ß-1,3-Glucan binding protein-based silver nanoparticles enhance the wound healing potential and disease resistance in Oreochromis mossambicus against Aeromonas hydrophilla.

Here we attempted to synthesize ß-1,3-glucan binding protein-based silver nanoparticles (Phß-GBP-AgNPs) and evaluate its wound healing and disease resistance prompting ability in Oreochromis mossambicus. Using a column chromatography technique, an immune molecule, Phß-GBP was purified from the haemolymph of rice field crab, Paratelphusa hydrodromus. Phß-GBP-AgNPs were synthesized and described through SDS-PAGE, UV-vis spectroscopy, HR-TEM, XRD and FTIR analysis. HR-TEM revealed that the synthesized Phß-GBP-AgNPs were spherical with a 20-40 nm size range and the particles were not aggregated. Wound and infection were experimentally generated in O. mossambicus and treated with Phß-GBP, chem-AgNPs and Phß-GBP-AgNPs for 20 days. The immune parameters (peroxidase, lysozyme and protease) and antioxidant enzymes (SOD and catalase) were examined in the serum of experimental fish. Phß-GBP-AgNPs elevated the immune and antioxidant enzymes during the healing process and enhanced the wound healing percentage in fish than Phß-GBP and chem-AgNPs. The immune parameters and antioxidant enzymes were declined in the serum of fish (treated with Phß-GBP-AgNPs) after the mid-period of wound healing. Compared to others, relative percentage survival was increased in experimentally wounded and infected fish treated with Phß-GBP-AgNPs against Aeromonas hydrophila. Moreover, Phß-GBP-AgNPs exhibited less toxicity towards Artemia salina than chem-AgNPs during 24 h exposure period. As a result, Phß-GBP-AgNPs may act as an alternative to commercial antibiotics and be considered an effective immunostimulant in treating skin lesions in intensive farming.

Control of biofilm forming clinically important bacteria by green synthesized ZnO nanoparticles and its ecotoxicity on Ceriodaphnia cornuta.

Zinc oxide nanoparticles were synthesized using the aqueous leaf extracts of Plectranthus barbatus (Plb-ZnO NPs) and characterized by UV-visible spectroscopy, XRD, FTIR, SEM and EDS. UV-Visible spectra recorded the absorbance peak of Plb-ZnO NPs at 343 nm. SEM analyses showed the spherical shape of Plb-ZnO NPs with the particle size between 30 and 60 nm. Plb-ZnO NPs exhibited antibacterial and antibiofilm activity against Gram positive Bacillus subtilis at all tested concentrations. In contrast, Plb-ZnO NPs showed antibacterial and antibiofilm activity against Gram negative Vibrio parahaemolyticus and Proteus vulgaris only at 100 µg/ml. The Atomic absorption spectrometry (AAS) revealed that Zn2+ dissolution was 1.87 and 8.8 µg/L at 10 and 160 µg/L of Plb-ZnO NPs respectively. The body accumulation of Zn2+ was increased from 0.8 µg/g body weight to 3.5 µg/g body weight when C. cornuta exposed to 10 µg/L and 160 µg/L respectively. Plb-ZnO NPs were toxic to Ceriodaphnia cornuta neonates (LC50: 28 µg/L). Plb-ZnO NPs caused 100% mortality of C. cornuta at 160 µg/L after 24 h. However, zinc acetate does not cause any mortality of C. cornuta upto 350 µg/L. The light and confocal laser scanning microscopic images evidenced the uptake and accumulation of Plb-ZnO NPs on the internal gut regions of C. cornuta at 160 µg/L after 5, 10, 15, 20 and 24 h. Abnormalities in the swimming behaviour such as erratic swimming (ERR), migration to bottom (BOT) and migration to water surface (SUR) of C. cornuta were noticed after treatment with different concentrations of Plb-ZnO NPs.

Antibacterial, antibiofilm and cytotoxic effects of Nigella sativa essential oil coated gold nanoparticles.

