Therapeutic effects of gold nanoparticles synthesized using Musa paradisiaca peel extract against multiple antibiotic resistant Enterococcus faecalis biofilms and human lung cancer cells (A549).

Botanical-mediated synthesis of nanomaterials is currently emerging as a cheap and eco-friendly nanotechnology, since it does not involve the use of toxic chemicals. In the present study, we focused on the synthesis of gold nanoparticles using the aqueous peel extract of Musa paradisiaca (MPPE-AuNPs) following a facile and cheap fabrication process. The green synthesized MPPE-AuNPs were bio-physically characterized by UV-Vis spectroscopy, FTIR, XRD, TEM, Zeta potential analysis and EDX. MPPE-AuNPs were crystalline in nature, spherical to triangular in shape, with particle size ranging within 50 nm. The biofilm inhibition activity of MPPE-AuNPs was higher against multiple antibiotic resistant (MARS) Gram-positive Enterococcus faecalis. Light and confocal laser scanning microscopic observations evidenced that the MPPE-AuNPs effectively inhibited the biofilm of E. faecalis when tested at 100 µg mL-1. Cytotoxicity studies demonstrated that MPPE-AuNPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 100 µg mL-1. The morphological changes in the MPPE-AuNPs treated A549 lung cancer cells were visualized under phase-contrast microscopy. Furthermore, the ecotoxicity of MPPE-AuNPs on the freshwater micro crustacean Ceriodaphnia cornuta were evaluated. Notably, no mortality was recorded in MPPE-AuNPs treated C. cornuta at 250 µg mL-1. This study concludes that MPPE-AuNPs are non-toxic, eco-friendly and act as a multipurpose potential biomaterial for biomedical applications.

Conformation of phylogenetic relationship of Penaeidae shrimp based on morphometric and molecular investigations.

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly amplified polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric analysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable.

cDNA cloning, characterization and expression of lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) gene from the Indian white shrimp Fenneropenaeus indicus.

A 1372bp full length cDNA of an LGBP gene was identified from the Indian white shrimp, Fenneropenaeus indicus. The open reading frame (ORF) of the F. indicus LGBP (Fein-LGBP) was 1164bp long, encoding a polypeptide of 388 amino acids. The Fein-LGBP deduced amino acid has conserved potential recognition motif for ß-1, 3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites. Phylogenetic analysis revealed that the Fein-LGBP was grouped together with LGBPs of other crustaceans such as F. chinensis (91%, AAZ41363), Penaeus monodon (88%, AAM21213) and Litopenaeus vannamei (88%, ABU92557). Quantitative RT-PCR results showed that Fein-LGBP gene expression was up-regulated in hemocytes following a 6h challenge in response to peptidoglycan (PG) and Vibrio parahaemolyticus. Messenger RNA transcripts of Fein-LGBP were measured in hemocytes of F. indicus from different molting stages. The highest Fein-LGBP mRNA expression was observed in pre-molting stages (D0/1) when compared to post-molt stages (A and B) and inter-molt stages (C). Fein-LGBP is involved in the regulation and activation of the prophenoloxidase cascade.

Antibacterial activity of silver nanoparticles (AgNps) synthesized by tea leaf extracts against pathogenic Vibrio harveyi and its protective efficacy on juvenile Feneropenaeus indicus.

AIMS: To determine the antibacterial potential of silver nanoparticles (AgNps) synthesized by tea leaf extract against Vibrio harveyi and its protective effect on juvenile Feneropenaeus indicus. METHODS AND RESULTS: AgNps were synthesized by a simple procedure using tea leaf extract as the reducing agent. Bacteriological tests were performed in Luria-Bertani medium on solid agar plates and in liquid systems supplemented with V. harveyi against different concentrations of AgNps. AgNps synthesized in the present study were shown to be effective against V. harveyi isolated from F. indicus. The combined results of long- and short-term treatment of AgNps synthesized by tea leaf extract showed a 71% reduction in accumulated mortality. CONCLUSIONS: The long-term administration of AgNps synthesized by tea leaf extracts at the concentration of 10 microg significantly reduced the mortalities in F. indicus from V. harveyi infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The AgNps synthesized by tea leaf extract may be an alternative to antibiotics in controlling V. harveyi infections.

RpoN gene, RAPD profile, antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon.

AIM: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis-infected Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Thirty-six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. CONCLUSIONS: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.

Microwave assisted green synthesis of Hydroxyapatite nanorods using Moringa oleifera flower extract and its antimicrobial applications.

