Culturable Streptomyces spp. from high-altitude, oligotrophic North Western Himalaya: a comprehensive study on the diversity, bioactivity and insights into the proteome of potential species.

The increasing global concern of antimicrobial resistance and shortage of new antimicrobials necessitates exploring untapped terrestrial environments for new bioactive microbiome diversity. The low-temperature and oligotrophic North Western Himalaya (NWH) region has a vast diversity of Streptomyces with potential antimicrobial properties that remain largely unexplored. This study evaluates the diversity of culturable Streptomyces from high-altitude NWH and their potential as a source of new antimicrobials through genus-specific isolation and identification. The results demonstrate a distinct phylogenetic clustering of Streptomyces from different sampling regions of NWH, site-specific variation in culturable ß-diversity and species commonness with varying intersite bioactivity among different sites. Further, the study optimized the media selection for large-scale culture cultivation in antibiotic production processes and demonstrated the antimicrobial efficacy of Streptomyces against a range of pathogens through in vitro bioassays using minimum inhibitory concentration determination and antibiofilm activity. Untargeted label-free proteomic profiling also revealed variable expression of stress-response proteins and antibiotic regulators as a competitive survival strategy for selective antagonistic Streptomyces. The findings highlight the potential of NWH in augmenting antimicrobial discovery and combating antimicrobial resistance through the isolation and study of novel bioactive Streptomyces.

Functional antagonism and insights into the biosynthetic potential of human gut-derived microbes.

The specialised small molecules encoded by commensal microbes mediate distinct functional interactions. However, there is a landscape of antagonistic interactions mediated by specialised strains and their small molecules. Herein, the antagonistic landscape within a collection of 330 human gut-derived commensal microbial strains was elucidated to evaluate antimicrobial interactions as a defensive contributor, and gain new insights into structure-related functions. The potential antagonistic gut-derived strains displayed strain-specific selective inhibition. This is in contrast to common antimicrobial drugs, which typically wipe out a broad range of species and are usually found in environmental microbes. Genome sequencing of representative gut strains revealed the presence of significant biosynthetic gene clusters (BGCs) encoding compound families that contribute to antagonistic activities, and are important in host defence and maintaining gut homeostasis. Subsets of these BGCs were abundant in metagenomic sequencing data from healthy individuals. Furthermore, the cell culture secretome of these strains revealed potential biomarkers linked to hallmark pathways. These microorganisms have biosynthetic novelty and are a source of biologically significant natural products. Such natural products are essential in the development of new antimicrobial agents to reduce the usage of broad-spectrum antibiotics and combat antimicrobial resistance.

Strategizing the human microbiome for small molecules: Approaches and perspectives.

Studies of the human microbiome are providing a deeper understanding of its significance to human health, and increasing evidence links the microbiota with several diseases. Nevertheless, the exact mechanisms involved in human-microbe interactions are mostly undefined. The genomic potential of the human microbiome to biosynthesize distinct molecules outmatches its known chemical space, and small-molecule discovery in this context remains in its infancy. The profiling of microbiome-derived small molecules and their contextualization through cause-effect mechanistic studies may provide a better understanding of host-microbe interactions, guide new therapeutic interventions, and modulate microbiome-based therapies. This review describes the advances, approaches, and allied challenges in mining new microbial scaffolds from the human microbiome using genomic, microbe cultivation, and chemical analytic platforms. In the future, the complete biological characterization of a single microbe-derived molecule that has a specific therapeutic application could resolve the current limitations of microbiota-modulating therapies.

New approaches for antituberculosis leads from Actinobacteria.

Bioactive metabolites derived from the phylum Actinobacteria represent many of the existing antimicrobial drugs. Compared with other bacterial pathogens, direct preliminary screening by diffusion assays is a limiting factor against Mycobacterium tuberculosis (Mtb) and different methodologies have been used to improve the search for new molecules. However, the concern remains that most of the previously discovered molecules replicate by conventional procedures. The combination of multidisciplinary approaches with new technologies could advance the discovery of new leads against Mtb like considering the unexplored Actinobacteria jointly with selective and integrative procedures.

