EFFECTS OF 1,3-1,6 ß-GLUCAN FEED INCLUSION ON CAUDAL FIN REGENERATION PERFORMANCES OF ZEBRAFISH Danio rerio

Zebrafish (Danio rerio) are considered an interesting animal model for studying the tissue regeneration mechanisms because able to regrow amputated fins. Neutrophils and macrophages have shown functionally important role in tissue repair. Macrophages appear to act as the key regulatory cells for skin repair by removing dead tissues and killing pathogens. Macrophages also produce growth factors that stimulate other cells involved in wound healing. Since β-glucans may "activate" immune cells and macrophages in particular, our hypothesis is that they may positively affect the tissue regeneration process. For these reasons, we investigated the effect 1,3-1,6 β-glucan (extract form yeast cell wall) on caudal fin regeneration in zebrafish. Ninety fish were distributed into 3 groups (3 replicates). Two products were used as source of 1,3-1,6 ß-glucan: MacroGard® and new MacroGard (Biorigin©), MI and MII, respectively. Fish daily feed intake was estimated in advance (3.75% BW) and both MI and MII included into the feed for reaching the dose of 12.5 mg kg-1 BW. Feeding treatment with MI and MII was started just after amputation. The fin regeneration process was observed and described by measuring the fin regenerated area (RA=(x dpa fin area/pre-amputation fin area)*100) and calculating the daily regenerated area (DRA=(x dpa fin area-x+1 dpa fin area)/pre-amputation fin area)*100). The fin regeneration process is also described by a quadratic equation. MacroGard® showed a significantly (P0.05) were observed between the two experimental groups. Further studies are necessary for understanding the mode of action of 1,3-1,6 ß-glucan on fin regeneration

Beta glucan enhances the wound healing process in zebrafish

β-glucans are natural compounds that interact with the innate immune system. Macrophages play an important role in wound healing process and appear to act as the key regulatory cells for skin repair by removing dead tissues and killing pathogens. Macrophages also produce growth factors that stimulate cells involved in wound healing (production of extracellular matrix). Discovering natural products which may enhance the wound healing process in fish, has numerous health benefits and 1,3-1,6 ß-glucans may reduce the negative effect of stress, inflammatory reactions, and secondary infections. In this study, the effects on wound healing process in zebrafish (Danio rerio) of 1,3-1,6 ß-glucan extracted from yeast cell wall was investigated. Ninety female fish were distributed into 3 groups (3 replicates). Two products were used as source of 1,3-1,6 ß-glucan: MacroGard® and new MacroGard (Biorigin©), MI and MII respectively; fish daily feed intake was estimated in advance (3.88% BW) and both MI and MII included into the feed for reaching the dose of 12.5 mg kg-1 BW. Treatments started 2 weeks before injuring. Afterwards, two circular shape wounds were made using a laser source on the dorsal edge of the abdomen. Wounds were then digitally photographed at 2, 4, 10, 16, 20, 30 days post wound (dpw). Wound area was measured by Image J® software and wound size given as a mean of the two wounds for each fish. The first clear differences of wound sizes (1.882, 1.725 and 1.665 mm2, for Control, MI and MII, respectively) were observed already at 4 dpw and differences were statistically significant between MI and MII compared to Control group (P<0.05); this early effect seems to suggest a rapid influx of immune cells to a wound in particular when "activated" by 1,3-1,6 ß-glucan (Paul and Fend 2009). Again, at 16 dpw groups MI (0.766 mm2) and MII (0.634 mm2) showed significantly (P<0.05) lower wound area than Control (0.994 mm2); at 20 dpw, wound area of MII (0.518 mm2) was significantly (P<0.05) different from Control (0.702 mm2) and MI (0.713 mm2). Analysis of final wound status (at 30 dpw) showed that 67.9% of fish from MII group had completely healed wounds while that was significantly (P<0.05) lower with 23.1% and 41.4% in control and MI groups, respectively suggesting that MII has better wound healing potential than MI. The present study demonstrate that 1,3-1,6 ß-glucan (MacroGard®) have potential to be applied as natural wound healing agent in fish. Particularly interesting seems mainly to be their early effect that may imply a higher protection from possible secondary infections. However, further studies are required to determine the optimal feed inclusion in diets for different fish species as well as to explain the slightly higher wound healing effects observed for MII than MI

Genome Characterization of Bacteriophage KPP-1, a Novel Member in the Subfamily <i>Vequintavirinae</i>, and Use of Its Endolysin for the Lysis of Multidrug-Resistant <i>Klebsiella variicola</i> In Vitro

