Broad-spectrum Antifungal Activity Research Articles

Synthesis of Myrtenal-Based Nanocellulose/Diacylhydrazine Complexes with Antifungal Activity for Plant Protection

In search of novel bioactive compounds with excellent and broad-spectrum antifungal activity and nanopesticides with sustained releasing property, a series of novel myrtenal-based diacylhydrazines were designed, synthesized, and characterized. The preliminary bioassay showed that myrtenal-based 2-picolinyl hydrazide exhibited better or comparable antifungal activity than that of the commercial fungicides boscalid and chlorothalonil against the tested fungi. Furthermore, myrtenal-based nanocellulose was designed as a nanopesticide carrier and prepared from two biomass materials, bleached bagasse pulp and turpentine oil. Drug-loading capacities (LCs) of these carriers and sustained releasing properties of corresponding complexes were also evaluated, and the results indicated that the esterification reaction in the different solvents would affect the micromorphology of carriers, which was the important influential factor for loading or releasing drugs. To our delight, complex VIII-3 (LC = 0.32, total releasing amount/time = 99.8%/168 h) showed a macroporous framework with the drug evenly distributed across the opening network and staged drug-releasing performance that deserved further study as a nanopesticide.

Novel Pyrimidine-Triazole Schiff Bases: Synthesis, Antifungal Activities, DFT and Molecular Docking

Seven 4-amino-5-substituted-1,2,4-triazole-3-thione Schiff base compounds were synthesized reacting 4-amino-5-substituted-1,2,4-triazole-3-thione with dichloro-substituted 5-pyrimidines, and the structures were verified by elemental analysis and spectroscopic techniques (FT-IR, 1H NMR). Additionally, in vitro antifungal activities of the compounds (named F1~F2; A1~A5) against Grape anthracnose and Wheat gibberellic have been evaluated. The compounds of F1, A4 and A5 were found to be potentially effective antifungal agents against Grape anthracnose, while the others showed the low bioactivity. The antifungal activity of all compounds against Wheat gibberellic were superior to that of fluconazole (standard drug, SD). Particularly, compounds of F1, A1, A4 and A5 exhibited a broad-spectrum antifungal activity against two fungus as compared to the others. Therefore, molecular docking study was carried out to explore the potential interaction between ligands and Fusarium graminearum (PDB ID: 5E9H). The results showed that four compounds had higher affinity compared with fluconazole and form the stable complex with the receptor. Besides, the frontier molecular orbitals (FMOs) and molecular electrostatic potentials (MEP) of four compounds with broad-spectrum antimicrobial activity were also calculated with DFT/ B3LYP /6-31G (d, p) method. The energy gap values (△ELUMO-HOMO) of all the synthesized compounds ranged from 3.307-3.375 eV, which was lower than that of SD (6.248 eV). Additionally, according to MEP the electrophile reaction of 5-substituted groups was beneficial to improving the biological activity against Wheat gibberellic and Grape anthracnose.

Construction and activity evaluation of novel benzodioxane derivatives as dual-target antifungal inhibitors

Ergosterol exert the important function in maintaining the fluidity and osmotic pressure of fungal cells, and its key biosynthesis enzymes (Squalene epoxidase, SE; 14 α-demethylase, CYP51) displayed the obvious synergistic effects. Therefore, we expected to discover the novel antifungal compounds with dual-target (SE/CYP51) inhibitory activity. In the progress, we screened the different kinds of potent fragments based on the dual-target (CYP51, SE) features, and the method of fragment-based drug discovery (FBDD) was used to guide the construction of three different series of benzodioxane compounds. Subsequently, their chemical structures were synthesized and evaluated. These compounds displayed the obvious biological activity against the pathogenic fungal strains. Notably, target compounds 10a-2 and 22a-2 possessed the excellent broad-spectrum anti-fungal activity (MIC50, 0.125–2.0 μg/mL) and the activity against drug-resistant strains (MIC50, 0.5–2.0 μg/mL). Preliminary mechanism studies have confirmed that these compounds effectively inhibited the dual-target (SE/CYP51) activity, they could cause fungal rupture and death by blocking the bio-synthetic pathway of ergosterol. Further experiments discovered that compounds 10a-2 and 22a-2 also maintained a certain of anti-fungal effect in vivo. In summary, this study not only provided the new dual-target drug design strategy and method, but also discover the potential antifungal compounds.

