Antibiofilm Activity of the Brown Alga Halidrys siliquosa against Clinically Relevant Human Pathogens

The marine brown alga Halidrys siliquosa is known to produce compounds with antifouling activity against several marine bacteria. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of organic extracts obtained from the marine brown alga H. siliquosa against a focused panel of clinically relevant human pathogens commonly associated with biofilm-related infections. The partially fractionated methanolic extract obtained from H. siliquosa collected along the shores of Co. Donegal; Ireland; displayed antimicrobial activity against bacteria of the genus Staphylococcus; Streptococcus; Enterococcus; Pseudomonas; Stenotrophomonas; and Chromobacterium with MIC and MBC values ranging from 0.0391 to 5 mg/mL. Biofilms of S. aureus MRSA were found to be susceptible to the algal methanolic extract with MBEC values ranging from 1.25 mg/mL to 5 mg/mL respectively. Confocal laser scanning microscopy using LIVE/DEAD staining confirmed the antimicrobial nature of the antibiofilm activity observed using the MBEC assay. A bioassay-guided fractionation method was developed yielding 10 active fractions from which to perform purification and structural elucidation of clinically-relevant antibiofilm compounds.     

Pachydictyols B and C: New Diterpenes from Dictyota dichotoma Hudson

Two new diterpenoids, pachydictyol B (1a/1b) and C (2), were isolated from the dichloromethane extract of the marine brown alga, Dictyota dichotoma, collected from the Red Sea coast of Egypt, along with the known metabolites, pachydictyol A (3a), dictyol E (4), cis-africanan-1α-ol (5a), fucosterol (6), tetrahydrothiophen-1,1-dioxide and poly-β-hydroxybutyric acid. GC-MS analysis of the nonpolar fractions also indicated the presence of β-bourbonene and nonanal, along with three hydrocarbons and five fatty acids or their simple derivatives, respectively. GC-MS analysis of the unsaponifiable algal petroleum ether extract revealed the presence of a further eight compounds, among them 2,2,6,7-tetramethyl-10-oxatricyclo[4.3.0.1(1,7)]decan-5-one (7), N-(4-bromo-n-butyl)-piperidin-2-one (8) and tert-hexadecanethiol. Structures 1–6 were assigned by 1D and 2D NMR, mass spectra (EI, CI, HREI and HRESI) and by comparison with data from related structures. The crude algal extract was potently active against the breast carcinoma tumor cell line, MCF7 (IC₅₀ = 0.6 µg mL⁻¹); pachydictyol B (1a) and dictyol E (4) showed weak antimicrobial properties, and the other compounds were inactive. Pachydictyols B (1a) and C (2) demonstrated a weak and unselective cytotoxicity against twelve human tumor cell lines with a mean IC₅₀ of >30.0 µM.     

Valorization of Sargassum muticum Biomass According to the Biorefinery Concept

The biorefinery concept integrates processes and technologies for an efficient biomass conversion using all components of a feedstock. Sargassum muticum is an invasive brown algae which could be regarded as a renewable resource susceptible of individual valorization of the constituent fractions into high added-value compounds. Microwave drying technology can be proposed before conventional ethanol extraction of algal biomass, and supercritical fluid extraction with CO₂ was useful to extract fucoxanthin and for the fractionation of crude ethanol extracts. Hydrothermal processing is proposed to fractionate the algal biomass and to solubilize the fucoidan and phlorotannin fractions. Membrane technology was proposed to concentrate these fractions and obtain salt- and arsenic-free saccharidic fractions. Based on these technologies, this study presents a multipurpose process to obtain six different products with potential applications for nutraceutical, cosmetic and pharmaceutical industries.     

