Oxepine-containing pyrazinopyrimidine alkaloids and quinolinone derivatives produced by Aspergillus versicolor AS-212, a deep-sea-derived endozoic fungus.

Four new oxepine-containing pyrazinopyrimidine alkaloids, versicoxepines A - D (1-4), two quinolinone alkaloid analogs including 3-hydroxy-6-methoxy-4-phenylquinolin-2(1H)-one (5) and 3-methoxy-6-hydroxy-4-phenylquinolin-2(1H)-one (6) which were new naturally occurring compounds, together with two known compounds (7 and 8) were isolated from Aspergillus versicolor AS-212, an endozoic fungus isolated from the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts in the Western Pacific Ocean. Their structures were determined by extensive analysis of the spectroscopic and X-ray crystallographic data as well as by chiral HPLC analysis, ECD calculation, and DP4+ probability prediction. Structurally, versicoxepines B and C (2 and 3) represent the first example of a new oxepine-containing pyrazinopyrimidine alkaloid whose cyclic dipeptide moiety is composed of the same type of amino acid (Val or Ile). Compound 5 displayed antibacterial activity against aquatic pathogens, Vibrio harveyi and V. alginolyticus, with MICs of 8 µg/mL.

Anti-vibrio and immune-enhancing activity of medicinal plants in shrimp: A comprehensive review.

Disease epidemics in shrimp aquaculture increase apace with the development of aquaculture systems throughout the world. The disease caused by Vibrio spp. (vibriosis) is considered the most devastating, which has made it the most feared bacterial disease in the shrimp sector. In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils; hence, there has been considerable attention for using plants in shrimp aquaculture to provide sustainable, eco-friendly and safe compounds, such as alkaloids, saponins, terpenoids and flavonoids for replacing chemical compounds and antibiotics in current aquaculture. Medicinal plants may also have immunostimulating activity, increase growth and resistance in shrimps. The present paper aims to review the inhibition of Vibrio spp. in shrimp by medicinal plants, using both in vitro or/and in vivo techniques. Several medicinal plants appear capable of inhibiting growth of Vibrio pathogens outside living shrimp or in the body of shrimp, through enhancing growth and immune capacity when shrimps are fed or injected with them. In the current review Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrio.

Molecular docking study of bio-inhibitors extracted from marine macro-alga Ulva fasciata against hemolysin protein of luminescence disease-causing Vibrio harveyi.

Shrimp grow-out and hatchery systems are being affected by bacterial disease particularly Vibrios. The use of chemotherapeutic agents in aquaculture practices has to lead to the development of resistance among aquatic bacteria. Thus, health management becomes of major importance in aquaculture. Under this situation, progressing bio-inhibitors from marine resources are most appropriate to be considered against pathogenic bacteria. Molecular docking is an appropriate tool in structural biology and computer-assisted drug design to predict and neutralize a target protein of known diseases. In this study, marine macro-alga Ulva fasciata was aimed at developing inhibitors against luminescence disease-causing pathogenic bacteria Vibrio harveyi. U. fasciata was collected from Thoothukudi, Tamil Nadu, India. Extract of U. fasciata was tested against growth and virulence factors of V. harveyi during Penaeus monodon larviculture. Further U. fasciata extract was subjected to GC-MS analysis to identify the biomolecules. The homology modeling of virulent protein, hemolysin of V. harveyi was designed in this study. Hence, it was aimed for molecular docking against the biomolecules identified from U. fasciata extract. During shrimp larviculture, the extract of U. fasciata (200 µg mL-1) exhibited reduction on Cumulative Percentage of Mortality (32.40%) in postlarvae against challenge of V. harveyi infection. Biomolecule Methyl dehydroabietate had showed highest binding affinity among the compounds was evaluated in molecular docking study. Statistical analysis had revealed significant differences (p < 0.05) in trials. Therefore, it was proved that the bio-inhibitors from U. fasciata will be a better option for controlling luminescence disease-causing V. harveyi in shrimp grow-out practices.

Cadinane and carotane derivatives from the marine algicolous fungus Trichoderma virens RR-dl-6-8.