Isolation and Evaluation of Rhizosphere Actinomycetes With Potential Application for Biocontrolling Fusarium Wilt of Banana Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) is globally one of the most destructive soil-borne fungal diseases. Biological control using environmental microorganisms is considered as an alternative and sustainable strategy. Actinomycetes have the potential to explore biocontrol agents due to their production of diverse metabolites. The isolation and identification of high-efficiency and broad-spectrum antagonistic actinomycetes are the key for the application of biocontrol agents. In the present study, 60 actinomycetes were obtained from the rhizosphere soil of Machilus pingii in the primitive ecological natural reserve of Hainan province, China. Seventeen isolates and their extracts exhibited significant antifungal activity against F. oxysporum TR4. Particularly, strain BITDG-11 with the strongest inhibition ability had a broad-spectrum antifungal activity. The assay of its physiological and biochemical profiles showed that strain BITDG-11 had the ability to produce IAA and siderophores and had a positive response to gelatin liquefaction and nitrate reduction. Enzyme activities of chitinase, β-1,3-glucanase, lipase, and urease were also detected. Average nucleotide identity calculated by comparison with the standard strain genome of Streptomyces albospinus JCM3399 was 86.55% below the novel species threshold, suggesting that the strain could be a novel species. In addition, Streptomyces BITDG-11 obviously reduced the disease index of banana plantlets and promoted plant growth at 45 days post inoculation. The higher and lasting expression levels of defense genes and activities of antioxidant enzymes were induced in the roots of banana. Genome sequencing revealed that the Streptomyces BITDG-11 chromosome contained large numbers of conserved biosynthesis gene clusters encoding terpenes, non-ribosomal peptides, polyketides, siderophores, and ectoines. Fifteen bioactive secondary metabolites were further identified from Streptomyces BITDG-11 extract by gas chromatography–mass spectrometry. Dibutyl phthalate demonstrating a strong antifungal activity was the major compound with the highest peak area. Hence, Streptomyces sp. BITDG-11 has a great potential to become an essential constituent of modern agricultural practice as biofertilizers and biocontrol agents.

Biocontrol Ability and Mechanism of a Broad-Spectrum Antifungal Strain Bacillus safensis sp. QN1NO-4 Against Strawberry Anthracnose Caused by Colletotrichum fragariae.

Strawberry is a very popular fruit with a special taste, color, and nutritional value. Anthracnose caused by Colletotrichum fragariae severely limits fruit shelf life during post-harvest storage. Use of traditional chemical fungicides leads to serious environment pollution and threatens food safety. Biocontrol is considered as a promising strategy to manage the post-harvest fruit diseases. Here, strain QN1NO-4 isolated from noni (Morinda citrifolia L.) fruit exhibited a high antifungal activity against C. fragariae. Based on its physicochemical profiles and phylogenetic tree of the 16S rRNA sequence, strain QN1NO-4 belonged to the genus Bacillus. The average nucleotide identity (ANI) calculated by comparing two standard strain genomes was below 95–96%, suggesting that the strain might be a novel species of the genus Bacillus and named as Bacillus safensis sp. QN1NO-4. Its extract effectively reduced the incidence of strawberry anthracnose of harvested fruit. Fruit weight and TSS contents were also maintained significantly. The antifungal mechanism assays indicated that the extract of the test antagonist inhibited mycelial growth and spore germination of C. fragariae in vitro. Cells of strain QN1NO-4 demonstrated the cytoplasmic heterogeneity, disappeared organelles, and ruptured ultrastructure. Notably, the strain extract also had a broad-spectrum antifungal activity. Compared with the whole genome of strain QN1NO-4, several functional gene clusters involved in the biosynthesis of active secondary metabolites were observed. Fifteen compounds were identified by gas chromatography–mass spectrometry (GC-MS). Hence, the fruit endophyte B. safensis sp. QN1NO-4 is a potential bio-agent identified for the management of post-harvest disease of strawberry fruit.

