Identification of Antimicrobial Metabolites from the Egyptian Soil-Derived Amycolatopsis keratiniphila Revealed by Untargeted Metabolomics and Molecular Docking.

Actinomycetes are prolific producers of bioactive secondary metabolites. The prevalence of multidrug-resistant (MDR) pathogens has prompted us to search for potential natural antimicrobial agents. Herein, we report the isolation of rare actinobacteria from Egyptian soil. The strain was identified as Amycolatopsis keratiniphila DPA04 using 16S rRNA gene sequencing. Cultivation profiling, followed by chemical and antimicrobial evaluation of crude extracts, revealed the activity of DPA04 ISP-2 and M1 culture extracts against Gram-positive bacteria. Minimum inhibitory concentrations (MIC) values ranged from 19.5 to 39 µg/mL. Chemical analysis of the crude extracts using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) led to the identification of 45 metabolites of different chemical classes. In addition, ECO-0501 was identified in the cultures with significant antimicrobial activity. Multidrug resistance in Staphylococcus aureus is reported to be related to the multidrug efflux pump (MATE). ECO-0501 and its related metabolites were subjected to molecular docking studies against the MATE receptor as a proposed mechanism of action. ECO-0501 and its derivatives (AK_1 and N-demethyl ECO-0501) had better binding scores (-12.93, -12.24, and -11.92 kcal/mol) than the co-crystallized 4HY inhibitor (-8.99 kcal/mol) making them promising candidates as MATE inhibitors. Finally, our work established that natural products from this strain could be useful therapeutic tools for controlling infectious diseases.

Cholinesterase Inhibitors from an Endophytic Fungus Aspergillus niveus Fv-er401: Metabolomics, Isolation and Molecular Docking.

Alzheimer's disease poses a global health concern with unmet demand requiring creative approaches to discover new medications. In this study, we investigated the chemical composition and the anticholinesterase activity of Aspergillus niveus Fv-er401 isolated from Foeniculum vulgare (Apiaceae) roots. Fifty-eight metabolites were identified using UHPLC-MS/MS analysis of the crude extract. The fungal extract showed acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory effects with IC50 53.44 ± 1.57 and 48.46 ± 0.41 µg/mL, respectively. Two known metabolites were isolated, terrequinone A and citrinin, showing moderate AChE and BuChE inhibitory activity using the Ellman's method (IC50 = 11.10 ± 0.38 µg/mL and 5.06 ± 0.15 µg/mL, respectively for AChE, and IC50 15.63 ± 1.27 µg/mL and 8.02 ± 0.08 µg/mL, respectively for BuChE). As evidenced by molecular docking, the isolated compounds and other structurally related metabolites identified by molecular networking had the required structural features for AChE and BuChE inhibition. Where varioxiranol G (-9.76 and -10.36 kcal/mol), penicitrinol B (-9.50 and -8.02 kcal/mol), dicitrinol A (-8.53 and -7.98 kcal/mol) and asterriquinone CT5 (-8.02 and -8.25 kcal/mol) showed better binding scores as AChE and BuChE inhibitors than the co-crystallized inhibitor (between -7.89 and 7.82 kcal/mol) making them promising candidates for the development of new drugs to treat Alzheimer's.

Genome-Based Analysis of the Potential Bioactivity of the Terrestrial Streptomyces vinaceusdrappus Strain AC-40.

Streptomyces are factories of antimicrobial secondary metabolites. We isolated a Streptomyces species associated with the Pelargonium graveolens rhizosphere. Its total metabolic extract exhibited potent antibacterial and antifungal properties against all the tested pathogenic microbes. Whole genome sequencing and genome analyses were performed to take a look at its main characteristics and to reconstruct the metabolic pathways that can be associated with biotechnologically useful traits. AntiSMASH was used to identify the secondary metabolite gene clusters. In addition, we searched for known genes associated with plant growth-promoting characteristics. Finally, a comparative and pan-genome analysis with three closely related genomes was conducted. It was identified as Streptomyces vinaceusdrappus strain AC-40. Genome mining indicated the presence of several secondary metabolite gene clusters. Some of them are identical or homologs to gene clusters of known metabolites with antimicrobial, antioxidant, and other bioactivities. It also showed the presence of several genes related to plant growth promotion traits. The comparative genome analysis indicated that at least five of these gene clusters are highly conserved through rochei group genomes. The genotypic and phenotypic characteristics of S. vinaceusdrappus strain AC-40 indicate that it is a promising source of beneficial secondary metabolites with pharmaceutical and biotechnological applications.

