Isolation of two new stereochemical variants of streptophenazine by cocultivation of Streptomyces NIIST-D31, Streptomyces NIIST-D47, and Streptomyces NIIST-D63 strains in 3C2 combinations.

Cocultivation of combinations of Streptomyces species isolated from the same soil was explored to isolate novel secondary metabolites. Recently, we reported the isolation of a novel vicinal diepoxide of alloaureothin along with three carboxamides, 4-aminobenzoic acid, and 1,6-dimethoxyphenazine from the individual culture of Streptomyces luteireticuli NIIST-D31. Herein, cocultivation of NIIST-D31 with Streptomyces luteoverticillatus NIIST-D47 afforded two new stereochemical variants of streptophenazine (S1 and S2), and 1-N-methylalbonoursin, where the individual culture of NIIST-D47 primarily produced carbazomycins A, D, and E. The new streptophenazines and 1-N-methylalbonoursin were also observed during cocultivation of NIIST-D31 with Streptomyces thioluteus NIIST-D63, where the individual culture of NIIST-D63 strain afforded for the first time 2,2'-bipyridines (caerulomycinamide and dipyrimicin B), picolinamide, 2,3-dimethoxybenzamide, 2-hydroxy-3-methoxybenzamide, and 6-amino-2-pyridone along with known natural products aureothin and 1,6-dimethoxyphenazine. Finally, cocultivation of NIIST-D47 and NIIST-D63 strains produced carbazomycins B and C, alloaureothin, cyclo-(Leu-Pro), investiamide, and 4-aminobenzoic acid. Some of the compounds observed in the individual cultures were also produced in cocultivations. Improvement in the yield of secondary metabolites during cocultivation compared to individual culturing is well-known, which is noted here for vicinal diepoxide of alloaureothin. The production of new streptophenazines by cocultivation combinations with NIIST-D31 suggests that NIIST-D47 and NIIST-D63 may function as inducers in activating cryptic secondary metabolite-biosynthetic gene clusters. Cytotoxicity of the new streptophenazines in cancerous (MCF7 and MDA-MB-231) or non-cancerous (WI-38) cells were tested, however, they exhibited no significant activity.

A novel aureothin diepoxide derivative from Streptomyces sp. NIIST-D31 strain.

A novel vicinal diepoxide of alloaureothin was isolated from Streptomyces sp. NIIST-D31 strain along with three carboxamides, p-aminobenzoic acid and 1,6-dimethoxyphenazine. Exhaustive 2D NMR analysis and analysis of experimental, theoretical CD spectra aided in establishing the structure of compound 1. Compound 1 inhibits adipogenesis and accumulation of lipid droplets during the differentiation of 3T3-L1 cells.

Induction of mitochondria-mediated apoptosis and suppression of tumor growth in zebrafish xenograft model by cyclic dipeptides identified from Exiguobacterium acetylicum.

Colorectal cancer is the most common type of gastrointestinal cancers with poor survival and limited therapeutic options. In this study, four structurally different cyclic dipeptides (or diketopiperazine) were isolated and identified as cyclo (L-Pro-L-Leu), cyclo (L-Pro-L-Val), cyclo (L-Pro-L-Phe) and cyclo (L-Pro-L-Tyr) from the ethyl acetate extract in the cell-free filtrate of Exiguobacterium acetylicum S01. The anticancer potential of identified DKPs on colorectal cancer HT-29 cells in vitro and in vivo zebrafish xenograft model was evaluated. The MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide)) assay showed that four DKPs exhibited significant inhibition of HT-29 cells viability in a dose-dependent manner whereas there were no cytotoxic effects on normal mouse fibroblast 3T3 cells. Also, we observed that all DKPs induce early and late apoptotic cell death in HT-29 cells. Moreover, the expression levels of apoptotic (cytochrome-c, caspase-3 and Bid) and anti-apoptotic (Bcl-2) markers were up- and down-regulated in HT-29 cells in response to DKPs treatments. Furthermore, these four DKPs remarkably inhibited the tumor progression in a zebrafish xenograft model within a nonlethal dose range. Overall, our findings suggest that cyclic dipeptides derived from E. acetylicum S01 could be promising chemopreventive/ therapeutic candidates against cancer.

Expedient Synthesis of Indolo[2,3-b]quinolines, Chromeno[2,3-b]indoles, and 3-Alkenyl-oxindoles from 3,3'-Diindolylmethanes and Evaluation of Their Antibiotic Activity against Methicillin-Resistant Staphylococcus aureus.

