Exploring the biocompatibility and healing activity of actinobacterial-enhanced reduced nano-graphene oxide in in vitro and in vivo model and induce bone regeneration through modulation of OPG/RANKL/RUNX2/ALP pathways.

BACKGROUND: The development of cost-effective, simple, environment-friendly biographene is an area of interest. To accomplish environmentally safe, benign culturing that has advantages over other methods to reduce the graphene oxide (GO), extracellular metabolites from actinobacteria associated with mushrooms were used for the first time. METHODS: Bactericidal effect of GO against methicillin-resistant Staphylococcus aureus, antioxidant activity, and hydroxyapatite-like bone layer formation, gene expression analysis and appropriate biodegradation of the microbe-mediated synthesis of graphene was studied. RESULTS: Isolated extracellular contents Streptomyces achromogenes sub sp rubradiris reduced nano-GO to graphene (rGO), which was further examined by spectrometry and suggested an efficient conversion and significant reduction in the intensity of all oxygen-containing moieties and shifted crystalline peaks. Electron microscopic results also suggested the reduction of GO layer. In addition, absence of significant toxicity in MG-63 cell line, intentional free radical scavenging prowess, liver and kidney histopathology, and Wistar rat bone regeneration through modulation of OPG/RANKL/RUNX2/ALP pathways show the feasibility of the prepared nano GO. CONCLUSIONS: The study demonstrates the successful synthesis of biographene from actinobacterial extracellular metabolites, its potential biomedical applications, and its promising role in addressing health and environmental concerns.

Mosquito larvicidal activity of silver nanoparticles synthesised using actinobacterium, Streptomyces sp. M25 against Anopheles subpictus, Culex quinquefasciatus and Aedes aegypti.

The present work reports the larvicidal potential of microbially synthesised silver nanoparticles (AgNPs) by using an actinobacterium, Streptomyces sp. M25 isolated from Western Ghats, Tamil Nadu, India. The biomass of Streptomyces sp. was exposed to 1 mM silver nitrate (AgNO3) solution. The synthesis of AgNPs was confirmed by visual inspection followed by instrumental analysis such as UV-vis spectroscopy, fourier transform infrared spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray spectroscopy, scanning electron microscopy, atomic force microscopy and transmission electron microscopy (TEM) with selected area electron diffraction. Based on the TEM and XRD analysis, the average size of the AgNPs was determined to be 10-35 nm. The biosynthesised AgNPs exhibited significant larvicidal activity against malarial vector, Anopheles subpictus (LC50 51.34 mg/L and χ (2) value of 8.228), filarial vector, Culex quinquefasciatus (LC50 48.98 mg/L and χ (2) value of 14.307) and dengue vector, Aedes aegypti (LC50 60.23 mg/L and χ (2) value of 4.042), respectively. Similarly, AgNO3 had larvicidal activity against malarial vector, A. subpictus (LC50 42.544 mg/L and χ (2) value of 2.561), filarial vector, C. quinquefasciatus (LC50 44.922 mg/L and χ (2) value of 1.693) and dengue vector, A. aegypti (LC50 39.664 mg/L and χ (2) value of 5.724), respectively. The current study is a rapid, cost effective, eco-friendly and single step approach. The Streptomyces sp. M25 is a newly added source for the synthesis of AgNPs with improved larvicidal activity.

Characterization of a potential ß-lactamase inhibitory metabolite from a marine Streptomyces sp. PM49 active against multidrug-resistant pathogens.

Actinobacteria is a prolific producer of complex natural products; we isolated a potential marine Streptomyces sp. PM49 strain from Bay of Bengal coastal area of India. The strain PM49 exhibited highly efficient antibacterial properties on multidrug-resistant pathogens with a zone of inhibition of 14-17 mm. SSF was adopted for the production of the secondary metabolites from PM49 with ISP2; utilizing agricultural wastes for compound extraction was also attempted. Bioactive fraction of Rf value 0.69 resolved using chloroform and ethyl acetate (1:1, v/v) was obtained and subjected to further analysis. Based on UV, IR, ESI-MS, and (1)H and (13)C NMR spectral analysis, it was revealed that the compound is closely similar to cyslabdan with a molecular mass of 467.66 corresponding to the molecular formula C25H41NO5S. ESBL and MBL production was screened in the hospital test isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Staphylococcus aureus. PCR amplification in the phenotypically positive strains was positive for bla IMP, bla SHV, bla CTX-M, and mec genes. The ß-lactamase enzyme from tested strains had cephalosporinase activity with a 31-kDa protein and isolated compound from the strain possessing ß-lactamase inhibitory potential. MIC of the active fraction was 16-32 µg/ml on ATCC strains; the ceftazidime and meropenem sensitive and resistant test strains showed MIC of 64-256 µg/ml. The Streptomyces sp. PM49 aerial mycelium was rectiflexibile; the 16S rRNA showed 99 % identity with Streptomyces rochei and submitted at Genbank with accession no JX904061.1.

Mechanism of drug resistance, characterization of plasmid-borne determinants and transformation study in P. aeruginosa from burn and ICU units-its susceptibility pattern.