Tasco(®), a product of Ascophyllum nodosum, imparts thermal stress tolerance in Caenorhabditis elegans

Tasco^®, a commercial product manufactured from the brown alga Ascophyllum nodosum, has been shown to impart thermal stress tolerance in animals. We investigated the physiological, biochemical and molecular bases of this induced thermal stress tolerance using the invertebrate animal model, Caenorhabiditis elegans. Tasco^® water extract (TWE) at 300 μg/mL significantly enhanced thermal stress tolerance as well as extended the life span of C. elegans. The mean survival rate of the model animals under thermal stress (35 °C) treated with 300 μg/mL and 600 μg/mL TWE, respectively, was 68% and 71% higher than the control animals. However, the TWE treatments did not affect the nematode body length, fertility or the cellular localization of daf-16. On the contrary, TWE under thermal stress significantly increased the pharyngeal pumping rate in treated animals compared to the control. Treatment with TWE also showed differential protein expression profiles over control following 2D gel-electrophoresis analysis. Furthermore, TWE significantly altered the expression of at least 40 proteins under thermal stress; among these proteins 34 were up-regulated while six were down-regulated. Mass spectroscopy analysis of the proteins altered by TWE treatment revealed that these proteins were related to heat stress tolerance, energy metabolism and a muscle structure related protein. Among them heat shock proteins, superoxide dismutase, glutathione peroxidase, aldehyde dehydrogenase, saposin-like proteins 20, myosin regulatory light chain 1, cytochrome c oxidase RAS-like, GTP-binding protein RHO A, OS were significantly up-regulated, while eukaryotic translation initiation factor 5A-1 OS, 60S ribosomal protein L18 OS, peroxiredoxin protein 2 were down regulated by TWE treatment. These results were further validated by gene expression and reporter gene expression analyses. Overall results indicate that the water soluble components of Tasco^® imparted thermal stress tolerance in the C. elegans by altering stress related biochemical pathways.     

Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.     

Cephalopods as Vectors of Harmful Algal Bloom Toxins in Marine Food Webs

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These mollusks have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg⁻¹ and palytoxin reaching 115 μg kg⁻¹ (the regulatory limit for PlTXs is 30 μg kg⁻¹ in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area.     

The Effect of Sulfated (1→3)-α-L-Fucan from the Brown Alga Saccharina cichorioides Miyabe on Resveratrol-Induced Apoptosis in Colon Carcinoma Cells

Accumulating data clearly indicate that the induction of apoptosis by nontoxic natural compounds is a potent defense against the development and progression of many malignancies, including colon cancer. Resveratrol and the fucoidans have been shown to possess potent anti-tumor activity in vitro and in vivo. The aim of the present study was to examine whether the combination of a fucoidan from the brown alga Saccharina cichorioides Miyabe and resveratrol would be an effective preventive and/or therapeutic strategy against colon cancer. Based on NMR spectroscopy and MALDI-TOF analysis, the fucoidan isolated and purified from Saccharina cichorioides Miyabe was (1→3)-α-L-fucan with sulfate groups at C2 and C4 of the α-L-fucopyranose residues. The fucoidan enhanced the antiproliferative activity of resveratrol at nontoxic doses and facilitated resveratrol-induced apoptosis in the HCT 116 human colon cancer cell line. Apoptosis was realized by the activation of initiator caspase-9 and effector caspase-7 and -3, followed by the cleavage of PARP. Furthermore, significant inhibition of HCT 116 colony formation was associated with the sensitization of cells to resveratrol by the fucoidan. Taken together, these results demonstrate that the combination of the algal fucoidan with resveratrol may provide a potential therapy against human colon cancer.     

Fucoxanthin and Its Metabolites in Edible Brown Algae Cultivated in Deep Seawater

Three metabolites of fucoxanthin were isolated from a brown alga, Scytosiphon lomentaria, and the structure of a new compound was determined by NMR. The content of fucoxanthin, a biologically active carotenoid, in four edible brown algae, cultivated in deep seawater, was studied.     

