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

Biomimetic synthesis of silver nanoparticles using endosymbiotic bacterium inhabiting euphorbia hirtal. 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 nanoparticle

Intra-specific differentiation of fungal endosymbiont AlternarialongissimaCLB44 using RNA secondary structure analysisand their anti-infective potential

New antimicrobial agents derived from endosymbio-tic fungi with unique and targeted mode of action are crucially rudimentary to combat multidrug-resistant infections. Most of the fungi isolated as endosymbionts show close morphological feature resemblance to plant pathogenic or free-living forms, and it is difficult to differentiate these different lifestyles. A fungal endosymbiont strain CLB44 was isolated from Combretum latifolium Blume (Combretaceae). CLB44 was then identified as Alternaria longissima based on morphological and internal transcribed spacer (ITS) intervening 5.8S rRNA gene sequence analysis. ITS2 RNA secondary structure analysis was carried out using mfold server with temperature 37 °C, and anti-infective potential was determined by MIC and disk diffusion methods. ITS2 RNA secondary structure analysis clearly distinguished endosymbiotic A. longissima CLB44 from free-living and pathogenic A. longissima members in the same monophyletic clade. Secondary metabolites produced effectively inhibited Pseudomonas aeruginosa (25 μg/ml), Escherichia coli (25 μg/ml), methicillin-resistant Staphylococcus aureus (50 μg/ml), Candida albicans (100 μg/ml), and other human pathogens. This study emerges as an innovative finding that explores newly revealed ITS2 RNAs that may be an insight as new markers for refining phylogenetic relations and to distinguish fungal endosymbionts with other free-living or pathogenic forms. A. longissima CLB44, in the emerging field of endosymbionts, will pave the way to a novel avenue in drug discovery to combat multidrug-resistant infections. The sequence data of this fungus is deposited in GenBank under the accession no. KU310611

Antimicrobial profiling and molecular identification of Alternaria arborescens CLB12, a Myco-endosymbiont inhabiting Combretum latifolium Blume

AbstractFungal endosymbionts are currently driving the therapeutic use of antimicrobial drugs to combat multi drug resistant infections. An endosymbiotic strain CLB12 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) collected from the Western Ghats of Southern India. Strain CLB12 was then identified as Alternaria arborescens by its characteristic cultural morphology with ITS rDNA and intervening 5.8S rRNA gene sequence. Antimicrobial profiling of ethyl acetate fraction of CLB12 was performed by disc diffusion method. Secondary metabolites produced were effectively inhibited a panel of test human pathogens including Pseudomonas aeruginosa (12.66 ± 0.33 mm), methicillin-resistant Staphylococcus aureus (12.33 ± 0.33 mm), Bacillus subtilis (16.66 ± 0.33 mm) and Candida albicans (12.00 ± 0.57 mm). TLC-bioautography assay confirmed the presence of antimicrobial compound as depicted by zone of inhibition on the intensive band. Effective inhibition of both bacterial and fungal pathogens suggests that metabolites have a broad spectrum of antimicrobial activity. The present research highlights the utility of A. arborescens CLB12 as prolific source for the biodiscovery of new antimicrobials to combat multidrug-resistant pathogens. The study also emphasizes the inter-habitat cycling of fungal endosymbiont and their possible benefits of A. arborescens to the host

Implication of PKS type I gene and chromatographic strategy for the biodiscovery of antimicrobial polyketide metabolites from endosymbiotic Nocardiopsis prasina CLA68