In vitro bactericidal activity of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant and drug-tolerant isolates of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) poses a serious global threat to humans. New bactericidal agents that can shorten treatment duration and target drug resistance still remain a top priority in the discovery of anti-TB drugs. The objective of this study was to investigate the bactericidal potential of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant clinical isolates and drug-tolerant Mycobacterium tuberculosis. METHODS: The minimum bactericidal concentration (MBC) was determined by colony-forming unit (CFU) enumeration. The kill curve analysis was done at different concentrations spanning over 16 days. Drug combination studies with antituberculosis drugs were done to investigate possible synergy. The potential against drug- resistant isolates of M. tuberculosis was done by broth dilution assay. CFU enumeration was done to determine its activity against nutrient-starved drug tolerants, and its feasibility for oral administration was tested by serum inhibitory titre. RESULTS: CHP displayed bactericidal activity with an MBC of 4 µg/mL against M. tuberculosis H37Rv. The kill curve analysis exhibited a biphasic pattern of killing. CHP showed synergy with rifampicin, isoniazid and amikacin but was indifferent towards ethambutol and levofloxacin. CHP retained its full activity against drug-susceptible, monoresistant and multidrug-resistant (MDR) clinical isolates. CHP showed very strong bactericidal activity against nondividing, drug-tolerant M. tuberculosis that on comparison was highly superior to rifampicin. Furthermore, CHP significantly improved the bactericidal activity of rifampicin and isoniazid in a combination study. The serum inhibitory titre in mice indicated its high oral bioavailability. CONCLUSION: Our results show strong bactericidal potential of CHP against M. tuberculosis that warrant its immediate mechanistic, pharmacokinetic and pharmacodynamic studies.

Identification of dinactin, a macrolide antibiotic, as a natural product-based small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells.

PURPOSE: Despite the fact that hyper-activation of Wnt/ß-catenin signaling pathway has been seen in many cancers, including liver, colorectal and lung carcinoma, no small molecule inhibitors are available that specifically target this pathway. In this study, we analyzed the impact of dinactin (DA), an antibiotic ionophore produced by Streptomyces species, as an effective small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells. METHODS: We performed MTT assays to investigate cell viability and proliferation after exposure to small molecules. Protein expression analysis was carried out by western blotting. Top-Flash reporter assays were used to score for ß-catenin signaling and cell cycle analysis was carried out by flow cytometry. RESULTS: In the first set of experiments, DA was seen to selectively inhibit the proliferation of HCT-116 and HepG2 cancer cells, unlike HEK-293 cells (a low tumorigenic cell line), in apoptosis-independent manner. Further, DA was seen to block the G1/S progression and decrease the expression of cyclin D1 in cancer cells. Since cyclin D1 is the downstream target gene of Wnt/ß-catenin signaling, we examined the impact of DA on TCF-dependent ß-catenin activity using Top-Flash reporter assay. Interestingly, DA significantly decreased Top-Flash activity at lower nano-molar concentrations when compared with salinomycin in HCT-116 and HepG2 cells. CONCLUSION: We report the identification of dinactin as a natural product-based small molecule that effectively blocks the Wnt/ß-catenin signaling pathway in cancer cells at nano-molar concentration. We anticipate that DA could be developed as a novel drug for anti-cancer therapy and for the management of neuropathic pain.

In vitro evaluation of dinactin, a potent microbial metabolite against Mycobacterium tuberculosis.

Current long duration treatment options and the emergence of drug resistance in tuberculosis (TB) have led to renewed interest in discovery of novel anti-tubercular agents or the scaffolds exhibiting enhanced efficacy with current anti-TB drugs. Herein, dinactin, a potent bioactive macrotetrolide isolated from Streptomyces puniceus AS13, was evaluated against Mycobacterium tuberculosis H37Rv and other susceptible and drug-resistant clinical isolates of M. tuberculosis. In vitro pharmacological assays showed that dinactin is bactericidal against laboratory standard strain M. tuberculosis H37Rv (minimum inhibitory concentration [MIC] 1 µg/mL and minimum bactericidal concentration [MBC] 4 µg/mL). Dinactin also retained its activity against various clinical isolates, including multidrug-resistant strains of M. tuberculosis. Whole cell interaction assays with standard first- and second-line anti-TB drugs showed the synergistic interaction of dinactin with rifampicin or amikacin, reflecting its suitability for use in combination regimens. The killing kinetics studies of dinactin against M. tuberculosis H37Rv revealed that it has strong concentration-dependent anti-TB activity that is also dependent on time. The kill curve also showed dynamic killing capacity of dinactin as it exhibited bactericidal potential at all concentrations tested. Kill curve data demonstrated that dinactin, like isoniazid, exerts its strong tuberculocidal activity within the first two days of exposure. This evidence strongly supports further evaluation of dinactin as a new option in the treatment of TB.

Streptomyces puniceus strain AS13., Production, characterization and evaluation of bioactive metabolites: A new face of dinactin as an antitumor antibiotic.