Gene Sequence Based Clustering Assists in Dereplication of Pseudoalteromonas luteoviolacea Strains with Identical Inhibitory Activity and Antibiotic Production

Some microbial species are chemically homogenous, and the same secondary metabolites are found in all strains. In contrast, we previously found that five strains of P. luteoviolacea were closely related by 16S rRNA gene sequence but produced two different antibiotic profiles. The purpose of the present study was to determine whether such bioactivity differences could be linked to genotypes allowing methods from phylogenetic analysis to aid in selection of strains for biodiscovery. Thirteen P. luteoviolacea strains divided into three chemotypes based on production of known antibiotics and four antibacterial profiles based on inhibition assays against Vibrio anguillarum and Staphylococcus aureus. To determine whether chemotype and inhibition profile are reflected by phylogenetic clustering we sequenced 16S rRNA, gyrB and recA genes. Clustering based on 16S rRNA gene sequences alone showed little correlation to chemotypes and inhibition profiles, while clustering based on concatenated 16S rRNA, gyrB, and recA gene sequences resulted in three clusters, two of which uniformly consisted of strains of identical chemotype and inhibition profile. A major time sink in natural products discovery is the effort spent rediscovering known compounds, and this study indicates that phylogeny clustering of bioactive species has the potential to be a useful dereplication tool in biodiscovery efforts.     

Comparative Studies on the Characteristic Fatty Acid Profiles of Four Different Chinese Medicinal Sargassum Seaweeds by GC-MS and Chemometrics

Sargassum seaweeds produce abundant biomass in China and have long been used as herbal medicine and food. Their characteristic fatty acid (FA) profiles and related potential function in promoting cardiovascular health (CVH) have not been systematically investigated. In this study, FA profiles of four medicinal Sargassum were characterized using GC-MS. Principal component analysis was used to discriminate the four medicinal Sargassum, and orthogonal projection to latent structures discriminant analysis was carried out between the two official species HAI ZAO and between the two folk medicine species HAI QIAN. In all of the algae investigated, the major SFA and MUFA were palmitic and stearic acid, respectively, while the major PUFAs were linoleic, arachidonic, and eicosapentaenoic acid. S. fusiforme and S. horneri had higher concentrations of PUFAs. With respect to CVH, all of the studied species, particularly S. fusiforme, exhibited satisfactory levels such as PUFA/SFA ratio and n-6/n-3 ratio. Each species possesses a unique FA profile and is discriminated clearly. Potential key FA markers (between the two Chinese official species, and between the two folk species) are assessed. The study provides characteristic fatty acid profiles of four Chinese medicinal Sargassum and their related potential function in promoting CVH.     

Quorum Sensing Inhibition by Asparagopsis taxiformis,a Marine Macro Alga: Separation of the Compound that Interrupts Bacterial Communication

The majority of the marine algal species, though completing their life cycle in seawater, are rarely susceptible to fouling, making them an important source of quorum sensing (QS) inhibitory substances. The separation and characterization of QS inhibitors are crucial for any potential application. Thirty marine macroalgae were tested for QS inhibition activity by using Chromobacterium violaceum CV026 as the reporter strain, and among them, Asparagopsis taxiformis showed antibacterial, as well as antiquorum, sensing activities. Cinnamaldehyde (75 mM) and methanol were used as positive and negative controls, respectively. The antiquorum sensing activity of A. taxiformis was further confirmed using the sensor strain, Serratia liquefaciens MG44, having green fluorescent protein (gfp). Methanolic extract of the alga was fractionated by solid phase extraction (SPE), and each fraction was tested for QS inhibition. Two types of activities were observed—zone of clearance (antibacterial activity) and zone of inhibition with or without finger-like projections (QS inhibition). Out of five SPE cartridges, Bond Elut PH showed clear separation of these two fractions. The Ion Cyclotron Resonance Fourier Transformation Mass Spectrometer (ICR-FT/MS) analysis of the fractions further supported the bioassay results. The presence of strong QS inhibitory compound in A. taxiformis indicates its potential use in antifouling preparations.     

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.     

Sporiolides A and B,New Cytotoxic Twelve-Membered Macrolides from a Marine-Derived Fungus Cladosporium Species

Two new cytotoxic twelve-membered macrolides, sporiolides A (1) and B (2), were isolated from the cultured broth of a fungus Cladosporium sp., which was separated from an Okinawan marine brown alga Actinotrichia fragilis, and the structures were elucidated by spectroscopic data. Sporiolides A (1) and B (2) exhibited cytotoxicity against murine lymphoma L1210 cells. Spoliolide A (1) showed antifungal activity against Cryptococcus neoformans and Neurospora crassa.     