Easily accessible 3,3'-diindolylmethanes (DIMs) were utilized to generate a focused library of indolo[2,3-b]quinolines (2), chromeno[2,3-b]indoles (3), and 3-alkenyl-oxindoles (4) under 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-mediated oxidative conditions. DIMs with ortho-NHTosyl (NHTs) phenyl group afforded indolo[2,3-b]quinolines (2), whereas DIMs with ortho-hydroxy phenyl groups yielded chromeno[2,3-b]indoles (3) and 3-alkenyl-oxindoles (4). The mild conditions and excellent yields of the products make this method a good choice to access a diverse library of bioactive molecules from a common starting material. Two optimized compounds 2a and 2n displayed excellent activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Compound 2a showed the minimum inhibitory concentration values in the concentration between 1 and 4 µg/mL, whereas compound 2n revealed the values of 1-2 µg/mL. Furthermore, both the compounds were highly bactericidal and capable to kill the MRSA completely within 360 min. Collectively, the results suggested that both compounds 2a and 2n possess enormous potential to be developed as anti-MRSA agents.

Synergistic combination of violacein and azoles that leads to enhanced killing of major human pathogenic dermatophytic fungi Trichophyton rubrum.

Superficial mycoses caused by dermatophytic fungi such as Trichophyton rubrum represent the most common type of worldwide human infection affecting various keratinized tissues in our body such as the skin, hair, and nails, etc. The dermatophytic infection is a significant public health threat due to its persistent nature and high recurrence rates, and thus alternative treatments to cure this fungal infection are urgently required. The present study mainly focused on the synergistic activity of violacein with four azole drugs (ketoconazole, fluconazole, clotrimazole, and itraconazole) against T. rubrum. The synergistic antifungal activities of violacein and azoles were measured by checkerboard microdilution and time-kill assays. In our study, combinations of violacein and azoles predominantly recorded synergistic effect (FIC index < 0.5). Significant synergistic value was recorded by the combination of violacein and clotrimazole. Time-kill assay by the combination of MIC concentration of violacein and azoles recorded that the growth of the T. rubrum was significantly arrested after 4-12 h of treatment. The combination of violacein and azoles leads to the enhanced inhibition of mycelial growth and conidial germination. Moreover combination enhanced the rate of release of intracellular materials. Morphological changes by SEM analysis were also prominent with the combination. A normal human cell line [Foreskin (FS) normal fibroblast] was used to check the cytotoxicity of violacein. Interestingly violacein recorded no cytotoxicity up to 100 µg/ml. The in vitro synergistic effect of violacein and azoles against clinically relevant fungi, T. rubrum, is reported here for the first time. Finally, our findings support the potential use of the violacein as an antifungal agent especially against dermatophytic fungi T. rubrum.

Characterization of the bioactive metabolites from a plant growth-promoting rhizobacteria and their exploitation as antimicrobial and plant growth-promoting agents.

A plant growth-promoting bacterial strain, PM 105, isolated from a tea plantation soil from the North Eastern region of India was identified as Pseudomonas aeruginosa through classical and 16S ribosomal DNA (rDNA) gene sequencing. Further studies with this strain confirmed broad spectrum antifungal activity against ten human and plant pathogenic fungal pathogens viz. Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus tubingensis, Candida albicans, Colletotrichum gloeosporioides, Fusarium oxysporum, Pencillium expansum, Rhizoctonia solani, Trichophyton rubrum besides growth-promoting property in cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan). However, no antibacterial property was exhibited by this strain against the four test bacterial pathogens tested in agar overlay method. The crude bioactive metabolites produced by this strain were isolated with three different solvents that exhibited significant antimicrobial and plant growth-promoting activity. Chloroform extract recorded significant antimicrobial and plant growth-promoting activity. Three major compounds viz. 1-hydroxyphenazine, pyocyanin, and phenazine-1-carboxamide were purified and characterized from crude extracts of this strain by various spectral data. The purified compounds recorded prominent antimicrobial activity but failed to establish the plant growth promotion activity in test crop plants under gnotobiotic conditions. Pyocyanin recorded significant antimicrobial activity, and best activity was recorded against T. rubrum (29 mm), followed by P. expansum (28 mm). These results suggest the use of PM 105 as plant growth-promoting agent in crop plants after successful field trials.

Asarones from Acorus calamus in combination with azoles and amphotericin B: a novel synergistic combination to compete against human pathogenic Candida species in vitro.

