Natural products from Streptomyces spp. as potential inhibitors of the major factors (holoRdRp and nsp13) for SARS-CoV-2 replication: an in silico approach.

The COVID-19 pandemic caused unprecedented damage to humanity, and while vaccines have been developed, they are not fully effective against the SARS-CoV-2 virus. Limited targeted drugs, such as Remdesivir and Paxlovid, are available against the virus. Hence, there is an urgent need to explore and develop new drugs to combat COVID-19. This study focuses on exploring microbial natural products from soil-isolated bacteria Streptomyces sp. strain 196 and RI.24 as a potential source of new targeted drugs against SARS-CoV-2. Molecular docking studies were performed on holoRdRp and nsp13, two key factors responsible for virus replication factor. Our in silico studies, K-252-C aglycone indolocarbazole alkaloid (K252C) and daunorubicin were found to have better binding affinities than the respective control drugs, with K252C exhibiting binding energy of - 9.1 kcal/mol with holoRdRp and - 9.2 kcal/mol with nsp13, and daunorubicin showing binding energy at - 8.1 kcal/mol with holoRdRp and - 9.3 kcal/mol with nsp13. ADMET analysis, MD simulation, and MM/GBSA studies indicated that K252C and daunorubicin have the potential to be developed as targeted drugs against SARS-CoV-2. The study concludes that K252C and daunorubicin are potential lead compounds that might suppress the inhibition of SARS-CoV-2 replication among the tested microbial compounds and could be developed as targeted drugs against COVID-19. In the future, further in vitro studies are required to validate these findings.

In vitro antifungal activity analysis of Streptomyces sp. strain 196 against Candida albicans and Aspergillus flavus.

Numerous bioactive compounds have been reported to be produced by the members of the genus Streptomyces. During our previous studies, Streptomyces sp. strain 196 was tested for its antimicrobial activity, and bioactive compounds produced by this strain were characterized LC-MS and 1H NMR. To examine the antifungal potential of strain 196 is the goal of the current investigation. Present investigation is focused on exploring antifungal activity of extract of strain 196 (196EA) on membrane disruption potential against two fungi Candida albicans ATCC 90028 and Aspergillus flavus ITCC 5599. Results revealed that the MIC value is higher for A. flavus than for C. albicans which is 450 µg/mL and 250 µg/mL, respectively. Disc diffusion and spot assay also correspond to the values of the MIC for their respective pathogen. In growth curve analysis, lag and log phase are significantly affected by the extract of strain 196. The effects of extract from strain 196 on plasma membrane disruption of Candida albicans and Aspergillus flavus were analyzed in terms of ergosterol quantification assay, cellular leakage, proton efflux measurement (PM-ATPase), plasma membrane integrity assay (PI), and DNA damage assay (DAPI). Results shown that the extract of strain 196 has the potential to inhibit the cell membrane of the both pathogenic fungi which was further confirmed with the help of scanning electron microscopic (SEM) studies.

Microbial Journey: Mount Everest to Mars.

A rigorous exploration of microbial diversity has revealed its presence on Earth, deep oceans, and vast space. The presence of microbial life in diverse environmental conditions, ranging from moderate to extreme temperature, pH, salinity, oxygen, radiations, and altitudes, has provided the necessary impetus to search for them by extending the limits of their habitats. Microbiology started as a distinct science in the mid-nineteenth century and has provided inputs for the betterment of mankind during the last 150 years. As beneficial microbes are assets and pathogens are detrimental, studying both have its own merits. Scientists are nowadays working on illustrating the microbial dynamics in Earth's subsurface, deep sea, and polar regions. In addition to studying the role of microbes in the environment, the microbe-host interactions in humans, animals and plants are also unearthing newer insights that can help us to improve the health of the host by modulating the microbiota. Microbes have the potential to remediate persistent organic pollutants. Antimicrobial resistance which is a serious concern can also be tackled only after monitoring the spread of resistant microbes using disciplines of genomics and metagenomics The cognizance of microbiology has reached the top of the world. Space Missions are now looking for signs of life on the planets (specifically Mars), the Moon and beyond them. Among the most potent pieces of evidence to support the existence of life is to look for microbial, plant, and animal fossils. There is also an urgent need to deliberate and communicate these findings to layman and policymakers that would help them to take an adequate decision for better health and the environment around us. Here, we present a glimpse of recent advancements by scientists from around the world, exploring and exploiting microbial diversity.