Hydroxyapatite is an important biomaterial and main mineral component found in bones for potential clinical applications. Moringa oleifera, a common plant in which all parts are edible and rich in iron content. This study reported the chemically synthesized Hydroxyapatite and green synthesis of Hydroxyapatite nanorods using the aqueous flower extract of Moringa oleifera by microwave assisted method. The synthesized Moringa oleifera flower extract Hydroxyapatite nanorods were characterized by UV-Vis spectroscopy (UV-vis), Fourier Transform Infra Red spectroscopy (FTIR), X-Ray Diffraction analysis (XRD), Transmission Electron Microscopy (TEM), Photo Luminescence spectroscopy (PL), Thermo Gravimetric analysis (TGA) and Energy Dispersive X-ray analysis (EDX). In addition, the antimicrobial activity of these nanorods was assessed. Moringa oleifera flower extract Hydroxyapatite nanorods were crystalline in nature, rod like structure with a mean particle size of 41 nm. The antibacterial activity of Moringa oleifera flower extract capped Hydroxyapatite nanorods was greater against Gram positive bacteria than Gram negative bacteria. Furthermore, Moringa oleifera extract capped Hydroxyapatite nanorods showed a very good antifungal activity against three common pathogenic fungi including; Candida albicans, Aspergillus fumigatus and Aspergillus niger.

In vitro susceptibility of antibiotics against Vibrio spp. and Aeromonas spp. isolated from Penaeus monodon hatcheries and ponds.

Susceptibility patterns to 16 different antibiotics were investigated against pathogenic Vibrio spp. and Aeromonas spp. isolated from shrimp culture hatcheries and ponds in India. Thirteen species of Vibrio (N = 90) and two species of Aeromonas (N = 7) isolates were tested by agar disk diffusion. The results show that 100% of the isolates were resistant to ampicillin, and that 43.2% and 47.4% were sensitive to chlortetracycline and erythromycin, respectively. Susceptibility patterns of another 160 isolates belonging to the genera Vibrio and Aeromonas obtained from the water samples of shrimp hatcheries and ponds were tested against six commonly used antibiotics. Results indicate that isolates from the hatcheries were more resistant to antimicrobials than isolates from the ponds. The minimum inhibitory concentrations of five antibiotics against the different Vibrio spp. and Aeromonas spp. were determined. Ciprofloxacin was found to be the most effective in controlling the isolates from hatcheries and ponds compared with the other antibiotics used in the study. Our results reveal that antibiotic-resistant bacteria are widespread in the shrimp culture hatcheries and ponds in India. Potential risk to human health was not addressed in this study and remains to be elucidated.

Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae.

In this study, zinc oxide nanoparticles were biologically synthesized using the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs). The synthesized Pam-ZnO NPs were characterized by UV-Vis spectrophotometer, FTIR, TEM and XRD analysis. TEM analysis of Pam-ZnO NPs showed the average size of about 20-50 nm. Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8-10 µg/ml. Confocal laser scanning microscope (CLSM) images revealed that Pam-ZnO NPs strongly inhibited the biofilm forming ability of S. aureus. In addition, Pam-ZnO NPs showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 µg/ml. The histopathological studies of Pam-ZnO NPs treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes including rupture and disintegration of epithelial layer and cellular vacuolization. The present study conclude that Pam-ZnO NPs showed effective control of S. aureus biofilms and mosquito larvae by damaging the mid gut cells.

Quorum-quenching activity of the AHL-lactonase from Bacillus licheniformis DAHB1 inhibits Vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality.

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.

cDNA cloning, characterization and expression analysis of a novel antimicrobial peptide gene penaeidin-3 (Fi-Pen3) from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

A new member of antimicrobial peptide genes of the penaeidin family, penaeidin 3, was cloned from the haemocytes of Indian white shrimp Fenneropeneaus indicus (F. indicus), by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR) methods. The complete nucleotide sequence of cDNA clone of Indian white shrimp F. indicus Penaeidin 3 (Fi-Pen3) was 243bp long and has an open reading frame which encodes 80 amino acid peptide. The homology analysis of Fi-Pen3 sequence with other Penaeidins 3 shows higher similarity with Penaeus monodon (92%). The theoretical 3D structure generated through ab initio modelling indicated the presence of two-disulphide bridges in the alpha-helix. The signal peptide sequence of Fi-Pen3 is almost entirely homologous to that of other Penaeidin 3 of crustaceans, while differing relatively in the N-terminal domain of the mature peptide. The mature peptide has a predicted molecular weight of 84.9kDa, and a theoretical pI of 9.38. Phylogenetic analysis of Fi-Pen3 shows high resemblance with other Pen-3 from P. monodon, Litopenaeus stylirostris, Litopenaeus vannamei and Litopenaeus setiferus. Fi-Pen3 found to be expressed in haemocytes, heart, hepatopancreas, muscles, gills, intestine, and eyestalk with higher expression in haemocytes. Microbial challenge resulted in mRNA up-regulation, up to 6h post injection of Vibrio parahemolyticus. The Fi-Pen3 mRNA expression of F. indicus in the premolt stage (D(01) and D(02)) was significantly up-regulated than the postmolt (A and B) and intermolt stages (C). The findings of the present paper underline the involvement of Fi-Pen3 in innate immune system of F. indicus.

Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines.