Antifungal Streptomyces spp., Plausible Partners for Brood-Caring of the Dung Beetle Copris tripartitus.

The dung beetle Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae) is a coprophagous insect that lives in and feeds primarily on the feces of mammalian herbivores and is known to protect their offspring from the pathogen-rich environment by performing parental care for brood balls. Brood balls under continuous management by dung beetle are rarely contaminated by entomopathogenic fungi compared to abandoned brood balls. On the supposition that dung beetles may benefit from mutualistic bacteria that protect their offspring against fungal pathogens, we evaluated the antifungal activities of bacteria isolated from the dung beetle and brood ball. As a result, bacterial isolates, mainly streptomycetes, manifested potent and broad-spectrum antifungal activity against various fungi, including entomopathogens. Of the isolates, Streptomyces sp. AT67 exhibited pronounced antifungal activities. Culture-dependent and independent approaches show that this strain has occurred continuously in dung beetles that were collected over three years. Moreover, metabolic profiling and chemical investigation demonstrated that the strain produced an antifungal polyene macrocyclic lactam, sceliphrolactam, as a major product. Our findings imply that specific symbiotic bacteria of C. tripartitus are likely to contribute brood ball hygiene by inhibiting fungal parasites in the environment.

Broad-spectrum antifungal activity of lipopeptide brevilaterin B and its inhibition effects against Fusariumoxysporum and Penicillium chrysogenum.

Brevilaterin B is a natural antimicrobial lipopeptide produced by Brevibacillus laterosporus S62-9. However, its antifungal spectrum and modes of action are still unclear. Herein, we investigated the detailed antifungal activity of brevilaterin B against 33 pathogenic fungi and the antifungal effects against two sensitive fungi in vitro and in vivo. Brevilaterin B exhibited inhibitory activity against 33 pathogenic fungi involved in plant disease and food spoilage at the minimum inhibitory concentrations (MICs) range of 16-128μgml-1 . The antifungal effects were further studied by Fusarium oxysporum and Penicillium chrysogenum. Both spore germination and mycelium growth were inhibited by brevilaterin B at sub-MIC. Transmission electron microscopy and fluorescent dye staining assays indicated brevilaterin B damaged cell integrity and induced apoptosis. In vivo tests, brevilaterin B inhibited the infection of F. oxysporum to Dendrobium officinale and P. chrysogenum to mandarin (Citrus reticulata) at 500μgml-1 , respectively. Brevilaterin B showed broad-spectrum antifungal activity against 33 pathogenic fungi. And its antifungal modes of action were proposed as damaging cell integrity and inducing cell apoptosis. The lipopeptide is promising to control F. oxysporum in the D. officinale and P. chrysogenum in the mandarin. The research provided insights into antifungal modes of action of brevilaterin B. The lipopeptide brevilaterin B is potential to be developed as a broad-spectrum antifungal agent for agricultural biocontrol and postharvest storage.

Bacillus siamensis CNE6- a multifaceted plant growth promoting endophyte of Cicer arietinum L. having broad spectrum antifungal activities and host colonizing potential

Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 μg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60–80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC–MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.

Antifungal Activity of an Endophytic Fungus Aspergillus versicolor DYSJ3 from Aphanamixis grandifolia Blume against Colletotrichum musae

An endophytic fungus strain DYSJ3 was isolated from a stem of Aphanamixis grandifolia Blume, which was identified as Aspergillus versicolor based on the morphological characteristics, internal transcribed spacer (ITS) and calmodulin gene sequences analyses. A. versicolor DYSJ3 exhibited strong antagonistic activity against Colletotrichum musae, C. gloeosporioides and Fusarium oxysporum f. sp. cubense with the inhibition rates of 61.9, 51.2 and 55.3% respectively. The antifungal metabolites mainly existed in the mycelium of A. versicolor DYSJ3, and its mycelial crude extract (CE) had broad-spectrum antifungal activities against plant pathogenic fungi. The CE had a good thermal stability, and the inhibition rate of 100 µg/mL CE against C. musae was above 70.0% after disposing at 120 °C for 1 h. Five secondary metabolites were isolated from the CE and identified as averufanin, ergosterol peroxide, versicolorin B, averythrin and sterigmatocystin. Activity evaluation showed versicolorin B exhibited inhibitory effects on the mycelial growth and conidial germination of C. musae, and sterigmatocystin had a weak inhibitory effect on the mycelial growth of C. musae.

