Antifungal Efficacy of Antimicrobial Peptide Octominin II against Candida albicans.

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.

The Aqueous Leaf Extract of the Medicinal Herb Costus speciosus Suppresses Influenza A H1N1 Viral Activity under In Vitro and In Vivo Conditions.

This study investigated the antiviral activity of aqueous leaf extract of Costus speciosus (TB100) against influenza A. Pretreatment of TB100 in RAW264.7 cells enhanced antiviral activity in an assay using the green fluorescence-expressing influenza A/Puerto Rico/8/1934 (H1N1) virus. The fifty percent effective concentration (EC50) and fifty percent cytotoxic concentration (CC50) were determined to be 15.19 ± 0.61 and 117.12 ± 18.31 µg/mL, respectively, for RAW264.7 cells. Based on fluorescent microscopy, green fluorescence protein (GFP) expression and viral copy number reduction confirmed that TB100 inhibited viral replication in murine RAW264.7 and human A549 and HEp2 cells. In vitro pretreatment with TB100 induced the phosphorylation of transcriptional activators TBK1, IRF3, STAT1, IKB-α, and p65 associated with interferon pathways, indicating the activation of antiviral defenses. The safety and protective efficacy of TB100 were assessed in BALB/c mice as an oral treatment and the results confirmed that it was safe and effective against influenza A/Puerto Rico/8/1934 (H1N1), A/Philippines/2/2008 (H3N2), and A/Chicken/Korea/116/2004 (H9N2). High-performance liquid chromatography of aqueous extracts led to the identification of cinnamic, caffeic, and chlorogenic acids as potential chemicals for antiviral responses. Further confirmatory studies using these acids revealed that each of them confers significant antiviral effects against influenza when used as pretreatment and enhances the antiviral response in a time-dependent manner. These findings suggest that TB100 has the potential to be developed into an antiviral agent that is effective against seasonal influenza.

Spirulina maxima Derived Pectin Nanoparticles Enhance the Immunomodulation, Stress Tolerance, and Wound Healing in Zebrafish.

In this study, Spirulina maxima derived pectin nanoparticles (SmPNPs) were synthesized and multiple biological effects were investigated using in vitro and in vivo models. SmPNPs were not toxic to Raw 264.7 cells and zebrafish embryos up to 1 mg/mL and 200 µg/mL, respectively. SmPNPs upregulated Il 10, Cat, Sod 2, Def 1, Def 2, and Muc 1 in Raw 264.7 cells and tlr2, tlr4b, tlr5b, il1ß, tnfα, cxcl8a, cxcl18b, ccl34a.4, ccl34b.4, muc5.1, muc5.2, muc5.3, hamp, cstd, hsp70, cat, and sod1 in the larvae and adult zebrafish, suggesting immunomodulatory activity. Exposure of larvae to SmPNPs followed by challenge with pathogenic bacterium Aeromonas hydrophila resulted a two-fold reduction of reactive oxygen species, indicating reduced oxidative stress compared to that in the control group. The cumulative percent survival of larvae exposed to SmPNPs (50 µg/mL) and adults fed diet supplemented with SmPNPs (4%) was 53.3% and 76.7%, respectively. Topical application of SmPNPs on adult zebrafish showed a higher wound healing percentage (48.9%) compared to that in the vehicle treated group (38.8%). Upregulated wound healing markers (tgfß1, timp2b, mmp9, tnfα, il1ß,ccl34a.4, and ccl34b.4), enhanced wound closure, and restored pigmentation indicated wound healing properties of SmPNPs. Overall, results uncover the multiple bioactivities of SmPNPs, which could be a promising biocompatible candidate for broad range of aquatic and human therapies.

Spirulina maxima derived marine pectin promotes the in vitro and in vivo regeneration and wound healing in zebrafish.

Purified bioactive components of marine algae have shown great pharmaceutical and biomedical potential, including wound healing activity. However, the activity of Spirulina maxima is the least documented with regard to wound healing potential. In the present study, we investigated the regenerative and wound healing activities of a Spirulina (Arthrospira) maxima based pectin (SmP) using in vitro human dermal fibroblasts (HDFs) and in vivo zebrafish model. SmP treated (12.5-50 µg/mL) HDFs showed increased cell proliferation by 20-40% compared to the untreated HDFs. Moreover, in vitro wound healing results in HDFs demonstrated that SmP decreased the open wound area % in concentration-dependent manner at 12.5 (32%) and 25 µg/mL (12%) compared to the control (44%). Further, zebrafish larvae displayed a greater fin regenerated area in the SmP exposed group at 25 (0.48 mm2) and 50 µg/mL (0.51 mm2), whereas the untreated group had the lowest regenerated area (0.40 mm2) at 3 days post amputation. However, fin regeneration was significantly (P < 0.001) higher only in the SmP treated group at 50 µg/mL. Furthermore, the open skin wound healing % in adult zebrafish was significantly higher (P < 0.05) after topical application (600 µg/fish) of SmP (46%) compared to the control (38%). Upregulation of genes such as tgfß1, timp2b, mmp9, tnf-α, and il-1ß, and chemokines such as cxcl18b, ccl34a.4, and ccl34b.4, in the muscle and kidney tissues of SmP treated fish compared to the respective control group was demonstrated using qRT-PCR. Histological analysis results further supported the rapid epidermal growth and tissue remodeling in SmP treated fish, suggesting that SmP exerts positive effects associated with wound healing. Therefore, SmP can be considered a potential regenerative and wound healing agent.