Evaluation of different bacterial honey isolates as probiotics and their efficient roles in cholesterol reduction.

Continue to hypothesize that honey is a storehouse of beneficial bacteria, and the majority of these isolates are levansucrase producers. Accordingly, ten bacterial strains were isolated from different honey sources. Four honey isolates that had the highest levansucrase production and levan yield were identified by the partial sequencing of the 16S rRNA gene as Achromobacter sp. (10A), Bacillus paralicheniformis (2M), Bacillus subtilis (9A), and Bacillus paranthracis (13M). The cytotoxicity of the selected isolates showed negative blood hemolysis. Also, they are sensitive to the tested antibiotics (Amoxicillin + Flucloxacillin, Ampicillin, Gentamicin, Benzathine benzylpenicillin, Epicephin, Vancomycin, Amikacin, and Zinol). The isolates had strong alkaline stability (pHs 9, 11) and were resistant to severe acidic conditions (29-100 percent). The tested isolates recorded complete tolerance to both H2O2 and the bile salt (0.3% Oxgall powder) after 24 h incubation. The cell-free supernatant of the examined strains had antifungal activities against C. Albicans with varying degrees. Also, isolates 2M and 13M showed strong activities against S. aureus. The isolates showed strong adhesion and auto-aggregation capacity. Isolate 10A showed the highest antioxidant activity (91.45%) followed by 2M (47.37%). The isolates recorded different catalase and protease activity. All isolates produced cholesterol oxidase and lipase with different levels. Besides, the four isolates reduced LDL (low-density lipoprotein) to different significant values. The cholesterol-reducing ability varied not only for strains but also for the time of incubation. The previous results recommended these isolates be used safely in solving the LDL problem.

Molecular characterization of endophytic and ectophytic plant growth promoting bacteria isolated from tomato plants (Solanum lycopersicum L.) grown in different soil types.

BACKGROUND: Successful rhizosphere colonization by plant growth promoting rhizobacteria (PGPR) is of crucial importance to perform the desired plant growth promoting activities. Since rhizocompetence is a dynamic process influenced by surrounding environmental conditions. In the present study, we hypothesized that bacterial isolates obtained from different tomato plant microhabitats (balk soil, rhizosphere, endorhiza, phyllosphere, and endoshoot) grown in different soils (sand, clay, and peat moss) will show different rhizocompetence abilities. RESULTS: To evaluate this hypothesis, bacterial isolates were obtained from different plant microhabitats and screened for their phosphate solubilizing and nitrogen fixing activates. BOX-PCR fingerprint profiles showed high genotypic diversity among the tested isolates and that same genotypes were shared between different soils and/or plant microhabitats. 16S rRNA gene sequences of 25 PGP isolates, representing different plant spheres and soil types, were affiliated to eight genera: Enterobacter, Paraburkholderia, Klebsiella, Bacillus, Paenibacillus, Stenotrophomonas, Pseudomonas, and Kosakonia. The rhizocompetence of each isolate was evaluated in the rhizosphere of tomato plants grown on a mixture of the three soils. Different genotypes of the same bacterial species displayed different rhizocompetence potentials. However, isolates obtained from the above-ground parts of the plant showed high rhizocompetence. In addition, biological control-related genes, ituD and srfC, were detected in the obtained spore forming bacterial isolates. CONCLUSION: This study evaluates, for the first time, the relationship between plant microhabitat and the rhizocompetence ability in tomato rhizosphere. The results indicated that soil type and plant sphere can influence both the genotypic diversity and rhizocompetence ability of the same bacterial species. Bacterial isolates obtained in this study are promising to be used as an environmentally friendly substitution of chemical fertilizers.

Biosynthesis and Characterization of Silver Nanoparticles Produced by Phormidium ambiguum and Desertifilum tharense Cyanobacteria.