Clerodane Diterpenoids Identified from Polyalthia longifolia Showing Antifungal Activity against Plant Pathogens.

In the search for new natural resources showing plant disease control effects, we found that the methanol extract of Polyalthia longifolia suppressed fungal disease development in plants. To identify the bioactive substances, the methanol extract of P. longifolia was extracted by organic solvents, and consequently, four new 2-oxo-clerodane diterpenes (1-4), a new 4(3 → 2)-abeo-clerodane diterpene (5), together with ten known compounds (6-16) were isolated and identified from the extracts. Of the new compounds, compound 2 showed a broad spectrum of antifungal activity with moderated minimum inhibitory concentration (MIC) values in a range of 50-100 μg/mL against tested fungal pathogens. Considering with the known compounds, compound 6 showed the most potent antifungal activity with an MIC value in the range of 6.3-12.5 μg/mL. When compound 6 was evaluated for an in vivo antifungal activity against rice blast, tomato late blight, and pepper anthracnose, compound 6 reduced the plant disease by at least 60% compared to the untreated control at concentrations of 250 and 500 μg/mL. Together, our results suggested that the methanol extract of twigs and leaves of P. longifolia and its major compound 6 could be used as a source for the development of eco-friendly plant protection agents.

Identification and delineation of action mechanism of antifungal agents: Reveromycin E and its new derivative isolated from Streptomyces sp. JCK-6141

Streptomyces species have been used as biocontrol agents for the management of fungal postharvest diseases. Reveromycin E (RE) and its new derivative, reveromycin E 1-methyl ester (REME), were isolated from Streptomyces sp. JCK-6141. Both compounds displayed broad-spectrum and extensive antifungal activities at acidic condition, but their activities were dramatically reduced at neutral or base conditions. These compounds effectively suppressed the cherry tomato gray mold and mandarin blue mold caused by Botrytis cinerea and Penicillium italicum, respectively, at 10 and 50 mg L−1. In in vitro and in vivo assays, RE showed stronger antifungal activity than REME. Reveromycin was known as an isoleucine tRNA synthetase (IleRS) inhibitor. However, there is unconsolidated result in confirming binding site of reveromycin in previous studies. A computational study revealed that RE had a lower binding energy than REME and both compounds tend to bind catalytic domain rather than editing domains of IleRS. Our results indicated that Streptomyces sp. JCK-6141 producing reveromycins can be widely used as a new microbial fungicide for the control of postharvest diseases on fruit.

Isolation, characterization and identification of novel broad spectrum bacterial antagonist(s) to control Fusarium wilt of eggplant

Soil bacteria are now-a-days familiar for plant disease prevention in comparison to chemical control measures in an eco-friendly way. Antagonistic soil bacteria have created tremendous interests among scientists to be an alternative means of plant disease control. The aim of our study was to isolate soil bacteria with a multifaceted role, both in the aspects of plant nutrition and disease prevention. To achieve our goal, rhizobacteria from crop plants, cultivated in different regions of West Bengal were isolated and from a total of 250 isolates, only eight were primarily selected based on their initial growth suppression capabilities against the phytopathogen Fusarium oxysporum. Finally, the two bacterial isolates, NC1 and NC2, showed in vitro broad-spectrum antifungal activity against seven potent phytopathogenic fungi, i.e., Fusarium oxysporum, Alternaria alternata, Colletotrichum capsici, Macrophomina phaseolina, Sclerotium hydrophilum, Rhizoctonia solani and Penicillium digitatum, where they suppressed mycelial growth up to 69% over untreated control. The two potent antagonists were screened in vitro for their plant growth promoting (PGP) and antifungal properties where they showed their efficacy to produce many lytic enzymes, indole acetic acid (IAA) and ammonia. The isolates were detected as Gram-positive, endospore, and capsule forming rod-shaped bacteria. On the basis of morphological, physiological, biochemical and BLAST analysis of 16s rDNA sequences, NC1 and NC2 were identified as Bacillus amyloliquefaciens KY568716 and Bacillus velezensis KY568715. When applied on eggplant, both the isolates suppressed Fusarium wilt symptoms and enhanced plant growth significantly. The effects were observed up to 30 days after pathogen inoculation and both the isolates were effective in reducing Fusarium wilt disease symptoms by 57.23% and 64.19% respectively, as compared to pathogen treated plants. They also showed significant enhancement of all plant growth parameters such as root and shoot length, biomass and chlorophyll content. Thus, the two isolates can be used to develop an effective dual function bio-control agents to control plant diseases as well as promoting plant growth.

