Marine-Derived Quorum-Sensing Inhibitory Activities Enhance the Antibacterial Efficacy of Tobramycin against Pseudomonas aeruginosa

Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (−63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (−33%). KS8 supernatant also caused a 0.97-log reduction (−89%) and a 2-log reduction (−99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments.     

Exploring Chemical Diversity of Phorbas Sponges as a Source of Novel Lead Compounds in Drug Discovery

Porifera, commonly referred to as marine sponges, are acknowledged as major producers of marine natural products (MNPs). Sponges of the genus Phorbas have attracted much attention over the years. They are widespread in all continents, and several structurally unique compounds have been identified from this species. Terpenes, mainly sesterterpenoids, are the major secondary metabolites isolated from Phorbas species, even though several alkaloids and steroids have also been reported. Many of these compounds have presented interesting biological activities. Particularly, Phorbas sponges have been demonstrated to be a source of cytotoxic metabolites. In addition, MNPs exhibiting cytostatic, antimicrobial, and anti-inflammatory activities have been isolated and structurally characterized. This review provides an overview of almost 130 secondary metabolites from Phorbas sponges and their biological activities, and it covers the literature since the first study published in 1993 until November 2021, including approximately 60 records. The synthetic routes to the most interesting compounds are briefly outlined.     

Assessment of the Antimicrobial Activity of Algae Extracts on Bacteria Responsible of External Otitis

External otitis is a diffuse inflammation around the external auditory canal and auricle, which is often occurred by microbial infection. This disease is generally treated using antibiotics, but the frequent occurrence of antibiotic resistance requires the development of new antibiotic agents. In this context, unexplored bioactive natural candidates could be a chance for the production of targeted drugs provided with antimicrobial activity. In this paper, microbial pathogens were isolated from patients with external otitis using ear swabs for over one year, and the antimicrobial activity of the two methanol extracts from selected marine (Dunaliella salina) and freshwater (Pseudokirchneriella subcapitata) microalgae was tested on the isolated pathogens. Totally, 114 bacterial and 11 fungal strains were isolated, of which Staphylococcus spp. (28.8%) and Pseudomonas aeruginosa (P. aeruginosa) (24.8%) were the major pathogens. Only three Staphylococcus aureus (S. aureus) strains and 11 coagulase-negative Staphylococci showed resistance to methicillin. The two algal extracts showed interesting antimicrobial properties, which mostly inhibited the growth of isolated S. aureus, P. aeruginosa, Escherichia coli, and Klebsiella spp. with MICs range of 1.4 × 10⁹ to 2.2 × 10¹⁰ cells/mL. These results suggest that the two algae have potential as resources for the development of antimicrobial agents.     

Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence^® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence^® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.     

Chemical Diversity and Biological Properties of Secondary Metabolites from Sea Hares of Aplysia Genus

The marine environment is an important source of structurally-diverse and biologically-active secondary metabolites. During the last two decades, thousands of compounds were discovered in marine organisms, several of them having inspired the development of new classes of therapeutic agents. Marine mollusks constitute a successful phyla in the discovery of new marine natural products (MNPs). Over a 50-year period from 1963, 116 genera of mollusks contributed innumerous compounds, Aplysia being the most studied genus by MNP chemists. This genus includes 36 valid species and should be distinguished from all mollusks as it yielded numerous new natural products. Aplysia sea hares are herbivorous mollusks, which have been proven to be a rich source of secondary metabolites, mostly of dietary origin. The majority of secondary metabolites isolated from sea hares of the genus Aplysia are halogenated terpenes; however, these animals are also a source of compounds from other chemical classes, such as macrolides, sterols and alkaloids, often exhibiting cytotoxic, antibacterial, antifungal, antiviral and/or antifeedant activities. This review focuses on the diverse structural classes of secondary metabolites found in Aplysia spp., including several compounds with pronounced biological properties.     

Marine-Source Quorum Quenching Enzyme YtnP to Improve Hygiene Quality in Dental Units

Biofilm in dental unit water lines may pose a health risk to patients and dental practitioners. An AdiC-like quorum quenching enzyme, YtnP, was cloned from a deep-sea probiotic Bacillus velezensis, and heterologously expressed in E. coli to examine the application on the improvement of hygiene problems caused by biofilm infection of Pseudomonas aeruginosa in dental units. Pseudomonas bacteria were isolated from dental chair units and used to grow static biofilms in the laboratory. A water filter system was designed to test the antifouling activity of YtnP in Laboratory, to simulate the biofilm contamination on water filter in dental unit water lines. The results demonstrated that the enzyme of YtnP was able to degrade the N-acyl homoserine lactones, significantly inhibited the EPS generation, biofilm formation, and virulence factors production (pyocyanin and rhamnolipid) of P. aeruginosa, and was efficient on the antifouling against P. aeruginosa. The findings in this study indicated the possibility of YtnP as novel disinfectant reagent for hygiene treatment in dental units.     