Protection of surplus food from fungal spoilage using Streptomyces spp.: a green approach.

Consortia of Streptomyces spp. (colonies 169, 194, 165 and 130) used in this study are an efficient producer of secondary metabolites like chitinases and antifungal compounds, which may help in the protection of surplus food from spoilage. Qualitative screening for chitinase production and taxonomy of these colonies were undertaken in our previous studies. In the current study, GC-MS analysis of extract produced from the consortia of Streptomyces strains was done for the identification of antifungal compounds. Treatment of surplus food with activated consortia of Streptomyces spp. has protected powdered food for a month, whereas fresh food (unpowdered) was preserved for two days. A control sample of surplus food (untreated) was kept to check the contamination, which resulted in the growth of three fungi (FP-1, FG-1, and FB-1). Taxonomic characterization of fungi and identification of toxic compounds produced from them were done by ITS amplification and GC-MS analysis, respectively. The study shows that the secondary metabolites from Streptomyces spp. have the potential to protect the food from mycotoxin contamination. Based on literature reports, this is for the first time that bioactive compounds and chitinases produced from Streptomyces are being used for the protection and management of surplus food.

Potential applications of extracellular enzymes from Streptomyces spp. in various industries.

Extracellular enzymes produced from Streptomyces have the potential to replace toxic chemicals that are being used in various industries. The endorsement of this replacement has not received a better platform in developing countries. In this review, we have discussed the impact of chemicals and conventional practices on environmental health, and the role of extracellular enzymes to replace these practices. Burning of fossil fuels and agriculture residue is a global issue, but the production of biofuel using extracellular enzymes may be the single key to solve all these issues. We have discussed the replacement of hazardous chemicals with the use of xylanase, cellulase, and pectinase in food industries. In paper industries, delignification was done by the chemical treatment, but xylanase and laccase have the efficient potential to remove the lignin from pulp. In textile industries, the conventional method includes the chemicals which affect the nervous system and other organs. The use of xylanase, cellulase, and pectinase in different processes can give a safe and environment-friendly option to textile industries. Hazardous chemical pesticides can be replaced by the use of chitinase as an insecticide and fungicide in agricultural practices.

Draft genome and secondary metabolite biosynthetic gene clusters of Streptomyces sp. strain 196.

Emergence of MDR 'superbugs' inflamed a severe sense of urgency amongst scientists aiming at the discovery of novel potential drug molecules. Bacteria of the genus Streptomyces are really worth investigating for their immense potential to produce natural compounds of pharmaceutical importance. In the present study, the genome of Streptomyces sp. strain 196 was sequenced, studied and secondary metabolite biosynthetic gene clusters (smBGCs) were detected. FAME analysis was used for taxonomic validation of strain 196. Genome of strain 196 was sequenced using the Illumina NextSeq system which has resulted in a draft genome of 7.4 Mb. Rapid annotation using subsystem technology (RAST) results revealed the presence of 6682 CDS, 64 tRNA genes and 7 rRNA genes. Comparative studies revealed that strain 196 have 93.5% nucleotide and 96% protein level similarities with Streptomyces rhizosphaericola 1AS2c. Genome mining using antiSMASH predicted the presence of BGCs responsible for diverse bioactive compound production. The detected gene clusters were two PKS-III, one PKS-I, five NRPS, two hybrid PKS-I/NRPS, one thiopeptide/LAP, and one bacteriocin types. Furthermore, many other types BGCs such as three ectoine, two siderophore, one arylpolyene, two butyrolactone, one lassopeptide, one lanthipeptide and one melanin were also found. The results of this study provides information about genome and BGCs of strain 196, this information is valuable for researchers who are interested in isolation of bioactive compounds and working on heterologous expression of cryptic BGCs for novel bioactive compounds production.