Multidrug-resistant members of the Klebsiella pneumoniae complex have become a threat to human lives and animals, including aquatic animals, owing to the limited choice of antimicrobial treatments. Bacteriophages are effective natural tools available to fight against multidrug-resistant bacteria. The bacteriophage KPP-1 was found to be strictly lytic against K. variicola, a multidrug-resistant isolate, producing clear plaques. The genome sequence analysis of KPP-1 revealed that it comprised 143,369 base pairs with 47% overall GC content. A total of 272 genes (forward 161, complementary 111) encode for 17 tRNAs and 255 open reading frames (ORFs). Among them, 32 ORFs could be functionally annotated using the National Center for Biotechnology Information (NCBI) Protein Basic Local Alignment Search Tool (BLASTp) algorithm while 223 were found to code for hypothetical proteins. Comparative genomic analysis revealed that the closest neighbor of KPP-1 can be found in the genus Mydovirus of the subfamily Vequintavirinae. KPP-1 not only markedly suppressed the growth of the host but also worked synergistically with ampicillin. Useful genes for pathogen control such as endolysin (locus tag: KPP_11591) were found to have activity against multidrug-resistant isolate of K. variicola. Further studies are necessary to develop a strategy to control the emerging pathogen K. variicola using bacteriophages such as KPP-1

Antifungal Efficacy of Antimicrobial Peptide Octominin II against <i>Candida albicans</i>

Most clinically isolated Candida albicans strains are drug-resistant, emphasizing the urgent need to discover alternative therapies. In this study, the previously characterized Octominin was modified into a shorter peptide with an 18 amino acid sequence (1GWLIRGAIHAGKAIHGLI18) and named Octominin II. The secondary structure of Octominin II is a random coil with a helical turn and a positive charge (+2.46) with a hydrophobic ratio of 0.46. Octominin II inhibited C. albicans, C. auris, and C. glabrata with minimum inhibitory and fungicidal concentrations against C. albicans of 80 and 120 µg/mL, respectively. Field emission scanning electron microscopy confirmed that Octominin II treatment caused ultra-structural changes in C. albicans cells. Furthermore, membrane permeability results for the fluorescent indicator propidium iodide revealed modifications in cell wall integrity in Octominin II-treated C. albicans. Octominin II treatment increases the production of reactive oxygen species (ROS) in C. albicans. Gene expression studies revealed that Octominin II suppresses virulence genes of C. albicans such as CDR1, TUP1, AGE3, GSC1, SAP2, and SAP9. In addition, a nucleic acid binding assay revealed that Octominin II degraded genomic DNA and total RNA in a concentration-dependent manner. Additionally, Octominin II inhibited and eradicated C. albicans biofilm formation. Octominin II showed relatively less cytotoxicity on raw 264.7 cells (0–200 µg/mL) and hemolysis activity on murine erythrocytes (6.25–100 µg/mL). In vivo studies confirmed that Octominin II reduced the pathogenicity of C. albicans. Overall, the data suggests that Octominin II inhibits C. albicans by employing different modes of action and can be a promising candidate for controlling multidrug-resistant Candida infections

Antimicrobial Peptide Octominin-Encapsulated Chitosan Nanoparticles Enhanced Antifungal and Antibacterial Activities

Antimicrobial peptides (AMPs) have become a key solution for controlling multi-drug-resistant (MDR) pathogens, and the nanoencapsulation of AMPs has been used as a strategy to overcome challenges, such as poor stability, adverse interactions, and toxicity. In previous studies, we have shown the potent antimicrobial activity of Octominin against Candida albicans and Acinetobacter baumannii. This study is focused on the nanoencapsulation of Octominin with chitosan (CS) and carboxymethyl chitosan (CMC) as a drug delivery system using the ionotropic gelation technique. Octominin-encapsulated CS nanoparticles (Octominin-CNPs) had an average diameter and zeta potential of 372.80 &plusmn; 2.31 nm and +51.23 &plusmn; 0.38 mV, respectively, while encapsulation efficiency and loading capacity were 96.49 and 40.20%, respectively. Furthermore, Octominin-CNPs showed an initial rapid and later sustained biphasic release profile, and up to 88.26 &plusmn; 3.26% of the total Octominin release until 96 h. Transmission electron microscopy data showed the irregular shape of the Octominin-CNPs with aggregations. In vitro and in vivo toxicity of Octominin-CNPs was significantly lower than the Octominin at higher concentrations. The antifungal and antibacterial activities of Octominin-CNPs were slightly higher than those of Octominin in both the time-kill kinetic and microbial viability assays against C. albicans and A. baumannii, respectively. Mode of action assessments of Octominin-CNPs revealed that morphological alterations, cell membrane permeability alterations, and reactive oxygen species generation were slightly higher than those of Octominin at the tested concentrations against both C. albicans and A. baumannii. In antibiofilm activity assays, Octominin-CNPs showed slightly higher biofilm inhibition and biofilm eradication activities compared to that of Octominin. In conclusion, Octominin was successfully encapsulated into CS, and Octominin-CNPs showed lower toxicity and greater antimicrobial activity against C. albicans and A. baumannii compared to Octominin

Octominin: A Novel Synthetic Anticandidal Peptide Derived from Defense Protein of Octopus minor