5-Episinuleptolide Decreases the Expression of the Extracellular Matrix in Early Biofilm Formation of Multi-Drug Resistant Acinetobacter baumannii

Nosocomial infections and increasing multi-drug resistance caused by Acinetobacter baumannii have been recognized as emerging problems worldwide. Moreover, A. baumannii is able to colonize various abiotic materials and medical devices, making it difficult to eradicate and leading to ventilator-associated pneumonia, and bacteremia. Development of novel molecules that inhibit bacterial biofilm formation may be an alternative prophylactic option for the treatment of biofilm-associated A. baumannii infections. Marine environments, which are unlike their terrestrial counterparts, harbor an abundant biodiversity of marine organisms that produce novel bioactive natural products with pharmaceutical potential. In this study, we identified 5-episinuleptolide, which was isolated from Sinularia leptoclados, as an inhibitor of biofilm formation in ATCC 19606 and three multi-drug resistant A. baumannii strains. In addition, the anti-biofilm activities of 5-episinuleptolide were observed for Gram-negative bacteria but not for Gram-positive bacteria, indicating that the inhibition mechanism of 5-episinuleptolide is effective against only Gram-negative bacteria. The mechanism of biofilm inhibition was demonstrated to correlate to decreased gene expression from the pgaABCD locus, which encodes the extracellular polysaccharide poly-β-(1,6)-N-acetylglucosamine (PNAG). Scanning electron microscopy (SEM) indicated that extracellular matrix of the biofilm was dramatically decreased by treatment with 5-episinuleptolide. Our study showed potentially synergistic activity of combination therapy with 5-episinuleptolide and levofloxacin against biofilm formation and biofilm cells. These data indicate that inhibition of biofilm formation via 5-episinuleptolide may represent another prophylactic option for solving the persistent problem of biofilm-associated A. baumannii infections.     

Anti-Microbial,Anti-Biofilm Activities and Cell Selectivity of the NRC-16 Peptide Derived from Witch Flounder,Glyptocephalus cynoglossus

Previous studies had identified novel antimicrobial peptides derived from witch flounder. In this work, we extended the search for the activity of peptide that showed antibacterial activity on clinically isolated bacterial cells and bacterial biofilm. Pseudomonas aeruginosa was obtained from otitis media and cholelithiasis patients, while Staphylococcus aureus was isolated from otitis media patients. We found that synthetic peptide NRC-16 displays antimicrobial activity and is not sensitive to salt during its bactericidal activity. Interestingly, this peptide also led to significant inhibition of biofilm formation at a concentration of 4–16 μM. NRC-16 peptide is able to block biofilm formation at concentrations just above its minimum inhibitory concentration while conventional antibiotics did not inhibit the biofilm formation except ciprofloxacin and piperacillin. It did not cause significant lysis of human RBC, and is not cytotoxic to HaCaT cells and RAW264.7 cells, thereby indicating its selective antimicrobial activity. In addition, the peptide’s binding and permeation activities were assessed by tryptophan fluorescence, calcein leakage and circular dichroism using model mammalian membranes composed of phosphatidylcholine (PC), PC/cholesterol (CH) and PC/sphingomyelin (SM). These experiments confirmed that NRC-16 does not interact with any of the liposomes but the control peptide melittin did. Taken together, we found that NRC-16 has potent antimicrobial and antibiofilm activities with less cytotoxicity, and thus can be considered for treatment of microbial infection in the future.     

