In Vitro Assessment of Marine Bacillus for Use as Livestock Probiotics

Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of β-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.     

Natural Marine and Synthetic Xenobiotics Get on Nematode’s Nerves: Neuro-Stimulating and Neurotoxic Findings in Caenorhabditis elegans

Marine algae release a plethora of organic halogenated compounds, many of them with unknown ecological impact if environmentally realistic concentrations are applied. One major compound is dibromoacetic acid (DBAA) which was tested for neurotoxicity in the invertebrate model organism Caenorhabditis elegans (C. elegans). This natural compound was compared with the widespread synthetic xenobiotic tetrabromobisphenol-A (TBBP-A) found in marine sediments and mussels. We found a neuro-stimulating effect for DBAA; this is contradictory to existing toxicological reports of mammals that applied comparatively high dosages. For TBBP-A, we found a hormetic concentration-effect relationship. As chemicals rarely occur isolated in the environment, a combination of both organobromines was also examined. Surprisingly, the presence of DBAA increased the toxicity of TBBP-A. Our results demonstrated that organohalogens have the potential to affect single organisms especially by altering the neurological processes, even with promoting effects on exposed organisms.     

Antiaging Potential of Peptides from Underused Marine Bioresources

Aging is a biological process that occurs under normal conditions and in several chronic degenerative diseases. Bioactive natural peptides have been shown to improve the effects of aging in cell and animal models and in clinical trials. However, few reports delve into the enormous diversity of peptides from marine organisms. This review provides recent information on the antiaging potential of bioactive peptides from underused marine resources, including examples that scavenge free radicals in vitro, inhibit cell apoptosis, prolong the lifespan of fruit flies and Caenorhabditis elegans, suppress aging in mice, and exert protective roles in aging humans. The underlying molecular mechanisms involved, such as upregulation of oxidase activity, inhibition of cell apoptosis and MMP-1 expression, restoring mitochondrial function, and regulating intestinal homeostasis, are also summarized. This work will help highlight the antiaging potential of peptides from underused marine organisms which could be used as antiaging foods and cosmetic ingredients in the near future.     

Genome Mining for Antimicrobial Compounds in Wild Marine Animals-Associated Enterococci

New ecosystems are being actively mined for new bioactive compounds. Because of the large amount of unexplored biodiversity, bacteria from marine environments are especially promising. Further, host-associated microbes are of special interest because of their low toxicity and compatibility with host health. Here, we identified and characterized biosynthetic gene clusters encoding antimicrobial compounds in host-associated enterococci recovered from fecal samples of wild marine animals remote from human-affected ecosystems. Putative biosynthetic gene clusters in the genomes of 22 Enterococcus strains of marine origin were predicted using antiSMASH5 and Bagel4 bioinformatic software. At least one gene cluster encoding a putative bioactive compound precursor was identified in each genome. Collectively, 73 putative antimicrobial compounds were identified, including 61 bacteriocins (83.56%), 10 terpenes (13.70%), and 2 (2.74%) related to putative nonribosomal peptides (NRPs). Two of the species studied, Enterococcus avium and Enterococcus mundtti, are rare causes of human disease and were found to lack any known pathogenic determinants but yet possessed bacteriocin biosynthetic genes, suggesting possible additional utility as probiotics. Wild marine animal-associated enterococci from human-remote ecosystems provide a potentially rich source for new antimicrobial compounds of therapeutic and industrial value and potential probiotic application.     

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.     

Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis

Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture.     

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3–0.5 µg/mL (1.4–2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca²⁺-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.     

Effects of temperature on copper resistance in daf-21 mutant and Hsp90 expression of Caenorhabditis elegans

To determine the effects of temperature alterations on copper toxicity and stress response in Caenorhabditis elegans, an aquatic acute toxicity test was utilized to combinations of four temperature levels (15, 20, 25, and 30°C) and four copper concentrations (0, 0.013, 0.13, and 1.3 mM). Endpoints used included 24-h median lethal concentrations (LC50) value, and 90-kDa heat shock proteins (Hsp90) induction. Results showed a significant acceleration in the mortality with increasing temperature and depressed expression in Hsp90 of the presence of copper. Data on LC50 revealed that simultaneous exposure to both copper and heat stress (30°C) was severe. In contrast, subdued reactions to copper were observed in adult nematode exposed to low temperature (15°C). We have discovered that daf-21 mutation adults result in decreased Cu²⁺ resistance in a 24 h toxicity assay. Significantly weak expression of Hsp90 in C. elegans was observed at high copper stress as LC50 concentration irrespective of temperatures. These results suggest acclimation temperatures caused variations in copper stress of C. elegans, and Hsp90 expression exhibited greater severity with increasing copper concentration. The present study confirms that temperature effectively confounds copper toxicity and needs to be considered for the accurate prediction and assessment of copper-induced toxicity in nematode.     