The world faces a challenge with the pervasion of multidrug-resistant bacteria that encourages scientists to develop and discover alternative, ecofriendly, and easy-to-produce new antibacterial agents. Our work is part of the greater effort of scientists around the world to achieve this goal by the biological synthesis of silver nanoparticles using cyanobacterial extracellular and intracellular components as nonchemical reducing agents. Two Egyptian cyanobacteria were isolated and identified according to 16S rRNA gene sequencing as Phormidium ambiguum and a novel species Desertifilum tharense. The sequences were deposited with accession numbers MW762709 and MW762710 for Desertifilum tharense and Phormidium ambiguum, respectively, in the GenBank. The results of UV-Vis analysis showed promising extracellular Ag-NPs synthesis by Desertifilum tharense and Phormidium ambiguum under light conditions. Therefore, these Ag-NPs were characterized and evaluated for antibacterial and antioxidant activity. TEM and SEM analyses revealed the spherical crystals with face-centered cubic structures and size range of 6.24-11.4 nm and 6.46-12.2 nm for Ag-NPs of Desertifilum tharense and Phormidium ambiguum, respectively. XRD and EDX results confirmed the successful synthesis of Ag-NPs in their oxide form or chloride form. The FTIR spectrum data confirmed the presence of hydroxyl and amide groups. Desertifilum tharense Ag-NPs displayed the largest inhibition zone that ranged from 9 mm against Micrococcus luteus ATCC 10240 to 25 mm against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. For Phormidium ambiguum Ag-NPs, the inhibition zone diameter was in the range of 9 mm to 18 mm. The biosynthesized Ag-NPs significantly inhibited the growth of medically important resistance-pathogenic Gram-positive and Gram-negative bacteria. The Ag-NPs of Phormidium ambiguum exhibited the highest scavenging activity of 48.7% when compared with that of Desertifilum tharense, which displayed 43.753%.

Potato plant spheres and to a lesser extent the soil type influence the proportion and diversity of bacterial isolates with in vitro antagonistic activity towards Ralstonia solanacearum.

Ralstonia solanacearum biovar2-race3 (Rs r3b2) is an epidemic soil-borne bacterial phytopathogen causing brown rot disease in potato. In this study, we assessed how three soil types stored at the same field site influenced the proportion and diversity of bacterial isolates with in vitro antagonistic activity towards Rs in bulk soil and different potato plant spheres (rhizosphere, endorhiza and endocaulosphere; ecto- and endosphere of seed and yield tubers). In general, the plate counts observed for each sample type were not significantly different. A total of 96 colonies per sample type was picked and screened for in vitro antagonistic activity against Rs. Antagonists were obtained from all bulk soils and plant spheres with the highest proportion obtained from the endorhiza and endocaulosphere of potato plants. BOX-PCR fingerprints of antagonists showed that some were specific for particular plant spheres independent of the soil type, while others originated from different plant spheres of a particular soil type. The majority of antagonists belonged to Pseudomonas. A high proportion of antagonists produced siderophores, and interestingly antagonists from potato tubers frequently carried multiple antibiotic production genes. Our data showed an enrichment of bacteria with genes or traits potentially involved in biocontrol in the rhizosphere and in endophytic compartments. We report that the proportion and diversity of in vitro antagonists towards Rs isolated from bulk soil and different spheres of potato plants grown under field conditions in three different soil types was mainly shaped by the plant sphere and to a lesser extent by the soil type. Bacteria with antagonistic activity towards Ralstonia solanacearum were isolated from all plant spheres and bulk soils but their proportion was highest in endophytic compartments.

Gene-targeted molecular phylogeny, phytochemical analysis, antibacterial and antifungal activities of some medicinal plant species cultivated in Egypt.

INTRODUCTION: Medicinal plants have been used in healthcare since time immemorial, as have their therapeutic activities and the production of plant-based medicines. OBJECTIVES: This study aims to use gene-targeted molecular markers for genetic diversity analysis of 16 medicinal plants. Besides, phytochemical analysis antibacterial and antifungal activities of some medicinal plant extracts commonly used in Egypt are compared to major compounds. METHODS: DNA-based classification of 16 medicinal species using Conserved DNA-Derived Polymorphism (CDDP) and Start Codon Targeted (SCoT) primers. Three species representing three orders (Pelargonium graveolens, Matricaria chamomilla, and Hyoscyamus muticus were analysed [high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS)] and evaluated for their antibacterial and antifungal activities against (Escherichia coli O157: H7 ATCC 93111, Salmonella typhimurium ATCC 14028, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Bacillus ceruse ATCC 33018, and Sclerotinia sclerotiorum in comparison with some of their antimicrobial components. RESULTS: Our results revealed 309 and 349 polymorphic bands with 100% polymorphism. Among them, 51 and 57 were unique loci for CDDP and SCoT, respectively. The 16 species were categorised into three groups depending on the similarity matrix. The results of antibacterial and antifungal activities revealed that Pelargonium oil showed significant antifungal and antibacterial activities against the tested pathogens. Gallic acid severely reduced all tested bacteria's growth, but atropine severely reduced the growth of the B. ceruse only. Molecular modelling revealed their activity against sclerotium development. CONCLUSION: The gene-targeted marker techniques were highly useful tools for the classification of the 16 medicinal plant species, despite displaying high similarities at morphological and phytochemical analyses but, have antifungal and antibacterial activities.

