Characterization of eDNA from the Clinical Strain Acinetobacter baumannii AIIMS 7 and Its Role in Biofilm Formation

Release of extracellular DNA (eDNA) was observed during in vitro growth of a clinical strain of Acinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20–200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation in A. baumannii. This is the first paper elucidating the characteristics and role of eDNA in A. baumannii biofilm, which may provide new insights into its pathogenesis

An MFS Transporter-Like ORF from MDR Acinetobacter baumannii AIIMS 7 Is Associated with Adherence and Biofilm Formation on Biotic/Abiotic Surface

A major facilitator superfamily (MFS) transporter-like open reading frame (ORF) of 453 bp was identified in a pathogenic strain Acinetobacter baumannii AIIMS 7, and its association with adherence and biofilm formation was investigated. Reverse transcription PCR (RT-PCR) showed differential expression in surface-attached biofilm cells than nonadherent cells. In vitro translation showed synthesis of a ~17 kDa protein, further confirmed by cloning and heterologous expression in E. coli DH5α. Up to 2.1-, 3.1-, and 4.1- fold biofilm augmentation was observed on abiotic (polystyrene) and biotic (S. cerevisiae/HeLa) surface, respectively. Scanning electron microscopy (SEM) and gfp-tagged fluorescence microscopy revealed increased adherence to abiotic (glass) and biotic (S. cerevisiae) surface. Extracellular DNA(eDNA) was found significantly during active growth; due to probable involvement of the protein in DNA export, strong sequence homology with MFS transporter proteins, and presence of transmembrane helices. In summary, our findings show that the putative MFS transporter-like ORF (pmt) is associated with adherence, biofilm formation, and probable eDNA release in A. baumannii AIIMS 7

Characterization of the algC

Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P<0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces

Adiantum philippense

Development of an ecofriendly, reliable, and rapid process for synthesis of nanoparticles using biological system is an important bulge in nanotechnology. Antioxidant potential and medicinal value of Adiantum philippense L. fascinated us to utilize it for biosynthesis of gold and silver nanoparticles (AuNPs and AgNPs). The current paper reports utility of aqueous extract of A. philippense L. fronds for the green synthesis of AuNPs and AgNPs. Effect of various parameters on synthesis of nanoparticles was monitored by UV-Vis spectrometry. Optimum conditions for AuNPs synthesis were 1 : 1 proportion of original extract at pH 11 and 5 mM tetrachloroauric acid, whereas optimum conditions for AgNPs synthesis were 1 : 1 proportion of original extract at pH 12 and 9 mM silver nitrate. Characterization of nanoparticles was done by TEM, SAED, XRD, EDS, FTIR, and DLS analyses. The results revealed that AuNPs and AgNPs were anisotropic. Monocrystalline AuNPs and polycrystalline AgNPs measured 10 to 18 nm in size. EDS and XRD analyses confirmed the presence of elemental gold and silver. FTIR analysis revealed a possible binding of extract to AuNPs through –NH2 group and to AgNPs through C=C group. These nanoparticles stabilized by a biological capping agent could further be utilized for biomedical applications

Synthesis of Gold Nanoanisotrops Using Dioscorea bulbifera

Biosynthesis of metal nanoparticles employing plant extracts and thereby development of an environmentally benign process is an important branch of nanotechnology. Here, the synthesis of gold nanoparticles using Dioscorea bulbifera tuber extract (DBTE) as the reducing agent is reported. Field emission scanning electron microscopy (FESEM), energy-dispersive spectroscopy (EDX), X-ray diffraction (XRD), and UV-visible absorption spectroscopy confirmed the reduction of gold ions to AuNPs. The anisotropic nanoparticles consist of a mixture of gold nanotriangles, nanoprisms, nanotrapezoid, and spheres. The kinetics of particle formation was time dependent and was enhanced by the increase of temperature from 6°C to 50°C, the optimum being 50°C. The optimum concentration of chloroauric acid was found to be 1 mM. Complete reduction of the metal ions within 5 hours by DBTE highlights the development of a novel ecofriendly route of biological synthesis of gold nanoparticles. This is the first paper on synthesis of gold nanoparticles using DBTE

Antidiabetic Activity of Gnidia glauca

Diabetes is a metabolic disorder affecting about 220 million people worldwide. One of the most critical complications of diabetes is post-prandial hyper-glycemia (PPHG). Glucosidase inhibitor and α-amylase inhibitors are class of compounds that help in managing PPHG. Low-cost herbal treatment is recommended due to their lesser side effect for treatment of diabetes. Two plants with significant traditional therapeutic potential, namely, Gnidia glauca and Dioscorea bulbifera, were tested for their efficiency to inhibit α-amylase and α-glucosidase. Stem, leaf, and flower of G. glauca and bulb of D. bulbifera were sequentially extracted with petroleum ether, ethyl acetate, and methanol as well as separately with 70% ethanol. Petroleum ether extract of flower of G. glauca was found to inhibit α-amylase significantly (78.56%). Extracts were further tested against crude murine pancreatic, small intestinal, and liver glucosidase enzyme which revealed excellent inhibitory properties. α-glucosidase inhibition provided a strong in vitro evidence for confirmation of both G. glauca and D. bulbifera as excellent antidiabetic remedy. This is the first report of its kind that provides a strong biochemical basis for management of type II diabetes using G. glauca and D. bulbifera. These results provide intense rationale for further in vivo and clinical study

potential

Choke point analysis of the metabolic pathways of Acinetobacter baylyi: A genomics approach to asses

Lignin peroxidase mediated silver nanoparticle synthesis in Acinetobacter sp.

Abstract Metals present in environment render the bacteria to attain certain resistance machinery to survive, one of which is transformation of metal ions to nano forms. Various enzymes and proteins have been suggested to play significant role in synthesis of silver nanoparticles (AgNPs) in bacteria. In present study, we have purified lignin peroxidase from secreted enzyme extract of Acinetobacter sp. employing diethyl aminoethyl cellulose ion exchange and Biogel P-150 gel filtration column chromatography. The purified fraction has a specific activity of 1.571 U/mg with substrate n-propanol and 6.5-fold purification. The tetrameric enzyme, with molecular weight of 99 kDa, consisted of dimers of two polypetides of 23.9 and 24.6 kDa as revealed by native and SDS-PAGE. On exposure to purified enzyme, spherical polydispersed AgNPs of ~ 50 nm were obtained as observed under transmission electron microscope. Optimum activity of the purified enzyme was obtained at pH 2 and 60 °C with n-propanol as substrate. This is the first report describing the reduction of extracellular silver ions by lignin peroxidase purified from Acinetobacter sp

Synthesis of an adenine nucleoside containing the (8'R) epimeric carbohydrate core of amipurimycin and its biological study

The (8'R) epimeric carbohydrate core 2 of amipurimycin was synthesized from D-glucose derived allylic alcohol 3 in 11 steps and 13% overall yield. The key steps involve an acid-catalyzed acetonide ring opening of 9 with concomitant formation of an unprecedented pyranose ring skeleton to give 2,7-dioxabicyclo[3.2.1]octane 10. The α-orientation of the furan ring in 10 readily allows the stereoselective β-glycosylation and opening of the furanose ring that on removal of protecting groups affords the pyranosyl adenine nucleoside 2. The antifungal and anticancer activities of 2 were studied