Agglutination of human and animal erythrocytes in marine unicellular algae

Ethanol extracts of 12 marine unicellular algae were assayed for agglutinating activity against native and enzyme-treated human and animal erythrocytes. All of the algae assayed agglutinated at least one group of normal erythrocytes from humans. Notably, all algal extracts agglutinated erythrocytes of hemophilia patients arising from coagulation disorders. Meanwhile, all algae had a strong reaction with monkey erythrocytes, but to a lesser extent or not at all with sheep erythrocytes. Both trypsin and pronase improved the detection of most algal agglutinins and caused a drastic increase in hemagglutinating activity after treatment for 2 h or more. However, hemagglutinating activity decreased or disappeared completely when two extracts of different algal species were combined. Journal of Industrial Microbiology & Biotechnology (2000) 24, 262–266. Received 24 September 1999/ Accepted in revised form 06 January 2000     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Screening and preliminary characterization of hemagglutinins in Vietnamese marine algae

Extracts from 44 species of Vietnamese marine algae, including 15 Chlorophyta, 18 Rhodophyta and 11 Phaeophyta species, were examined for hemagglutination activity with a variety of different animal and human erythrocytes that were untreated or treated with enzymes. Almost all extracts showed activity toward at least one type of erythrocytes, although those from three Chlorophyta and two Rhodophyta species showed no hemagglutination with any type of erythrocytes examined. Strong activity was detected in extracts from two Chlorophyta (Anadyomene plicata and Avrainvillea erecta) and four Rhodophyta species (Gracilaria eucheumatoides, Gracilaria salicornia, Kappaphycus alvarezii, and Kappaphycus striatum) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of seven Chlorophyta and eight Rhodophyta species were examined for sugar-binding specificity, pH- and temperature-stability, and divalent cation-independency of hemagglutination using ammonium sulfate-precipitates prepared from their extracts. In a hemagglutination-inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides, except the Codium arabicum and Gracilaria euchematoides hemagglutinins, whose activities were inhibited by both N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. On the other hand, all of the hemagglutinins activities were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The activities of these algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations. These results indicate that Vietnamese marine algae are a good source of novel and useful lectins.     

A new screening for hemagglutinins from Vietnamese marine macroalgae

Aqueous extracts from 42 species of Vietnamese marine macroalgae, including 17 Chlorophyta, 22 Rhodophyta, and three Phaeophyta species, were examined for hemagglutination activity using native and enzyme-treated different animal and human erythrocytes. All extracts agglutinated at least one type of erythrocytes tested. Strong activity was detected in extracts from four Chlorophyta (Caulerpa serulata var. boryana, Caulerpa sertularioides f. longipes, Halimeda velasquezii, and Halimeda discoidea) and two Rhodophyta species (Gelidiella acerosa and Titanophora pulchra) with enzyme-treated rabbit and horse erythrocytes. The hemagglutinins of some active species were examined for sugar-binding specificity, pH, temperature stability, and divalent cation independency using ammonium sulfate precipitates prepared from their extracts. In a hemagglutination–inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides. The activity of the hemagglutinins was inhibited by some glycoproteins tested. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, suggesting the presence of lectins specific for complex N-glycans, high mannose N-glycans or O-glycans. On the other hand, the activities of almost all algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations, except Gelidiopsis scoparia hemagglutinin, its activity was dependent on the presence of divalent cations. These results suggest that Vietnamese marine macroalgae may be good sources of useful lectins for many biological applications.     

Inhibition of algal spore germination by the marine bacterium Pseudoalteromonas tunicata

A collection of 56 bacteria isolated from different surfaces in the marine environment were assayed for their effects on the germination of spores from the common green alga Ulva lactuca. Thirteen bacterial isolates were shown to inhibit spore germination. Of these bacteria, Pseudoalteromonas tunicata displayed the most pronounced effects against algal spores. Further characterisation of the anti-algal activity of P. tunicata was performed and it was found that this bacterium produces an extracellular component with specific activity toward algal spores that is heat-sensitive, polar and between 3 and 10 kDa in size. This biologically active compound was also found to prevent the germination of spores from the red alga Polysiphonia sp. and, given the widespread occurrence of P. tunicata in a range of marine habitats, this may suggest that it is effective against a variety of marine algae.     