Biocontrol of Bacterial Wilt Disease Through Complex Interaction Between Tomato Plant, Antagonists, the Indigenous Rhizosphere Microbiota, and Ralstonia solanacearum.

Ralstonia solanacearum (biovar2, race3) is the causal agent of bacterial wilt and this quarantine phytopathogen is responsible for massive losses in several commercially important crops. Biological control of this pathogen might become a suitable plant protection measure in areas where R. solanacearum is endemic. Two bacterial strains, Bacillus velezensis (B63) and Pseudomonas fluorescens (P142) with in vitro antagonistic activity toward R. solanacearum (B3B) were tested for rhizosphere competence, efficient biological control of wilt symptoms on greenhouse-grown tomato, and effects on the indigenous rhizosphere prokaryotic communities. The population densities of B3B and the antagonists were estimated in rhizosphere community DNA by selective plating, real-time quantitative PCR, and R. solanacearum-specific fliC PCR-Southern blot hybridization. Moreover, we investigated how the pathogen and/or the antagonists altered the composition of the tomato rhizosphere prokaryotic community by 16S rRNA gene amplicon sequencing. B. velezensis (B63) and P. fluorescens (P142)-inoculated plants showed drastically reduced wilt disease symptoms, accompanied by significantly lower abundance of the B3B population compared to the non-inoculated pathogen control. Pronounced shifts in prokaryotic community compositions were observed in response to the inoculation of B63 or P142 in the presence or absence of the pathogen B3B and numerous dynamic taxa were identified. Confocal laser scanning microscopy (CLSM) visualization of the gfp-tagged antagonist P142 revealed heterogeneous colonization patterns and P142 was detected in lateral roots, root hairs, epidermal cells, and within xylem vessels. Although competitive niche exclusion cannot be excluded, it is more likely that the inoculation of P142 or B63 and the corresponding microbiome shifts primed the plant defense against the pathogen B3B. Both inoculants are promising biological agents for efficient control of R. solanacearum under field conditions.

Comparable dynamics of linuron catabolic genes and IncP-1 plasmids in biopurification systems (BPSs) as a response to linuron spiking.

On-farm biopurification systems (BPSs) represent an efficient technology for treating pesticide-contaminated wastewater. Biodegradation by genetically adapted bacteria has been suggested to perform a major contribution to the removal of pesticides in BPSs. Recently, several studies pointed to the role of IncP-1 plasmids in the degradation of pesticides in BPSs but this was never linked with catabolic markers. Therefore, a microcosm experiment was conducted in order to examine whether changes in mobile genetic element (MGE) abundances in response to the application of phenylurea herbicide linuron are linked with changes in catabolic genes. Denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S ribosomal RNA gene fragments amplified from total community (TC)-DNA suggested significant shifts in the bacterial community composition. PCR-Southern blot-based detection of genes involved in linuron hydrolysis (libA and hylA) or degradation of its metabolite 3,4-dichloroaniline (dcaQ I , dcaQ II , and ccdC) in TC-DNA showed that the abundance of the hylA gene was increased faster and stronger in response to linuron application than that of the libA gene, and that the dcaQ II gene was more abundant than the isofunctional gene dcaQ I 20 and 60 days after linuron addition. Furthermore, a significant increase in the relative abundance of the IncP-1-specific korB gene in response to linuron was recorded. Our data suggest that different bacterial populations bearing isofunctional genes coding for enzymes degrading linuron seemed to be enriched in BPSs in response to linuron and that IncP-1 plasmids might be involved in their dissemination.