Application of phage therapy: Synergistic effect of phage EcSw (ΦEcSw) and antibiotic combination towards antibiotic-resistant Escherichia coli.

Bacteriophage therapy is acknowledged as a potential tool to prevent or treat multidrug-resistant bacterial infections. In this study, our major focus was on the bacteriolytic activity of phage EcSw (ΦEcSw) against the emergence of the clinically important Escherichia coli Sw1 and E. coli O157:H7. The amount of the antibiotics was changed in a concentration-dependent manner, and the ΦEcSw susceptibility to antibiotics was determined. The kanamycin and chloramphenicol inhibited the titre of phage, but ampicillin did not show phage inhibition. Though the kanamycin and chloramphenicol controlled the growth of Sw1 in a concentration-dependent manner, the ampicillin did not due to the resistance. The combined activity of the ΦEcSw with antibiotics (kanamycin and chloramphenicol) compared with the antibiotics alone showed significant lytic activity p < .001). In addition, phage-based therapy was evaluated for controlling the multidrug-resistant E. coli Sw1 and E. coli O157:H7 in zebrafish and BALB/c mice, respectively. Our results provide novel advantages of phage therapy and phage-antibiotic therapy to control antibiotic-resistant bacteria.

Marine Microalgae, Spirulina maxima-Derived Modified Pectin and Modified Pectin Nanoparticles Modulate the Gut Microbiota and Trigger Immune Responses in Mice.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm2) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.

Candida albicans Infection Model in Zebrafish (Danio rerio) for Screening Anticandidal Drugs.

BACKGROUND: Candida albicans is an opportunistic fungal pathogen which causes systemic infections in human. In this study, C. albicans infection model was developed in zebrafish to understand the host-pathogen interactions for straightforward anticandidal drug screening. METHODS: To develop the infection, 1 × 106 cells of C. albicans suspended in phosphate-buffered saline were deposited in zebrafish dorsal muscle by manually operated syringe. The infection progression was externally assessed by a scale of wound-healing events, based on visible changes of yeast deposited in the muscle tissues. Chemotherapy was carried out with known antifungal drugs (fluconazole, nystatin, and amphotericin B) and a potential antifungal agent, chitosan silver nanocomposites (CAgNC), after the infection as direct exposure in the water. Histopathological analysis was performed to identify the pathogen virulence and the host-pathogen interaction during the infection. RESULTS: The light microscopic observations and histopathological analysis revealed the yeast-hyphae transition at the site of infection (at 72 hpi) and progression of the infection in the host tissues. The larval survival rate under fluconazole (up to 80 µg mL-1) and nystatin (up to 20 µg mL-1) was > 90% and for CAgNC it was 40% at 36 h post-exposure (hpe). The infection progression was suppressed with the fungicidal treatments. Among inflammatory genes, il-1ß has been highly upregulated (14.68-fold) at 24 h post infection (hpi). Both il-1ß and tnf-α were moderately upregulated in infected fish gills at 72 hpi. Among the C. albicans antioxidant genes, cat1 and sod2 have been upregulated during the infection, and relative expression folds were increased from low to moderate levels with the time. DISCUSSION: We demonstrate the approach for the development of artificial infection model of zebrafish with C. albicans. By this mini vertebrate zebrafish model, researchers will be able to study novel anticandidal compounds in vivo with respect to the host, pathogen, and their interactions.

Novel pectin isolated from Spirulina maxima enhances the disease resistance and immune responses in zebrafish against Edwardsiella piscicida and Aeromonas hydrophila.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 µg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 µg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1ß: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1ß) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.

In vitro and in vivo antifungal efficacy of plant based lawsone against Fusarium oxysporum species complex.

Fusarium oxysporum is an ascomycete facultative fungus which generally affects to plants. However, it is recently known as a serious emerging opportunistic pathogen of human and other animals. F. oxysporum shows broad resistance to commonly used antifungal agents and therefore development of alternative therapeutic agents is required. In this study, we investigated the antifungal efficacy of plant based natural lawsone against pathogenic F. oxysporum. Antifungal susceptibility test determined the concentration dependent growth inhibition of lawsone against F. oxysporum with minimum inhibitory concentration (MIC) at 100µg/mL. Ultra-structural analysis indicates the prominent damage on cell wall of the mycelium after lawsone treatment, and suggests that it could increase the membrane permeability and disintegration of cells leading to cellular death. Propidium iodide (PI) uptake assay results showed the higher level of cell death in lawsone treated F. oxysporum which further confirms the loss of plasma membrane integrity. Also, detection of reactive oxygen species (ROS) using DCFH-DA has clearly indicated that lawsone (100µg/mL) can induce the ROS level in the filaments of F. oxysporum. MTT assay results showed the loss of viability and germination capacity of F. oxysporum spores by lawsone in concentration dependent manner. Moreover, lawsone treatment induced the mRNA expression of two autophagy related genes (ATG1 and ATG8) indicating that lawsone may activate the autophagy related pathways in F. oxysporum due to the oxidative stress generated by ROS. F. oxysporum infected zebrafish has recovered after lawsone therapy as a topical treatment suggesting that lawsone is a potential natural antifusariosis agent.

