Amit Kumar Mandal

Green synthesis of silver nanoparticles using glucan from mushroom and study of antibacterial activity

This work demonstrates synthesis of silver nanoparticles (AgNPs) using glucan isolated from a mushroom Pleurotus florida blue variant. UV-vis spectroscopy showed maximum absorbance at 425 nm due to surface plasmon resonance of AgNPs. Average diameter of the synthesized AgNPs was 2.445 ± 1.08 nm as revealed from TEM analysis. XRD analysis confirmed the face-centered cubic (fcc) crystalline structure of metallic silver. The synthesized AgNPs-glucan conjugates exhibited antibacterial activity against multiple antibiotic resistant (MAR) bacterium Klebsiella pneumoniae YSI6A and the activity was possibly due to damage of cellular macromolecules by the generation of reactive oxygen species (ROS) which was supported by observed degradation of bacterial DNA. Decrease of bactericidal effect of AgNPs-glucan conjugates in dose-dependent manner in presence of a ROS scavenger histidine further ascertained the involvement of ROS in antibacterial activity. AgNPs-glucan conjugates at LD50 dose caused least damage (0.68% hemolysis) to human RBCs. This particular dose of AgNPs-glucan conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, cefepime and tetracycline) to which K. pneumoniae YSI6A was resistant, showed synergistic effect to inhibit almost 100% bacterial growth. It thus opens an avenue to use antibiotics in combination with minimum dosages of AgNPs-glucan conjugates to control MAR bacteria.

Bactericidal effect of polyethyleneimine capped ZnO nanoparticles on multiple antibiotic resistant bacteria harboring genes of high-pathogenicity island

Zinc oxide nanoparticles (ZnO-NP) were synthesized by alcoholic route using zinc acetate as the precursor material and lithium hydroxide as hydrolyzing agent. Further ZnO-PEI NP (derivative of ZnO-NP) was made in aqueous medium using the capping agent polyethyleneimine (PEI). The nanoparticles were characterized by XRD measurements, TEM and other techniques; the weight % of coating shell in the polymer-capped particles was determined by TGA. ZnO-PEI NP is more soluble in water than the uncapped ZnO-NP, and forms a colloidal suspension in water. PEI-capped ZnO-NP exhibited better antibacterial activity when compared with that of uncapped ZnO-NP against a range of multiple-antibiotic-resistant (MAR) Gram-negative bacterial strains harboring genes of high-pathogenicity island. ZnO-NP effectively killed these microorganisms by generating reactive oxygen species (ROS) and damaging bacterial membrane. ZnO-PEI NP at LD50 dose in combination with tetracycline showed synergistic effect to inhibit tetracycline-resistant Escherichia coli MREC33 growth by 80%. These results open up a new vista in therapeutics to use antibiotics (which have otherwise been rendered useless against MAR bacteria) in combination with minimized dosage of nanoparticles for the more effective control of MAR pathogenic bacteria.

Partial characterization and flocculating behavior of an exopolysaccharide produced in nutrient-poor medium by a facultative oligotroph Klebsiella sp. PB12.

A facultative oligotrophic strain from the water sample of River Mahananda, Siliguri India was selected for its property to produce exopolysaccharide (EPS) in nutrient-poor (oligotrophic) medium. Viability assay of the strain was performed in sterile liquid LB, R2A, river water and diluted (10(-3)) LB at 30°C and pH 7 to understand oligotrophy. The selected strain was identified by 16S rRNA gene sequencing and designated as Klebsiella sp. PB12. Phylogenetic analysis showed its closest relationship with Klebsiella variicola ATCC BAA-830(T). Purification of EPS was performed by ethanol precipitation, dialysis and freeze-drying. Chemical analysis revealed that purified EPS was mainly composed of 72.32% (w/w) neutral sugar and 14.12% (w/w) uronic acids. Fourier transform infrared (FT-IR) spectroscopy indicated the presence of hydroxyl, carboxylic and methoxyl functional groups. The optimal dosages for flocculation of activated carbon suspension were 17 mg/l EPS and 4 mM CaCl(2). EPS showed flocculating rate of above 80% over a wide range of pH (pH 3-10) whereas, more than 90% rate was noted in the temperature range (10-50°C) tested in presence of CaCl(2). Moreover, EPS showed characteristic emulsifying activity with toluene (66.6%), n-hexadecane (65%), olive oil (63.3%) and kerosene (50%). The apparent molecular weight of the EPS was ~2 × 10(5) Da.