Antimicrobial activity of marine organisms collected off the coast of South East India

In vitro antimicrobial screening of nine marine sponges (Porifera) and two seaweeds, collected from south east coast of India, against selected clinical isolates of bacteria and fungi was conducted in this study. Methanolic extracts of all the marine organisms demonstrated activity against one or more of microbes tested. Sigmadocia carnosa was the most active exhibiting a broad spectrum antimicrobial activity against each of the microbe tested with the exception of Fusarium species. Contrary to this, the genus Echinogorgia did not show any detectable bactericidal activity but, Echninogorgia reticulata was weakly fungicidal against Rhodotorula species and E. compecta against Fusarium and Nocardia species. Considerable antibacterial activity was exhibited by Haliclona cribricutis and Chrotella australiensis against Klebsiella species and Vibrio cholerae, respectively. Petrocia testudinaria showed equally good activity against the bacterium V. chlorae and the fungus Cryptococcus neoformans. The sponges Callyspongia fibrosa, Ircinia species and the seaweed Stoecheospermum margilatum are totally inactive against fungi. The extracts showing good antimicrobial activity are undergoing further analysis to identify the active constituents.     

Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.     

Analysis of the agglutinating activity from unicellular algae

Agglutinating activity often varies both between and within the algal species assayed. However, it is difficulty to interpret such variation without further analysis. We report a statistical analysis of agglutinating activities against human, cow, sheep, and pig erythrocytes, using cell extracts from 43 taxa (strains) of freshwater microalgae. Most of the extracts agglutinated erythrocytes from at least one of the sources, but pig erythrocytes appeared to be most suitable for the detection of agglutination reactions. Chlorella cell extracts preferentially agglutinated human erythrocytes, whereas extracts of other taxa were less active against mammalian erythrocytes. Cluster analysis generated four distinct subclusters of taxa, characterized by different specificities for antigens or carbohydrate receptors on the erythrocytes. Principal component analysis further separated the agglutination characteristics of Chlamydomonas from Chlorella on the first two components. Specificity for pig erythrocytes accounted for most of the clustering or grouping of algal taxa in multivariate analysis. However, clustering or grouping patterns of Chlorella species on haemagglutinating activity resembled that based on DNA sequences, revealing a possible genetic connection of agglutinins and their biochemical characteristics in algal cells. Variability of agglutination reactions among the algae investigated is simplified and interpreted most easily using multivariate analysis.     

Antibacterial activity of<Emphasis Type="Italic">Ulva lactuca</Emphasis> against methicillin-resistant<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA)

Thein vitro antimicrobial activity of the marine green algaeUlva lactuca was examined against gram-positive bacteria, gram-negative bacteria, and a fungus. The ethyl-ether extract of algae exhibited a broad-spectrum of antibacterial activity. but not antifungal activity againstCandida albicans. In particular, theU. lactuca extract showed strong activity aganst the bacterium methicillin-resistantStaphylococcus aureus (MRSA). This result confirms the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting food for aquaculture.     

Antibacterial substances from marine algae isolated from Jeddah coast of Red sea,Saudi Arabia

Marine algae are known to produce a wide variety of bioactive secondary metabolites and several compounds have been derived from them for prospective development of novel drugs by the pharmaceutical industries. However algae of the Red sea have not been adequately explored for their potential as a source of bioactive substances. In this context Ulva reticulata, Caulerpa occidentalis, Cladophora socialis, Dictyota ciliolata, and Gracilaria dendroides isolated from Red sea coastal waters of Jeddah, Saudi Arabia, were evaluated for their potential for bioactivity. Extracts of the algae selected for the study were prepared using ethanol, chloroform, petroleum ether and water, and assayed for antibacterial activity against Escherichia coli ATCC 25322, Pseudomonas aeruginosa ATCC 27853, Stapylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212. It was found that chloroform was most effective followed by ethanol, petroleum ether and water for the preparation of algal extract with significant antibacterial activities, respectively. Results also indicated that the extracts of red alga G. dendroides were more efficient against the tested bacterial strains followed by green alga U. reticulata, and brown algae D. ciliolata. Chemical analyses showed that G. dendroides recorded the highest percentages of the total fats and total proteins, followed by U. reticulata, and D. ciliolate. Among the bioflavonoids determined Rutin, Quercetin and Kaempherol were present in high percentages in G. dendroides, U. reticulata, and D. ciliolate. Estimation of saturated and unsaturated fatty acids revealed that palmitic acid was present in highest percentage in all the algal species analyzed. Amino acid analyses indicated the presence of free amino acids in moderate contents in all the species of algae. The results indicated scope for utilizing these algae as a source of antibacterial substances.     