Feeding of nano scale oats ß-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 ß-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-1ß, ß-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.

Functional characterization of a novel lytic phage EcSw isolated from Sus scrofa domesticus and its potential for phage therapy.

In this study, multi-drug resistant Escherichia coli Sw1 (E. coli Sw1) and active lytic phage EcSw was isolated from feces samples of Sus scrofa domesticus (piglet) suffering from diarrhea. Transmission electron microscopy (TEM) indicated that isolated EcSw belongs to the Myoviridae family with an icosahedral head (80 ± 4) and a long tail (180 ± 5 nm). The EcSw phage genome size was estimated to be approximately 75 Kb of double-stranded DNA (dsDNA). Phage dynamic studies show that the latent period and burst size of EcSw were approximately 20 min and 28 PFU per cell, respectively. Interestingly, the EcSw phage can tolerate a wide range of environmental conditions, such as temperature, pH and ions (Ca(2+) and Mg(2+)). Furthermore, genome sequence analysis revealed that the lytic genes of the EcSw phage are notably similar to those of enterobacteria phages. In addition, phage-antibiotic synergy has notable effects compared with the effects of phages or antibiotics alone. Inhibition of E. coli Sw1 and 0157:H7 strains showed that the limitations of host specificity and infectivity of EcSw. Even though, it has considerable potential for phage therapy for handling the problem of the emergence of multidrug resistant pathogens.

Bacteriophage therapy of a Vibrio parahaemolyticus infection caused by a multiple-antibiotic-resistant O3:K6 pandemic clinical strain.

BACKGROUND: Recently isolated Vibrio parahaemolyticus strains have displayed multiple antibiotic resistance. Alternatives to conventional antibiotics are needed, especially for the multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. METHODS: A bacteriophage, designated pVp-1, showed effective infectivity for multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic strains. The therapeutic potential of the phage was studied in a mouse model of experimental infection using a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. We monitored the survivability and histopathological changes, quantified the bacterial and phage titers during phage therapy, and observed the immune response induced by phage induction. RESULTS: Phage-treated mice displayed protection from a V. parahaemolyticus infection and survived lethal oral and intraperitoneal bacterial challenges. CONCLUSIONS: To the best of our knowledge, this is the first report of phage therapy in a mouse model against a multiple-antibiotic­resistant V. parahaemolyticus pandemic strain infection.

Characterization and expression analysis of EF hand domain-containing calcium-regulatory gene from disk abalone: calcium homeostasis and its role in immunity.

The complete amino acid sequence of a calcium-regulatory gene (denoted as Ab-CaReg I) was identified from the disk abalone Haliotis discus discus cDNA library. The Ab-CaReg I is composed of 176 amino acids and the calculated molecular mass and isoelectric point were 20 and 4.2, respectively. The sequence homology of Ab-CaReg I was 28-30 and 18-27% of known calmodulin and troponin C, respectively. Four characteristic calcium-binding EF hand motifs with some modifications at conserved positions of known homologous calmodulin genes were observed in the sequence. The tissue-specific transcription analysis and variation of mRNA transcription level of Ab-CaReg I in gills and mantle after animals were immersed in seawater containing 2000 ppm CaCl(2) was quantified by SYBR Green real-time PCR analysis. Transcription variation of Ab-CaReg I in hemocytes and gills followed by bacteria challenge (Vibrio alginolyticus, Vibrio parahaemolyticus and Listeria monocytogenes) was used to investigate Ab-CaReg I in immune responses. Transcripts of Ab-CaReg I mRNA were mainly detected in hemocytes, mantle, muscle, gills, digestive tract and hepatopancreas with highest expression in hemocytes. The CaCl(2) immersion significantly altered the Ab-CaReg I mRNA transcription level by 3 h, compared to animals in normal seawater (control). The mRNA expression of Ab-CaReg I in gills and hemocytes was upregulated significantly to 11-fold and 4-fold in 3 h compared to control (uninfected), respectively, in bacteria-challenged abalones. The results suggest that Ab-CaReg I could be effectively induced to maintain internal Ca(2+) homeostasis of the animal due to influx of Ca(2+) in the cells by external stimuli such as a high dose of Ca(2+) and pathogens like bacteria.

Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H(2)O(2) oxidative stress and Vibrio alginolyticus bacterial infection.

Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at (223)TGA(225) that corresponds to selenocysteine (Sec) amino acid as U(75). The full length cDNA consists of 675 bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 ((97)LGFPCNQF(104)), an active site motif ((183)WNFEKF(188)) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3' UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H(2)O(2) injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1,254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H(2)O(2) oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H(2)O(2) oxidative stress and immune defence against bacterial infection.