Anti-virulence properties of catechin-in-cyclodextrin-in-phospholipid liposome through down-regulation of gene expression in MRSA strains.

Methicillin resistant Staphylococcus aureus (MRSA) is a prime pathogen responsible for various infections in human beings. Expression of virulence factors is a biggest challenge in MRSA, which results in failure of conventional antibiotic therapy. In connection to the search for natural and safe anti-virulence compounds, the present study focused to evaluate the anti-virulence potential of catechin-in-cyclodextrin-in-phospholipid liposome (CCPL) on MRSA strains. CCPL inhibited young biofilm (64.15-72.70%) as well degraded mature biofilm (55.60-63.65%) at ½ and » MIC doses, which was further confirmed by scanning electron microscopy and confocal laser scanning microscope studies. CCPL was capable enough to modify the surface hydrophobicity (40.26-48.59%), reduce the EPS production (1.71-2.25 folds) and bacterial motility. In addition, CCPL inhibited the synthesis of virulence factors like slime production (0.40-0.50 folds), DNase production, hemolytic activity (28.08-49.07%), proteolytic production (14.65-18.04%), lipase production, autolysis and cell auto-aggregation. CCPL prevented the staphyloxanthin production and thereby increased the susceptibility of MRSA strains towards H2O2. Further, CCPL significantly down-regulated the virulence genes (agrA, agrC, clfA, clfB, fnbA, fnbB, icaA, icaD, hla, hld, rna III, atlA, sarA, sigB & geh). Thus, the results of present study revealed that the CCPL can effectively reduce the virulence properties and its application could inhibit the pathogenicity and also prevents the development of drug-resistance in MRSA strains.

Antioxidant and cytoprotective properties of loganic acid isolated from seeds of Strychnos potatorum L. against heavy metal induced toxicity in PBMC model.

Present work carried out with the objectives to isolate active component of S. potatorum and also to evaluate its free radical scavenging activity and preventing capacity against heavy metal toxicity. Solvents of different polarity were used to prepare crude extracts of S. potatorum seeds and screened for antioxidant activity. Among the crude extracts, methanolic extract was found to exhibit higher antioxidant activity (81.22%) which was fractionated by liquid-liquid partitioning method. Among the different fractions (LF1-LF4), LF-2 showed higher antioxidant activity (98.24%) as compared to other three liquid fractions and hence LF-2 was further purified by column chromatography. Among nine column fractions (CF1-CF9), fraction CF-7 was found to have higher antioxidant activity (92.14%), which was further analyzed using LC-MS and NMR and identified as loganic acid. In vitro radical scavenging assays showed remarkable antioxidant activity of loganic acid in terms of DPPH scavenging (IC50 149 µg/ml), superoxide radical scavenging (IC50 632.43 µg/ml) and hydroxyl radical scavenging (IC50 29.78 µg/ml). Loganic acid exhibited 81% prevention of heavy metal toxicity through the mechanism of inhibiting ROS generation (2046 AU vs. 5264 AU in control) and lipid peroxidation (95.01%). Thus, the active compound (loganic acid) isolated from S. potatorum has strong free radical scavenging activity and remarkable cyto-protective effect against heavy metal mediated toxicity.

Development and characterization of catechin-in-cyclodextrin-in-phospholipid liposome to eradicate MRSA-mediated surgical site infection: Investigation of their anti-infective efficacy through in vitro and in vivo studies.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the prime pathogens responsible for surgical site infection (SSI). Treatment of SSI remains challenging because of resistant nature of MRSA, which is a major threat in recent years. Our previous work revealed the antibacterial potential of catechin isolated from cashewnut shell against MRSA. However, the application of catechin to treat MRSA-mediated SSI is hampered because of its poor solubility and low trans-dermal delivery. Hence, the present study focused on developing catechin-in-cyclodextrin-in-phospholipid liposome (CCPL) and evaluating its physicochemical characteristics and anti-infective efficacy through in vitro and in vivo models. Encapsulation of catechin with ß-cyclodextrin and soybean lecithin was confirmed through UV-Vis spectroscopy, FTIR, and XRD techniques, while TEM imaging revealed the size of CCPL (206 nm). The CCPL displayed a higher level of water solubility (25.13%) and in vitro permeability (42.14%) compared to pure catechin. A higher level of encapsulation efficiency (98.9%) and antibacterial activity (19.8 mm of ZOI and 31.25 µg/mL of MIC) were noted in CCPL compared to the catechin/cyclodextrin complex. CCPL recorded significant and dose-dependent healing of the incision, significant reduction of bacterial count, improved epithelization, and effective prevention of inflammation in skin samples of SSI-induced Balb/c mice. Data of the present work suggest that the CCPL could be considered as a novel and potential candidate to mitigate MRSA-mediated SSI after clinical trials.

Catechin isolated from cashew nut shell exhibits antibacterial activity against clinical isolates of MRSA through ROS-mediated oxidative stress.