Factors affecting hemolysin production byVibrio vulnificus

Factors affecting the hemolytic activity ofVibrio vulnificus cultures supernatant fluids against sheep erythrocytes were studied in order to optimize conditions and develop an assay system to assess the pathogenic potential of marineV. vulnificus strains. The heat-labile hemolysin produced during logarithmic growth ofV. vulnificus was produced optimally in heart infusion broth (HIB). Lesser amounts of hemolysin were produced in brain-heart infusion and tryptic soy broths. Hemolytic activity of HIB cultures decreased with increasing NaCl concentrations. Higher NaCl concentrations were found to affect hemolysin production but not its activity. The assay system described herein is a simple and rapid method that is being applied to the study of the pathogenic potential ofV. vulnificus strains from marine environments.     

Inhibition of <Emphasis Type="Italic">Vibrio</Emphasis> biofilm formation by a marine actinomycete strain A66

China remains by far the largest aquaculture producer in the world. However, biofilms formed by pathogenic Vibrio strains pose serious problems to marine aquaculture. To provide a strategy for biofilm prevention, control, and eradication, extracts from 88 marine actinomycetes were screened. Thirty-five inhibited the biofilm formation of Vibrio harveyi, Vibrio vulnificus, and Vibrio anguillarum at a concentration of 2.5% (v/v). Thirty-three of the actinomycete extracts dispersed the mature biofilm. Six extracts inhibited the quorum-sensing system of V. harveyi by attenuating the signal molecules N-acylated homoserine lactones’ activity. Strain A66, which was identified as Steptomyces albus, both attenuated the biofilms and inhibited their quorum-sensing system. It is suggested that strain A66 is a promising candidate to be used in future marine aquaculture.     

Telomerase inhibiting activity in vitro from natural resources, marine algae extracts

We attempted to screen for telomerase inhibitingactivity in vitro from a total of 304 marine algae samples which were collected from various Japancoasts by nonradioisotope telomeric repeatamplification protocol (Non-RI TRAP) assay using humanleukemia MOLT-4 cells. Ten of the MeOH extracts andtwo of the PBS extracts from those algal samplesshowed telomerase inhibiting activity. In particular,the MeOH extract from a green alga, Caulerpasertularioides strongly inhibited telomerase activitywhen added to MOLT-4 cell culture at a level of 1.25%(v/v), suggesting that it may be possible to developa novel anti-cancer agent in view of its specificanti-telomeric property.     

Competitive Induction and Enhancement of Indole and a Diketopiperazine in Marine Bacteria

Thirteen bacterial strains isolated from a sample of Ulva californica were cultured, extracted, and assayed for antibiotic activity. The target strains were 2 gram-positive isolates from the same algal sample. When cultured in monoculture, 2 of the 13 isolates produced extracts with antimicrobial activity. More significantly, one Bacillus isolate (UA-094) produced indole and cyclo(Phe-Pro) at active levels upon challenge with a different Bacillus target strain. These mildly antibiotic compounds were barely detectable in extracts without challenge. This technique of competitive induction and enhancement could be a valuable tool in the search for new antibiotics.     

Phlorotannins as Radical Scavengers from the Extract of Sargassum ringgoldianum

To screen algal phlorotannins with antioxidative activities, 50% ethanol extracts of 25 Japanese marine algae were evaluated. Scavenging activity against superoxide anion radicals was frequently found with a high content of total phenolic compounds. Among these, the extract from the brown seaweed, Sargassum ringgoldianum, showed the strongest scavenging activity. The active fraction contained a mixture of high molecular weight polyphenols, phlorotannins that were found to be polymerized bifuhalol, as analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The scavenging activity of the fraction against superoxide anion radicals was estimated to be 1.0 μg/ml (IC₅₀), which were approximately five times stronger than that of catechin.     

Algae of Peter the Great Bay of the Sea of Japan as a source of lectins

Algae of Peter the Great Bay (Sea of Japan) were for the first time bioassayed as a source of lectins. From 28 algal species of three orders, only some extracts from brown (Phaeophyta) and red (Rhodophyta) seaweeds were found to cause agglutination of human erythrocytes. The hemagglutinating activity of extracts from three species of brown algae and the red alga Tichocarpus crinitus was caused by lectins; for a majority of extracts from the investigated algae, this activity was due to the presence of substances of non-lectin nature.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Antifouling activity of sessile bacilli derived from marine surfaces

