Devaraju Rakshith

Extracellular synthesis of silver nanoparticles by novel Pseudomonas veronii AS41G inhabiting Annona squamosa L. and their bactericidal activity

In present investigation extracellular synthesis of silver nanoparticles were synthesized using cell free supernatant of Pseudomonas veronii AS41G isolated from Annona squamosa L. The bacterium significantly reduced silver nitrate to generate silver nanoparticles which was characterized with hyphenated techniques. Synthesis of silver nanoparticles preliminary confirmed by UV-Visible spectrophotometry with the intense peak at 410nm, Further FTIR analysis revealed the possible role of biomolecules in the supernatant responsible for mediating the nanoparticles formation. The XRD spectra exhibited the characteristic Bragg peaks of 100, 111, 200, and 220 facets of the face centred cubic symmetry of nanoparticles suggesting that these nanoparticles were crystalline in nature. TEM microgram showed polydispersity of nanoparticles with size ranging from 5 to 50nm. Synthesized silver nanoparticles showed antibacterial activity against human and environmental pathogens including MRSA. The study enlightens the role of biosynthesized silver nanoparticles as an emerging alternative for drug resistant microorganisms. The obtained results are promising enough to pave the environmentally benign nanoparticle synthesis processes without use of any toxic chemicals and also envision the emerging role of endophytes towards synthesis of nanoparticles. With scanty reports available on P.veronii species, a new role has been reported in this study which will be very valuable for future researchers working on it.

Mycosynthesis of silver nanoparticles bearing antibacterial activity.

Mycosynthesis of silver nanoparticles was achieved by endophytic Colletotrichum sp. ALF2-6 inhabiting Andrographis paniculata. Well dispersed nanoparticles were characterized using UV–Visible spectrometry with maximum absorption conferring at 420 nm. FTIR analysis revealed possible biomolecules reducing the metal salt and stabilization of nanoparticles. XRD analysis depicted the diffraction intensities exhibiting between 20 and 80 °C at 2theta angle thus conferring the crystalline nature of nanoparticles. Morphological characteristic using TEM revealed the polydispersity of nanoparticles with size ranging from 20 to 50 nm. Synthesized nanoparticles exhibited bactericidal activity against selected human pathogens. Nanoparticles mode of action was carried out to reveal DNA damage activity. Thus the present investigation reports facile fabrication of silver nanoparticles from endophytic fungi.

Molecular characterization of an endophytic Phomopsisliquidambaris CBR-15 from Cryptolepis buchanani Roem. and impact of culture media on biosynthesis of antimicrobial metabolites

An endophytic fungus Phomopsis liquidambaris CBR-15, was isolated from Cryptolepis buchanani Roem. (Asclepiadaceae) and identified by its characteristic culture morphology and molecular analysis of the ITS region of rDNA and intervening 5.8S rRNA gene. The impact of different culture media on biosynthesis of antimicrobial metabolites was tested by disc diffusion assay. Polyketide synthase gene (PKS) of the endophytic fungus was investigated using three pairs of degenerate primers LC1–LC2c, LC3–LC5c and KS3–KS4c by PCR. TLC-bioautography method was employed to detect the antimicrobial metabolites. Antimicrobial metabolites fractionated with ethyl acetate extract showed significant antimicrobial activity against the test bacteria and fungi. Biosynthesis of antimicrobial metabolites was optimum as depicted by zone of inhibition from ethyl acetate extract cultured in potato dextrose broth. Strain CBR-15 was identified as Phomopsisliquidambaris and PKS genes of the fungus were amplified with LC3–LC5c and KS3–KS4c sets of degenerate primers. These findings suggest that endophytic P.liquidambaris CBR-15 harbor iterative type I fungal PKS gene domain which indicates the biosynthetic potential of endophytic fungi as producers of natural antimicrobial metabolites. The study also demonstrates the utilization and optimization of different culture media which best supports for the biosynthesis of the antimicrobial metabolites from P.liquidambaris.