Eight cadinane derivatives, trichocadinins H - N (1-7) and methylhydroheptelidate (8), and two carotane derivatives, 14-O-methyltrichocarotin G (9) and 14-O-methyl CAF-603 (10), including eight new ones (1-6, 9, and 10), were isolated from the culture of Trichoderma virens RR-dl-6-8 obtained from the organohalogen-enriched marine red alga Rhodomela confervoides. Their structures and relative configurations were established by analysis of NMR and mass spectroscopic data, and the absolute configurations were assigned on the basis of ECD curves, highlighted by the ECD diversity of carboxylic acid derivatives. Among the isolates, 1 with a halogen atom and 8, a new naturally occurring compound, are 2,3-seco-cadinane sesquiterpenes, and the epimeric 2 and 3 feature a 2-nor-cadinane skeleton. A commercially-sourced compound with the same planar structure as that of 7 has been reported in a patent, but its configuration was not given. Compounds 1-10 exhibited growth inhibition of some marine phytoplankton species.

Effects of aqueous and ethanolic leaf extracts from drumstick tree (Moringa oleifera) on gilthead seabream (Sparus aurata L.) leucocytes, and their cytotoxic, antitumor, bactericidal and antioxidant activities.

Aqueous and ethanolic extracts of drumstick, Moringa oleifera, leaves were evaluated in vitro to ascertain their principal active components and determine their immunostimulant, cytotoxic, antitumoral, bactericidal and antioxidant activities. Phytochemical screening of M. oleifera leaf extracts showed a greater abundance of phenolic and cyanogenic glycosides in aqueous than in ethanolic extracts, characterized by several flavonoids, condensed tannins and saponins. No significant effects on gilthead seabream (Sparus aurata) head-kidney leucocyte activities (phagocytic ability and capacity, respiratory burst and peroxidase) were detected after incubation for 24 h with different concentrations (0.001/1 mg mL-1) of either extract. In addition, the aqueous extract showed a marked cytotoxic effect on both SAF-1 (at doses above 0.01 mg mL-1) and PLHC-1 (at doses above 0.25 mg mL-1) cell lines. The ethanolic extract improved the viability of SAF-1 cells and decreased the viability of PLHC-1 cells when used at higher concentrations. Both the ethanolic and, particularly, the aqueous extracts showed significant bactericidal activity on pathogenic Vibrio anguillarum and Photobacterium damselae strains. The antiradical activity of M. oleifera, as determined by the ABTS assay, increased in a linear dose-response with increasing extract concentrations. The results as a whole for the cytotoxic, bactericidal and antioxidant activities of M. oleifera leaf extracts point to their possible use as additives in functional diets for farmed fish.

Bactericidal effect on skin mucosa of dietary guava (Psidium guajava L.) leaves in hybrid tilapia (Oreochromis niloticus × O. mossambicus).

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the intensification practices in global aquaculture, fish are often confined in small volumes, which can results in outbreak diseases. In this context, the use of antibiotics is very usual. Thus, looking for natural substance able to reduce the use of the antibiotics is imperative. Among them, there is a great interest at present in the study of medicinal plants such as guava (Psidium guajava L.). These plants could help to develop a more sustainable aquaculture all over the world. The application of guava in traditional medicine dates for centuries and it is widely used in tropical countries for the treatment of diseases in human and animals. AIM OF THE STUDY: The purpose of this work was to study the effects of the dietary administration of dried leaves of Psidium guajava on the skin mucosal immunity of hybrid tilapia (Oreochromis niloticus × O. mossambicus). Furthermore, the ability of this plant to inhibit the bacterial load in different tissues after an experimental infection with Vibrio harveyi was studied. MATERIALS AND METHODS: P. guajava leaves collection and the experimentation was carried out in Dominican Republic. Fish were fed with a commercial diet supplemented with guava leaf at different concentrations (0%, 1.5% and 3%) for 21 days before being intraperitoneally injected with V. harveyi (1 × 104 cells mL-1). Thereafter, several immune activities were measured in fish skin mucus and after 48 h of injection, the skin, spleen and liver were collected to analyse the bactericidal activity of guava leaf and the gene expression of some immune related genes. RESULTS: The administration of P. guajava leaves significantly modulated some immune-related enzymes (protease, antiprotease and peroxidase) in the skin mucus of hybrid tilapia. In addition, the bacterial load after V. harveyi infection in skin, spleen and liver significantly reduced in fish supplemented with guava leaves. Finally, the expression profile of hepcidin gene in skin and liver was modulated in fish feed with control diet after V. harveyi infection. CONCLUSION: These results demonstrate that the dietary intake of guava leaves increases the skin mucosal barrier defences of hybrid tilapia and confers protection against V. harveyi colonization.