First Total Syntheses and Antimicrobial Evaluation of Penicimonoterpene,a Marine-Derived Monoterpenoid,and Its Various Derivatives

The first total synthesis of marine-derived penicimonoterpene (±)-1 has been achieved in four steps from 6-methylhept-5-en-2-one using a Reformatsky reaction as the key step to construct the basic carbon skeleton. A total of 24 new derivatives of 1 have also been designed and synthesized. Their structures were characterized by analysis of their ¹H NMR, ¹³C NMR and HRESIMS data. Some of them showed significant antibacterial activity against Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Vibrio anguillarum, V. harveyi and/or V. parahaemolyticus, and some showed activity against plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosporioides and/or Fusarium graminearum). Some of the derivatives exhibited antimicrobial MIC values ranging from 0.25 to 4 μg/mL, which were stronger than those of the positive control. Notably, Compounds 3b and 10 showed extremely high selectively against plant-pathogenic fungus F. graminearum (MIC 0.25 μg/mL) and pathogenic bacteria E. coli (MIC 1 μg/mL), implying their potential as antimicrobial agents. SAR analysis of 1 and its derivatives indicated that modification of the carbon-carbon double bond at C-6/7, of groups on the allylic methylene unit and of the carbonyl group at C-1, effectively enhanced the antimicrobial activity.     

Marennine,Promising Blue Pigments from a Widespread Haslea Diatom Species Complex

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.     

Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.     

Coexistence mechanisms at multiple scales in mosquito assemblages

Background

Although the nematode Caenorhabditis elegans is a major model organism in diverse biological areas and well studied under laboratory conditions, little is known about its ecology. Therefore, characterization of the species’ natural habitats should provide a new perspective on its otherwise well-studied biology. The currently best characterized populations are in France, demonstrating that C. elegans prefers nutrient- and microorganism-rich substrates such as rotting fruits and decomposing plant matter. In order to extend these findings, we sampled C. elegans continuously across 1.5 years from rotting apples and compost heaps in three North German locations.

Results

C. elegans was found throughout summer and autumn in both years. It shares its habitat with the related nematode species C. remanei, which could thus represent an important competitor for a similar ecological niche. The two species were isolated from the same site, but rarely the same substrate sample. In fact, C. elegans was mainly found on compost and C. remanei on rotten apples, possibly suggesting niche separation. The occurrence of C. elegans itself was related to environmental humidity and rain, although the correlation was significant for only one sampling site each. Additional associations between nematode prevalence and abiotic parameters could not be established.

Conclusions

Taken together, our findings vary from the previous results for French C. elegans populations in that the considered German populations always coexisted with the congeneric species C. remanei (rather than C. briggsae as in France) and that C. elegans prevalence can associate with humidity and rain (rather than temperature, as suggested for French populations). Consideration of additional locations and time points is thus essential for full appreciation of the nematode's natural ecology.     

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.     

Large-Scale Plasma Peptidomic Profiling Reveals a Novel,Nontoxic, Crassostrea hongkongensis-Derived Antimicrobial Peptide against Foodborne Pathogens

Antimicrobial peptides are a fundamental component of mollusks’ defense systems, though they remain a thinly investigated subject. Here, infection by Vibrio parahemolyticus triggered a significant increase in antimicrobial activity in oyster plasma. By using PBS-challenged oysters as a control, plasma peptides from immunologically challenged oysters were subjected to peptidomic profiling and in silico data mining to identify bioactive peptides. Thirty-five identified plasma peptides were up-regulated post infection, among which, six up-regulated peptides (URPs) showed a relatively high positive charge. URP20 was validated with significant antibacterial activity. Virtually, URP20 triggered aggregation of bacterial cells, accompanied by their membrane permeabilization. Interestingly, URP20 was found to be active against Gram-positive and Gram-negative foodborne pathogens as well as Candida albicans, with no cytotoxicity to mammalian cells and mice. Our study provides the first large-scale plasma peptidomic dataset that identifies novel bioactive peptides in marine mollusks. Further exploration of peptide diversity in marine invertebrates should prove a fruitful pursuit for designing novel AMPs with broad applications.     

A Lymphoid Organ Specific Anti-Lipopolysaccharide Factor from Litopenaeus vannamei Exhibits Strong Antimicrobial Activities

Different shrimp species are known to possess apparent distinct resistance to different pathogens in aquaculture. However, the molecular mechanism underlying this finding still remains unknown. One kind of important antimicrobial peptides, anti-lipopolysaccharide factors (ALF), exhibit broad-spectrum antimicrobial activities. Here, we reported a newly identified ALF from the shrimp Litopenaeus vannamei and compared the immune function with its counterpart in the shrimp Fenneropenaeus chinensis. The ALF, designated as LvALF8, was specifically expressed in the lymphoid organ of L. vannamei. The expression level of LvALF8 was apparently changed after white spot syndrome virus (WSSV) or Vibrio parahaemolyticus challenges. The synthetic LBD peptide of LvALF8 (LvALF8-LBD) showed strong antibacterial activities against most tested Gram-negative and Gram-positive bacteria. LvALF8-LBD could also inhibit the in vivo propagation of WSSV similar as FcALF8-LBD, the LBD of LvALF8 counterpart in F. chinensis. However, LvALF8-LBD and FcALF8-LBD exhibited apparently different antibacterial activity against V. parahaemolyticus, the main pathogen causing acute hepatopancreatic necrosis disease (AHPND) of affected shrimp. A structural analysis showed that the positive net charge and amphipathicity characteristics of LvALF8-LBD peptide were speculated as two important components for its enhanced antimicrobial activity compared to those of FcALF8-LBD. These new findings may not only provide some evidence to explain the distinct disease resistance among different shrimp species, but also lay out new research ground for the testing and development of LBD-originated antimicrobial peptides to control of shrimp diseases.     