Molecular profiling and antimicrobial potential of endophytic Gliomastix polychroma CLB32 inhabiting Combretum latifolium Blume

Fungal endophytes as a source of bioactive metabolites have led to the development of pharmaceutical products finding new applications. In a survey of endophytic fungal biodiversity, an antimicrobial endophytic strain CLB32 was isolated from the leaf of Combretum latifolium Blume (Combretaceae) from the Western Ghats of Southern India. CLB32 was then identified as Gliomastix polychroma (KR704576) by morphological and phylogenetic analysis based on internal transcribed spacer (ITS) nuclear rDNA and intervening 5.8S rRNA gene. CLB32 here constituted the first report on incidence of endophytic fungi from C. latifolium Blume. Ethyl acetate fraction of strain CLB32 was evaluated for antimicrobial activity by disc diffusion assay. Secondary metabolites produced effectively inhibited methicillin-resistant Staphylococcus aureus (18.33 ± 0.33 mm), Pseudomonas aeruginosa (14.66 ± 0.33 mm) and Candida albicans (14.00 ± 0.57 mm). Biosynthesis of these antimicrobial compounds was detected by analytical TLC-bioautography method as depicted by zone of inhibition on intensive the band. These findings suggest that G. polychroma CLB32, as a producer of natural antimicrobial drugs, could help to combat against multidrug-resistant infections and also provide baseline information for industrial applications.

Antimicrobial properties of endophytic actinomycetes isolated from Combretum latifolium Blume, a medicinal shrub from Western Ghats of India

Endophytic actinomycetes were isolated from Combretum latifolium Blume (Combretaceae),Western Ghats of Southern India and identified by its characteristic culture morphology and molecular analysis of 16S rRNA gene sequences. In this survey of endophytic actinomycetes, a total of 117 isolates representing 9 different genera of endophytic actinomycetes were obtained using four different isolation media and several of them seemed to be novel taxa. Streptomyces genera (35%) was the most frequently isolated strains, followed by Nocordiopsis (17%) and Micromonospora (13%). ISP-4 medium recovered more isolates (47%) when compared to rest of the media used. Preliminary antibacterial activity of the isolates was carried out by confrontation test. Ethyl acetate fraction of selected isolates in disc diffusion assay exhibited broad spectrum antimicrobial activity against test human pathogens. All Streptomyces spp. strains displayed significant antimicrobial activity against test pathogens. Strain CLA-66 and CLA-68 which are Nocordipsis spp. inhibited both bacterial and fungal pathogens where as other isolates inhibited atleast three test human pathogens in disc diffusion assay. Antimicrobial screening of endophytic actinomycetes from this host may represent a unique potential niche for antimicrobial compounds of industrial and pharmaceutical applications. This work is the first comprehensive report on incidence of potential endophytic actinomycetes inhabiting C. latifolium Blume.

Dereplication Strategy for Antimicrobial Metabolite Using Thin-Layer Chromatography-Bioautography and LC-PDA-MS Analysis

Rapid identification of known compounds, i.e., dereplication, has become a strategically important area for the natural-product chemists involved in bioprospecting of microbes for novel bioactive metabolites. Among microbial biodiversity, endophytic fungi represent an abundant and dependable source of structurally diverse bioactive metabolites. During the course of screening for antimicrobial secondary metabolites from endophytic fungi, an antimicrobial metabolite was identified from the ethyl acetate extract obtained from the culture broth of Xylaria sp., an endophytic fungus from Ficus pumila Linn. (Moraceae) that exhibited a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. Chemical investigation of the ethyl acetate fraction using thin-layer chromatography (TLC) bioautography and LC-hyphenated techniques led to the identification of a known benzoic acid derivative. Here, we describe the application of analytical strategies (TLC-bioautography) and hyphenated spectroscopic techniques (liquid chromatography-photodiode array detector-mass spectrometry [LC-PDA-MS]) for the dereplication of antimicrobial metabolites.

Biomimetic Synthesis of Silver Nanoparticles Using Endosymbiotic Bacterium Inhabiting Euphorbia hirta L. and Their Bactericidal Potential

The present investigation aims to evaluate biomimetic synthesis of silver nanoparticles using endophytic bacterium EH 419 inhabiting Euphorbia hirta L. The synthesized nanoparticles were initially confirmed with change in color from the reaction mixture to brown indicating the synthesis of nanoparticles. Further confirmation was achieved with the characteristic absorption peak at 440 nm using UV-Visible spectroscopy. The synthesized silver nanoparticles were subjected to biophysical characterization using hyphenated techniques. The possible role of biomolecules in mediating the synthesis was depicted with FTIR analysis. Further crystalline nature of synthesized nanoparticles was confirmed using X-ray diffraction (XRD) with prominent diffraction peaks at 2θ which can be indexed to the (111), (200), (220), and (311) reflections of face centered cubic structure (fcc) of metallic silver. Transmission electron microscopy (TEM) revealed morphological characteristics of synthesized silver nanoparticles to be polydisperse in nature with size ranging from 10 to 60 nm and different morphological characteristics such as spherical, oval, hexagonal, and cubic shapes. Further silver nanoparticles exhibited bactericidal activity against panel of significant pathogenic bacteria among which Pseudomonas aeruginosa was most sensitive compared to other pathogens. To the best of our knowledge, present study forms first report of bacterial endophyte inhabiting Euphorbia hirta L. in mediating synthesizing silver nanoparticles.