The increase in drug resistance to current antifungal drugs brings enormous challenges to the management of Candida infection. Therefore, there is a continuous need for the discovery of new antimicrobial agents that are effective against Candida infections especially from natural source especially from medical plants. The present investigation describes the synergistic anticandidal activity of two asarones (∞ and ß) purified from Acorus calamus in combination with three clinically used antifungal drugs (fluconazole, clotrimazole, and amphotericin B). The synergistic anticandidal activities of asarones and drugs were assessed using the checkerboard microdilution and time-kill assays. The results of the present study showed that the combined effects of asarones and drugs principally recorded substantial synergistic activity (fractional inhibitory concentration index (FICI) <0.5). Time-kill study by combination of the minimal inhibitory concentration (MIC) of asarones and drugs (1:1) recorded that the growth of the Candida species was significantly arrested between 0 and 2 h and almost completely attenuated between 2 and 6 h of treatment. These findings have potential implications in adjourning the development of resistance as the anticandidal activity is achieved with lower concentrations of asarones and drugs. The combination of asarones and drugs also significantly inhibit the biofilm formation by Candida species, and this would also help to fight against drug resistance because biofilms formed by Candida species are ubiquitous in nature and are characterized by their recalcitrance toward antimicrobial treatment. The in vitro synergistic activity of asarones and drugs against pathogenic Candida species is reported here for the first time.

Purification and characterization of antifungal phenazines from a fluorescent Pseudomonas strain FPO4 against medically important fungi.

The strain FPO4 was isolated from the rhizoplane of rice plant root and identified as a fluorescent Pseudomonas aeruginosa on the basis of 16S rDNA sequences and BLAST analysis. The extracellular metabolites produced by this strain were purified by silica gel column chromatography and isolated four pure compounds. Based on the spectral data the four compounds were identified as phenazin-1-ol, phenazine-1-carboxylic acid (PCA), 2-heptyl-3-hydroxyl-4(1H)-quinolone (PQS), and phenazine-1-carboxamide (PCN), respectively. Phenazin-1-ol and PCA were active against all the eight fungi tested. The highest activity of 4 µg/mL by PCA was recorded against Trichophyton rubrum, a human pathogen responsible for causing athlete's foot, jock itch, ringworm and fingernail fungus infections, followed by Candida albicans and Candida tropicalis. The activity of phenazin-1-ol, PCA against Candida spp. was found to be better than the standard antifungal agent amphotericin B. Furthermore, the present study reports the antimicrobial activity of the purified phenazines on major human pathogen, T. rubrum for the first time.

In vitro antibacterial screening of six proline-based cyclic dipeptides in combination with ß-lactam antibiotics against medically important bacteria.

The in vitro synergistic antibacterial activity of six proline-based cyclic dipeptides [cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-D-Tyr)] in combination imipenem and ceftazidime was investigated in the present manuscript. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the cyclic dipeptides were compared with those of the standard antibiotics (imipenem and ceftazidime). The synergistic antibacterial activities of cyclic dipeptides with imipenem or ceftazidime were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effect of six cyclic dipeptides with imipenem predominantly recorded synergistic interaction (FIC index <0.5), whereas combination of certain cyclic dipeptides with ceftazidime recorded additive. The time-kill curve showed that the growth of the test bacteria was completely attenuated after 12-24 h of treatment with a 50:50 ratio of proline-based cyclic dipeptides and antibiotics. These synergistic effects have a potential role in delaying the development of resistance as the antibacterial activity is achieved with the very low concentrations of cyclic dipeptides and antibiotics. The cytotoxicity of cyclic dipeptides was tested against VERO cell line (African green monkey kidney cell line), and no cytotoxicity was recorded for cyclic dipeptides up to 100 µg/mL. These findings suggest that combination of cyclic dipeptides and antibiotics might be a good strategy for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for antimicrobial chemotherapy. Moreover, these combinations may lead to the development of a new and vital antimicrobial combination against the infections caused by pathogenic bacteria. The in vitro synergistic activity of cyclic dipeptides with antibiotics against medically important bacteria is reported here for the first time.

Antioxidant and anticancer activity of 3,5-dihydroxy-4-isopropylstilbene produced by Bacillus sp. N strain isolated from entomopathogenic nematode.

