Sustainable use of marine biodiversity as source of novel anti-biofilm agents in industrial and clinical settings

Amongst marine bacteria, cold-adapted microorganisms represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire able to synthesize a broad range of potentially valuable bioactive compounds, including antimicrobial activity. The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics. One of the main causes of antibiotic resistance is the capability of microorganisms to associate into communities of cells called biofilms. These complex structures provide protection from potential stressors, including the lack of water, high or low pH, or the presence of substances toxic to microorganisms such as antibiotics, antimicrobials or heavy metals. Therefore, coordinated efforts to implement the arsenal of novel anti-infective treatments are greatly needed. In this contest, my PhD project aimed to the sustainable exploitation of Polar marine biodiversity in an attempt to find viable sources of novel anti-biofilm agents, in particular acting against Staphylococcus epidermidis, one of the most common causes of infections associated with medical devices. In detail, during the first part of my project, I focused on the study of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 and of its ability to produce anti-biofilm molecules, then, on the purification, identification and characterization of the active molecule produced. By setting up of a strategy for the large scale biofilm cultivation of the Antarctic bacterium, the production yield of P. haloplanktis TAC125 anti-biofilm agent was improved, so as to allow the purification and the identification of the active molecule, the pentadecanal. However, as the pentadecanal is a chemically reactive agent, it could easily undergo oxidation reactions, therefore it could not be suitable for all possible anti-biofilm strategies. Therefore, some chemical analogues were synthesized and characterized for their anti-biofilm activity and their possible use in combination with antibiotics were investigated. Then, as a possible clinical application, an anti-biofilm coating system, active against S. epidermidis, was developed, by physical adsorption of pentadecanal and its analogues on polydimethylsiloxane (PDMS), a silicon-based material commonly used for the manufacturing of medical devices. Finally, some physiological studies were dedicated to P. haloplanktis TAC125 biofilm formation in relation with environmental adaptations, with the purpose to explore the potentiality of P. haloplanktis TAC125 in biotechnological field. In the second part of my PhD project, given their only partially explored potential, I have also studied other Polar bacteria belonging to different genera, looking for novel anti-biofilm agents against S. epidermidis. Through the screening of small metabolites and proteins/peptides libraries designed starting from planktonic cultures of Polar bacteria, some promising producer strains were identified and their anti-biofilm activities were characterized. Preliminary purification protocols were set up for each kind of molecules, according to their physico-chemical characteristics. Further studies are still ongoing to identify the structure of the active molecules

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

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

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

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

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

Bacteriophage-Resistant <i>Salmonella rissen</i>: 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

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

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

Biofilm protects bacteria against the host&rsquo;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 Xcc&phi;1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-&alpha;-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a &phi;HA6PP 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 Xcc&phi;1, 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