This study reports the biological synthesis of gold nanoparticles using essential oil of Nigella sativa (NsEO-AuNPs). The synthesized NsEO-AuNPs were characterized by UV-visible spectra, X-ray diffraction (XRD), FTIR and Transmission electron microscopy (TEM). UV-vis spectra of NsEO-AuNPs showed strong absorption peak at 540 nm. The X-ray diffraction analysis revealed crystalline nature of nanoparticle with distinctive facets (111, 200, 220 and 311 planes) of NsEO-AuNPs. The FTIR spectra recorded peaks at 3388, 2842, 1685, 1607, 1391 and 1018 cm(-1). TEM studies showed the spherical shape of nanoparticles and the particle size ranges between 15.6 and 28.4 nm. The antibacterial activity of NsEO-AuNPs was greater against Gram positive Staphylococcus aureus MTCC 9542 (16 mm) than Gram negative Vibrio harveyi MTCC 7771 (5 mm) at the concentration of 10 µg ml(-1). NsEO-AuNPs effectively inhibited the biofilm formation of S. aureus and V. harveyi by decreasing the hydrophobicity index (78% and 46% respectively). The in-vitro anti-lung cancer activity confirmed by MTT assay on the cell line of A549 carcinoma cells showed IC50 values of bulk Au at 87.2 µg ml(-1), N. sativa essential oil at 64.15 µg ml(-1) and NsEO-AuNPs at 28.37 µg ml(-1). The IC50 value showed that NsEO-AuNPs was highly effective in inhibiting the A549 lung cancer cells compared to bulk Au and N. sativa essential oil.

Biosynthesis of silver nanoparticles using a probiotic Bacillus licheniformis Dahb1 and their antibiofilm activity and toxicity effects in Ceriodaphnia cornuta.

In the present study, we synthesized and characterized a probiotic Bacillus licheniformis cell free extract (BLCFE) coated silver nanoparticles (BLCFE-AgNPs). These BLCFE-AgNPs were characterized by UV-visible spectrophotometer, XRD, EDX, FTIR, TEM and AFM. A strong surface plasmon resonance centered at 422 nm in UV-visible spectrum indicates the formation of AgNPs. The XRD spectrum of silver nanoparticles exhibited 2θ values corresponding to the silver nanocrystal. TEM and AFM showed the AgNPs were spherical in shape within the range of 18.69-63.42 nm and the presence of silver was confirmed by EDX analysis. Light and Confocal Laser Scanning Microscope (CLSM) images showed a weak adherence and disintegrated biofilm formation of Vibrio parahaemolyticus Dav1 treated with BLCFE-AgNPs compared to control. This result suggests that BLCFE-AgNps may be used for the control of biofilm forming bacterial populations in the biomedical field. In addition, acute toxicity results concluded that BLCFE-AgNPs were less toxic to the fresh water crustacean Ceriodaphnia cornuta (50 µg/ml) when compared to AgNO3 (22 µg/ml). This study also reports a short term analysis (24 h) of uptake and depuration of BLCFE-AgNPs in C. cornuta.

Chitosan coated Ag/ZnO nanocomposite and their antibiofilm, antifungal and cytotoxic effects on murine macrophages.

In the present study, chitosan coated Ag/ZnO (CS/Ag/ZnO) nanocomposite was synthesized and characterized by UV-Vis spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The CS/Ag/ZnO nanocomposite exhibited antibacterial activity against Gram positive (B. licheniformis and B. cereus) bacteria at 8 µg mL-1 compared to Gram negative (V. parahaemolyticus. and P. vulgaris) bacteria. CS/Ag/ZnO nanocomposite effectively inhibited the biofilm growth of Gram positive bacteria compared to Gram negative bacteria at 30 µg mL-1. The hydrophobicity index and EPS (extracellular polysaccharide) production of both Gram positive and Gram negative bacteria was decreased after treatment with 30 µg mL-1 of CS/Ag/ZnO nanocomposite. CS/Ag/ZnO nanocomposite showed effective control of fungal C. albicans biofilm (92%) at 50 µg mL-1. The inhibition of bacterial and fungal biofilms was clearly visualized under light and confocal laser scanning microscopy (CLSM). CS/Ag/ZnO nanocomposite was observed to be non toxic to RAW264.7 murine macrophages and no changes in the morphology of macrophages was observed under phase contrast microscopy. The study concludes that CS/Ag/ZnO nanocomposite is the promising candidate to be used as biomaterial against bacterial and fungal infections without any toxicity risk.

GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus.

In this study, the pathogenicity of GFP tagged Vibrio parahaemolyticus Dahv2 and the protective effect of the probiotic strain, Bacillus licheniformis Dahb1 was studied on the Asian catfish, Pangasius hypophthalmus. The experiment was carried out for 24 days with three groups and one group served as the control (without treatment). In the first group, P. hypophthalmus was orally infected with 1 mL of GFP tagged V. parahaemolyticus Dahv2 at two different doses (10(5) and 10(7) cfu mL(-1)). In the second group, P. hypophthalmus was orally administrated with 1 ml of the probiotic B. licheniformis Dahb1 at two different doses (10(5) and 10(7) cfu mL(-1)). In the third group, P. hypophthalmus was orally infected first with 1 mL of GFP tagged V. parahaemolyticus Dahv2 followed by the administration of 1 mL of B. licheniformis Dahb1 (combined treatment) at two different doses (10(5) and 10(7) cfu mL(-1)). The growth, immune (myeloperoxidase, respiratory burst, natural complement haemolytic and lysozyme activity) and antioxidant (glutathione-S-transferase, reduced glutathione and total glutathione) responses of P. hypophthalmus were reduced after post infection of GFP tagged V. parahaemolyticus Dahv2 compared to control. However, after administration with the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1), P. hypophthalmus showed significant increase in the growth, immune and antioxidant responses compared to 10(7) cfu mL(-1). On the otherhand, the growth, immune and antioxidant responses of P. hypophthalmus infected and administrated with combined GFP tagged Vibrio + Bacillus at 10(5) cfu mL(-1) were relatively higher than that of GFP tagged V. parahaemolyticus Dahv2 and control groups but lower than that of probiotic B. licheniformis Dahb1 groups. The results of the present study conclude that the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1) has the potential to protect the P. hypophthalmus against V. parahaemolyticus Dahv2 infection by enhancing the growth, immune and antioxidant responses. The probiotic B. licheniformis Dahb1 would be effectively used in the treatment of aquatic diseases for improvement of aquaculture industry.

N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection.

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish.

Exopolysaccharides-Mediated ZnO Nanoparticles for the Treatment of Aquatic Diseases in Freshwater Fish Oreochromis mossambicus.

Bacterial fish disease outbreaks are a key concern for aquaculture. Complementary feed additives such as immunostimulants can serve as an ideal solution for disease prevention. Herein, we scrutinized the efficacy of exopolysaccharides (EPSs) from probiotic Bacillus licheniformis and EPS-mediated zinc oxide nanoparticles (EPS-ZnO NPs) for a diet to evaluate growth parameters, antioxidant enzyme activities, and immune stimulation together with disease resistance against Aeromonas hydrophila and Vibrio parahaemolyticus in Mozambique tilapia Oreochromis mossambicus. Fish were separated into seven groups, with six experimental groups fed with EPS and EPS-ZnO NPs at 2, 5, and 10 mg/g and a control fed a basal diet. The fish ingesting feed supplemented with EPS and EPS-ZnO NPs at 10 mg/g showed improved growth performance. Cellular and humoral-immunological parameters were tested in serum and mucus after 15 and 30 days of feeding. These parameters were substantially enriched with a 10 mg/g diet (p < 0.05) of EPS and EPS-ZnO NPs in comparison with the control. Furthermore, the EPS and EPS-ZnO NP supplemental diet actively enhanced the antioxidant response (glutathione peroxidase, superoxide dismutase, and catalase). In addition, the supplemental diet of EPS and EPS-ZnO NPs lowered the death rate and improved the disease resistance of O. mossambicus following assessment with A. hydrophila and V. parahaemolyticus at 50 µL. Hence, the overall results suggest that the supplemental diet of EPS and EPS-ZnO NPs might be used to ensure aquaculture feed additives.