3,5-Dihydroxy-4-isopropylstilbene is a natural phytoalexin and was first identified as bacterial secondary metabolites. The aim of this study is to investigate in vitro antioxidant and anticancer activity of 3,5-dihydroxy-4-isopropystilbene purified from the cell free culture filtrate of Bacillus sp. N strain associated with rhabditid entomopathogenic nematode. Antioxidant activity was evaluated by five separate methods: free radical scavenging, reducing power assay, chelating effects on ferrous ions, NBT superoxide radical scavenging assay and hydroxyl radical scavenging activity. The stilbene recorded powerful antioxidant activity at various antioxidant systems in vitro. The superoxide radical scavenging (92.1 %) and hydroxyl radical scavenging (83.4 %) activities of the stilbenes at 100 µg/ml were higher than the butylated hydroxyanisole, the known antioxidant agent. Anticancer activity of stilbene was tested against breast cancer (MDAM B-231), cervical cancer (HeLa), lung cancer (A 549), colon cancer (HTL 116) cell lines using MTT method. The induction of apoptosis was studied by morphological analysis, apoptotic cell staining, caspase 3 activation assay and cell cycle analysis using flow cytometry. Stilbene induced significant morphological changes and DNA fragmentation associated with apoptosis in HeLa cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by stilbene. Up-regulation of caspase 3 activity was also found in cells treated with stilbene. Flow cytometry analysis showed an increase in the percentage of apoptotic cells in sub G0 phase (2.4 % in control plates to 11.4 % in 25 µg/ml of stilbene) confirming the stilbene induced apoptosis. The results of the present study showed that stilbene demonstrated a strong antioxidant and anticancer effects. These suggest that stilbene may be used as possible natural antioxidant and anticancer agents to control various human diseases.

Rhizoctonia wilt suppression of brinjal (Solanum melongena L) and plant growth activity by Bacillus BS2.

An antibiotic-producing and hydrogen-cyanide-producing rhizobacteria strain Bacillus BS2 showed a wide range of antifungal activity against many Fusarium sp. and brinjal wilt disease pathogen Rhizoctonia solani. Seed bacterization with the strain BS2 promoted seed germination and plant growth in leguminous plants Phaseolus vulgaris and non-leguminous plants Solanum melongena L, Brassica oleracea var. capitata, B. oleraceae var. gongylodes and Lycopersicon esculentum Mill in terms of relative growth rate, shoot height, root length, total biomass production and total chlorophyll content of leaves. Yield of bacterized plants were increased by 10 to 49% compared to uninoculated control plants. Brinjal sapling raised through seed bacterization by the strain BS2 showed a significantly reduced wilt syndrome of brinjal caused by Rhizoctonia solani. Control of wilt disease by the bacterium was clue to the production of antibiotic-like substances, whereas plant growth-promotion was due to the activity of hydrogen cyanide. Root colonization study confirmed that the introduced bacteria colonized the roots and occupied 23-25% of total aerobic bacteria, which was confirmed using dual antibiotic (nalidixic acid and streptomycin sulphate) resistant mutant strain. The results obtained through this investigation suggested the potentiality of the strain BS2 to be used as a plant growth promoter and suppressor of wilt pathogen.

Biological activity of secondary metabolites produced by a strain of Pseudomonas fluorescens.

Biological activity of secondary metabolites produced by a plant-growth-promoting Pseudomonas fluorescens was evaluated. The strain produced antibiotics phenazine (PHE), 2,4-diacetylphloroglucinol (PHL) and siderophore pyoverdin (PYO) in standard King's B and succinic acid media, respectively. After extraction, PYO was identified by comparing the UV-spectra and moss-green color development after 'diazotized sulfanilic acid' (DSA) spray in TLC. PHE and PHL were identified by comparing standard compounds on TLC and orange-color development immediately after DSA spray. In vitro antibiosis study of the metabolites revealed their antibacterial and antifungal activity against bacterial test organisms Corynebacterium sp., Mycobacterium phlei and M. smegmatis and test fungi Fusarium moniliforme, F. oxysporum, F. semitectum, F. solani and Rhizoctonia solani. A statistically significantly higher plant growth was recorded in siderophore-amended plantlets under gnotobiotic conditions whereas PHE and PHL did not show any plant-growth-promoting activity. These results support the importance of the secondary metabolites produced by the strain P. fluorescens in enhancing plant growth and in controlling fungal and bacterial pathogens.

Plant disease suppression and growth promotion by a fluorescent Pseudomonas strain.

An antibiotic- and siderophore-producing Pseudomonas strain isolated from virgin soils (with forest trees) displayed in vitro antibiosis against many plant pathogenic fungi. The presence of iron had no effect on this in vitro antibiosis. Seed bacterization improved germination, shoot height, root length, fresh and dry mass, enhanced yield and chlorophyll content of leaves in the five test crop plants under field conditions. Seed bacterization also reduced the number of infected brinjal plants grown in soil infested with Rhizoctonia solani. The strain produced a yellowish green siderophore in the standard succinate medium and both siderophore and a yellow viscous antibiotic compound in King's B medium. The results confirmed that the plant growth promotion was due to siderophore production whereas the disease suppression was due to the antibiotic substance.