In-silico and in-vitro evaluation of antifungal bioactive compounds from Streptomyces sp. strain 130 against Aspergillus flavus.

Streptomyces spp. are considered excellent reservoirs of natural bioactive compounds. The study evaluated the bioactive potential of secondary metabolites from Streptomyces sp. strain 130 through PKS-I and NRPS gene-clusters screening. GC-MS analysis was done for metabolic profiling of bioactive compounds from strain 130 in the next set of experiments. Identified antifungal compounds underwent ADMET analyses to screen their toxicity. All compounds' molecular docking was done with the structural gene products of the aflatoxin biosynthetic pathway of Aspergillus flavus. MD simulations were utilized to evaluate the stability of protein-ligand complexes under physiological conditions. Based on the in-silico studies, compound 2,4-di-tert butyl-phenol (DTBP) was selected for in-vitro studies against Aspergillus flavus. Simultaneously, bioactive compounds were extracted from strain 130 in two different solvents (ethyl-acetate and methanol) and used for similar assays. The MIC value of DTBP was found to be 314 µg/mL, whereas in ethyl-acetate extract and methanol-extract, it was 250 and 350 µg/mL, respectively. A mycelium growth assay was done to analyze the effect of compounds/extracts on the mycelium formation of Aspergillus flavus. In agar diffusion assay, zone of inhibitions in DTBP, ethyl-acetate extract, and methanol extract were observed with diameters of 11.3, 13.3, and 7.6 mm, respectively. In the growth curve assay, treated samples have delayed the growth of fungi, which signified that the compounds have a fungistatic nature. Spot assay has determined the fungal sensitivity to a sub-minimum inhibitory concentration of antifungal compounds. The study's results suggested that DTBP can be exploited for antifungal-drug development.Communicated by Ramaswamy H. Sarma.

Streptomyces antibioticalis, a Novel Species from a Sanitary Landfill Soil.

A novel isolate belonging to the genus Streptomyces, strain SL-4(T), was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4(T) were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4(T) contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4(T) and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357 (T) (DQ026672), S. albogriseolus NRRLB 1305 (T) (AJ494865), S viridodiastaticus NBRC 13106 (T) (AB184317), S. caelestis NRRL 2418 (T) (X80824), S. flavoviridis NBRC 12772 (T) (AB184842), S. pilosus NBRC 12807 (T) (AB184161) and S. longispororuber NBRC 13488 (T) (AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA-DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4(T) and its relatives S. atrovirens, S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4(T) representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4(T) (=CCM 7434(T)=MTCC 8588(T)).

In vitro and in silico anticancer potential analysis of Streptomyces sp. extract against human lung cancer cell line, A549.

During our previous investigation, bioactive compounds present in the extract of Streptomyces sp. strain 196 were characterized using LC-MS/MS and 1H NMR studies. These compounds were K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide; the study of these natural drugs against lung carcinoma is still limited. Focus of the current investigation was to study the anticancer effect of strain 196 extract on lung cancer cells (A549). During in vitro studies, anti-proliferative effect of extract was studied using MTT assay in A549 cells. Effect of extract on cell survival was further evaluated using colony assay. Cell death was qualitatively assessed using apoptosis assay. The aftereffect of extract treatment on metastatic potential of cancerous cells was studied using wound closure assay. Effect of extract on the morphology and cytoskeletal arrangement of A549 cells was studied using phalloidin staining. The extract demonstrated concentration and time-dependent cytotoxicity with IC50 value at 0.5 mg/ml (6 h) and 0.15 mg/ml (24 h). The proliferation and metastatic potential of cells, as characterized by MTT and migration assay, decreased over time in a concentration-dependent manner. Discrete changes in cellular morphology were noted as a result of the induced cytotoxicity. Apoptosis assay demonstrated 98.7% cell death at highest concentration of extract (1 mg/ml). During in silico studies, molecular docking revealed that strain 196 compounds are efficiently binding to mutant EGFR form (T790M/L858R) with release of binding energy (∆G) between - 5 and - 6.9 kcal/Mol. In conclusion, strain 196 extract could be a source of therapeutic drugs to treat lung carcinoma.