Green synthesis of silver nanoparticles using Pongamia pinnata seed: Characterization, antibacterial property, and spectroscopic investigation of interaction with human serum albumin

In recent years, green synthesized nanoparticles from plant extract have drawn a great interest due to their prospective nanomedicinal application. This study investigates a proficient, safer, and sustainable way for the preparation of AgNPs using medicinal plant Pongamia pinnata (family: Leguminoseae, species: Pinnata) seeds extract without using any external reducing and stabilizing agent. Both ultraviolet‐visible spectrum at λmax = 439 nm and energy dispersive X‐ray spectra proof the formation of AgNPs. An average diameter of the AgNPs was 16.4 nm as revealed from transmission electron microscope. Hydrodynamic size (d = ~19.6 nm) was determined by dynamic light scattering (DLS). Zeta potential of AgNPs was found to be −23.7 mV, which supports its dispersion and stability. Fourier transform infrared study revealed that the O ─ H, C ═ O, and C‐O‐C groups were responsible for the formation of AgNPs. The antibacterial activity of the synthesized AgNPs was checked against Escherichia coli ATCC 25922. AgNPs at its LD50 dose exhibited synergistic effect with ampicillin. Because protein‐AgNPs association greatly affects its adsorption, distribution, and functionality and can also influence the functions of biomolecules. So in order to understand the adsorption and bioavailability, we investigated by fluorescence, ultraviolet‐visible, and circular dichroism spectroscopic methods the interaction of synthesized AgNPs toward human serum albumin. The binding affinity and binding sites of human serum albumin toward AgNPs were measured by using the fluorescence quenching data. The circular dichroism spectroscopic results revealed that there was a negligible change of α‐helical content in their native structure. Overall, these AgNPs show versatile biological activities and may be applied in the field of nanomedicine.

A water soluble β-glucan of an edible mushroom Termitomyces heimii: Structural and biological investigation.

A water soluble β-glucan (PS-I) with an average molecular weight ∼ 1.48 × 10(5)Da was isolated from the alkaline extract of an edible mushroom Termitomyces heimii. PS-I contained (1 → 3)-, (1 → 6)-, (1 → 3, 6)-linked and terminal β-d-glucopyranosyl moieties in a ratio of nearly 2:1:1:1. Based on the total hydrolysis, methylation analysis, periodate oxidation, Smith degradation, partial hydrolysis and 1D/2D NMR experiments the structure of the PS-I was elucidated. On the basis of these experiments, the repeating unit of the polysaccharide was found to consist of a backbone chain of two (1 → 6)-β-D-glucopyranosyl residues, one of which was branched at O-3 position with the side chain consisting of two (1 → 3)-β-D-glucopyranosyl and a terminal β-D-glucopyranosyl residue. Cytotoxic effect of PS-I on human blood lymphocytes at varied concentrations was studied. Moreover, it also exhibited potent antioxidant activities by diminishing the ROS and NO in the nicotine stimulated lymphocytes up to 200 μg/ml.

Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A.

A water-soluble heteropolysaccharide (PS) was isolated from extracellular polymeric substances (EPS) produced by a novel metal tolerant bacterium, Acinetobacter junii BB1A. Sugar analysis showed that the PS was composed of mannose, galactose and arabinose in a molar ratio of nearly 3:1:1. Structural characterization of the PS was carried out using methylation analysis, periodate oxidation, Smith degradation and 1D/2D NMR experiments. Methylation analysis revealed that the PS was consisted of 2,4-linked-mannopyranosyl, 3,4-linked-mannopyranosyl, 2-linked-galactopyranosyl, terminal mannopyranosyl and arabinopyranosyl residues in a relative proportion of nearly 1:1:1:1:1. Smith degradation of the PS showed the presence of hydrated glyceraldehyde containing disaccharide unit consisting of α-d-Manp-(1→and→4)-α-d-Manp-(1→residues where the later was directly attached to a hydrated glyceraldehyde moiety. This polysaccharide showed significant in vitro splenocyte, thymocyte, and macrophage activations with optimum dose of 100 μg/mL for macrophage and 25 μg/mL both for the splenocyte and thymocyte.

