Chemical Analysis and Antimicrobial Activity of Moringa oleifera Lam. Leaves and Seeds.

Moringa oleifera is a traditional food crop widespread in Asiatic, African, and South American continents. The plant, able to grow in harsh conditions, shows a high nutritional value and medicinal potential evidencing cardioprotective, anti-inflammatory, antioxidant, and antimicrobial properties. The purpose of this study was the phytochemical analysis of M. oleifera and the identification of the antimicrobial compounds by combining a chemical approach with in vitro tests. The metabolite profile of M. oleifera polar and apolar extracts of leaves and seeds were investigated by using Nuclear Magnetic Resonance spectroscopy and Gas Chromatography-Mass Spectrometry. The antimicrobial activity of all of the obtained extract was evaluated against four bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella enterica). The chemical analysis provided a wide set of metabolites that were identified and quantified. Moreover, apolar extracts from seeds showed a significant concentration-dependent antimicrobial activity against S. aureus and S. epidermidis, (4 mg/mL reduced the viability up to 50%) that was associated to the content of specific fatty acids. Our results remarked the advantages of an integrated approach for the identification of plant metabolites and its use in association with biological tests to recognize the compounds responsible for bioactivity without compounds purification.

Cell-wall associated polysaccharide from the psychrotolerant bacterium Psychrobacter arcticus 273-4: isolation, purification and structural elucidation.

In this paper, the structure of the capsular polysaccharide isolated from the psychrotolerant bacterium Psychrobacter arcticus 273-4 is reported. The polymer was purified by gel filtration chromatography and the structure was elucidated by means of one- and two-dimensional NMR spectroscopy, in combination with chemical analyses. The polysaccharide consists of a trisaccharidic repeating unit containing two residues of glucose and a residue of a N,N-diacetyl-pseudaminic acid.

Pentadecanal inspired molecules as new anti-biofilm agents against Staphylococcus epidermidis.

Staphylococcus epidermidis, a harmless human skin colonizer, is a significant nosocomial pathogen in predisposed hosts because of its capability to form a biofilm on indwelling medical devices. In a recent paper, the purification and identification of the pentadecanal produced by the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, able to impair S. epidermidis biofilm formation, were reported. Here the authors report on the chemical synthesis of pentadecanal derivatives, their anti-biofilm activity on S. epidermidis, and their action in combination with antibiotics. The results clearly indicate that the pentadecanal derivatives were able to prevent, to a different extent, biofilm formation and that pentadecanoic acid positively modulated the antimicrobial activity of the vancomycin. The cytotoxicity of these new anti-biofilm molecules was tested on two different immortalized eukaryotic cell lines in view of their potential applications.

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces.

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.

Large-scale biofilm cultivation of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 for physiologic studies and drug discovery.

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air-liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.

Harmful and beneficial properties of cyanotoxins: Two sides of the same coin.

Cyanotoxins are by definition "harmful agents" produced by cyanobacteria. Their toxicity has been extensively studied and reviewed over the years. Cyanotoxins have been commonly classified, based on their poisonous effects on mammals, into three main classes, neurotoxins, hepatotoxins and dermatotoxins, and, considering their chemical features, mainly identified as peptides, alkaloids and lipopolysaccharides. Here we propose a broader subdivision of cyanotoxins into eight distinct classes, taking into account their molecular structures, biosynthesis and modes of action: alkaloids, non-ribosomal peptides, polyketides, non-protein amino acids, indole alkaloids, organophosphates, lipopeptides and lipoglycans. For each class, the structures and primary mechanisms of toxicity of the main representative cyanotoxins are reported. Despite their powerful biological activities, only recently scientists have considered the biotechnological potential of cyanotoxins, and their applications both in medical and in industrial settings, even if only a few of these have reached the biotech market. In this perspective, we discuss the potential uses of cyanotoxins as anticancer, antimicrobial, and biocidal agents, as common applications for cytotoxic compounds. Furthermore, taking into account their mechanisms of action, we describe peculiar potential bioactivities for several cyanotoxin classes, such as local anaesthetics, antithrombotics, neuroplasticity promoters, immunomodulating and antifouling agents. In this review, we aim to stimulate research on the potential beneficial roles of cyanotoxins, which require interdisciplinary cooperation to facilitate the discovery of innovative biotechnologies.

Reviewing the state of biosensors and lab-on-a- chip technologies: opportunities for extreme environments and space exploration.