Exploitation of potential bioactive compounds from two soil derived actinomycetes, Streptomyces sp. strain 196 and RI.24.

Due to emergence of drug resistant pathogens, nearly all available medicines are becoming ineffective against these life threatening pathogens so there is dire need for the discovery of compounds having unique modes of action. During our previous studies, actinomycetes designated as 196 and RI.24 were isolated, screened for bioactive compounds production and characterized using 16S rRNA gene sequencing. Colony 196 was identified as strain of Streptomyces albolongus (100% sequence similarity) and RI.24 as strain of Streptomyces enissocaesilis (100% sequence similarity). In current study, potential bioactive compounds produced by these strains were characterized. Cold extraction method was applied for taking out of bioactive compounds from actinomycetes. Minimum inhibitory concentration (MIC) determination of compounds from these strains showed activity nearly in the range of commercial antibiotics (strain 196 0.0075 mg/ml, RI.24 0.25 mg/ml and chloramphenicol 0.0075 mg/ml, ampicillin 0.025 mg/ml). Structural elucidation of these compounds was carried out using spectroscopic techniques of LC-MS/MS and 1H NMR. Compounds K-252-C-Aglycone, indolocarbazole alkaloid, decoyinine, cycloheximide were detected from strain 196 whereas daunorubicin, hygromycin B, agecorynin F, indinavir-N-glucuronide and minocycline were identified from strain RI.24.Current study reports these compounds for the first time from strains of Streptomyces albolongus and Streptomyces enissocaesilis. Present investigation also suggests that strains 196 and RI.24 contain polyketide synthase-I (PKS-I) and non-ribosomal peptide synthetase (NRPS) gene clusters which are responsible for the production of bioactive compounds. The results of this study can be used by the scientific world or pharmaceutical industries for the development of new drugs/formulations by applying more advanced techniques.

Bioactive compounds from marine actinomycetes.

Actinomycetes are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Among its various genera, Streptomyces, Saccharopolyspora, Amycolatopsis, Micromonospora and Actinoplanes are the major producers of commercially important biomolecules. Several species have been isolated and screened from the soil in the past decades. Consequently the chance of isolating a novel actinomycete strain from a terrestrial habitat, which would produce new biologically active metabolites, has reduced. The most relevant reason for discovering novel secondary metabolites is to circumvent the problem of resistant pathogens, which are no longer susceptible to the currently used drugs. Existence of actinomycetes has been reported in the hitherto untapped marine ecosystem. Marine actinomycetes are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, insecticidal and enzyme inhibition. Bioactive compounds from marine actinomycetes possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens.

RP-HPLC Method for Simultaneous Estimation of Frusemide and Amiloride Hydrochloride in Tablet Formulation.

A new reverse phase high performance liquid chromatography method for the simultaneous estimation of frusemide and amiloride hydrochloride in tablet formulation is developed. The determination was carried out on a HIQ SIL, C18 (250×4.6 mm, 5 µm) column using a mobile phase of 50 mM phosphate buffer solution:acetonitrile (50:50 v/v, pH 3.0). The flow rate was 1.0 ml/min with detection at 283 nm. The retention time for frusemide was 3.038 min and for amiloride hydrochloride 10.002 min. Frusemide and amiloride hydrochloride showed a linear response in the concentration range of 20-200 µg/ml and 10-100 µg/ml, respectively. The results of analysis have been validated statistically and by recovery studies. The mean recoveries found for frusemide was 99.98% and for amiloride hydrochloride was 100.09%. Developed method was found to be simple, accurate, precise and selective for simultaneous estimation of frusemide and amiloride hydrochloride in tablets.