Effect of double tailed cationic surfactants on the physicochemical behavior of hybrid vesicles

Hybrid vesicles, prepared from soy phosphatidylcholine (SPC), ion pair amphiphile (IPA), cholesterol (CHOL) and dihexadecyldimethylammmonium bromide (DHDAB), were investigated to assess their potential as novel drug delivery systems for indomethacin (IMC), a nonsteroidal anti inflammatory drug (NSAID). The size, polydispersity index and zeta potential values of the vesicles were monitored with respect to time using dynamic light scattering (DLS) measurements which confirmed the profound effect of both DHDAB and the drug. Incorporation of DHDAB, although resulting in the size enhancement of the liposome, however, enhanced vesicle stability induced by electrostatic repulsion. Both conventional and freeze-fractured transmission electron microscopic (TEM) studies revealed the spherical morphology of the vesicles. FTIR studies confirmed perceptible interaction among the drug and the lipid–surfactant mixtures. Thermal behavior of the vesicles was assessed by means of differential scanning calorimetric (DSC) studies in order to understand the interaction between the incorporated NSAID and lipids. The state of polarity of IMC and another fluorescent molecular probe (7-hydroxycoumarin) were examined via absorption and emission spectroscopy respectively. Results on the fluorescence anisotropy studies helped in understanding the head group packing of the amphiphiles in the vesicles. An in vitro release study of the NSAID from vesicles revealed that incorporation of DHDAB promoted the release of the drug. Among different proposed models, the Korsmeyer–Peppas model was found to be the best one and the release mechanism was predominantly Fickian diffusion. The formulations, in the absence and presence of IMC showed no cytotoxicity in healthy human blood cell lymphocyte as well as in the human breast adenocarcinoma cell line (MCF 7). The aforementioned studies provided deeper insight into the interaction pattern of DHDAB with hybrid vesicles thereby exploring the usefulness of such systems as effective drug carriers.

Antibacterial and DNA degradation potential of silver nanoparticles synthesized via green route.

Silver nanoparticles (AgNPs) were synthesized using a hetero polysaccharide (PS) isolated from Lentinus squarrosulus (Mont.) Singer. The polysaccharide fraction (consisting of glucose, fucose and galactose) serves the role of both reducing as well as stabilizing agent. UV-vis spectroscopy showed maximum absorbance at 407 nm due to surface plasmon resonance. High resolution transmission electron microscopy (HRTEM) exhibited that the average diameter of the nanoparticles was 2.78±1.47 nm. The XRD analysis revealed face-centered cubic (fcc) geometry of silver nanoparticles. Antibacterial activity of the AgNPs-PS conjugate was tested against multiple antibiotics resistant (MAR) Escherichia coli strain MREC33 and found that the killing was due to generation of reactive oxygen species (ROS). Internalization of AgNPs-PS conjugate along with its DNA degradation capability was demonstrated using flow cytometry. AgNPs-PS conjugates showed negligible toxicity to human RBCs. This LD50 dosage of AgNPs-PS conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, kanamycin and netilmicin) to which E. coli MREC33 was resistant, showed synergistic effect to inhibit complete bacterial growth.

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India

Background In this study a large random collection (n = 2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007–2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products. Methodology/Principal Findings Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ2) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6′-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. Conclusions Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.

Spectroscopic investigation on interaction of biogenic, Croton bonplandianum leaves extract mediated potential bactericidal silver nanoparticles with human hemoglobin and human serum albumin

The green synthesis of nanoparticles has received increasing attention due to the growing demand to produce safe, cost-effective, and eco-friendly technology for nanomaterials synthesis. We report on the use of aqueous Croton bonplandianum (Family: Euphorbiaceae, Genus: Croton) leaves extract for the preparation of silver nanoparticles (AgNPs) without using any external reducing and stabilizing agent. Ultraviolet–visible spectroscopy showed maximum absorbance at 446 nm due to surface plasmon resonance of AgNPs. Energy dispersive X-ray spectra also supported the existence of AgNPs. An average diameter (d = ~17.4 nm) of the spherical AgNPs was determined from the transmission electron microscopic images. Hydrodynamic size (d = ~21.1 nm) was determined by dynamic light scattering. Fourier transform infrared analysis designed that the functional groups like O–H, N–H, , CONH2, and COOH participated in the AgNPs formation. The negative zeta potential value (−19.3 mV) of the AgNPs indicated its dispersion and stability. The AgNPs exhibited strong antibacterial activity against Escherichia coli ATCC 25922 and 1.5 nM proved to be minimum inhibitory concentration for it. Hemolysis assay demonstrated the blood compatibility of the AgNPs toward human RBCs. The binding affinity of the AgNPs toward human hemoglobin and human serum albumin (HSA) was also determined by means of fluorescence spectroscopy. The circular dichroism spectroscopy revealed that the native structures of human hemoglobin and HSA remain unchanged, but its secondary structures were slightly changed upon interaction with AgNPs. Overall, it can be concluded that the AgNPs may be applied in the area of nanomedicines.