Diverse bacterial PKS sequences derived from okadaic acid-producing dinoflagellates

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.     

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters,Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m³) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (F_v/F_m) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.     

A Stable-Isotope Mass Spectrometry-Based Metabolic Footprinting Approach to Analyze Exudates from Phytoplankton

Phytoplankton exudates play an important role in pelagic ecology and biogeochemical cycles of elements. Exuded compounds fuel the microbial food web and often encompass bioactive secondary metabolites like sex pheromones, allelochemicals, antibiotics, or feeding attractants that mediate biological interactions. Despite this importance, little is known about the bioactive compounds present in phytoplankton exudates. We report a stable-isotope metabolic footprinting method to characterise exudates from aquatic autotrophs. Exudates from ¹³C-enriched alga were concentrated by solid phase extraction and analysed by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. We used the harmful algal bloom forming dinoflagellate Alexandrium tamarense to prove the method. An algorithm was developed to automatically pinpoint just those metabolites with highly ¹³C-enriched isotope signatures, allowing us to discover algal exudates from the complex seawater background. The stable-isotope pattern (SIP) of the detected metabolites then allowed for more accurate assignment to an empirical formula, a critical first step in their identification. This automated workflow provides an effective way to explore the chemical nature of the solutes exuded from phytoplankton cells and will facilitate the discovery of novel dissolved bioactive compounds.     

Two New Bromophenols with Radical Scavenging Activity from Marine Red Alga Symphyocladia latiuscula

Chemical investigation of a Chinese collection of marine red alga Symphyocladia latiuscula yielded two new highly brominated phenols. The structures of the new compounds were elucidated by detailed spectroscopic analysis, including HRMS, 1D and 2D NMR and MS methods. Compounds 1 and 2 were evaluated for radical scavenging capability by 1,1-diphenyl-2-picrylhydrazuyl (DPPH) radical with the IC₅₀ value of 14.5 and 20.5 μg/mL, respectively.     

Larvicidal Potential of the Halogenated Sesquiterpene (+)-Obtusol,Isolated from the Alga Laurencia dendroidea J. Agardh (Ceramiales: Rhodomelaceae), against the Dengue Vector Mosquito Aedes aegypti (Linnaeus) (Diptera: Culicidae)

Dengue is considered a serious public health problem in many tropical regions of the world including Brazil. At the moment, there is no viable alternative to reduce dengue infections other than controlling the insect vector, Aedes aegypti Linnaeus. In the continuing search for new sources of chemicals targeted at vector control, natural products are a promising alternative to synthetic pesticides. In our work, we investigated the toxicity of a bioactive compound extracted from the red alga Laurencia dendroidea J. Agardh. The initial results demonstrated that crude extracts, at a concentration of 5 ppm, caused pronounced mortality of second instar A. aegypti larvae. Two molecules, identified as (−)-elatol and (+)-obtusol were subsequently isolated from crude extract and further evaluated. Assays with (−)-elatol showed moderate larvicidal activity, whereas (+)-obtusol presented higher toxic activity than (−)-elatol, with a LC₅₀ value of 3.5 ppm. Histological analysis of the larvae exposed to (+)-obtusol revealed damage to the intestinal epithelium. Moreover, (+)-obtusol-treated larvae incubated with 2 µM CM-H₂DCFDA showed the presence of reactive oxygen species, leading us to suggest that epithelial damage might be related to redox imbalance. These results demonstrate the potential of (+)-obtusol as a larvicide for use against A. aegypti and the possible mode of action of this compound.     

Isolation and Structure Elucidation of Three New Dolastanes from the Brown Alga Dilophus spiralis

Three new dolastane diterpenes (1–3) and five previously reported perhydroazulenes were isolated from the organic extracts of the brown alga Dilophus spiralis. The structure elucidation and the assignment of the relative configurations of the isolated natural products were based on extensive analyses of their spectroscopic data, whereas the absolute configuration of metabolite 2 was determined through its chemical conversion to a previously isolated compound of known configuration.     

Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.