A Novel Antifungal Actinomycete Streptomyces sp. Strain H3-2 Effectively Controls Banana Fusarium Wilt.

Banana Fusarium wilt disease caused by Fusarium oxyspoum f. sp. cubense (Foc) seriously threatens the banana industry. Foc tropical race 4 (Foc TR4) can infect almost all banana cultivars. Compared with traditional physical and chemical practices, biocontrol strategy using beneficial microbes is considered as an environmentally sound option to manage fungal disease. In this study, a strain, H3-2, isolated from a non-infected banana orchard, exhibited high antifungal activity against Foc TR4. According to its morphological, physiological, and biochemical characteristics, the strain H3-2 was identified as Streptomyces sp. and convinced by the polymorphic phylogenic analysis of 16S rRNA sequences. Extracts of the strain H3-2 suppressed the growth and spore germination of Foc TR4 in vitro by destroying cell membrane integrity and mycelial ultrastructure. Notably, the strain and its extracts showed broad-spectrum antifungal activity against the selected seven fungal phytopathogens. Fourteen chemical compounds in the extracts were identified by gas chromatography–mass spectrometer (GC-MS), primarily phenolic compounds. Additional pot inoculation experiment demonstrated that the fermentation broth of the strain H3-2 promoted the growth of banana seedlings by efficiently inhibiting the spread of banana Fusarium wilt disease. This study demonstrated the potential application of the novel Streptomyces sp. H3-2 for the management of banana Fusarium wilt.

Characterization and Antifungal Activity of Limonoid Constituents Isolated from Meliaceae Plants Melia dubia, Aphanamixis polystachya, and Swietenia macrophylla against Plant Pathogenic Fungi In Vitro

The plants of Meliaceae are native to tropical and subtropical regions as the Americas, west India, Southeast Asia, and Southern China. Many species of the genera Khaya, Swietenia, Aphanamixis, and Melia in this family are known as medicinal plants and have biological activities such as antiviral, antimicrobial, antifeeding, insecticidal, and cytotoxic properties. The objectives of this research are to characterize and evaluate the bioactive limonoids from several plants of Meliaceae against phytopathogenic fungi. During the search of antifungal compounds from the plants of Meliaceae, the three methanol extracts of Melia dubia, Aphanamixis polystachya, and Swietenia macrophylla were found to suppress the mycelial growth of several phytopathogenic fungi. Nine limonoids isolated from M. dubia (1–2), A. polystachya (3–5), and S. macrophylla (6–9) were evaluated, for the first time, their antifungal effectiveness against nine phytopathogenic fungi Fusarium oxysporum, Magnaporthe oryzae, Sclerotium rolfsii, Rhizoctonia solani, Alternaria spp., and Botrytis cinerea, and three oomycetes Phytophthora species. Limonoids 2, 3, 6, and 8 displayed a remarkable broad-spectrum antifungal activity against all the test fungi. Sclerotium rolfsii was highly sensitive to the four limonoids with IC50 values ranging from 79.4 to 128.0 µg/mL. Notably, chisocheton compound G (3) isolated from A. polystachya and khayanolide B (8) isolated from S. macrophylla were the most potent antifungal limonoids and exhibited a dose-dependent activity against Phytophthora species. Compounds 2 and 9 displayed moderate activity against M. oryzae. Our study results demonstrated the discovery of antifungal and lead compounds from the group of limonoids for application in the control of fungal plant diseases.

