Mohammad Azam Ansari

Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital.

Purpose: Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red Agar methods. Materials and Methods: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. Results: The minimum inhibitory concentration (MIC) was found to be in the range of 11.25-45 μg/ml. The AgNPs coated surfaces effectively restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as low as 50 μg/ml. Conclusions: Our findings suggested that AgNPs can be exploited towards the development of potential anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm.

Interaction of Al2O3 nanoparticles with Escherichia coli and their cell envelope biomolecules

The aim of this study is to investigate the antibacterial activity of aluminium oxide nanoparticles (Al2O3 NPs) against multidrug‐resistant clinical isolates of Escherichia coli and their interaction with cell envelope biomolecules.

Interaction of silver nanoparticles with Escherichia coli and their cell envelope biomolecules

The antibacterial effect of AgNPs was investigated by determining MIC/MBC and growth kinetics assay. The lowest MIC/MBC was found to be in the range of 11.25–22.5u2009µgu2009ml−1. The growth kinetics curve shows that 25u2009µgu2009ml−1 AgNPs strongly inhibits the bacterial growth. Confocal laser scanning electron microscopy (CLSM) shows that as the concentration of NPs increases, reduction in the number of cells was observed and at 50u2009µgu2009ml−1 of NPs, 100% death was noticed. Scanning electron microscopy (SEM) shows cells were severely damaged with pits, multiple depressions, and indentation on cell surface and original rod shape has swollen into bigger size. High resolution‐transmission electron microscopic (HR‐TEM) micrograph shows that cells were severely ruptured. The damaged cells showed either localized or complete separation of the cell membrane. The NPs that anchor onto cell surface and penetrating the cells may cause membrane damage, which could result in cell lysis. The interaction of AgNPs to membrane biomolecules; lipopolysaccharide (LPS) and L‐α‐phosphatidyl‐ethanolamine (PE) were investigated by attenuated total reflectance–fourier transform infrared (ATR–FTIR) spectroscopy. LPS and PE showed IR spectral changes after AgNPs exposure. The O‐antigen part of LPS was responsible for interaction of NPs through hydrogen bonding. The phosphodiester bond of PE was broken by AgNPs, forming phosphate monoesters and resulting in the highly disordered alkyl chain. The AgNPs‐induced structural changes in phospholipid may lead to the loss of amphiphilic properties, destruction of the membrane and cell leaking. The biomolecular changes in bacterial cell envelope revealed by ATR–FTIR provide a deeper understanding of cytotoxicity of AgNPs.

Gum arabic capped-silver nanoparticles inhibit biofilm formation by multi-drug resistant strains of Pseudomonas aeruginosa

Clinical isolates (nu2009=u200955) of Pseudomonas aeruginosa were screened for the extended spectrum β‐lactamases and metallo‐β‐lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped‐silver nanoparticles (GA‐AgNPs) against the multi‐drug resistant (MDR) biofilm forming P. aeruginosa. The GA‐AgNPs were characterized by UV‐spectroscopy, X‐ray diffraction, and high resolution‐transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA‐AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA‐AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A‐fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA‐AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA‐AgNPs at 50u2009µgu2009ml−1 has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA‐AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA‐AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings.

Synthesis and characterization of Schiff base octaazamacrocyclic complexes and their biological studies

A condensation reaction between 1,2-diphenylethane-1,2-dione dihydrazone (DPEDDH) and dimethyl or diethyloxalate in methanol resulted in a novel Schiff base octaazamacrocyclic ligand, (L): (6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-octaazacyclohexadecane-1,3,9,11-tetraene). Subsequently metal complexes of the type [MLX2] and [CuL]X2; (M=Mn(II), Co(II), Ni(II) and Zn(II); X=Cl or NO3) were synthesized by the reaction of the free macrocyclic ligand (L) with the corresponding metal salts in 1:1 molar ratio. These complexes were characterized on the basis of analytical data, molar conductivity and magnetic susceptibility measurements, ESI-mass, IR, NMR ((1)H and (13)C), EPR and electronic spectral studies. The thermal stability of the complexes was also studied by TGA and DTA analyses. These studies show that all the complexes have octahedral arrangement around the metal ions except copper complexes which are square planar. The ligand and its complexes were screened for their antibacterial activity in vitro against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and were also studied for their anticancer activity against the human cancer cells lines: HeLa (Human cervical carcinoma), MCF7 (Human breast adenocarcinoma) and Hep3B (Human Hepatocellular carcinoma). The recorded IC50 values for the tested compounds show moderate to good cytotoxicity against these cancer cell lines. The copper complex, [CuL]Cl2, showed excellent antimicrobial activity against tested microorganisms which is almost equivalent to the standard drug ciprofloxacin.