Unraveling the anti-biofilm potential of green algal sulfated polysaccharides against Salmonella enterica and Vibrio harveyi.

One of the main reasons for the bacterial resistance to antibiotics is caused by biofilm formation of microbial pathogens during bacterial infections. Salmonella enterica and Vibrio harveyi are known to form biofilms and represent a major health concern worldwide, causing human infections responsible for morbidity and mortality. The current study aims to investigate the effect of purified sulfated polysaccharides (SPs) from Chlamydomonas reinhardtii (Cr) on planktonic and biofilm growth of these bacteria. The effect of Cr-SPs on bacterial planktonic growth was assessed by using the agar well diffusion method, which showed clear zones ranging from 13 to 26 mm in diameter from 0.5 to 8 mg/mL of Cr-SPs against both the bacteria. Time-kill activity and reduction in clonogenic propagation further help to understand the anti-microbial potential of Cr-SPs. The minimum inhibitory concentration of Cr-SPs against S. enterica and V. harveyi was as low as 440 µg/mL and 490 µg/mL respectively. Cr-SPs inhibited bacterial cell attachment up to 34.65-100% at 0.5-8 mg/mL in S. enterica and V. harveyi respectively. Cr-SPs also showed 2-fold decrease in the cell surface hydrophobicity, indicating their potential to prevent bacterial adherence. Interestingly, Cr-SPs efficiently eradicated the preformed biofilms. Increased reduction in total extracellular polysaccharide (EPS) and extracellular DNA (eDNA) content in a dose-dependent manner demonstrates Cr-SPs ability to interact and destroy the bacterial EPS layer. SEM analysis showed that Cr-SPs effectively distorted preformed biofilms and also induced morphological changes. Furthermore, Cr-SPs also showed anti-quorum-sensing potential by reducing bacterial urease and protease activities. These results indicate the potential of Cr-SPs as an anti-biofilm agent and will help to develop them as alternative therapeutics against biofilm-forming bacterial infections. KEY POINTS: • Cr-SPs not only inhibited biofilm formation but also eradicated preformed biofilms. • Cr-SPs altered bacterial cell surface hydrophobicity preventing biofilm formation. • Cr-SPs efficiently degraded eDNA of the EPS layer disrupting mature biofilms. • Cr-SPs reduced activity of quorum-sensing-mediated enzymes like protease and urease.

Rheum officinale extract promotes the innate immunity of orange-spotted grouper (Epinephelus coioides) and exerts strong bactericidal activity against six aquatic pathogens.