Staphylococcus aureus causes severe infections and among all methicillin-resistant S. aureus (MRSA) remains a great challenge in spite of decade research of antibacterial compounds. Even though some synthetic antibiotics have been developed, they are not effective against MRSA, and hence, there is a search for natural, alternative and plant-based antibacterial compound. In this connection, catechin isolated from cashew nut shell was investigated for its antibacterial potential against MRSA. Catechin exhibited zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) in a range of 15.1-19.5 mm and 78.1-156.2 µg/ml, respectively, against ATCC and clinical isolates of MRSA. Among all clinical isolates, clinical isolate-3 exhibited highest sensitivity to catechin. Catechin has arrested the growth of MRSA strains and also caused toxicity by membrane disruption which was illustrated by AO/EB fluorescence staining. Increased nucleic acid leakage (1.58-28.6-fold) and protein leakage (1.40-23.50-fold) was noticed in MRSA due to catechin treatment when compared to methicillin. Bacteria treated with catechin at its MIC showed 1.52-, 1.87- and 1.74-fold increase of ROS production in methicillin susceptible S. aureus (MSSA), MRSA and clinical isolate-3 strains, respectively, as compared to control. Superoxide dismutase (5.31-9.63 U/mg protein) and catalase (1573-3930 U/mg protein) were significantly decreased as compared to control in catechin-treated S. aureus. Thus, catechin exhibited antibacterial activity through oxidative stress by increased production of ROS and decreased antioxidant enzymes. Altogether results suggest that catechin is a promising lead compound with antibacterial potential against MRSA. KEY POINTS: • Catechin was isolated and identified as active compound in cashew nut shell. • Catechin exhibited antimicrobial activity against clinical isolates of MRSA. • Bacterial cell wall damage was caused by catechin in MRSA strains. • Catechin increased the oxidative stress in MRSA by intracellular ROS production.

Gallic Acid an Agricultural Byproduct Modulates the Biofilm Matrix Exopolysaccharides of the Phytopathogen Ralstonia solanacearum.

Ralstonia solanacearum is a soil-borne plant pathogen which causes wilt disease in economically important crops of the Solanaceae family in tropical and temperate regions. As biofilm formation is the major virulence factor in R. solanacearum, research inputs are necessary to identify natural biofilm inhibitors to mitigate virulence of this bacterium. Hence in the present work, the anti-biofilm potential of phytochemical compound gallic acid (GA) isolated from an agricultural byproduct (cashewnut shell) was investigated. Initially the Minimum inhibitory concentration (MIC) of crude extracts of cashewnut shell and coconut shell against R. solanacearum were investigated. The MIC of both the extracts were 400 µg/ml and their sub-MIC (200 µg/ml) inhibited biofilms in the range of 62-70% and 49-57%, respectively. As the cashewnut shell extract have higher biofilm inhibitory effect compared to coconut shell extract, we proceeded our further study by isolating the major compound GA from cashewnut shell by acid hydrolysate method. The sub-MIC of crude cashewnut shell extract inhibited 85% of young biofilms. The MIC of GA were observed at 3 mg/ml and sub-MIC (1.5 mg/ml) was found to eradicate 85% of mature biofilms which was confirmed by standard crystal violet assay and the biofilm reduction was further visualized under light microscopy and scanning electron microscopic images. Toxicity of GA was evaluated against R. solanacearum through XTT cell viability assay and found no antibacterial effect at sub-MIC. Additionally, it is confirmed with growth curve and time kill assays. Swimming and twitching motility were considered as an important virulence factors to invade plants and to block the xylem vessels. Therefore, sub-MIC of GA was found to inhibit both swimming and twitching motility of about 93% and 63% respectively. Anti-biofilm efficacy of GA was also worked well with tomato plant model where remarkable biofilm inhibition was found on treatment with GA before and after 24 h of infection with R. solanacearum. Hence GA will be an alternative, cheap source which is eco-friendly as well as novel source for the treatment of R. solanacearum biofilms and to prevent wilt disease in important crops.

Use of agricultural waste (coconut shell) for the synthesis of silver nanoparticles and evaluation of their antibacterial activity against selected human pathogens.

Green synthesis of silver nanoparticles (AgNPs) is environmentally satisfactory because of their low cost and safe to nature. In the present study, extract of an agricultural waste, coconut (Cocos nucifera) shell is used to synthesize AgNPs and their antibacterial effect was investigated against selected human pathogens Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium. The AgNPs synthesized using coconut shell extract (CSE-AgNPs) were characterized using UV-Visible spectroscopy (absorption peak at 432 nm), Transmission Electron Microscopy (spherical shaped particles size of 14.2-22.96 nm), Fourier-Transform Infrared Spectroscopy indicating the CSE capping around the AgNPs (Peaks 1384, 1609 and 3418 corresponds to organic molecules) and X-Ray Diffraction (Peak at 32.078 and 2-Theta). CSE-AgNPs exhibited zone of inhibition against S. aureus (15 mm), E. coli (13 mm), S. typhimurium (13 mm) and L. monocytogenes (10 mm) and minimum inhibitory concentration (MIC) of 26, 53, 106 and 212 µg/ml, respectively. Growth curve assay showed the effectiveness of CSE-AgNPs to inhibit the selected pathogens when compared to amphicillin control and extract. Scanning electron microscopy results indicated that the cell wall degradation might be the possible mechanism of antibacterial action of CSE-AgNPs. Different concentrations of AgNPs (0.078-2.5 mg/ml) showed no toxicity against human PBMC cell line. Hence, such highly effective CSE-AgNPs could be explored as antibacterial agent.