Nanohybrids of silver particles immobilized on silicate platelet for infected wound healing

Silver nanoparticles supported on nanoscale silicate platelets (AgNP/NSP) possess interesting properties, including a large surface area and high biocide effectiveness. The nanohybrid of AgNP/NSP at a weight ratio 7/93 contains 5-nm Ag particles supported on the surface of platelets with dimensions of approximately 80×80×1 nm(3). The nanohybrid expresses a trend of lower cytotoxicity at the concentration of 8.75 ppm Ag and low genotoxicity. Compared with conventional silver ions and the organically dispersed AgNPs, the nanohybrid promotes wound healing. We investigated overall wound healing by using acute burn and excision wound healing models. Tests on both infected wound models of mice were compared among the AgNP/NSP, polymer-dispersed AgNPs, the commercially available Aquacel, and silver sulfadiazine. The AgNP/NSP nanohybrid was superior for wound appearance, but had similar wound healing rates, vascular endothelial growth factor (VEGF)-A levels and transforming growth factor (TGF)-β1 expressions to Aquacel and silver sulfadiazine.     

Proteomic identification of the Cus system as a major determinant of constitutive Escherichia coli silver resistance of chromosomal origin

Although silver is one of the most potent and rapidly acting toxic metals to bacteria, silver-resistant bacteria do exist with low incidence. A proteomic approach was employed to identify the silver resistance determinants of a silver-resistant Escherichia coli strain isolated from stepwise selection against increasing concentrations of silver (Li et al. J. Bacteriol 1997, 179, 6127-32). Two-dimensional gel electrophoresis and mass spectrometry analysis revealed that members of the CusCFBA copper/silver chemiosmotic efflux system were highly expressed in the silver-resistant strain but undetectable in the parental silver-sensitive strain. Disruption of the cus locus of the silver-resistant strain resulted in a decrease of the minimum inhibitory concentration of Ag (+) from more than 1 mM to 12 microM. These results suggest that the chromosomally encoded Cus system, which naturally controls the periplasmic copper concentrations, is selectable to confer a constitutive silver resistance phenotype.     

Silver-resistant Enterobacteriaceae from hospital patients

The inclusion of agar medium containing 0.5 mM AgNO3 in the hospital laboratory replicating system for routine antibiotic-susceptibility determinations resulted in identification of species of Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, and Citrobacter freundii) with silver resistance. Since the study began in October, 1975, 11 in-hospital patients receiving silver sulfadiazine for burn wound prophylaxis have yielded silver-resistant bacteria from their infected burns. During this treatment routine burn-site cultures from these patients yielded 230 isolates of Enterobacteriaceae, including 211 which were sulfonamide-resistant, 97 of which were also silver-resistant, and 38 of which were untested for silver resistance. Seven silver-resistant but sulfonamide-sensitive isolates were incidentally recovered from respiratory specimens from four nonburn patients with silver tracheostomy tubes, one silver-resistant sulfonamide-sensitive isolate was recovered from a small infected burn on the foot of an Emergency Room patient. Previous treatment of this burn was unknown. Representative AgNO3-resistant E. coli isolates from four patients were serologically untypable. Serotyping of representative isolates of K. pneumoniae showed a diversity of types except from two patients who had been in the same ward at the same time.     

The isolation and characterisation of bacteria capable of accumulating silver

Summary The isolation of silver-resistant, silver-accumulating bacteria is reported. Following the screening of a number of environmental sources, silver-resistant Enterobacteriaceae were isolated from both sewage and photographic processing effluent. The level of resistance to silver and other heavy metals was determined for a selection of these isolates and, together with preliminary accumulation data derived from batch culture studies, one isolate, a strain of Citrobacter intermedius, was selected for further examination. The effect of silver concentration on batch culture growth of this organism was also investigated.     

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins

Neutrophils contain high levels of chymotrypsin‐like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so‐called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non‐covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non‐proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.     