We investigated the antimicrobial properties and the effects of Rheum officinale extract (ROE) on nonspecific immune parameters of orange-spotted grouper (Epinephelus coioides) in vitro and in vivo. The in vitro analysis was conducted by treating grouper primary head kidney leukocytes with various concentrations of ROE. The phagocytic rate of the leukocytes was elevated in a dose-dependent manner from 0.01 to 0.1 mg/ml, but decreased with higher concentrations of ROE (0.5 and 1.0 mg/ml). The production of reactive oxygen species (ROS) was strongly enhanced in a dose-dependent manner by treatment with ROE doses of 0.1-10.0 mg/ml. However, morphological changes (e.g., rounding and shrinkage of cells, chromatin condensation, fragmentation, and appearance of apoptotic bodies) were observed in the leukocytes after incubation with higher concentrations of ROE (1.0 and 10.0 mg/ml). A 28-day feeding trial was performed to assess the impact of dietary administration of ROE on grouper innate immunity parameters. Fish were fed with feed supplemented with 0, 0.1, 1.0, or 5.0 g ROE per kg of feed. The phagocytic activity of the animals' leukocytes was significantly elevated in all ROE-fed groups on day 1 and in groups fed with ROE at 0.1 or 1.0 g/kg on day 14. Production of ROS was substantially increased on day 1 in fish fed with ROE at 1.0 and 5.0 g/kg, but decreased steadily later on. The ability to generate ROS increased steadily until day 7 in fish fed the lowest concentration of ROE (0.1 mg/ml), but decreased thereafter. ROE showed excellent antibacterial activity against six pathogens of aquatic animals: Vibrio parahaemolyticus, V. vulnificus, V. alginolyticus, V. carchariae, Aeromonas hydrophila, and Edwardsiella tarda. The minimum inhibitory and bactericidal concentrations of measured ROE-derived anthraquinones were 10.57-84.53 µg/ml and 10.57-169.05 µg/ml, respectively.

The antioxidant power of horseradish, Armoracia rusticana, underlies antimicrobial and antiradical effects, exerted in vitro.

Armoracia rusticana (AR) was tested for antimicrobial and antioxidants power. The compound demonstrated to inhibit fish pathogens such as Vibrio anguillarum, V. harvey, V. alginolyticus, Aeromonas hydrophila, A. salmonicida, Photobacterium damselae subspecie piscicida, Tenacibaculum marinum and Pseudomonas anguilliseptica,. The total phenolic content and the reducing power resulted higher in the water extract of AR, respect to the hydroalcoolic. In vitro test demonstrated that AR significantly protect cells against death, induced by oxidative stress.

Burden of Shigella spp and Vibrio spp, and their antibiotic sensitivity pattern in the patients with acute gastroenteritis in tertiary care hospital in Nepal.

OBJECTIVES: The present study aims to investigate the etiology, clinical profile and resistance pattern of the isolated pathogens in Nepalese adults with acute gastroenteritis. This cross-sectional study was conducted at Sukraraj Tropical and Infectious Disease Hospital, from April 2016 to Sep 2017. Subjects' ages 14 or above, presenting with gastroenteritis with positive stool culture were enrolled for analysis. RESULTS: Of total 153 patients, 47.72% subjects confirmed the presence of bacterial infection. Vibrio cholerae spp and Shigella spp were detected in 36.6% and 23.28% respectively. The most common resistance among Vibrio cholerae was to nitrofurantoin (92.8%), cotrimoxazole (92.8%) and nalidixic acid (92.8%). Among 17 isolates of Shigella spp, the most frequent drug resistant was observed in ampicillin (64.7%), nalidixic acid (58.8%), ceftriaxone (47%). Chloramphenicol (94.1%), tetracycline (88.2%), and cotrimoxazole (82.3%) were found to be the most sensitive towards this pathogen. High rate of diarrhea due to bacterial infection, especially Shigella spp and Vibrio spp and their high rate of drug resistance emphasize an urgent need of designing a surveillance system for antimicrobial resistance in Nepalese setting.

H-Aquil: a chemically defined cell culture medium for trace metal studies in Vibrios and other marine heterotrophic bacteria.

A variety of trace metals, including prominently iron (Fe) are necessary for marine microorganisms. Chemically defined medium recipes have been used for several decades to study phytoplankton, but similar methods have not been adopted as widely in studies of marine heterotrophic bacteria. Medium recipes for these organisms frequently include tryptone, casamino acids, as well as yeast and animal extracts. These components introduce unknown concentrations of trace elements and organic compounds, complicating metal speciation. Minimal medium recipes utilizing known carbon and nitrogen sources do exist but often have high background trace metal concentrations. Here we present H-Aquil, a version of the phytoplankton medium Aquil adapted for marine heterotrophic bacteria. This medium consists of artificial seawater supplemented with a carbon source, phosphate, amino acids, and vitamins. As in Aquil, trace metals are controlled using the synthetic chelator EDTA. We also address concerns of EDTA toxicity, showing that concentrations up to 100 µM EDTA do not lead to growth defects in the copiotrophic bacterium Vibrio harveyi or the oligotrophic bacterium Candidatus Pelagibacter ubique HTCC1062, a member of the SAR11 clade. H-Aquil is used successfully to culture species of Vibrio, Phaeobacter, and Silicibacter, as well as several environmental isolates. We report a substantial decrease in growth rate between cultures grown with or without added Fe, making the medium suitable for conducting Fe-limitation studies in a variety of marine heterotrophic bacteria.