The most practical approach to reduce morbidity and mortality of cancer is to delay the process of carcinogenesis by usage of anticancer agents. This necessitates that safer compounds are to be critically examined for anticancer activity especially, those derived from natural sources. A spice commonly found in India and the surrounding regions, is turmeric, derived from the rhizome of Curcuma longa and the major active component is a phytochemical termed curcumin. Green tea is one of the most popular beverages used worldwide, produced from the leaves of evergreen plant Camellia sinensis and the major active ingredients are polyphenolic compounds known as catechins. In this study, synergistic anticancer activity of curcumin and catechin was evaluated in human colon adenocarcinoma HCT 15, HCT 116, and human larynx carcinoma Hep G-2 cell lines. Although, both curcumin or catechin inhibited the growth of above cell lines, interestingly, in combination of both these compounds highest level of growth control was observed. The anticancer activity shown is due to cytotoxicity, nuclear fragmentation as well as condensation, and DNA fragmentation associated with the appearance of apoptosis. These results suggest that curcumin and catechin in combination can inhibit the proliferation of HCT 15, HCT 116, as well as Hep G-2 cells efficiently through induction of apoptosis.

Effect of copper on morphology, weight, and chromosomal aberrations in the spiny lobster, Panulirus homarus (Linnaeus, 1758).

Spiny lobster Panulirus homarus which had been exposed to cupric ion at 9.55 and 19.1 µg/l for 28 days was examined for sub-lethal effects including morphology, wet weight, and induced genotoxic effect on the chromosome. Following cupric exposure, the color of lobster P. homarus changed from yellowish-brown to greenish black in the hepatopancreas, changed from normal creamy white to yellowish white in the muscle, and changed to greenish black in the gill. A significant change in the percentage of wet weight of muscle (28.70 ± 0.41-23.47 ± 0.45), hepatopancreas (4.03 ± 0.12-2.63 ± 0.17), and gills (3.63 ± 0.45-3.87 ± 0.12) were observed in the copper-treated lobsters. The diploid number of chromosomes of P. homarus was over 200 metaphases from ten lobsters, as 2n = 58, and consisted of 16 acrocentric, seven metacentric, and six sub-metacentric chromosomes. The lobsters exposed to cupric ion at 9.55 and 19.1 µg/l showed different types of chromosomal aberrations such as centromeric gaps, chromatid breaks, centromeric fusion, stickiness, ring chromosomes, and acrocentric association region. The frequency of aberrations increased with duration of exposure. In conclusion, it was suggested that cupric ion interacts with the spindle formation and consequently distorts the normal karyomorphology, indicating cytogenetic effect on lobster.

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India.

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.

Shrimp vaccination trials with the VP292 protein of white spot syndrome virus.

AIMS: Construction of a recombinant vector that expresses VP292 protein of white spot syndrome virus (WSSV) and to exploit the possibility of obtaining the vaccine conferring protection against WSSV infection in shrimps. METHODS AND RESULTS: VP292 protein of WSSV was amplified from WSSV genomic DNA by PCR. The target 814 bp amplified product specific for VP292 protein was inserted in to pQE30 expression vector. The recombinant plasmid of VP292 protein was transformed and expressed in Escherichia coli under induction of isopropyl-1-1-thio-beta-D-galactoside (IPTG) and the immunoreactivity of the fusion protein was detected by Western blot. Shrimp were vaccinated by intramuscular injection of the purified protein VP292 of WSSV and challenged for 0-30 days. Vaccination trial experiments show that two injections with recombinant VP292 (rVP292) protein induced a higher resistance, with 52% relative percentage survival value, in the shrimp at the 30th day postvaccination. CONCLUSIONS: The expression system of protein VP292 of WSSV with a high efficiency has been successfully constructed. Vaccination trials show significant resistance in the shrimp vaccinated twice with recombinant VP292. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study prosper the development of WSSV protein vaccine against WSSV infection in shrimps.

Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon.

AIMS: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios. METHODS AND RESULTS: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The probiotic effect of Bacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 106-108 cfu ml-1 for 6 d before a challenge with V. harveyi at 103-104 cfu ml-1 for 1 h infection. The combined results of long- and short-term probiotic treatment of B. subtilis BT23 showed a 90% reduction in accumulated mortality. CONCLUSIONS: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.

PCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India.

AIMS: The occurrence and distribution of white spot syndrome virus (WSSV) among cultured and captured penaeid shrimps and crustaceans in the east coast of India was determined from November 1999 to April 2002 using PCR as a diagnostic tool. METHODS AND RESULTS: A total of 630 cultured samples consisting of 280 postlarvae collected from nine different hatcheries and 350 juvenile shrimps (40-60-day-old) collected from 18 different culture ponds were screened for WSSV. Of these cultured samples tested 53% were found to be single-step PCR positive. A total of 419 samples of captured crustaceans viz., Penaeus monodon brooders, P. indicus juveniles, Metapenaeus spp., crab Scylla serrata and Squilla mantis were also screened for WSSV by PCR, 23% of them were infected with WSSV. CONCLUSIONS: This study concluded that WSSV could be widespread in cultured and captured shrimps and other crustaceans in India. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that PCR screening of WSSV infection and rejection of infected stocks greatly assists shrimp aquaculture farmers for successful production and harvest.