Effect of the size and shape of silver nanoparticles on bacterial growth and metabolism by monitoring optical density and fluorescence intensity

In this study, we demonstrate the antibacterial activity of silver nanoparticles (AgNPs), depending on their size and shape, on green fluorescent protein (GFP)-expressing E. coli, which provides a facile, rapid, and noninvasive monitoring system. By measuring optical density and fluorescence intensity in the recombinant E. coli, we found that smaller sized plate-shaped AgNPs presented higher antibacterial activity than larger sized, cubic and spherical AgNPs. In the case of 10 nm spherical AgNPs, the optical density was detectable at 15 ng/mL after 12 h incubation, but the fluorescence intensity was not. On the other hand, smaller-sized AgNPs showed higher toxicity than plate-shaped AgNPs based on the measurement of the optical density and fluorescence intensity. The combined analysis of optical density and fluorescence intensity may be helpful for understanding the effect of various materials, including nano- and organic materials, on recombinant bacteria.     

Silver nanoparticles: partial oxidation and antibacterial activities

The physical and chemical properties of silver nanoparticles that are responsible for their antimicrobial activities have been studied with spherical silver nanoparticles (average diameter approximately 9 nm) synthesized by the borohydride reduction of Ag+ ions, in relation to their sensitivity to oxidation, activities towards silver-resistant bacteria, size-dependent activities, and dispersal in electrolytic solutions. Partially (surface) oxidized silver nanoparticles have antibacterial activities, but zero-valent nanoparticles do not. The levels of chemisorbed Ag+ that form on the particle's surface, as revealed by changes in the surface plasmon resonance absorption during oxidation and reduction, correlate well with the observed antibacterial activities. Silver nanoparticles, like Ag+ in the form of AgNO3 solution, are tolerated by the bacteria strains resistant to Ag+. The antibacterial activities of silver nanoparticles are related to their size, with the smaller particles having higher activities on the basis of equivalent silver mass content. The silver nanoparticles aggregate in media with a high electrolyte content, resulting in a loss of antibacterial activities. However, complexation with albumin can stabilize the silver nanoparticles against aggregation, leading to a retention of the antibacterial activities. Taken together, the results show that the antibacterial activities of silver nanoparticles are dependent on chemisorbed Ag+, which is readily formed owing to extreme sensitivity to oxygen. The antibacterial activities of silver nanoparticles are dependent on optimally displayed oxidized surfaces, which are present in well-dispersed suspensions.     

Ultrastructural Analysis of Candida albicans When Exposed to Silver Nanoparticles

Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC₅₀ was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.     

Phytosynthesis of silver nanoparticles using Mangifera indica flower extract as bioreductant and their broad-spectrum antibacterial activity

The present study focused on the evaluation of antibacterial property of silver nanoparticles (AgNPs) synthesized using mango flower extract. The morphology of the synthesized AgNPs was observed under transmission electron microscopy and the particles have shown spherical shape in the range of 10–20 nm. X-ray powder diffraction analysis confirmed the crystalline nature of the AgNPs. The atomic percentage of the Ag element in the nanoparticles was about 7.58% which is greater than the other elements present in the sample. The AgNPs showed extensive lethal effect on both Gram-positive (Staphylococcus sp.) and Gram-negative (Klebsiella sp., Pantoea agglomerans, and Rahnella sp.) bacteria. The extensive lethal effect of AgNPs against clinically important pathogens demonstrated that the mango flower mediated AgNPs could be applied as potential antibacterial agent to control the bacterial population in the respective industries.     