Irritable Bowel Syndrome Therapeutic Has Broad-Spectrum Antimicrobial Activity.

Otilonium bromide is a poorly absorbed oral medication used to control irritable bowel syndrome. It is thought to act as a muscle relaxant in the intestine. Here, we show that otilonium bromide has broad-spectrum antibacterial and antifungal activity, including against multidrug-resistant strains. Our results suggest otilonium bromide acts on enteric pathogens and may offer a new scaffold for poorly absorbed intestinal antimicrobial therapy.

Antimicrobial Activity of the Peptide LfcinB15 against Candida albicans.

Lactoferricin (Lfcin) is an amphipathic, cationic peptide derived from proteolytic cleavage of the N-lobe of lactoferrin (Lf). Lfcin and its derivatives possess broad-spectrum antibacterial and antifungal activities. However, unlike their antibacterial functions, the modes of action of Lfcin and its derivatives against pathogenic fungi are less well understood. In this study, the mechanisms of LfcinB15, a derivative of bovine Lfcin, against Candida albicans were, therefore, extensively investigated. LfcinB15 exhibited inhibitory activity against planktonic cells, biofilm cells, and clinical isolates of C. albicans and non-albicans Candida species. We further demonstrated that LfcinB15 is localized on the cell surface and vacuoles of C. albicans cells. Moreover, LfcinB15 uses several different methods to kill C. albicans, including disturbing the cell membrane, inducing reactive oxygen species (ROS) generation, and causing mitochondrial dysfunction. Finally, the Hog1 and Mkc1 mitogen-activated protein kinases were both activated in C. albicans cells in response to LfcinB15. These findings help us to obtain more insight into the complex mechanisms used by LfcinB15 and other Lfcin-derived peptides to fight fungal pathogens.

Preparation and Characterization of Liposomes Double-loaded with Amphotericin B and Amphotericin B/hydroxypropyl-beta-cyclodextrin Inclusion Complex.

Amphotericin B (AMB) is water-insoluble polyene, which has a broad spectrum of antifungal activity. The hydrophobic drug only exits in the phospholipid bilayer, leading to a low-drug liposomal loading capacity. This study is designed to prepare water-soluble inclusion complex (IC) between AMB and cyclodextrin (CD) to formulate liposomal vesicles, double-loaded with drug molecules in the phospholipid bilayer and AMB/CD IC in the aqueous core. Water-soluble AMB/CD IC was prepared by pH adjustment of the aqueous media and consequently characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Liposomes double-loaded with AMB were formulated by the thin-film hydration method and accordingly evaluated for vesicle size, polydispersity index, entrapment efficiency, zeta potential, and in vitro drug leakage. Hydroxypropyl β cyclodextrin (HP-β-CD) better solubilized AMB than both α-CD and β- CD e.g., the concentration of water-soluble AMB/HP-β-CD IC could reach 465 μg/mL. Both DSC and SEM data illustrated that the drug no longer existed in its crystalline form, in AMB/HP-β-CD IC. Liposomes double-loaded with hydrophilic AMB/HP-β-CD IC and hydrophobic AMB had a diameter of 270 nm, polydispersity index less than 0.27, and zeta potential ca.-42.8 mV. Moreover, liposomes double-loaded with AMB enhanced drug-liposomal loading capacity by 25%, less leaked drug in phosphate buffer pH 7.4 at 37°C in comparison to liposomes loaded with only hydrophobic AMB. Liposomes double-loaded with AMB and AMB/HP-β-CD IC increased drug-encapsulation ability and in vitro stability, suggesting potential drug delivery systems.

