities for the sector. In this context, nanocrystaline (nc) alloys are considered relevant materials due to their special features, such as their exceptional physical and mechanical properties. However, another important point that needs to be considered with newly developed alloys is the potential toxicological impact that these materials may have in humans and other living organisms. The aim of this work was to perform a preliminary toxicological evaluation of three nc metal alloys (WCu, WAl and TiAl) in powder form produced by mechanical alloying, applying different in vitro assays, including a mix of W-Cu powders with standard grain size in the experiments to stablish comparisons. The effects of the direct exposure to powder suspensions and/or to their derived leachates were analysed in three model organisms representative of human and environmental exposures (the adenocarcinomic human alveolar basal epithelial cell line A549, the yeast Saccharomyces cerevisiae and the Gram negative bacterium Vibrio fischeri). Altogether, the results obtained provide new insights about the potential harmful effects of the selected nc alloys, showing that, from a toxicological perspective, nc TiAl is the safest candidate in the model organisms and conditions tested.

Comparative toxicological assessment of three soils polluted with different levels of hydrocarbons and heavy metals using in vitro and in vivo approaches.

The biological effects induced by the pollutants present in soils, together with the chemical and physical characterizations, are good indicators to provide a general overview of their quality. However, the existence of studies where the toxicity associated to soils contaminated with mixtures of pollutants applying both in vitro and in vivo models are scarce. In this work, three soils (namely, Soil 001, Soil 002 and Soil 013) polluted with different concentrations of hydrocarbons and heavy metals were evaluated using different organisms representative of human (HepG2 human cell line) and environmental exposure (the yeast Saccharomyces cerevisiae, the Gram-negative bacterium Pseudomonas putida and, for the in vivo evaluation, the annelid Enchytraeus crypticus). In vitro assays showed that the soluble fraction of the Soil 001, which presented the highest levels of heavy metals, represented a great impact in the viability of the HepG2 cells and S. cerevisiae, while organic extracts from Soils 002 and 013 caused a slight decrease in the viability of HepG2 cells. In addition, in vivo experiments showed that Soils 001 and 013 affected the survival and the reproduction of E. crypticus. Altogether, these results provide a general overview of the potential hazards associated to three specific contaminated sites in a variety of organisms, showing how different concentrations of similar pollutants affect them, and highlights the relevance of testing both organic and soluble extracts when in vitro safety assays of soils are performed.

Evaluation of biostimulation, bioaugmentation, and organic amendments application on the bioremediation of recalcitrant hydrocarbons of soil.

In the present work, the operational conditions for improving the degradation rates of Total Petroleum Hydrocarbons (TPHs) in contaminated soil from a machinery park were optimized at a microcosms scale along a 90-days incubation period. In this study, bioremediation strategies and an organic amendment have been tested to verify the remediation of soil contaminated with different hydrocarbons, mineral oils, and heavy metals. Specifically, designed biostimulation and bioaugmentation strategies were compared with and without adding vermicompost. The polluted soil harboring multiple contaminants, partially attenuated for years, was used. The initial profile showed enrichment in heavy linear alkanes, suggesting a previous moderate weathering. The application of vermicompost increased five and two times the amounts of available phosphorus (P) and exchangeable potassium (K), respectively, as a direct consequence of the organic amendment addition. The microbial activity increased due to soil acidification, which influenced the solubility of P and other micronutrients. It also impacted the predominance and variability of the different microbial groups and the incubation, as reflected by phospholipid fatty acid (PLFA) results. An increase in the alkaline phosphatases and proteases linked to bacterial growth was displayed. This stimulation of microbial metabolism correlated with the degradation rates since TPHs degradation' efficiency after vermicompost addition reached 32.5% and 34.4% of the initial hydrocarbon levels for biostimulation and bioaugmentation, respectively. Although Polycyclic Aromatic Hydrocarbons (PAHs) were less abundant in this soil, results also decreased, especially for the most abundant, the phenanthrene. Despite improving the degradation rates, results revealed that recalcitrant and hydrophobic petroleum compounds remained unchanged, indicating that mobility, linked to bioavailability, probably represents the limiting step for further soil recovery.

Toxicology assessment of manganese oxide nanomaterials with enhanced electrochemical properties using human in vitro models representing different exposure routes.

In the present study, a comparative human toxicity assessment between newly developed Mn3O4 nanoparticles with enhanced electrochemical properties (GNA35) and their precursor material (Mn3O4) was performed, employing different in vitro cellular models representing main exposure routes (inhalation, intestinal and dermal contact), namely the human alveolar carcinoma epithelial cell line (A549), the human colorectal adenocarcinoma cell line (HT29), and the reconstructed 3D human epidermal model EpiDerm. The obtained results showed that Mn3O4 and GNA35 harbour similar morphological characteristics, whereas differences were observed in relation to their surface area and electrochemical properties. In regard to their toxicological properties, both nanomaterials induced ROS in the A549 and HT29 cell lines, while cell viability reduction was only observed in the A549 cells. Concerning their skin irritation potential, the studied nanomaterials did not cause a reduction of the skin tissue viability in the test conditions nor interleukin 1 alpha (IL- 1 α) release. Therefore, they can be considered as not irritant nanomaterials according to EU and Globally Harmonized System of Classification and Labelling Chemicals. Our findings provide new insights about the potential harmful effects of Mn3O4 nanomaterials with different properties, demonstrating that the hazard assessment using different human in vitro models is a critical aspect to increase the knowledge on their potential impact upon different exposure routes.

Proteomic and functional analyses reveal a unique lifestyle for Acinetobacter baumannii biofilms and a key role for histidine metabolism.

Biofilm formation is one of the main causes for the persistence of Acinetobacter baumannii, a pathogen associated with severe infections and outbreaks in hospitals. Here, we performed comparative proteomic analyses (2D-DIGE and MALDI-TOF/TOF and iTRAQ/SCX-LC-MS/MS) of cells at three different conditions: exponential, late stationary phase, and biofilms. These results were compared with alterations in the proteome resulting from exposure to a biofilm inhibitory compound (salicylate). Using this multiple-approach strategy, proteomic patterns showed a unique lifestyle for A. baumannii biofilms and novel associated proteins. Several cell surface proteins (such as CarO, OmpA, OprD-like, DcaP-like, PstS, LysM, and Omp33), as well as those involved in histidine metabolism (like Urocanase), were found to be implicated in biofilm formation, this being confirmed by gene disruption. Although l-His uptake triggered biofilms efficiently in wild-type A. baumannii, no effect was observed in Urocanase and OmpA mutants, while a slight increase was observed in a CarO deficient strain. We conclude that Urocanase plays a crucial role in histidine metabolism leading to biofilm formation and that OmpA and CarO can act as channels for L-His uptake. Finally, we propose a model in which novel proteins are suggested for the first time as targets for preventing the formation of A. baumannii biofilms.

Horizontal transfer of the OXA-24 carbapenemase gene via outer membrane vesicles: a new mechanism of dissemination of carbapenem resistance genes in Acinetobacter baumannii.

The resistance of Acinetobacter baumannii strains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of ß-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes among A. baumannii strains are not fully understood. In this study we used two carbapenem-resistant clinical strains of A. baumannii (AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borne bla(OXA-24) gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate that A. baumannii releases outer membrane vesicles (OMVs) during in vitro growth. The use of hybridization studies enabled us to show that these OMVs harbored the bla(OXA-24) gene. The incubation of these OMVs with the carbapenem-susceptible A. baumannii ATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring the bla(OXA-24) gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboring bla(OXA-24) were released by A. baumannii ATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates of A. baumannii may release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surrounding A. baumannii bacterial isolates.