Bacterial Endo-Symbiont Inhabiting Tridax procumbens L. and Their Antimicrobial Potential

Bacterial symbionts inhabiting Tridax procumbens L. were screened for antimicrobial potential with the aim to isolate potent bacteria bearing significant activity against test pathogens. The selected isolate was subjected to large scale fermentation to extract antimicrobial metabolite. The organic phase was reduced under vacuum pressure and crude ethyl acetate extract (10 mg/mL) was evaluated for antimicrobial activity against panel of test pathogens. The antibacterial activity was measured as a zone of inhibition and compared with standard antibiotics, gentamicin and tetracycline. Similarly, antifungal activity was compared with miconazole and bavistin. Significant activity was conferred against Shigella flexneri (MTCC 731) with 27±1.5 mm zone across the disc. Partially, purification of antimicrobial metabolite with TLC-bioautography and HPLC resulted in active fraction bearing activity at Rf 0.65 and eluting between 4 and 5 retention times. The obtained results are promising enough for future purification and characterization of antimicrobial metabolite. Thus, the study attributes to the growing knowledge on endophytes as one of the rich sources of antimicrobial potentials.

Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis

In the postgenomic era, a new strategy for chemical dereplication of polyketide anti-infective drugs requires novel genomics and chromatographic strategies. An endosymbiotic fungal strain CLB38 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) which was collected from the Western Ghats of India. The isolate CLB38 was then identified as Emericella variecolor by its characteristic stellate ascospores culture morphology and molecular analysis of ITS nuclear rDNA and intervening 5.8S rRNA gene sequence. ITS2 RNA secondary structure modeling clearly distinguished fungal endosymbiont E. variecolor CLB38 with other lifestyles in the same monophyletic clade. Ethyl acetate fraction of CLB38 explored a broad spectrum of antimicrobial activity against multidrug resistant pathogens. Biosynthetic PKS type-I gene and chromatographic approach afford two polyketide antimicrobial compounds which identified as evariquinone and isoindolones derivative emerimidine A. MIC of purified compounds against test microorganisms ranged between 3.12 μg/ml and 12.5 μg/ml. This research highlights the utility of E. variecolor CLB38 as an anticipate source for anti-infective polyketide metabolites evariquinone and emerimidine A to combat multidrug resistant microorganisms. Here we demonstrates a chemogenomics strategy via the feasibility of PKS type-I gene and chromatographic approach as a proficient method for the rapid prediction and discovery of new polyketides compounds from fungal endosymbionts.

Application of Bioassay-Guided Fractionation Coupled with a Molecular Approach for the Dereplication of Antimicrobial Metabolites

A systematically delineated dereplication approach was described based on genome mining and bioassay-guided fractionation using endophytic fungus Xylaria psidii FPL-52(S) isolated from leaves of Ficus pumila Linn., (Moraceae). A polyketide synthase gene-based molecular screening strategy by a degenerate oligonucleotide primer polymerase chain reaction technique coupled with a bioinformatic phylogenomic approach revealed the presence of an iterative polyketide synthase gene within the genome of Xylaria psidii FPL-52(S). Chemical dereplication of ethyl acetate extract derived from a submerged fermentation culture broth of Xylaria psidii FPL-52(S) by bioassay-guided chromatographic and hyphenated analytical spectroscopic techniques led to the identification of polyketide mycoalexin 3-O-methylmellein. Antimicrobial profiling and minimal inhibitory concentration values for 3-O-methylmellein were determined by disc diffusion and microbroth dilution techniques. Gram-positive bacteria, dermatophytic and phytopathogenic fungi were susceptible in terms of inhibition zone and minimum inhibitory concentration values when compared to co-assayed standards. Herein, we highlight and demonstrate an improved approach which facilitates efficient dereplication and effect-guided fractionation of antimicrobial metabolite(s). The present work flow serves as a promising dereplication tool to survey the biosynthetic potential of endophytic fungal diversity, thereby identifying the most promising strains and prioritizing them for novel polyketide-derived antimicrobial metabolite discovery.