Agro food by-products and essential oil constituents curtail virulence and biofilm of Vibrio harveyi.

Vibrio harveyi causes severe loss to the aquaculture industry due to its virulence, which is mediated by Quorum sensing (QS) and biofilm formation. In the current study, we have explored the anti-virulent properties and biofilm disruption ability of luteolin (extracted from coconut shell) and linalool against this important aquaculture pathogen. HPLC analysis of the methanolic extract of coconut shells revealed a single major peak which matched to the standard luteolin which was further elucidated by NMR studies. Further, luteolin and linalool were screened for their ability to inhibit biofilms and various quorum sensing mediated virulence factors of V. harveyi. The Minimum Inhibitory Concentration (MIC) of the two compounds was determined and the sub-inhibitory concentrations of the compounds were able to inhibit biofilm formation. Both the compounds disrupted about 60-70% mature biofilms, which was also visually observed by light microscopy. Both linalool and luteolin exhibited a significant reduction in the production of EPS and alginate in the biofilms matrix of V. harveyi which was confirmed by Scanning Electron Microscopy (SEM). Both compounds inhibited the swarming and swimming motility, the crucial quorum sensing (QS) mediated virulence of V. harveyi. The present study shows the presence of valuable polyphenolic compound like luteolin in coconut shells that are discarded as a waste. From the present study we envisage that luteolin and linalool can serve as potent anti-virulent agents to combat QS mediated infections against aquaculture pathogens.

Ethanolic extract of red seaweed Gracilaria fisheri and furanone eradicate Vibrio harveyi and Vibrio parahaemolyticus biofilms and ameliorate the bacterial infection in shrimp.

Bacteria respond to host immunity for their proliferation and survival by cell-cell communications such as biofilm formation, bioluminescence, and secreting virulence factors. In the biofilm form, bacteria are more resistant to various antimicrobial treatments and withstand the host's immune system. The approaches of deciphering biofilm formation for treating bacterial infections are therefore highly desirable. Recently, we have reported that the ethanolic extract of the red seaweed Gracilaria fisheri (G. fisheri) enhanced immune activities and inhibited growth of the luminescent bacteria Vibrio harveyi in shrimp. We undertook the present research study in order to evaluate and compare the effectiveness of the ethanolic extract from G. fisheri and furanone, a known biofilm inhibitor, in inhibiting the formation of clinically important Vibrio biofilms. The results showed that sub-lethal concentrations of both the ethanolic extracts (5, 10 and 100 µg ml-1) and furanone (5 µM) inhibited biofilm formation by V. harveyi and Vibrio parahaemolyticus and also light production (luminescence) in V. harveyi. It is known that V. harveyi mediated light production via autoinducer AI-2 pathway, we further determined whether the inhibitory effect of the extract was involved the AI-2 signaling. The bioluminescence assay was conducted in an AI-2 deletion mutant V. harveyi. Supplementation of the AI-2 containing media with the extract or furanone impaired the light production in the mutant V. harveyi suggesting that the extract interfered AI-2 mediated light production similar to furanone. In vivo challenge study showed that the low concentrations (Sub MICs) of the ethanolic extract and furanone decreased bacterial adhesion and colonization in the surfaces of stomach lumen, down-regulated expression of a virulence factor, and protected shrimp against mortality from V. harveyi and V. parahaemolyticus infection. In conclusion, the present results suggest a potential application of the low concentrations of the ethanolic extract of G. fisheri as an efficient approach for treating biofilm-associated Vibrio diseases in aquacultures.