The space race is entering a new era of exploration, in which the number of robotic and human missions to various places in our solar system is rapidly increasing. Despite the recent advances in propulsion and life support technologies, there is a growing need to perform analytical measurements and laboratory experiments across diverse domains of science, while keeping low payload requirements. In this context, lab-on-a-chip nanobiosensors appear to be an emerging technology capable of revolutionizing space exploration, given their low footprint, high accuracy, and low payload requirements. To date, only some approaches for monitoring astronaut health in spacecraft environments have been reported. Although non-invasive molecular diagnostics, like lab-on-a-chip technology, are expected to improve the quality of long-term space missions, their application to monitor microbiological and environmental variables is rarely reported, even for analogous extreme environments on Earth. The possibility of evaluating the occurrence of unknown or unexpected species, identifying redox gradients relevant to microbial metabolism, or testing for specific possible biosignatures, will play a key role in the future of space microbiology. In this review, we will examine the current and potential roles of lab-on-a-chip technology in space exploration and in extreme environment investigation, reporting what has been tested so far, and clarifying the direction toward which the newly developed technologies of portable lab-on-a-chip sensors are heading for exploration in extreme environments and in space.

Oxidoreductases and metal cofactors in the functioning of the earth.

Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic-protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles.

Surface Bacterioplankton Community Structure Crossing the Antarctic Circumpolar Current Fronts.

The Antarctic Circumpolar Current (ACC) is the major current in the Southern Ocean, isolating the warm stratified subtropical waters from the more homogeneous cold polar waters. The ACC flows from west to east around Antarctica and generates an overturning circulation by fostering deep-cold water upwelling and the formation of new water masses, thus affecting the Earth's heat balance and the global distribution of carbon. The ACC is characterized by several water mass boundaries or fronts, known as the Subtropical Front (STF), Subantarctic Front (SAF), Polar Front (PF), and South Antarctic Circumpolar Current Front (SACCF), identified by typical physical and chemical properties. While the physical characteristics of these fronts have been characterized, there is still poor information regarding the microbial diversity of this area. Here we present the surface water bacterioplankton community structure based on 16S rRNA sequencing from 13 stations sampled in 2017 between New Zealand to the Ross Sea crossing the ACC Fronts. Our results show a distinct succession in the dominant bacterial phylotypes present in the different water masses and suggest a strong role of sea surface temperatures and the availability of Carbon and Nitrogen in controlling community composition. This work represents an important baseline for future studies on the response of Southern Ocean epipelagic microbial communities to climate change.

Genome analysis of a new biosurfactants source: The Antarctic bacterium Psychrobacter sp. TAE2020.

Biosurfactants are considered a possible green alternative to chemical surfactants for countless commercial products including detergents and cleaners, personal care products, cosmetics, pharmaceuticals and therapeutics, food additives, emulsifiers, and dispersants for bioremediation. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants. In this study, we report the genome analysis of Psychrobacter sp. TAE2020, an aerobic Æ´-proteobacterium isolated from an Antarctic coastal seawater sample collected in the vicinity of the French Antarctic station Dumont d'Urville, Terre Adelie (66°40' S; 140° 01' E) which has been shown to produce biosurfactants. Biochemical assays indicate that Psychrobacter sp. TAE2020 can produce one or more excellent emulsifiers and a biosurfactant which is able to reduce the surface tension of a Gut medium. Next generation sequencing and genome mining allowed the identification of a plethora of biosynthetic gene clusters possibly involved in the production of emulsifying agents, just waiting to be isolated and characterized. This study paves the way for a more thorough investigation into the potential biotechnological applications of this new Antarctic strain.

Bacteriophage-Resistant Salmonella rissen: An In Vitro Mitigated Inflammatory Response.

Non-typhoid Salmonella (NTS) represents one of the major causes of foodborne diseases, which are made worse by the increasing emergence of antibiotic resistance. Thus, NTS are a significant and common public health concern. The purpose of this study is to investigate whether selection for phage-resistance alters bacterial phenotype, making this approach suitable for candidate vaccine preparation. We therefore compared two strains of Salmonella enterica serovar Rissen: RR (the phage-resistant strain) and RW (the phage-sensitive strain) in order to investigate a potential cost associated with the bacterium virulence. We tested the ability of both RR and RW to infect phagocytic and non-phagocytic cell lines, the activity of virulence factors associated with the main Type-3 secretory system (T3SS), as well as the canonic inflammatory mediators. The mutant RR strain-compared to the wildtype RW strain-induced in the host a weaker innate immune response. We suggest that the mitigated inflammatory response very likely is due to structural modifications of the lipopolysaccharide (LPS). Our results indicate that phage-resistance might be exploited as a means for the development of LPS-based antibacterial vaccines.

Whole-genome sequencing of Pseudomonas sp. TAE6080, a strain capable of inhibiting Staphylococcus epidermidis biofilm.

