In vitro and in vivo effect of 2,6-Di-tert-butyl-4-methylphenol as an antibiofilm agent against quorum sensing mediated biofilm formation of Vibrio spp.

This study unveils the in vitro and in vivo antibiofilm potential of 2,6-Di-tert-butyl-4-methylphenol (DTBMP) from Chroococcus turgidus against Vibrio spp. In the preliminary study, cell free culture supernatant (CFCS) of C. turgidus inhibited the violacein production in biomarker strain Chromobacterium violaceum and its mutant strain CV026 in a dose dependent manner. The effective biofilm inhibitory concentration (BIC) of pure compound DTBMP from C. turgidus was identified as 250 µg/ml concentration in tested Vibrio species. Furthermore, DTBMP proved to effectively inhibit the bioluminescence production in V. harveyi and other biofilm related virulence traits such as exopolysaccharides (EPS) production, hydrophobicity index, swimming and swarming motility at its BIC concentration in three major pathogenic vibrios: V. harveyi, V. parahaemolyticus and V. vulnificus. The antibiofilm potential of DTBMP was validated through light, confocal laser scanning and scanning electron microscopic analyses. In addition, the non-bactericidal effect of DTBMP was determined through growth curve and 2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Real-time PCR studies revealed the down-regulation of master quorum sensing (QS) regulator genes of V. harveyi such as luxR, luxS, luxP, luxQ and luxO on treatment with DTBMP. In vivo results confirmed that DTBMP augmented the survival rate of Litopenaeus vannamei larvae up to 75, 88 and 66% upon infection with V. harveyi, V. parahaemolyticus and V. vulnificus, respectively. The results of this study ascertain the promising effects of DTBMP as an antibiofilm agent, which could be positively explored to treat biofilm-associated vibrios infections in aquaculture.

Biological synthesis of silver nanoparticles using ß-1, 3 glucan binding protein and their antibacterial, antibiofilm and cytotoxic potential.

The present study reports the biological synthesis of silver nanoparticles using crustacean immune molecule ß-1, 3 glucan binding protein (ß-GBP) purified from the haemolymph of blue swimmer crab Portunus pelagicus. The characterization of synthesized ß-GBP based silver nanoparticles (Ppß-GBP-AgNPs) was made by UV-Vis spectroscopy, XRD, FTIR and TEM analysis. UV-Vis spectra recorded the strong absorbance peak at 420 nm due to its surface plasmon resonance. The XRD analysis revealed the crystalline nature of synthesized nanoparticles with Bragg's reflection peaks at (111), (200), (220), (311) planes. FTIR analysis showed the possible functional groups at 3422, 2926, 2847, 1648, 1556, 1407, 1016 and 669 cm-1. The mean particle size of Ppß-GBP-AgNPs was 33-47 nm revealed by TEM analysis. Ppß-GBP-AgNPs exhibit appreciable antibacterial activity against Enterococcus faecalis and Pseudomonas aeruginosa when compared to chemical based AgNPs (Chem-AgNPs). The antibiofilm property of Ppß-GBP-AgNPs was assessed through light microscopy and confocal laser scanning microscopy analysis (CLSM), which clearly demonstrates, thickness of E. faecalis and P. aeruginosa preformed biofilm was reduced to 11 µm & 8 µm from 47 µm & 45 µm respectively. Moreover, exopolysaccharide (EPS) quantification and cell surface hydrophobicity (CSH) index exhibited that, Ppß-GBP-AgNPs had the potential to disturb structural integrity of biofilm by upset EPS matrix and bacterial adhesion to hydrocarbons. In addition, the cytotoxic effect of Ppß-GBP-AgNPs was evaluated against human cervical cancer cells (HeLa). Ppß-GBP-AgNPs effectively inhibit the viability of HeLa cells at 50 µg/ml concentration and the morphological changes in Ppß-GBP-AgNPs treated HeLa cells were observed under phase contrast microscopy.

Two potential uses for silver nanoparticles coated with Solanum nigrum unripe fruit extract: Biofilm inhibition and photodegradation of dye effluent.

Silver nanoparticle was green synthesized involving the unripe fruit extracts of Solanum nigrum (Sn-AgNPs). The synthesized Sn-AgNPs was bio-physically characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Transmission electron microscopy (TEM). UV-Vis recorded the absorbance spectra at 443 nm. XRD analysis clearly demonstrated the crystalline nature of Sn-AgNPs with Bragg's reflection peaks at 111, 200, 220 and 311 lattice planes. The FTIR spectrum of Sn-AgNPs showed strong bands at 3432, 1555, 1455, 862 and 406 cm-1 which corresponds at O-H, C-H, C-C, C-OH and C-N groups respectively. TEM exhibited the spherical shape of Sn-AgNPs with particle size between 20 and 30 nm. The antibacterial effects of Sn-AgNPs were tested on clinically important biofilm forming Gram positive (Bacillus pumulis and Enterococcus faecalis) and Gram negative (Proteus vulgaris and Vibrio parahaemolyticus) bacteria. The greater inhibition of B. pumulis and E. faecalis was observed at 100 µg mL-1 of Sn-AgNPs compared to P. vulgaris and V. parahaemolyticus. The biofilm inhibition potential of Sn-AgNPs was greater against Gram positive bacteria than that of Gram negative bacteria. Furthermore, Sn-AgNPs effectively degraded the industrial effluent methyl orange dye by photocatalysis. It is concluded that Sn-AgNPs could be used as an effective therapeutics against the biofilm of clinically important bacteria. The green synthesized Sn-AgNPs can be employed to degrade dye effluents and prevent environmental pollution as well.

Exploitation of chemical, herbal and nanoformulated acaricides to control the cattle tick, Rhipicephalus (Boophilus) microplus - A review.