Neuroprotective Effects of the Cultivated Chondrus crispus in a C. elegans Model of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the elderly people, currently with no cure. Its mechanisms are not well understood, thus studies targeting cause-directed therapy or prevention are needed. This study uses the transgenic Caenorhabditis elegans PD model. We demonstrated that dietary supplementation of the worms with an extract from the cultivated red seaweed Chondrus crispus decreased the accumulation of α-synulein and protected the worms from the neuronal toxin-, 6-OHDA, induced dopaminergic neurodegeneration. These effects were associated with a corrected slowness of movement. We also showed that the enhancement of oxidative stress tolerance and an up-regulation of the stress response genes, sod-3 and skn-1, may have served as the molecular mechanism for the C. crispus-extract-mediated protection against PD pathology. Altogether, apart from its potential as a functional food, the tested red seaweed, C. crispus, might find promising pharmaceutical applications for the development of potential novel anti-neurodegenerative drugs for humans.     

Functional feed assessment on Litopenaeus vannamei using 100% fish meal replacement by soybean meal, high levels of complex carbohydrates and Bacillus probiotic strains

Functional feed supplemented with alternative-economic nutrient sources (protein, carbohydrates, lipids) and probiotics are being considered in shrimp/fish aquaculture production systems as an option to increase yield and profits and to reduce water pollution. In this study the probiotic potential to formulate functional feeds have been evaluated using four dietary treatments: Treatment 1 (B + Bs); Bacillus subtilis potential probiotic strain was supplemented to a soybeanmeal (SBM)-carbohydrates (CHO) basal feed. Treatment 2 (B + Bm); Bacillus megaterium potential probiotic strain was supplemented to the same SBM-CHO basal feed. In Treatment 3 (B); SBM-CHO basal feed was not supplemented with probiotic strains. Treatment 4 (C); fishmeal commercial feed (FM) was utilized as positive control. Feeding trials evaluated the survival, growth, and food conversion ratio and stress tolerance of juvenile Litopenaeus vannamei (Boone) Pacific white shrimp. Best overall shrimp performance was observed for animals fed with Treatment 1 (B+Bs); additionally, stress tolerance and hemolymph metabolites also showed the best performance in this treatment. SBM-CHO basal feed not supplemented with probiotic strains (B) presented smaller growth and lower feed conversion ratio (FCR). Shrimps fed with the fishmeal commercial feed (C) presented the lowest stress tolerance to high ammonia and low oxygen levels. Specifically selected B. subtilis strains are recommended to formulate functional and economical feeds containing high levels of vegetable; protein and carbohydrates as main dietary sources in L. vannamei cultures.     

Synthesis and Neuroprotective Action of Xyloketal Derivatives in Parkinson’s Disease Models

Parkinson’s disease (PD) is the second most common neurodegenerative disease affecting people over age 55. Oxidative stress actively participates in the dopaminergic (DA) neuron degeneration of PD. Xyloketals are a series of natural compounds from marine mangrove fungus strain No. 2508 that have been reported to protect against neurotoxicity through their antioxidant properties. However, their protection versus 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity is only modest, and appropriate structural modifications are necessary to discover better candidates for treating PD. In this work, we designed and synthesized 39 novel xyloketal derivatives (1–39) in addition to the previously reported compound, xyloketal B. The neuroprotective activities of all 40 compounds were evaluated in vivo via respiratory burst assays and longevity-extending assays. During the zebrafish respiratory burst assay, compounds 1, 9, 23, 24, 36 and 39 strongly attenuated reactive oxygen species (ROS) generation at 50 μM. In the Caenorhabditis elegans longevity-extending assay, compounds 1, 8, 15, 16 and 36 significantly extended the survival rates (p < 0.005 vs. dimethyl sulfoxide (DMSO)). A total of 15 compounds were tested for the treatment of Parkinson’s disease using the MPP+-induced C. elegans model, and compounds 1 and 8 exhibited the highest activities (p < 0.005 vs. MPP⁺). In the MPP+-induced C57BL/6 mouse PD model, 40 mg/kg of 1 and 8 protected against MPP+-induced dopaminergic neurodegeneration and increased the number of DA neurons from 53% for the MPP+ group to 78% and 74%, respectively (p < 0.001 vs. MPP+ group). Thus, these derivatives are novel candidates for the treatment of PD.