New Toxicity Mechanism of Silver Nanoparticles: Promoting Apoptosis and Inhibiting Proliferation

Silver nanoparticles are increasingly recognized for their utility in biological applications, especially antibacterial effects. Herein, we confirmed the antibacterial effect of silver nanoparticles on Escherichia coli using the conventional optical density (OD) and colony-forming units (CFU) method and used flow cytometry (FC), TEM and BrdU ELISA to investigate the mechanisms of this effect. From the results, we conclude that AgNPs can simultaneously induce apoptosis and inhibit new DNA synthesis in the cells in a positive concentration-dependent manner. This study presents the first induction of apoptosis in these bacteria by AgNPs in this field. Our findings may provide a new strategy for the use of silver nanoparticles in antibacterial applications.     

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3

Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK₃. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV–visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.     

Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine.

A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine. It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance. Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45.     

Development and evaluation of different strategies for the clean synthesis of silver nanoparticles using Yarrowia lipolytica and their antibacterial activity

An environment-friendly, cheap method, biogenic synthesis of silver nanoparticles (AgNPs) is interesting as compared to physical and chemical synthesis methods. The aim of the present study was to utilize the inherent capability of Yarrowia lipolytica as a novel biocatalyst for green production of AgNPs using different strategies, including growing cells, resting cells, and cell-free extracts (CFE) under optimized reaction conditions. The produced AgNPs were evaluated with UV–vis spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and Fourier transform infrared spectrometry. In the growing cells strategy, Y. lipolytica produced spherical AgNPs under the optimized conditions, 2.5 mM of silver ions, 7.5 g/l of yeast biomass, a temperature of 30 °C, a pH of 6, and a shaking rate of 50 rpm after 48 h. The sizes and monodispersity of the AgNPs in the resting cells strategy were better than those in the other two. However, the AgNPs were produced faster in the CFE strategy. The antibacterial activity and minimal inhibitory concentration of the AgNPs against certain Gram-positive and Gram-negative bacteria were determined by the agar well diffusion and broth microdilution methods. The AgNPs had a considerable antibacterial effect compared to chloramphenicol as a broad-spectrum antibiotic.     

Antibiofilm and antimicrobial activities of green synthesized silver nanoparticles using marine red algae Gelidium corneum

In our study, green synthesis of silver nanoparticles was carried out using a red algae Gelidium corneum extract as reducing agent. The obtained silver nanoparticles were characterized by UV–vis, TEM, XRD, FTIR and ICP-MS measurements. FTIR measurements indicated the possible functional groups responsible for the stabilization and reduction of nanoparticles, while XRD analysis results explained the crystalline structure of the particles with centric cubic geometry. TEM micrographs showed that the size of the nanoparticles was between 20–50 nm. According to the broth microdilution test results, AgNPs showed a high antimicrobial activity with very low MIC values (0.51 μg/ml for Candida albicans yeast and 0.26 μg/ml for Escherichia coli bacteria). The different ultrastructural effects of silver nanoparticles on yeast and bacterial cells were observed by TEM. Antibiofilm efficacy studies were also examined in two stages as prebiofilm and postbiofilm effect. In prebiofilm effect studies, AgNPs (0.51 μg/ ml) exhibited 81% reducing effect on biofilm formation. The highest reduction rate in postbiofilm studies was 73.5% and this was achieved with 2.04 μg/ml AgNPs. Our data support that the silver nanoparticles obtained by this environmentally friendly process have potential to be used for industrial and therapeutic purposes.     

Germanium and silver resistance, accumulation, and toxicity in microorganisms.

Germanium is an inert metal with no known biological function in prokaryotic or eukaryotic organisms. Its toxicity is low compared to that of silver. Germanium is accumulated in certain bacterial strains by either energy-independent passive binding or an energy-dependent mechanism. Little is known about the molecular aspects of silver resistance, toxicity, and accumulation in bacterial strains. This is surprising because silver has been used as an antimicrobial agent in the medical field for centuries. It is likely that silver ions are excluded (resulting in decreased silver accumulation) from certain bacterial strains or immobilized intracellularly to prevent toxic effects from being exerted. These mechanisms of silver resistance have not been fully elucidated. This review examines the toxicity and accumulation of germanium and silver in selected microbial species. In addition, resistance mechanisms to these biologically nonessential metals is discussed, with more emphasis placed on silver-resistant bacteria due to the knowledge available.     