A highly active actinobacterial strain isolated from untapped areas of Northwestern Himalayas and characterised as Streptomyces puniceus strain AS13 by 16S rRNA gene sequencing was selected for production of bioactive metabolites. The bioassay-guided fractionation of microbial cultured ethyl acetate extract of the strain, led to isolation of macrotetrolide compound 1 (Dinactin) and compound 2 (1-(2,4-dihydroxy-6-methylphenyl)-ethanone). Structures of the isolated compounds were elucidated by [corrected] interpretation of NMR and other spectroscopic data including HR-ESI-MS, FT-IR. These compounds are reported for first time from Streptomyces Puniceus. Compound 1 exhibited strong anti-microbial activity against all tested bacterial pathogens including Mycobacterium tuberculosis. The MIC values of compound 1 against Gram negative and Gram positive bacterial pathogens ranged between 0.019 - 0.156µgml-1 and 1µgml-1 against Mycobacterium tuberculosis H37Rv. Dinactin exhibited marked anti-tumor potential with IC50 of 1.1- 9.7µM in various human cancerous cell lines and showed least cytotoxicity (IC50∼80µM) in normal cells (HEK-293). Dinactin inhabited cellular proliferation in cancer cells, reduced their clonogenic survival as validated by clonogenic assay and also inhabited cell migration and invasion characteristics in colon cancer (HCT-116) cells. Our results expressed the antimicrobial potential of dinactin and also spotted its prospective as an antitumor antibiotic.

Bovine mastitis: An appraisal of its alternative herbal cure.

Bovine mastitis is globally recognized as the most common and costly disease affecting dairy herds. The disease causes huge financial losses to dairy industries by reduced yield and milk quality, deaths and culling of affected cows and also by associated treatment costs. The disease occurs due to invasion of the mammary glands by pathogenic bacteria followed by their multiplication in the milk producing tissues. The most common treatment method available against bovine mastitis is the intra-mammary infusion of antibiotics. However, their use is associated with the problem of antimicrobial resistance. This scenario has made search for alternative treatment approaches necessary. Medicinal plants with their well-established history are an excellent natural product resource used as an alternative therapy. Antibacterial agents from plants can act as important sources of novel antibiotics, efflux pump inhibitors, compounds that target bacterial virulence or can be used in combination with existing drugs. The plants form an essential component of ethno-veterinary medicine used in the treatment of different diseases like bovine mastitis. This review article attempts to provide an overview of the different medicinal plants used in the treatment of bovine mastitis. Antimicrobial studies of these plant species and some of their isolated constituents have been reviewed in detail. It highlights the logic and precedence behind mining this important natural product resource. Our own research findings in this direction and future scope of research are also discussed briefly.

Chemoprofile and functional diversity of fungal and bacterial endophytes and role of ecofactors - A review.

Endophytes represent a hidden world within plants. Almost all plants that are studied harbor one or more endophytes, which help their host to survive against pathogens and changing adverse environmental conditions. Fungal and bacterial endophytes with distinct ecological niches show important biological activities and ecological functions. Their unique physiological and biochemical characteristics lead to the production of niche specific secondary metabolites that may have pharmacological potential. Identification of specific secondary metabolites in adverse environment can also help us in understanding mechanisms of host tolerance against stress condition such as biological invasions, salt, drought, temperature. These metabolites include micro as well as macromolecules, which they produce through least studied yet surprising mechanisms like xenohormesis, toxin-antitoxin system, quorum sensing. Therefore, future studies should focus on unfolding all the underlying molecular mechanisms as well as the impact of physical and biochemical environment of a specific host over endophytic function and metabolite elicitation. Need of the hour is to reshape the focus of research over endophytes and scientifically drive their ecological role toward prospective pharmacological as well as eco-friendly biological applications. This may help to manage these endophytes especially from untapped ecoregions as a useful undying biological tool to meet the present challenges as well as lay a strong and logical basis for any impending challenges.

Antimicrobial investigation of selected soil actinomycetes isolated from unexplored regions of Kashmir Himalayas, India.

The aim of the present study was to isolate and evaluate the antimicrobial potential of soil actinomycetes of Kashmir Himalayas. The secondary metabolites of actinomycetes are the prominent source of antibiotics. A total of 121 morphologically different actinomycete strains were isolated and screened for antimicrobial activity against various human pathogens. The ethyl acetate extract of fermented broth an actinomycete strain, identified as Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus ATCC 29213 with MIC 0.25 µg/ml and Mycobacterium tuberculosis Strain H37Rv with MIC 0.062 µg/ml. The strain S. pratensis IIIM06 was grown on large scale and their broth was extracted with ethyl acetate. The extract was subjected to various chromatography techniques which led to the isolation of four compounds whose structures were established as actinomycin C1, actinomycin C2, actinomycin C3 and actiphenol on the basis of spectral data analysis. Actinomycin C1, C2 and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The isolated indigenous actinomycetes exhibited good antibacterial activity and the study reveals that IIIM06 is a promising strain and could be of great potential for industrial applications.

Novel bioactive molecules from Lentzea violacea strain AS 08 using one strain-many compounds (OSMAC) approach.