Crataeva nurvala nanoparticles inhibit virulence factors and biofilm formation in clinical isolates of Pseudomonas aeruginosa

Green synthesized nanoparticles have gained great attention due to their non‐toxic and non‐hazardous nature. In the present study, bark extract of the medicinal plant in Ayurveda Crataeva nurvala (Buch–Ham) (CN) was chosen for the biosynthesis of silver nanoparticles (AgNPs). These NPs were characterized by Ultra violet visible spectroscopy, Fourier Transform Infra Red, Atomic Force Microscopy, and Transmission Electron Microscopy (TEM). The average particle size of green synthesized CN‐AgNPs was 15.2u2009±u20091.01u2009nm. Gas chromatography‐ mass spectrometry analysis of methanolic bark extract involved in the formation of CN‐AgNPs revealed lupeol as a major active component. In this study, CN‐AgNPs (15u2009μgu2009ml−1) efficiently suppressed the production of quorum sensing mediated virulence factors viz. pyocyanin, protease, hemolysin, and biofilm formation in Pseudomonas aeruginosa. The pyocyanin production was strongly inhibited (74.64%) followed by proteolysis (47.3%) and hemolysin production (47.7%). However, the biofilm forming ability was maximally reduced up to 79.70%. Moreover, the Confocal Laser Scanning Microscopic Analysis showed that CN‐AgNPs inhibit colonization of P. aeruginosa on to the surface. Furthermore, TEM analysis revealed internalization of CN‐AgNPs inside the bacterial cell. It is concluded that green synthesized AgNPs have great potential to inhibit virulence factors and biofilm forming ability of drug‐resistant clinical isolates of P. aeruginosa.

Green synthesis and antifungal activity of Al2O3 NPs against fluconazole-resistant Candida spp isolated from a tertiary care hospital

Eco-friendly and cost effective Al2O3 NPs were synthesized by a green method using a leaf extract of Cymbopogon citratus. The characterization of the synthesized NPs was performed by XRD, HR-TEM, EDS, AFM, DLS, and zeta potential. To our knowledge, for the first time, the antifungal activity of Al2O3 NPs against various Candida spp isolated from oropharyngeal mucosa of HIV+ patients has been investigated. The antifungal potential of Al2O3 NPs has been assessed by determining minimum inhibitory concentrations (MIC), minimum fungicidal concentration (MFC), growth kinetics, and well diffusion methods. The minimum concentration at which Al2O3 NPs inhibit the growth of Candida spp were 250–500 μg ml−1 and more or less no growth has been observed at higher concentration (i.e., 1500 μg ml−1) when incubated for 48 h. The mechanisms of Al2O3 NPs–Candida cell biointerface interaction and its intracellular localization were further characterized by SEM and HR-TEM. The electron microscopic analysis shows that Al2O3 NPs not only attach to the surface but also penetrate inside the Candida cells and disrupt the morphological as well as physiological activity that led to the death of the cells. Due to the scavenging potential of Al2O3 NPs, the antifungal activity of Al2O3 NPs is mild towards tested yeast spp and that is why these NPs suppressed the growth of yeast cells at higher concentration. The result obtained in this study shows that Al2O3 NPs inhibit the growth of fluconazole susceptible and fluconazole resistant C. albicans and C. dubliniensis in a similar manner regardless of their drug resistant mechanisms. Thus, the present study revealed that Al2O3 NPs may be a promising remedy for the development of anticandidal agents and play an important role for sustained controlled drug delivery in medical implants and for therapeutic coatings on implantable devices used in orthopedics and dentistry.

Electron microscopic ultrastructural study on the toxicological effects of AgNPs on the liver, kidney and spleen tissues of albino mice.