Effects of Moringa oleifera leaf extract on growth performance, physiological and immune response, and related immune gene expression of Macrobrachium rosenbergii with Vibrio anguillarum and ammonia stress.

In order to study the effects of Moringa oleifera leaf extract on Macrobrachium rosenbergii under high ammonia exposure, freshwater prawns were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 0.25%, 0.5% and 1.0% M. oleifera leaf extract and 0.025% Enrofloxacin for 60 days, respectively. Then, freshwater prawns were exposed to high ammonia stress for 72 h and Vibro anguillarum infection. The growth, antioxidant capabilities, related immune genes as well as resistance to infection by V. anguillarum were determined. The results showed that compared with the control group, the weight gain, specific growth rate and protein efficiency rate, haemolymph catalase (CAT), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) increased while feed conversion ratio, haemolymph aspartate aminotransferase, alanine aminotransferase, nitrogen oxide (NO), hepatopancreas heat shock proteins (HSP70), immune deficiency (IMD) expression levels decreased in the group of 0.5% M. oleifera leaf extract before the stress. After ammonia stress, the group of 0.5% M. oleifera leaf extract also could improve the haemolymph SOD, glutathione peroxidase, NO, iNOS, hepatopancreas HSP70 expression levels and reduce haemolymph CAT, hepatopancreas peroxiredoxin 5 and NF kappa B inhibitor alpha expression level compared with the control group. The rate of mortality of the prawns challenged with V. anguillarum was lower in the supplemented groups in comparison with the control group with the lowest being in the group of 0.5% M. oleifera leaf extract. Antioxidant activities as well as biochemical parameters in the enrofloxacin group (0.025%E) were not significantly enhanced both pre and post challenge in comparison with the M. oleifera leaf extract groups, showing the superiority of the natural herb over the synthetic antibiotic. In summary, this study suggested that at an inclusion rate of 0.5%, M. oleifera leaf extract could increase the growth performance, even has positive effects on physiological and immune function and prevents high ammonia stress in the Freshwater prawn, M.rosenbergii.

Efficacy assessment of commercially available natural products and antibiotics, commonly used for mitigation of pathogenic Vibrio outbreaks in Ecuadorian Penaeus (Litopenaeus) vannamei hatcheries.

Bacterial diseases cause high mortality in Penaeus (Litopenaeus) vannamei postlarvae. Therefore, appropriate application of efficient therapeutic products is of vital importance for disease control. This study evaluated through in vitro analyses the antimicrobial effectiveness of commercial therapeutic products used for P. vannamei bacterial diseases and antibiotics against pathogenic Vibrio strains circulating in Ecuadorian hatcheries. Twenty strains were isolated from 31 larvae samples with high bacterial counts from 10 hatcheries collected during mortality events. The strains virulence was verified through challenge tests with Artemia franciscana nauplii and P. vannamei postlarvae. Through 16S rRNA sequence analysis, strains showed a great similarity to the Vibrio sequences reported as pathogens, with 95% belonging to the Harveyi clade. Through antibiograms and minimal inhibitory concentration (MIC) in vitro tests we found that furazolidone, ciprofloxacin, chloramphenicol, norfloxacin, nalidixic acid, florfenicol, fosfomycin and enrofloxacin inhibited the growth of all or most of the strains. Less efficient antibiotics were penicillin, oxytetracycline and tetracycline. A multiple antibiotic resistance (MAR) index of 0.23 showed some level of resistance to antibiotics, with two MAR prevalent patterns (Penicillin-Oxytetracycline and Penicillin-Oxytetracycline-Tetracycline). From a total of 16 natural products (five probiotics, nine organic acids and two essential oils), only three (one probiotic, one organic acid and one essential oil) were effective to control most of the strains. Shrimp producers can apply relatively simple in vitro analyses, such as those employed in this study, to help take adequate management decisions to reduce the impact of bacterial diseases and increase profit.

Antibacterial activity of Indonesian red algae Gracilaria edulis against bacterial fish pathogens and characterization of active fractions.