Metabolite Fingerprinting of Novel Streptomyces UK-238 from the Himalayan Forest

Aims: Characterization of antimicrobial metabolites of novel Streptomyces sp. UK-238. Background: Novel antimicrobial drug discovery is urgently needed due to emerging multi antimicrobial drug resistance among pathogens. Since many years, natural products have provided the basic skeletons for many therapeutic compounds including antibiotics. Bioprospection of un/under explored habitats and focussing on selective isolation of actinobacteria as a major reservoir of bio and chemodiversity has yielded good results. Objective: The main objectives of the study were the identification of UK-238 by 16S rDNA sequencing and antimicrobial metabolite fingerprinting of culture extracts. Methods: In the present study, a promising isolate, UK-238, has been screened for antimicrobial activity and metabolite fingerprinting from the Himalayan Thano Reserve forest. It was identified by 16S rDNA sequencing. Ethyl acetate extract was partially purified by column chromatography. The pooled active fractions were fingerprinted by GC-MS and compounds were tentatively identified by collated data analysis based on Similarity Index, observed Retention Index from Databases and calculated Retention Index. Results: UK-238 was identified as Streptomyces sp. with 98.4% similarity to S. niveiscabiei. It exhibited broad-spectrum antibacterial and antifungal activity. GC-MS analysis of active fractions of ethyl acetate extract showed the presence of eighteen novel antimicrobial compounds belonging to four major categories- alcohols, alkaloid, esters and peptide. Conclusion: The study confirms that bioprospection of underexplored habitats can elaborate novel bio and chemodiversity.

Structural studies on the O-specific polysaccharide of the lipopolysaccharide from Pseudomonas donghuensis strain SVBP6, with antifungal activity against the phytopathogenic fungus Macrophomina phaseolina

An O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide (LPS) of Pseudomonas donghuensis SVBP6, a bacterium with a broad-spectrum antifungal activity in vitro, particularly that against Macrophomina phaseolina. This latter is one of the most virulent and dangerous pathogens of plants, including soybean which is an economically important crop in Argentina today. The OPS was studied by sugar analysis and spectroscopy (1D and 2D 1H and 13C NMR) showing the following trisaccharide repeating unit:→6)-ɑ-D-ManpNAc-(1 → 3)-β-l-Rhap-(1 → 4)-β-D-Glcp-(1→.The crude LPS, the purified LPS and the O-chain were assayed for their antifungal activity against M. phaseolina at 25, 50, 100, and 200 μg plug−1. The results showed that the crude LPS best inhibition was at 200 μg plug−1, able to inhibit the fungus growth by about 45%, while purified LPS and the corresponding OPS, in the same condition, reduced fungus growth by 65%, and 75%, respectively. Furthermore, the purified LPS and OPS significantly reduced the growth of M. phaseolina already at 100 μg plug−1 compared to the crude LPS. The structure of the O-chain is unique among the bacterial LPS and this is the first time that both the antifungal activity of a bacterial LPS and its corresponding O-chain were described.

Efficient preparation of the chiral intermediate of luliconazole with Lactobacillus kefir alcohol dehydrogenase through rational rearrangement of the substrate binding pocket

Abstract Luliconazole has been approved by the FDA for fungal infection therapy because of its broad-spectrum antifungal activity. However, low efficiency and harsh reaction conditions in the current synthesis of the luliconazole chiral intermediate (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol ((S)-TCPE) limits its application. In the present study, a novel biocatalytic process for producing (S)-TCPE was developed. Based on a structural analysis at the atomic level on the wild-type (WT) Lactobacillus kefir ketoreductase (L. kefir KRED), a rational design of L. kefir KRED was performed with 2,2’,4’-trichloroacetophenone (TCAP) as the substrate. An enantioselective triple mutant A94S/S96E/E145A displaying a 117-fold increase in its relative activity compared to the WT was evolved, and the catalytic efficiency was raised to 5.17 s−1mM−1 from non-detectable. With 106 kg of wet cells of the triple variant loading, 275 kg TCAP was asymmetrically reduced with the yield of (S)-TCPE achieving 91% within 12 h, thus showing great potential for industrial applications. Furthermore, the molecular mechanism supporting the promotion of activity was revealed to be related to the rearrangement of the substrate binding pocket of the L. kefir KRED. By engineering an enzyme that serves as a biocatalyst for the efficient preparation of enantiomerically pure (S)-TCPE, the current study presents a reproducible strategy for the promotion of catalytic activity based on rational design.