Staphylococcus aureus protects its immune‐evasion proteins against degradation by neutrophil serine proteases

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti‐staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune‐evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune‐escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.     

Analysis of non-structural proteins,NSPs of SARS-CoV-2 as targets for computational drug designing

BackgroundThe ORF1ab of Severe Acute Respiratory Syndrome, SARS Corona Virus, SARS-CoV-2 genome is processed into 15 non-structural proteins, NSPs by proteases and each NSP has a specific role in the life cycle and pathogenicity of the virus. This research analyzes possible drugs for these proteins as targets in computational drug designing using already available experimental drugs from the drug bank database.MethodsOut of 471 proteins and 8820 drugs download from Protein Data Bank, PDB and Drug Bank database respectively, 16 proteins similar to NSP 1–15 and 31 drugs as per the “Rule of three” were selected for docking. Out of 88 docking results using PyRx, 18 proteins/chains with three promising drugs, DB01977, DB07132 and DB07535 were analyzed using PyMOL for final results.ResultsNSPs 3, 5, 11, 14 and 15 were identified as targets for the drugs, DB01977, BD07132 and DB07535. Drugs, DB01977 and DB07535 bind in the same binding pockets of NSP 5 and NSP 15. Drug, DB07132 binds with more number of residues when compared with the other two drugs and this indicates that the strength of protein-drug association is more by this drug with the NSPs than other drugs. Binding pockets of NSPs for these three drugs are very close with many sharing residues in common suggesting of similarity of pharmacophore of these drugs with the target binding pockets.ConclusionThe binding pockets of NSPs are well matched with the pharmacophore of drugs and with polar surface of drugs less than or equal to 100 A², drugs, DB01977, DB07132 and DB07535 bind individually and effectively with NSPs 3, 5, 11, 14 and 15 of ORF1ab of SARS-CoV-2 genome to bring changes in the activity of SARS-CoV-2 which may be useful for biological and clinical considerations.     

Exopolysaccharide-mediated silver nanoparticles produced by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> NM101-1 as antibiotic adjuvant

A green, simple and effective approach was performed to synthesize potent silver nanoparticles using bacterial exopolysaccharide as both a reducing and stabilizing agent. The formation of nanoparticles was first screened by measuring the surface plasmon resonance peak around 400 nm using UV-vis spectroscopy. The morphology of the synthesized AgNPs was determined using TEM, which indicated that the AgNPs were spherical in shape and with an average size of 11–25 nm. The presence of elemental silver of the AgNPs was confirmed by EDX analysis. The possible functional groups of EPS responsible for the reduction and stabilization of AgNPs were evaluated using FTIR. The EPS reduced AgNPs showed excellent antibacterial, and antibiofilm activities against various human pathogenic bacteria. In addition, the efficiency of AgNPs with various broad-spectrum antibiotics against the tested strains was evaluated. It is evident that, the antibacterial and antibiofilm activities of the selected antibiotics were increased in the presence of AgNPs. The increase in activity was more pronounced for gram-negative bacteria Pseudomonas aeruginosa and E. coli. Interestingly, the combination of antibiotics with AgNPs has significantly increased the membrane protein leakage and ROS generation than antibiotics or AgNPs alone. This work supports that AgNPs can be used to enhance the activity of existing antibiotics against gram-negative and gram-positive bacteria for the treatment of infectious diseases.     

Silver resistance in Pseudomonas stutzeri

Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P. stutzeri JM303 strain. Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s). Both the silver-resistant and silver-sensitive P. stutzeri strains produced H₂S, with the silver-resistant AG259 strain producing lower amounts of H₂S than the silver-sensitive JM303 strain. However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P. stutzeri AG259 strain. Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P. stutzeri AG259 strain.     

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.