Phloroglucinol-Gold and -Zinc Oxide Nanoparticles: Antibiofilm and Antivirulence Activities towards Pseudomonas aeruginosa PAO1

With the advancement of nanotechnology, several nanoparticles have been synthesized as antimicrobial agents by utilizing biologically derived materials. In most cases, the materials used for the synthesis of nanoparticles from natural sources are extracts. Natural extracts contain a wide range of bioactive components, making it difficult to pinpoint the exact component responsible for nanoparticle synthesis. Furthermore, the bioactive component present in the extract changes according to numerous environmental factors. As a result, the current work intended to synthesize gold (AuNPs) and zinc oxide (ZnONPs) nanoparticles using pure phloroglucinol (PG). The synthesized PG-AuNPs and PG-ZnONPs were characterized using a UV–Vis absorption spectrophotometer, FTIR, DLS, FE-TEM, zeta potential, EDS, and energy-dispersive X-ray diffraction. The characterized PG-AuNPs and PG-ZnONPs have been employed to combat the pathogenesis of Pseudomonas aeruginosa. P. aeruginosa is recognized as one of the most prevalent pathogens responsible for the common cause of nosocomial infection in humans. Antimicrobial resistance in P. aeruginosa has been linked to the development of recalcitrant phenotypic characteristics, such as biofilm, which has been identified as one of the major obstacles to antimicrobial therapy. Furthermore, P. aeruginosa generates various virulence factors that are a major cause of chronic infection. These PG-AuNPs and PG-ZnONPs significantly inhibit early stage biofilm and eradicate mature biofilm. Furthermore, these NPs reduce P. aeruginosa virulence factors such as pyoverdine, pyocyanin, protease, rhamnolipid, and hemolytic capabilities. In addition, these NPs significantly reduce P. aeruginosa swarming, swimming, and twitching motility. PG-AuNPs and PG-ZnONPs can be used as control agents for infections caused by the biofilm-forming human pathogenic bacterium P. aeruginosa.     

Characterisation of Non-Autoinducing Tropodithietic Acid (TDA) Production from Marine Sponge Pseudovibrio Species

The search for new antimicrobial compounds has gained added momentum in recent years, paralleled by the exponential rise in resistance to most known classes of current antibiotics. While modifications of existing drugs have brought some limited clinical success, there remains a critical need for new classes of antimicrobial compound to which key clinical pathogens will be naive. This has provided the context and impetus to marine biodiscovery programmes that seek to isolate and characterize new activities from the aquatic ecosystem. One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule.     

Cloning,Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A (exo A) and killed whole cell. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin (fliC) fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections. Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Key Words: Pseudomonas aeruginosa, Exotoxin A (exoA), Flagellin (fliC), Vaccines     

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P. aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.     

Anti-Quorum-Sensing Activity of Tryptophan-Containing Cyclic Dipeptides

Quorum sensing (QS) can regulate the pathogenicity of bacteria and the production of some virulence factors. It is a promising target for screening to find anti-virulence agents in the coming post-antibiotics era. Cyclo (L-Trp-L-Ser), one variety of cyclic dipeptides (CDPs), isolated from a marine bacterium Rheinheimera aquimaris, exhibited anti-QS activity against Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1. Unlike the CDPs composed of phenylalanine or tyrosine, the anti-QS activity has been widely studied; however, cyclo (L-Trp-L-Ser) and derivatives, containing one tryptophan unit and one non-aromatic amino acid, have not been systematically explored. Herein, the cyclo (L-Trp-L-Ser) and seven derivatives were synthesized and evaluated. All tryptophane-contained CDPs were able to decrease the production of violacein in C. violaceum CV026 and predicted as binding within the same pocket of receptor protein CviR, but in lower binding energy compared with the natural ligand C₆HSL. As for P. aeruginosa PAO1, owning more complicated QS systems, these CDPs also exhibited inhibitory effects on pyocyanin production, swimming motility, biofilm formation, and adhesion. These investigations suggested a promising way to keep the tryptophan untouched and make modifications on the non-aromatic unit to increase the anti-QS activity and decrease the cytotoxicity, thus developing a novel CDP-based anti-virulence agent.     

New Antimicrobial Bromotyrosine Analogues from the Sponge Pseudoceratina purpurea and Its Predator Tylodina corticalis

Bioassay-guided fractionation of extracts from temperate Australian collections of the marine sponge Pseudoceratina purpurea resulted in the isolation and characterisation of two new and six known bromotyrosine-derived alkaloids with antibiotic activity. Surprisingly, a single specimen of the mollusc Tylodina corticalis, which was collected while feeding on P. purpurea, contained only a few of the compounds found in the sponge suggesting selective accumulation and chemical modification of sponge metabolites.     