Antarctic bacteria are able to survive under extreme environmental conditions and have adapted to exploit some of the most ephemeral nutrient pockets. Importantly, such strains have been often shown to be capable of synthesizing compounds of valuable biotechnological importance. Here we show that Pseudomonas sp. TAE6080, a possibly new bacterium isolated in 1994 during water column samplings near the French Antarctic station Dumont d'Urville, is capable of inhibiting the formation of Staphylococcus epidermidis biofilm, known to be an important opportunistic pathogen in infections associated to medical devices. A better understanding of this bacterium can therefore provide useful insight on new bioactive molecules that could play a role against chronic infections. To this end, the anti-biofilm effect of cell-free supernatant of Pseudomonas sp. TAE6080 was evaluated on S. epidermidis RP62A biofilm formation, demonstrating that it significantly reduced its aggregation. Furthermore, genome sequencing, assembly and mining revealed a plethora of putative biosynthetic gene clusters that might be involved in biofilm disruption. The experimental and genomic data presented here open the venue to further investigations on the molecular basis underlying biofilm inhibition.

Development of anti-bacterial surfaces using a hydrophobin chimeric protein.

The search for new approaches for developing antimicrobial surfaces is a challenge of great urgency to prevent and control microbial growth on surfaces. The strategy herein proposed relies on the design of a new, simple and general tool for creating antimicrobial surfaces, based on a hydrophobin chimeric protein which fuses the adhesive self-assembling class I hydrophobin Vmh2 from Pleurotus ostreatus to the human antimicrobial peptide LL-37. The recombinant LL37-Vmh2 protein displayed both the adhesive and the antimicrobic properties of its members, and when deposited on polystyrene surface, a positive effect due to the fusion was observed in terms of both efficacy and versatility of the coating. Indeed, the chimeric protein significantly enlarges the range of pathogens affected by Vmh2 layer rendering it able to inhibit three Gram-positive and two Gram-negative pathogens, selected among the renowned biofilm producer bacteria. Confocal Laser Scanning Microscopy analysis performed on Staphylococcus epidermidis biofilms formed on coated surfaces proved that, besides inhibiting biofilm formation, the LL37-Vmh2 coating also displayed biocidal activity, since dead cells were present in the biofilm layer. The reported results open new perspectives in various fields of application of LL37, and of antimicrobial peptides in general. LL37-Vmh2 increases the inventory of chimeric hydrophobins, further proving their effectiveness and versatility in surface functionalization.

Antibiofilm Activity of a Trichoderma Metabolite against Xanthomonas campestris pv. campestris, Alone and in Association with a Phage.

Biofilm protects bacteria against the host's immune system and adverse environmental conditions. Several studies highlight the efficacy of lytic phages in the prevention and eradication of bacterial biofilms. In this study, the lytic activity of Xccφ1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-α-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a φHA6PP complex, was investigated using confocal laser microscopy under static and dynamic conditions. Additionally, the mechanism used by the complex to modulate the genes (rpf, gumB, clp and manA) involved in the biofilm formation and stability was also studied. Our results demonstrated that Xccφ1, alone or in combination with 6PP and HA, interfered with the gene pathways involved in the formation of biofilm. This approach can be used as a model for other biofilm-producing bacteria.

Pentadecanal and pentadecanoic acid coatings reduce biofilm formation of Staphylococcus epidermidis on PDMS.

Staphylococcus epidermidis is well known to be one of the major causes of infections related to medical devices, mostly due to its strong capacity to form device-associated biofilms. Nowadays, these infections represent a severe burden to the public health system and the necessity of novel antibacterial strategies for the treatment of these difficult-to-eradicate infections is urgent. The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was found to be able to produce an anti-biofilm molecule, the pentadecanal, active against S. epidermidis. In this work, we modified one of the most widely used silicone-based polymers, polydimethylsiloxane (PDMS), by adsorption of pentadecanal and its most promising derivative, pentadecanoic acid, on the PDMS surface. The biofilm formation of S. epidermidis RP62A on both untreated and modified PDMS was performed in a parallel plate flow chamber system, demonstrating the capability of the proposed anti-biofilm coatings to strongly reduce the biofilm formation. Furthermore, drug-release capacity and long-term efficacy (21 days) were also proven for the pentadecanoic acid coating.

The Union Is Strength: The Synergic Action of Long Fatty Acids and a Bacteriophage against Xanthomonas campestris Biofilm.

Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xccφ1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored.

Environmental conditions shape the biofilm of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.

Cold-adapted bacterial extracts as a source of anti-infective and antimicrobial compounds against Staphylococcus aureus.

Aim: The dramatic emergence of antibiotic resistance has directed the interest of research toward the discovery of novel antimicrobial molecules. In this context, cold-adapted marine bacteria living in polar regions represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire. The aim of this work was to identify new antimicrobials and/or antibiofilm molecules produced by cold-adapted bacteria. Materials & methods: Organic extracts obtained from polar marine bacteria were tested against Staphylococcus aureus. Most promising samples were subjected to suitable purification strategies. Results: Results obtained led to the identification of a novel lipopeptide able to effectively inhibit the biofilm formation of S. aureus. Conclusion: New lipopeptide may be potentially useful in a wide variety of biotechnological and medical applications.

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.