Marine biofilms are a virtually untapped source of bioactive molecules that may find application as novel antifoulants in the marine paint industry. This study aimed at determining the potential of marine biofilm bacteria to produce novel biomolecules with potential application as natural antifoulants. Nine representative strains were isolated from a range of surfaces and were grown in YEB medium and harvested during the late exponential growth phase. Bacterial biomass and spent culture medium were extracted with ethanol and ethyl acetate, respectively. Extracts were assayed for their antifouling activity using two tests: (1) antimicrobial well diffusion test against a common fouling bacterium, Halomonas marina, and (2) anti-crustacean activity test using Artemia salina. Our results showed that none of the ethanolic extracts (bacterial biomass) were active in either test. In contrast, most of the organic extracts had antimicrobial activity (88%) and were toxic towards A. salina (67%). Sequencing of full 16 S ribosomal DNA analysis showed that the isolates were related to Bacillus mojavensis and Bacillus firmus. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) profiling of ethyl acetate extracts of culture supernatants showed that these species produce the bioactive lipopeptides surfactin A, mycosubtilin and bacillomycin D.     

Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis.

The ethanolic extracts of a number of Turkish freshwater macrophytes (Potamogeton perfoliatus, Ranunculus tricophyllus and Cladophora glomerata) and marine macroalgae (Dictyota dichotoma, Halopteris scoparia, Posidonia oceanica, Scinaia furcellata, Sargassum natans and Ulva lactuca) were assayed for their in vitro antiprotozoal activity. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum were used as test organisms. The cytotoxicity of the extracts was also assessed against primary rat skeletal myoblasts (L6 cells). Whereas none of the extracts were active against T. cruzi, all crude extracts displayed appreciable trypanocidal activity against T. brucei rhodesiense, with S. natans being the most active one (IC(50) 7.4microg/ml). Except for the marine alga H. scoparia, all extracts also possessed leishmanicidal potential. The best antileishmanial activity was exerted by U. lactuca and P. oceanica (IC(50)'s 5.9 and 8.0microg/ml, respectively). Five extracts that demonstrated inhibitory activity towards P. falciparum (IC(50)'s 18.1-48.8microg/ml) were simultaneously assayed against FabI, a crucial enzyme of the fatty acid system of P. falciparum, to find out whether FabI was their target. The extracts of C. glomerata and U. lactuca efficiently inhibited the FabI enzyme with IC(50) values of 1.0 and 4.0microg/ml, respectively. None of the extracts were cytotoxic towards mammalian L6 cells. This work reports for the first time antiprotozoal activity of some Turkish marine and freshwater algae, as well as a target-based antiplasmodial screening for the identification of P. falciparum FabI inhibitors from aquatic and marine macrophytes.     

Macroalgal palatability and the flux of ciguatera toxins through marine food webs

The benthic dinoflagellate Gambierdiscus toxicus produces polyether toxins that cause ciguatera fish poisoning in humans. The toxins initially enter food webs when fish forage on macroalgae, or other substrates, hosting this epiphytic dinoflagellate. Population studies of G. toxicus and risk assessments in ciguatera-prone regions often rely on quantifying dinoflagellates on macroalgae. Underlying these studies is the assumption that the algae sampled represent a readily consumable resource equally available for benthic grazers. However, many algal hosts of G. toxicus possess a variety of defenses against grazing, and host–dinoflagellate associations may act as toxin sources or sinks depending on their palatability. Marine macroalgae may tolerate or avoid herbivory by exhibiting fast growth, by having poor nutritional quality, by utilizing spatial or temporal escapes or by using chemical or structural defenses. Thus, rapidly consumed algae that cope with herbivores by growing fast, such as many filamentous turfs, could be responsible for a high toxin flux even at low dinoflagellate densities. In contrast, ubiquitous unpalatable algae with much higher dinoflagellate densities might contribute little to toxin flux, and effectively act as refuges for G. toxicus. To date, G. toxicus has been reported from 56 algal genera, two cyanobacteria, one diatom, and one seagrass; 63% of these contain species that are defended from fish grazing and other grazers via chemical, morphological or structural defenses, by low nutritional quality, or by a combination of defensive strategies. High dinoflagellate densities on unpalatable macroalgae could indicate passive accumulation of cells on undisturbed hosts, rather than population explosions or active toxin sources for food webs. Understanding the flow of ciguatoxins in nature requires consideration of the ecology of both G. toxicus and its algal hosts. The complexity of marine algal–herbivore interactions also has consequences for other benthic dinoflagellates that produce toxins, which accumulate in consumers.