The rapid emergence of multidrug-resistant pathogens makes an urgent need for discovering novel antimicrobial agents as alternatives to conventional antibiotics. Towards this end, we designed and synthesized a synthetic peptide of 23 amino acids (AAs) (1GWLIRGAIHAGKAIHGLIHRRRH23) from a defense protein 3 cDNA sequence of Octopus minor. The sequence of the peptide, which was named Octominin, had characteristic features of known antimicrobial peptides (AMPs) such as a positive charge (+5), high hydrophobic residue ratio (43%), and 1.86 kcal/mol of Boman index. Octominin was predicted to have an alpha-helix secondary structure. The synthesized Octominin was 2625.2 Da with 92.5% purity. The peptide showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 50 and 200 &mu;g/mL, respectively, against Candida albicans. Field emission scanning electron microscopy observation confirmed that Octominin caused ultrastructural cell wall deformities in C. albicans. In addition, propidium iodide penetrated the Octominin-treated C. albicans cells, further demonstrating loss of cell membrane integrity that caused cell death at both MIC and MFC. Octominin treatment increased the production of intracellular reactive oxygen species and decreased cell viability in a concentration dependent manner. Cytotoxicity assays revealed no significant influence of Octominin on the viability of human embryonic kidney 293T cell line, with over 95% live cells in the Octominin-treated group observed up to 100 &micro;g/mL. Moreover, we confirmed the antifungal action of Octominin in vivo using a zebrafish experimental infection model. Overall, our results demonstrate the Octominin is a lead compound for further studies, which exerts its effects by inducing cell wall damage, causing loss of cell membrane integrity, and elevating oxidative stress

Effect of hydrolysed fish protein and autolysed yeast as alternative nitrogen sources on gilthead sea bream (Sparus aurata) growth performances and gut morphology

The aim of the present study was to compare the effects of two different nitrogen-rich ingredients such as hydrolysed fish protein and autolysed yeast, on gilthead sea bream (Sparus aurata) growth performances and histological gut morphology. Animals were allocated to three experimental groups: the first received a fishmeal-based diet (FM), the second and the third received hydrolysed fish protein (HFP) and autolysed yeast (AY), respectively, to replace an equal amount of fishmeal. The experiment lasted 92 days. No significant differences in body weight and mortality were observed among the different groups. Villus branching, intraepithelial lymphocytes and inflammatory infiltrate of the submucosa were more prominent in AY than HFP and FM. The gut absorbent surface ratio was 5.94, 6.44 and 7.28 for group FM, HFP and AY, respectively, with statistical significant difference between FM and AY and between HFP and AY. A significant increment in the goblet cell density was observed for HFP and AY in comparison to FM. A statistically significant increase in small-sized goblet cells was observed in AY compared to FM. All our findings suggest that it is possible to use either HFP or AY, as partial replacer of FM in the S. aurata diet.Highlights Yeast-derived products as a suitable alternative nitrogen source. Autolyzed yeast as a novel approach in the use of yeast products. Use of autolysed yeast for replacing fishmeal in aqua feed. Use of hydrolysed fish protein for replacing fishmeal in aqua feed

Feeding of nano scale oats β-glucan enhances the host resistance against Edwardsiella tarda and protective immune modulation in zebrafish larvae

In this study, we prepared and characterized the oats origin of nano scale β-glucan (NBG) and investigated the immunomodulatory properties in zebrafish larvae. Newly prepared NBG (average particle size of 465 nm) was fully soluble in water. Zebrafish larvae survival rate was increased against pathogenic bacteria Edwardsiella tarda, when NBG was added to the water (500 μg/mL) compared to NBG non-exposed controls. Moreover, quantitative real time PCR (qRT-PCR) results showed up-regulation of immune functional genes including TNF-α, IL-1β, β-defensin, lysozyme, IL 10, IL 12 and C-Rel indicating higher survival rate could be due to stronger immunomodulatory function of NBG (500 μg/mL). Thus, non-toxic, water soluble and biodegradable NBG from oats could be considered as the potential immunostimulant for larval aquaculture

Antimicrobial Activity of Thymol against Pathogenic Gram-negative Bacteria of Fishes

This work investigated the antimicrobial effects of thymol in vitro against seven species of Gram-negative fish pathogenic bacteria namely, Aeromonas salmonicida subsp. masoucida, A. salmonicida subsp. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio vulnificus, V parahaemolyticus and V anguillarum using disk diffusion, minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) tests. In the disk diffusion test, thymol inhibited growth of all bacteria including those known to be resistant to amoxicillin or lincomycin or both. MIC or MBC of thymol against all bacteria were in the range of 0.01 mg/ml to 0.32 mg/ml. The most sensitive was A. salmonicida subsp. salmonicida (0.01 mg/ml for MIC and 0.02 mg/ml for MBC), followed by A. salmonicida subsp. masoucida (0.04 mg/ml for MIC and 0.08 mg/ml for MBC). Based on the present results, thymol has the potential of controlling bacterial pathogens in the aquaculture industry.N