Metabolites from Marine-Derived Fungi as Potential Antimicrobial Adjuvants

Marine-derived fungi constitute an interesting source of bioactive compounds, several of which exhibit antibacterial activity. These acquire special importance, considering that antimicrobial resistance is becoming more widespread. The overexpression of efflux pumps, capable of expelling antimicrobials out of bacterial cells, is one of the most worrisome mechanisms. There has been an ongoing effort to find not only new antimicrobials, but also compounds that can block resistance mechanisms which can be used in combination with approved antimicrobial drugs. In this work, a library of nineteen marine natural products, isolated from marine-derived fungi of the genera Neosartorya and Aspergillus, was evaluated for their potential as bacterial efflux pump inhibitors as well as the antimicrobial-related mechanisms, such as inhibition of biofilm formation and quorum-sensing. Docking studies were performed to predict their efflux pump action. These compounds were also tested for their cytotoxicity in mouse fibroblast cell line NIH/3T3. The results obtained suggest that the marine-derived fungal metabolites are a promising source of compounds with potential to revert antimicrobial resistance and serve as an inspiration for the synthesis of new antimicrobial drugs.     

Recent Updates on Marine Cancer-Preventive Compounds

The natural compounds derived from marine organisms often exhibit unique chemical structures and potent biological activities. Cancer-preventive activity is one of the rather new activities that has emerged and been extensively studied over the last decades. This review summarizes the recent updates on the marine chemopreventive compounds covering the relevant literature published in 2013–2021 and following the previous comprehensive review by Stonik and Fedorov (Marine Drugs 2014, 12, 636–671). In the current article, only the molecules having an effect on malignant transformation (or related pathway and molecules), cancer stem cells, or carcinogen-induced in vivo tumor development were considered to be “true” cancer-preventive compounds and were, therefore, reviewed. Additionally, particular attention has been given to the molecular mechanisms of chemoprevention, executed by the reported marine compounds.     

Adding Zooplankton to the OSMAC Toolkit: Effect of Grazing Stress on the Metabolic Profile and Bioactivity of a Diatom

“One strain many compounds” (OSMAC) based approaches have been widely used in the search for bioactive compounds. Introducing stress factors like nutrient limitation, UV-light or cocultivation with competing organisms has successfully been used in prokaryote cultivation. It is known that diatom physiology is affected by changed cultivation conditions such as temperature, nutrient concentration and light conditions. Cocultivation, though, is less explored. Hence, we wanted to investigate whether grazing pressure can affect the metabolome of the marine diatom Porosira glacialis, and if the stress reaction could be detected as changes in bioactivity. P. glacialis cultures were mass cultivated in large volume bioreactor (6000 L), first as a monoculture and then as a coculture with live zooplankton. Extracts of the diatom biomass were screened in a selection of bioactivity assays: inhibition of biofilm formation, antibacterial and cell viability assay on human cells. Bioactivity was found in all bioassays performed. The viability assay towards normal lung fibroblasts revealed that P. glacialis had higher bioactivity when cocultivated with zooplankton than in monoculture. Cocultivation with diatoms had no noticeable effect on the activity against biofilm formation or bacterial growth. The metabolic profiles were analyzed showing the differences in diatom metabolomes between the two culture conditions. The experiment demonstrates that grazing stress affects the biochemistry of P. glacialis and thus represents a potential tool in the OSMAC toolkit.     

Anthraquinones,Diphenyl Ethers,and Their Derivatives from the Culture of the Marine Sponge-Associated Fungus Neosartorya spinosa KUFA 1047

Previously unreported anthraquinone, acetylpenipurdin A (4), biphenyl ether, neospinosic acid (6), dibenzodioxepinone, and spinolactone (7) were isolated, together with (R)-6-hydroxymellein (1), penipurdin A (2), acetylquestinol (3), tenellic acid C (5), and vermixocin A (8) from the culture of a marine sponge-associated fungus Neosartorya spinosa KUFA1047. The structures of the previously unreported compounds were established based on an extensive analysis of 1D and 2D NMR spectra as well as HRMS data. The absolute configurations of the stereogenic centers of 5 and 7 were established unambiguously by comparing their calculated and experimental electronic circular dichroism (ECD) spectra. Compounds 2 and 5–8 were tested for their in vitro acetylcholinesterase and tyrosinase inhibitory activities as well as their antibacterial activity against Gram-positive and Gram-negative reference, and multidrug-resistant strains isolated from the environment. The tested compounds were also evaluated for their capacity to inhibit biofilm formation in the reference strains.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.