A new eudesmane sesquiterpenoid (1), and a new homologue of virginiae butanolide E (2) along with butyl isobutyl phthalate (3) were isolated from, actinomycete-Lentzea violacea strain AS08 isolated from north western Himalayas by stressing on modified one strain-many compounds (OSMAC) method. The structures of the new compounds were elucidated by extensive spectroscopic analyses including 1D, 2D NMR along with HR-ESI-MS and FT-IR data. Herein, a distinctive method was added for inspecting secretory profile of the strain by quantification of extract value of cell free supernatant in different types of culture media fallowed by HPLC profiling of respective extracts, which revealed a highly altered metabolic profile of the strain and formed the base for the selection of media. The compounds 1 and 2 showed moderate activity against Gram negative (MIC ∼32-64µgml-1) in comparison to Gram positive bacterial pathogens. Compound 1 exhibited significant activity in human cancerous cell lines (IC50 ∼19.2µM).

Isolation and characterization of alborixin from Streptomyces scabrisporus: A potent cytotoxic agent against human colon (HCT-116) cancer cells.

The ethyl acetate extract from the fermentation broth of an actinomycete strain, identified as Streptomyces scabrisporus isolated from soil of Kashmir Himalayas - India, exhibited significant cytotoxic activity against a panel of human cancer cell lines. The active fraction subjected to column chromatography led to the isolation of pharmacologically potent anticancer compound whose structure was established to be alborixin on the basis of spectral data analysis. The compound exhibited antiproliferative activity against panel of cell lines N2a, MCF-7, MiaPaca-2, PC-3, HCT-116, MDA-MB-231, HL-60 and A-549 cells with IC50 of 9.7, 15.4, 7.2, 8.1, 3.2, 9.7, 7.5 and 11.5 µM respectively. Alborixin displayed the maximum cytotoxic activity against HCT-116 human colon carcinoma cells and therefore further studies were carried on this cell line. Alborixin decreased the clonogenic potential of HCT-116 cells in a dose dependent manner. It induced apoptotic cell death in HCT116 cells that were confirmed by Flow cytometric analysis of Annexin V/PI staining and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by mitochondrial membrane potential loss, decreasing the expression profile of anti-apoptotic protein Bcl-2, whereas it augments cleavage of caspase-3 and PARP-1, activates caspase-8 and 9 with concomitant increase in expression of proapoptotic protein Bax in a dose dependent manner. These results indicate that alborixin obtained from Streptomyces scabrisporus IIIM55 induces apoptotic cell death in colon cancer cells HCT-116 and can be further evaluated for its potential as an anticancer agent.

Isolation, characterization and HPLC quantification of compounds from Aquilegia fragrans Benth: Their in vitro antibacterial activities against bovine mastitis pathogens.

ETHNO-PHARMACOLOGICAL RELEVANCE: The underground parts of Aquilegia fragrans are traditionally used for the treatment of wounds and various inflammatory diseases like bovine mastitis. However, there are no reports on the phytochemical characterization and antibacterial studies of A. fragrans. AIM OF THE STUDY: To isolate compounds from the methanol extract of the underground parts of A. fragrans and determine their antibacterial activity against the pathogens of bovine mastitis. The study was undertaken in order to scientifically validate the traditional use of A. fragrans. MATERIALS AND METHODS: Five compounds were isolated from the methanol extract of the underground parts of A. fragrans using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of isolated compounds in the crude methanol extract. The methanol extract and isolated compounds were evaluated for antibacterial activities against mastitis pathogens using broth micro-dilution technique. RESULTS: The five isolated compounds were identified as (1) 2, 4-dihydroxyphenylacetic acid methyl ester (2) ß-sitosterol (3) Aquilegiolide (4) Glochidionolactone-A and (5) Magnoflorine. A quick and sensitive HPLC method was developed for the first time for qualitative and quantitative determination of four isolated marker compounds from A. fragrans. The crude methanol extract and compound 5 exhibited weak antibacterial activities that varied between the bacterial species (MIC=500-3000 µg/ml). CONCLUSIONS: The above results show that the crude methanol extract and isolated compounds from A. fragrans exhibit weak antibacterial activities. Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species in the treatment of various inflammatory diseases like bovine mastitis.

Inflammation in ischemic stroke: mechanisms, consequences and possible drug targets.

Ischemic stroke is caused when blood flow to the brain is hampered, leading to instant deficiency of nutrients and oxygen required for normal brain functioning. Reperfusion can alleviate damage from stroke if performed immediately after the onset of ischemia however the efficacy of reperfusion is tempered by secondary injury mechanisms. This multifarious sequence of events leads to the commencement of deleterious cycles of inflammation, oxidant stress and apoptosis that finally culminate in delayed death of neuronal cells even when the brain is effectively reperfused. Wealth of data from clinical as well as experimental studies points to a prominent role of inflammation in secondary injury. In this review we will discuss, in detail, the cellular and molecular mediators of inflammation and their possible therapeutic targets in both experimental and clinical forms of stroke.