In this study the crude ethyl acetate extracts and fractions from red algae Gracilaria edulis were studied against fish pathogens. G. edulis was extracted with ethyl acetate, fractionated in silica gel coloum chromatography and gradient hexane-ethyl acetate. The extract and fractions were subjected for bioassay guided isolation against fish pathogen Vibrio spp and Aeromonas hydrophila. Bioassays were accomplished by diffusion agar, bioauthography, and 96 well microplates format. Profiling chemical constituent was done by thin layer chromatography and GC-MS. Metabolites analysis using GC-MS yielded chemical constituents of active fractions as 9-hexadecenoic acid, hexadecanoic acid, 13-octadecenoic acid, 10-octadecenoic acid, eicosanoic acid, cholest-8-en-3-ol. The results provide in vitro scientific support to the possible application of Gracilaria edulis as antimicrobial agent that may contribute for treatment of bacterial infections in aquaculture.

Aspergixanthones I⁻K, New Anti-Vibrio Prenylxanthones from the Marine-Derived Fungus Aspergillus sp. ZA-01.

Marine-derived fungi are a rich source of structurally diverse metabolites. Fungi produce an array of compounds when grown under different cultivation conditions. In the present work, different media were used to cultivate the fungus Aspergillus sp. ZA-01, which was previously studied for the production of bioactive compounds, and three new prenylxanthone derivatives, aspergixanthones I⁻K (1⁻3), and four known analogues (4⁻7) were obtained. The absolute configuration of 1 was assigned by ECD experiment and the Mo2(AcO)4 ICD spectrum of its methanolysis derivative (1a). All the compounds (1⁻7) were evaluated for their anti-Vibrio activities. Aspergixanthone I (1) showed the strongest anti-Vibrio activity against Vibrio parahemolyticus (MIC = 1.56 µM), Vibrio anguillarum (MIC = 1.56 µM), and Vibrio alginolyticus (MIC = 3.12 µM).

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities.

Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.

Antimicrobial activity and molecular docking studies of a novel anthraquinone from a marine-derived fungus Aspergillus versicolor.

A novel anthraquinone, 2-(dimethoxymethyl)-1-hydroxyanthracene-9,10-dione (1), together with nine known compounds (2-10), were isolated from the fermentation of Aspergillus versicolor derived from deep sea sediment. Their structures were established through spectroscopic methods. Compound 1 exhibited strong inhibitory activities against MRSA ATCC 43300 and MRSA CGMCC 1.12409 (with MIC values of 3.9 and 7.8 µg/mL respectively) and moderate activities against tested strains of Vibrio (with MIC values ranging from 15.6 to 62.5 µg/mL). Compound 1 was subjected to molecular docking studies for inhibition of topoisomerase IV and AmpC ß-lactamase enzymes indicating its usefulness as antimicrobial agent.

Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection.

Many bacteria, including Vibrio pathogens of shrimp, need to colonize and/or form biofilms in hosts or the environment to cause disease. Thus, one possible control strategy for shrimp Vibriosis is biofilm inhibition. With this objective, an extract from the Japanese fermented soybean product, Natto was tested with the luminescent shrimp pathogen Vibrio harveyi (VH) for its ability to inhibit or degrade biofilm and to interfere with cell growth in broth. Natto is a traditional fermentation product of Bacillus subtilis var Natto (BSN1). Using 96 well microtiter plates coated with 0.4% chitosan, we found that biofilm formation by VH was inhibited, while growth in parallel broth cultures was not. When an extract from Natto prepared using BSN1 was mixed with feed for the whiteleg shrimp Penaeus vannamei before immersion challenge with V. harveyi at 106 cfu/ml, survival was significantly higher (p≤0.05) than for control shrimp given feed without these additives. Further work done to test whether d-amino acids were involved in biofilm formation as previously reported for B. subtilis, Staphylococus aureus and Pseudomonas aeruginosa gave negative results. In conclusion, we discovered that Natto extract can inhibit Vibrio biofilm formation and that it or BSN1 alone added to shrimp feed can significantly reduce shrimp mortality in immersion challenges with pathogenic VH. This shows some promise for possible application against Vibriosis in shrimp since Natto is generally regarded as safe (GRAS) for human consumption.