The present study deals with the intraperitoneal administration of 500, 1000, 3000, and 5000mg/kg of AgNPs in albino mice for 28 days to evaluate the potential toxicological effects of AgNPs on blood biochemical parameters and to investigate the light and electron microscopic histopathological alterations on three major targets organs i.e., liver, kidney and spleen. The AgNPs was well tolerated and no mortality was observed even at the highest dose i.e., 5000mg/kg. Mice treated with 500 and 1000mg/kg AgNPs did not show significant behavioral, biochemical and ultrastructural pathological changes. Mice treated with 1000mg/kg AgNPs produces little ultrastructural alteration in liver, kidney and spleen. However, mice treated with 3000 and 5000mg/kg AgNPs revealed significant changes in biochemical parameters. Electron microscopic ultrastructural investigation of liver and kidney shows that the administration of 3000 and 5000mg/kg AgNPs revealed irregularity in the nuclear membrane, nuclear chromatin condensations, degenerated hepatocytes, swollen and pleomorphic mitochondria with distorted cristae, extensive dilation of rough endoplasmic reticulum, destructed cytoplasm, hypertrophied and fused podocytes and thickened basement membrane in the endothelial cells of the proximal tubules. The spleen sections at 3000 and 5000mg/kg AgNPs revealed megakaryocytes hyperplasia, lobulations, invaginations and folding of nuclei and nuclear membrane. The present research indicates that AgNPs were well tolerated at the lower doses, but significant alterations in liver, kidney and spleen were observed at the higher doses tested. It is, therefore, suggested that further studies are needed for the minimization of the observed side effects, especially at higher doses before AgNPs being applied in pharmaceutical application.

Biochemical, histopathological, and transmission electron microscopic ultrastructural changes in mice after exposure to silver nanoparticles

Four‐week‐old mice, weighing about 25–35 g were divided into five groups (8 mice in each group): vehicle control, low‐ (0.5 g/kg), middle‐ (1 g/kg), high‐ (3 g/kg), and exceptionally high‐dose (5 g/kg). After first and second weeks of intraperitoneal exposure to AgNPs, biochemical, histopathological, and electron microscopic ultrastructural changes were investigated. No significant changes were observed in SGOT and ALP levels after first week of exposure, while the level of SGPT significantly increased (pu2009<u20090.05) in 2nd week treated mice, indicating that inflammatory of liver might be induced by high‐dose (3 and 5 g/kg) of AgNPs. No obvious changes were observed for UA and BUN in all groups of treated mice. However, significant (pu2009<u20090.05) decrease in CR level was noticed in all groups of treated mice only at high‐dose (3 and 5 g/kg). No remarkable changes in lipid profile were observed. Light microscopic histopathological investigation shows that first week treatment had not perceptible effect on the cytoarchitecture on liver, kidney, and spleen; while, second week treatment had only sporadic mild effects on these organs. However, no ultrastructural electron microscopic changes were observed in liver, kidney, and spleen of mice treated with 0.5, 1, and 3 g/kg of AgNPs when sacrificed on first and second week; while, exceptionally high‐dose (5 g/kg) of AgNPs resulted in slight nuclear chromatin condensation and irregularities in nuclear membrane. The results suggested that AgNPs could be well tolerated in mice when given intraperitoneally and no death has been found during the experiment in any groups of treated mice. Interestingly, significant (<0.05) decrease in glucose levels in all experiment group is suggestive of curious hypoglycemic role of AgNPs warranting further study to explore its possible therapeutic potential in hyperglycemic conditions as well as its mechanism of action at molecular level.

Biochemical and histopathological ultrastructural changes caused by ZnO nanoparticles in mice

Five week-old mice were divided into a vehicle control group, and groups exposed to ZnO nanoparticles at low (0.5 g/kg), middle (1 g/kg), high (3 g/kg), and exceptionally high-dose (5 g/kg). After the first, second, third, and fourth weeks’ of exposure, blood biochemistry, histopathology, and electron microscopic ultrastructural changes in liver, kidney and spleen were investigated. Increased alkaline phosphatase activities were observed in all treated mice being statistically significant at higher dose. No changes were observed in the serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, creatinine, blood urea nitrogen, and lipid levels. During the first and second weeks of the treatment, effects on the cytoarchitecture of liver, kidney, and spleen were not perceived while during the third and fouth weeks of treatment sporadic mild effects were seen. Ultrastructural electron microscopic changes in liver, kidney, and spleen were not observed for the low-dose group on the first, second, third, and fourth weeks, suggesting that exposure to ZnO nanoparticles at low dose is safe. Long-term (i.e., more than 28 days) exposure to the exceptionally high-dose resulted in sporadic changes in nuclear chromatin condensation, irregular nuclear membrane, polymorphic mitochondria, mitochondrial swelling, and vacuolation. ZnO nanoparticles could be well tolerated and no death occurred in any group of treated mice.