The tick Rhipicephalus (Boophilus) microplus is a key vector of bacterial and protozoan diseases causing heavy economic losses directly and indirectly in animal husbandry. In the past decades, the control of ticks faced some major issues, such as the rapid development of resistance in targeted vectors and non-target effects on human health and the environment, due to the employ of synthetic acaricides and repellents. Eco-friendly pesticides for treating and controlling animal parasites such as ticks were mainly from medicinal plants and thus they form the richest entity for manufacturing resources for drugs. Even though there are efforts made to discover reliable plant-based acaricides to control ectoparasites in animal husbandry, the effective control of R. (B.) microplus ticks still represent a major challenge in current veterinary entomology. Recently, a wide number of promising attempts have been conducted to use herbal preparations and green-fabricated nanoparticles for the control of R. (B.) microplus. The aim of this review is to critically summarize and discuss the use of herbal preparations used in ethno-veterinary as well as green-fabricated nanoparticles as novel acaricides for the control of the cattle tick R. (B.) microplus.

In vitro antioxidant and antibacterial activity of sulfated polysaccharides isolated from Spatoglossum asperum.

Fucoidan was extracted productively from Spatoglossum asperum and its antioxidant and antibacterial potential against Aeromonos hydrophila was investigated. The isolated fucoidan was characterized by HPLC, FTIR, 1H and 13C NMR spectrum. The isolated fucoidan contained 60.9% fucose, 21.35±0.81% sulfate and 4.2±0.56% protein, respectively. The in vitro antioxidant activity was evaluated by DPPH radical scavenging, reducing power and total antioxidant activities. The extracted fucoidan showed antioxidant properties in a dose-dependent manner. The antibacterial activity of isolated fucoidan was tested against A. hydrophila using agar bioassay, agar well diffusion and confocal assays. In this study, the minimum inhibitory concentration was obtained at 100µg/ml and the maximum zone of inhibition (33mm) was observed at 150µg/ml of fucoidan concentration in agar bioassay and agar well diffusion assay. The experimental results proved that the extracted fucoidan from S. asperum possesses admirable antioxidant and antibacterial properties.

Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity.

In this study antioxidant and anticancer effect of fucoidan isolated from brown seaweed Sargassum polycystum was investigated. The total yield of fucoidan was 4.51±0.24%, of these, 46.8% of fucose and 22.35±0.23% of sulphate respectively. The structural characteristic of fucoidan was analyzed by Fourier transform infrared spectroscopy and nuclear magnetic resonance. The antioxidant properties were determined by DPPH scavenging, reducing power and total antioxidant assays. The maximum DPPH scavenging activity (61.2±0.33%), reducing ability (67.56±0.26%) and total antioxidant activity (65.3±0.66%) were obtained at 1000µg/ml of fucoidan. The cytotoxicity effect of fucoidan showed a higher percentage (90.4±0.25%) of inhibition against the MCF-7 cell line at 150µg/ml with an estimated IC50 at 50µg/ml. Further, cytomorphological and apoptosis changes of fucoidan treated cells were observed under inverted light microscope and confocal laser scanning microscope (CLSM). The results demonstrated that the isolated fucoidan from S. polycystum possessed potent antioxidant and anticancer properties.

In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila.

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 µg ml-1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.

Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities.

The present study was aimed at biosynthesis of silver nanoparticles (AgNPs) using ethanolic extract of rose (Rosa indica) petals and testing their potential antibacterial activity using selective human pathogenic microbes, anticancer activity using human colon adenocarcinoma cancer cell line HCT 15 as well as anti-inflammatory activity using rat peritoneal macrophages in vitro. The biologically synthesized AgNPs were also characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The characterized AgNPs showed an effective antibacterial activity against Gram negative (Escherichia coli, Klebsiella pneumoniae) than Gram positive (Streptococcus mutans, Enterococcus faecalis) bacteria. MTT assay, analysis of nuclear morphology, mRNA expression of Bcl-2, Bax and protein expression of caspase 3 as well as 9, indicated potential anticancer activity. In addition, green synthesized AgNPs also attenuated cytotoxicity, nuclear morphology and free radical generation (O2(-) and NO) by rat peritoneal macrophages in vitro. The results of our study show the potential green synthesis of silver nanoparticles in mitigating their toxicity while retaining their antibacterial activities.

Ameliorative effect of ferulic acid against renal injuries mediated by nuclear factor-kappaB during glycerol-induced nephrotoxicity in Wistar rats.

The pathogenesis of glycerol-induced myoglobinuric acute renal failure involves ischemia, vascular congestion and reactive oxygen metabolites. In this study, we have investigated for the first time, the role of ferulic acid in attenuating glycerol-induced nephrotoxicity. Male Wistar rats were injected intramuscularly with 8 mL/kg body weight of 50% glycerol, glycerol + ferulic acid at the dose of 5, 10, 15, 20 and 25 mg/kg body weight. After 24 h, the rats were sacrificed and the kidneys were removed for histological and immunohistochemical studies. Furthermore, determinations of lipid peroxidation (LPO) as well as antioxidant enzymes were also analyzed; blood, urine samples were collected in order to quantify renal function and nitric oxide generation, respectively. Glycerol-induced rats showed a significant increase in the level of urinary markers assessed in serum as well as kidney and these were reversed upon ferulic acid treatment. A significant increase in urine nitric oxide, serum as well as kidney LPO, decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione were observed in glycerol-induced rats. Immunohistochemical study in glycerol-induced rats demonstrated an increase in the level of nuclear factor-kappaB (NF-κB). All these effects induced by glycerol were reduced upon treatment with ferulic acid in a dose-dependent manner. To conclude, ferulic acid enhances antioxidants and decreases NF-κB, thereby protecting the cells against stress induced by glycerol.