Purification, characterization and larvicidal activity of a potent bioactive compound asarone from leaves of Acorus calamus against the culician larval mosquitoes.

Mosquitoes are potent vectors by serving as agents to life-threatening diseases in humans. Increasing resistance in mosquitoes against existing insecticides and repellents brings new challenges and an opportunity to explore sustainable compounds. We chose six medicinal plants to screen potential bioactive compounds that could act as an insecticide. Among these, crude hexane leaf extract of Acorus calamus showed higher mortality percentage against Aedes aegypti and Culex quinquefasciatus. The LC50 and LC90 values were 151.86 ppm and 536.36 ppm, respectively, for the third instar A. aegypti larvae, and 174.70 ppm and 696.73 ppm, respectively, for C. quinquefasciatus. The treated larvae of both species showed morphological and physiological variations when compared to control. The GC-MS profile of purified fractions showed a single peak. Further, FT-IR and NMR analyses confirmed the propensity of the purified compound as trans asarone (phenylpropanoid; C12H16O3. LC50 and LC90 values of purified asasone-treated larvae were 2.35 ppm and 12.58 ppm, respectively, for A. aegypti and 2.15 ppm and 11.58 ppm, respectively, for C. quinquefasciatus. Treatment of different sub-lethal doses of asarone to mosquito larvae at various time intervals showed disruption of intestinal layers. By showing negligible toxicity to non-target organism, purified asarone has a great potential in vector management.

Scanometric Detection of Tomato Leaf Curl New Delhi Viral DNA Using Mono- and Bifunctional AuNP-Conjugated Oligonucleotide Probes.

Scanometric detection of tomato leaf curl New Delhi viral DNA using AuNP-conjugated mono- and bifunctional oligo probes through direct DNA hybridization assay (DDH assay) and sandwich DNA hybridization assay (SDH assay) with silver enhancement was developed. Tomato leaf curl New Delhi virus (ToLCNDV) coat protein gene-specific thiol-modified ssoligo probes were used for the preparation of mono- and bifunctional AuNP-ssoligo probe conjugates (signal probes). ssDNA arrays were prepared using polymerase chain reaction (PCR), rolling circle amplification (RCA), genomic DNAs fragments, and phosphate-modified positive control/capture probes through 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-methylimidazole conjugation on the amine-modified glass slide (GS) surface. In the DDH assay, signal probes were directly hybridized with ssDNA array of positive control and ToLCNDV DNA samples and the detection signals were amplified by silver enhancement. Dark black/gray colors were developed on the GS by the result of Ag enhancement, which can be visualized and discriminated by the naked eye. The images were captured using a simple flatbed scanner, and the determined amounts of signal probes were hybridized with their target DNA. Similarly, the SDH assay also performed through two rounds of hybridization between capture probes and target DNA; target DNA and signal probes followed by silver enhancement. The detection signals were found higher in the PCR sample than the RCA and genomic DNA samples because of the presence of increased copy numbers of complementary DNAs in PCR samples. Further, bifunctional AuNP-ssoligo probe shows higher intensity of detection signal than monofunctional probes because it can be hybridized with both strands of dsDNA targets. Moreover, the DDH-based scanometric method showed higher detection sensitivity than the SDH assay-based scanometric method. Overall, bifunctional signal probes showed more detection sensitivity than monofunctional probes in scanometric methods based on both DDH and SDH assays. The limit of detection of this developed scanometric method was optimized (100 zM to 100 pM concentration). Further, DDH assay-based scanometric method shows significant advantages over the SDH assay method, such as cost-effectiveness, because it requires only single probes (signal probes), less time-consuming by the need of only single-step hybridization, and higher detection sensitivity (up to zM). To the best of our knowledge, this is the first attempt made to develop a scanometric-based nanoassay method for the detection of plant viral DNA. This approach will be a remarkable milestone for the application of nanotechnology in the development of nanobiosensor for plant pathogen detection.