The transfer of drug resistance between hospital pathogens has led to alarming increase of multidrug resistant strains imposing therapeutic challenges. These resistant isolates harbor various mechanisms to counteract the drugs administered and have been reported to deliver these factors to sensitive strains in hostile environment. The present study aimed to screen for multidrug resistant Pseudomonas aeruginosa strains for the production of extended-spectrum ß-lactamases, metallo-ß-lactamases, AmpC ß-lactamase, drug efflux phenotypes and co-transfer the resistance for cephalosporin and other non-beta lactam antibiotics in CaCl2 treated drug sensitive E. coli strains. From the 87 samples processed about 23 isolates of P. aeruginosa were ESBL and MBL positive, 5 (20%) were found to be AmpC ß-lactamase producers, efflux mechanism was observed in 8 isolates, 15 isolates had MIC of 16 µg/ml. A putative efflux mechanism was observed in 8 out of 23 isolates that showed decrease in the MIC of meropenem with reserpine. The plasmid profile was characterized for all the common isolates obtained from burn and ICU units. About 69.66% of E. coli recombinants scored positive for both beta lactam and non-beta lactam antibiotics is due to co transfer of resistant plasmid obtained from P. aeruginosa.

Phylogenetic study of sponge associated bacteria from the Lakshadweep archipelago and the antimicrobial activities of their secondary metabolites.

Marine ecosystem of the Lakshadweep archipelago is unique and known to have a very high degree of biodiversity with a number of endemic flora and fauna. The present study focuses to isolate the endosymbiotic microorganism from sponges and its effectiveness against marine ornamental fish pathogens. The sponges were collected from Agatti island of Lakshadweep archipelago and identified as Clathria procera, Sigmadocia fibulata and Dysidea granulosa. In which, 15 different types of bacteria were isolated and screened against marine ornamental fish pathogens (A. hydrophila, Vibrio alginolyticus, V. harveyii, V. parahaemolyticus and Pseudomonas fluorescens). The strain S25 was found as potential bacteria based on their antimicrobial activity against the fish pathogens. Molecular identification of the potential strain (S25) of the 16S rRNA gene showed 99% identity with Acinetobacter sp. The sequenced 16 s rRNA gene with 1,081 bp in length was submitted in NCBI Genbank and Accession was obtained (GenBank Accession number HM004071). The strain exhibited high similarity (99%) with the 16S rRNA gene of Acinetobacter calcoaceticus from GenBank database. Crude extract obtained with acetone and ethyl acetate from extracellular products of S25 showed significant antimicrobial activity by disc diffusion assay using 1,500 µg/ml of crude extract. Extracellular metobolite of A. calcoaceticus was extracted by shake flask method and the crude extract was partially purified by thin layer chromatography. Partially purified crude extract showed significant inhibition zone of antimicrobial activity (A. hydrophila, V. alginolyticus, V. parahaemolyticus) and less similar activity against V. harveyii and P. fluorescens. This is the first report on A. calcoaceticus isolated from sponges of Lakshadweep archipelago and the studies are underway to characterize and purify the antimicrobial compounds of the potential bacteria.

Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10.

Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.

Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii.

This study aims in identifying MBLs particularly Zn requiring Molecular Class B enzymes produced by Pseudomonas aeruginosa and Acinetobacter baumannii .The resistance by these organisms are in a rise against all antibiotics including carbapenems and no prescribed CLSI guidelines is available for detecting them. Clinical isolates antibiotic susceptibility was determined by number of phenotypic tests by addition of 50mM of 10 µl zinc as cofactor for metallo beta lactamase production along with 0.5M ETDA of 5 µl (930 µg per disk) plain disks. Increase in zone size of the meropenem -EDTA disk compared to the meropenem disk without EDTA was recorded positive. For Zn requiring MBLs zone towards both disks of EDTA and Zn along with meropenem is detected by DDST.

Antibiotic pigment from desert soil actinomycetes; biological activity, purification and chemical screening.

An actinomycete strain, Streptomyces hygroscopicus subsp. ossamyceticus (strain D10) was isolated from Thar Desert soil, Rajasthan during the year 2006 and found to produce a yellow color pigment with antibiotic activity. Crude pigment was produced from strain D10 by solid state fermentation using wheat bran medium followed by extraction with ethyl acetate. The antimicrobial activity of the crude pigment was evaluated against drug resistant pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, extended spectrum beta-lactamase producing cultures of Escherichia coli, Pseudomonas aeruginosa and Klebsiella sp. About 420 mg of crude pigment was produced per 10 g of wheat bran medium. In the disc diffusion method the crude ethyl acetate extract showed a minimum of 10 mm inhibition against Klebsiella sp. and maximum of 19 mm of inhibition against Escherichia coli. The crude pigment was partially purified using thin layer chromatography with the solvent system chloroform:methanol (30:70) and the Rf value was calculated as 0.768. Antimicrobial activity of the partially purified compound from thin layer chromatography was determined using the bioautography method. The purified pigment showed minimum of 15 mm inhibition against Klebsiella sp. and a maximum of 23 mm of inhibition against vancomycin-resistant Staphylococcus aureus in the disc diffusion method. Based on the results of chemical screening, the pigment was tentatively identified as group of sugar containing molecules.