Advanced approach in probing for polyketide antimicrobials requires novel genomics and chromatographic strategies. An endophytic strain CLA68 was isolated from the root of Combretum latifolium Blume (Combretaceae) collected from the Western Ghats of Southern India. Strain CLA68 was then identified as Nocardiopsis prasina by its characteristic culture morphology and analysis of 16S rRNA gene sequence. Biosynthetic polyketide synthase genes were investigated using two pairs of degenerate primers. Ethyl acetate extract of CLA68 exhibited broad spectrum activity against a panel of test human pathogens. PKS type-I gene detection and chromatographic strategy yielded a robust polyketide antimicrobial compound which identified as nocapyrone E. Minimum inhibitory concentration of the purified compound against MRSA and other human pathogens ranged between 25 and 100 μg/ml. The present work highlights the utility of N. prasina CLA68 as potential source for antimicrobial polyketide nocapyrone E which could help to combat multidrug-resistant pathogens. This study demonstrates feasibility of PKS type-I gene-based molecular approach and chemical investigation by chromatographic approach is the best method for prediction and rapid discovery of novel polyketides from endosymbiotic actinomycetes. The sequence data of this endosymbiotic actinomycete is deposited in GenBank under the accession no. KP269077

Hydrothermal combustion based ZnO nanoparticles from Croton bonplandianum: Characterization and evaluation of antibacterial and antioxidant potential

The present study reports facile phytomediated synthesis of ZnO nanoparticles (ZnO NPs) using hydrothermal combustion from Croton bonplandianum Bail. The characterization studies revealed that an average 44 nm size spherical ZnO NPs with high zinc composition having good crystalline nature. FTIR analysis confirmed the presence of functional groups O-H, sp3 C-H bend, and alkoxy C-O functional groups which might be involved in capping and reduction for ZnO NPs synthesis. The biosynthesized ZnO NPs exhibited potent antibacterial activity against Gram-positive and Gram-negative bacteria. The MIC results revealed that ZnO NPs were most effective against Bacillus cereus whereas least sensitive towards Pseudomonas aeruginosa and Vibrio parahaemolyticus. This investigation suggests that biosynthesized ZnO NPs are promising agents for antibacterial and antioxidant applications in the biomedical field

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

Bioactivity-guided isolation of antimicrobial metabolite from Xylaria sp.

Symbiotic plant-microbe metabolic interactions not only have beneficial effects on plants but also contribute to rich, unmatched and complex chemical biodiversity with biological potential. Systematic delineated bioprospecting of fungal diversity associated with Ficus pumila Linn (Moraceae) for antimicrobial metabolite revealed Xylaria sp. FPL-25(M). The present study describes bioactivity guided fractionation prioritized for antimicrobial potential. Thus, chemical investigation of culture broth of Xylaria sp. FPL-25(M) by bioactivity guided fractionation with spectroscopic techniques revealed bioactive metabolite xylobovide-9-methyl ester. The xylobovide-9-methyl ester exhibited broad-spectrum antimicrobial activity. However, Gram-positive bacteria and fungi were more susceptible than Gram-negative bacteria. The present study results represent bioassay-based screening strategy which facilitates rapid, efficient and reliable approach for endophytic strain prioritization for novel bioactive molecules

Comparative Phytochemical Investigation of the Sources of Ayurvedic Drug Patha: A Chromatographic Fingerprinting Analysis

Standardization of herbal drugs based on their chemical and biological activity profile is an important prerequisite for acquiring the herbal market. The main problem encountered in standardization of Ayurvedic drugs is proper identification of the source plant. The present study was aimed to establish identification characters, quality control parameters, chemical and biological parameters for roots of three plants Cissampelos pareira, Cyclea peltata and Stephania japonica (Fam. Menispermaceae) which are being used as source of Patha, in the market. All the three plant were subjected for evaluation of quality control parameters as per WHO guidelines and root extracts and total alkaloidal fractions were subjected for HPTLC and HPLC fingerprinting analysis using a marker compound Bebeerine isolated from roots of Cissampelos pareira. The parameters studied clearly indicated the significant differences among the three plant materials. The roots of Cissampelos pareira can be distinguished from other two plants by presence of high concentration of alkaloids especially the presence of high concentration of pharmacologically active alkaloid bebeerine, which was found to be present in very low concentration in Stephania japonica and absent in roots of Cyclea peltata. The roots of Cyclea peltata were found to contain high concentration of saponins and comparatively in low concentration in Cissampelos pareira where as it was found to be absent in roots of Stephania japonica