Growth inhibition and antibiofilm potential of Ag nanoparticles coated with lectin, an arthropod immune molecule.

Lectins from the haemolymph of arthropods, including crustaceans, are molecules potentially involved in the immune recognition and phagocytosis. Here, lectin was purified from the haemolymph of blue swimmer crab Portunus pelagicus, using mannose-coupled sepharose CL-4B affinity column chromatography. In SDS-PAGE analysis, lectin showed a molecular mass of approximately 155kDa. The synthesis of lectin-coated silver nanoparticles (lectin-coated AgNPs) was confirmed by UV-Vis spectroscopy, XRD, FTIR, SEM, TEM, SAED, and EDX analysis. TEM analysis revealed that lectin-coated AgNPs were spherical in shape with size of 30-57nm. Their antibacterial activity against human pathogenic Gram negative and Gram-positive bacteria was determined by agar well diffusion method. Lectin-coated AgNPs showed significant antimicrobial activity when compared to lectin and silver nitrate tested alone. The antibiofilm properties of lectin-coated AgNPs were also investigated on human pathogenic Gram-negative Proteus vulgaris, Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis and Bacillus pumilus. Lectin-coated AgNPs showed antibiofilm activity on the bacteria as well as on Candida albicans. Lectin-coated AgNPs reduced the biofilm architecture interfering with cell adhesion and polysaccharide matrix. This was additionally confirmed by exopolysaccharide (EPS) quantification index revealing the trouble in the structural reliability of biofilm by decrease in EPS and bacterial adhesion to hydrocarbons.

Molecular cloning of peroxinectin gene and its expression in response to peptidoglycan and Vibrio harveyi in Indian white shrimp Fenneropenaeus indicus.

The cDNA sequence of peroxinectin was obtained from the haemocytes of Indian white shrimp Fenneropenaeus indicus using RT-PCR and RACE. Fenneropenaeus indicus peroxinectin (Fi-Pxn) sequence has an open reading frame (ORF) of 2415 bp encoding a protein of 804 amino acids with 21 residues signal sequence. The mature protein has molecular mass of 89.8 kDa with an estimated pI of 8.6. Two putative integrin-binding motifs, RGD and KGD, were observed at the basic N-terminal and C-terminal part of the mature aminoacid sequence. Fi-Pxn nucleotide sequence comparison showed high homology to mud crab Scylla serrata (89%) and to various vertebrate and invertebrate species. qRT-PCR showed peroxinectin mRNA transcript in haemocytes of F. indicus increased at 6 h post injection of peptidoglycan and Vibrio harveyi. The Fi-Pxn was mainly expressed in the tissues of haemocytes and the heart. The moulting stage responses showed Fi-Pxn expression in premoult stages D0/1 and D0/2.

Molecular cloning, sequence analysis and expression of Fein-Penaeidin from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

Penaeidins are members of a special family of antimicrobial peptide existing in penaeid shrimp and play an important role in the immunological defense of shrimp. Here, we report a penaeidin sequence cloned from the Indian white shrimp Fenneropenaus indicus (Fein-Penaeidin). The Fein-Penaeidin open reading frame encodes a 77 amino acid peptide including a 19 amino acid signal peptide. The deduced amino acid sequences of Fein-Penaeidin include a proline rich N-terminal domain and a carboxyl-domain that contains six cysteine residues. Structural analysis revealed an alpha-helix in its secondary structure and the predicted 3D structure indicated two-disulphide bridges in the alpha-helix. Phylogenetic analysis and sequence comparison with other known peaneidin suggest the gene shows high similarity to that of penaeidin from Peneaus monodon (95%), F. indicus (80%) and Fenneropenaeus chinensis (74%). Fein-Penaeidin was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in haemocytes, Heart, gills, muscles, intestine, hepatopancreas and eyestalk. Bacterial challenge resulted in mRNA up-regulation, inducing expression at 6 h post injection indicating the penaeidin involved in the innate immunity.