Soil Bacterium Yields Compounds Active Against Pathogens and Cancer Cell Lines

A research team based at Cairo University in Giza, Egypt, has isolated and characterized a soil-derived actinomycete bacterium named Streptomyces enissocaesilis strain SMS712, discovering that its secondary metabolites exhibit antibacterial, antioxidant, and selective anticancer properties in laboratory evaluations. The escalating global prevalence of multidrug-resistant bacterial strains alongside rising cancer rates has intensified scientific efforts to uncover novel natural therapeutics. Actinomycetes found in terrestrial soils serve as a primary natural repository of therapeutic molecules, historically supplying approximately two-thirds of known clinical antibiotics. In this investigation, the scientists set out to screen indigenous soil microflora for active biological agents capable of suppressing pathogenic microbes and mitigating cellular oxidative stress.

The bacterial strain was harvested from sandy, dark-colored soil collected at a depth between 10 and 12.5 centimeters on the grounds of Cairo University, situated at geographic coordinates 30°1’40.7202″N and 31°12’23.7823″E. Environmental measurements of the collection site recorded an ambient temperature of 24 degrees Celsius and an alkaline pH of 7.6. Following collection, three separate soil samples underwent thermal pretreatment at 60 degrees Celsius for two hours to suppress non-spore-forming microbial contaminants while promoting the growth of resilient actinomycete spores. Suspensions of the heat-treated soil were subsequently plated onto starch casein nitrate agar media and incubated at 30 degrees Celsius for seven days, yielding colonies characterized by light-yellow vegetative mycelia, light-gray aerial mycelia, and a distinct earthy odor.

Definitive taxonomic classification of the isolated organism was established through genetic extraction and comprehensive molecular sequencing of its 16S ribosomal RNA gene using specific universal primer sets. Comparative nucleotide alignment via the National Center for Biotechnology Information BLAST database established that the isolate shared 99 percent sequence identity—matching 1463 out of 1464 base pairs—with the reference type strain of Streptomyces enissocaesilis. Phylogenetic tree reconstruction using the neighbor-joining method confirmed its placement within a distinct evolutionary clade alongside related Streptomyces species. The identified gene sequence was formally registered in the GenBank database under accession number PV464955, solidifying the molecular identification of the strain as Streptomyces enissocaesilis SMS712.

To generate secondary metabolites for biological screening, the bacterial strain was cultivated in liquid glucose malt broth medium within shaking flasks at 28 degrees Celsius and 180 revolutions per minute for eight days. The resulting culture broth was subjected to triple solvent extraction using ethyl acetate, followed by rotary evaporation to recover concentrated organic residues. Disk diffusion antibacterial assays demonstrated that the crude extract exhibited broad-spectrum inhibitory actions against both Gram-negative and Gram-positive test bacteria. At an extract concentration of 100 milligrams per milliliter, the largest clear zone of inhibition was observed against Escherichia coli at 14.33 millimeters, followed by Staphylococcus aureus at 12.0 millimeters, Enterococcus faecalis at 10.67 millimeters, and Pseudomonas aeruginosa at 10.33 millimeters. Under identical experimental conditions, the crude organic extract produced no detectable inhibitory activity against the tested fungal strains Aspergillus niger and Candida albicans.

The crude bacterial extract was also evaluated for cytotoxic effects against established human malignant cell lines using the sulforhodamine B cell viability assay over a 48-hour exposure window. In these in vitro trials, the extract displayed marked cytotoxic potency against the MCF-7 human breast cancer line, yielding a half-maximal inhibitory concentration of 21.9 micrograms per milliliter. When challenged against the HCT-116 colon carcinoma cell line, cytotoxic effects were only observed at higher extract concentrations that simultaneously compromised the viability of non-cancerous BHK standard control cells. Parallel assessment of free radical scavenging capacity using the DPPH assay revealed concentration-dependent antioxidant activity, where radical scavenging percentages increased progressively from 37.0 percent at 12.5 milligrams per milliliter up to 53.5 percent at 100 milligrams per milliliter.

Chemical profiling of the crude extract using gas chromatography-mass spectrometry cataloged 29 individual organic constituents, dominated by long-chain fatty acid derivatives and complex phenolic structures. The primary detected constituent was the unsaturated fatty acid amide (Z)-13-docosenamide, commonly known as erucylamide, representing 24.11 percent of the total chromatographic area. The second most abundant compound was identified as (Z)-9-octadecenoic acid, or oleic acid, comprising 17.91 percent. Additional significant constituents identified in the organic mixture included 2,2’-methylenebis[6-tert-butyl-p-cresol] at 11.71 percent, a diazatricyclic derivative at 7.93 percent, an oxaspiro derivative at 7.80 percent, a polycyclic polyphenol at 6.47 percent, 2-ethyl-2-(hydroxymethyl)propane-1,3-diol at 5.36 percent, the flavonoid 3’,4’,7-trimethylquercetin at 3.28 percent, methyl octadecanoate at 2.31 percent, and the fatty alcohol 2-methylhexadecan-1-ol at 2.07 percent.

Preparative thin-layer chromatography using a mobile phase of ethyl acetate and chloroform in a three-to-one volume ratio separated the crude organic extract into four distinct resolved fractions. Band 1, isolated at a retention factor of 0.32, yielded 1.64 milligrams and produced the strongest antibacterial inhibition zone against Staphylococcus aureus measuring 21.0 millimeters, accompanied by 39.5 percent DPPH radical scavenging. Band 2, migrating at a retention factor of 0.74, yielded 12.1 milligrams and showed highest activity against Enterococcus faecalis with a 21.76-millimeter inhibition zone, alongside 16.9 percent radical scavenging. Band 3, obtained at a retention factor of 0.87, yielded 2.68 milligrams, displaying potent antioxidant scavenging of 52.9 percent. Band 4, isolated at a retention factor of 0.94, yielded 8.96 milligrams and produced the highest overall antioxidant activity at 55.3 percent, alongside antimicrobial activity against Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger.

Minimum inhibitory concentration evaluations performed with microdilution plates confirmed differential antimicrobial susceptibility profiles across the purified chromatographic fractions. Microbial species exposed to Band 1 showed complete growth cessation at 45 milligrams per milliliter for Staphylococcus aureus, Aspergillus niger, and Candida albicans, while Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa exhibited minimum inhibitory values of 90 milligrams per milliliter. Band 2 completely inhibited Enterococcus faecalis and Staphylococcus aureus at 45 milligrams per milliliter, whereas Candida albicans proved the most resistant organism, requiring 180 milligrams per milliliter. Band 3 inhibited the fungal species at 90 milligrams per milliliter but required up to 720 milligrams per milliliter against Gram-negative bacteria. Band 4 inhibited Aspergillus niger and Candida albicans at 45 and 90 milligrams per milliliter, respectively, while bacterial species required 360 milligrams per milliliter.

Structural elucidation of the purified fractions was performed using 500-megahertz proton nuclear magnetic resonance spectroscopy, correlating observed chemical shift signatures with chromatographic fragmentation data. Band 1 exhibited characteristic aliphatic signals alongside hydroxyl proton resonances at 2.502, 3.363, and 3.578 parts per million, confirming the structure of the long-chain fatty alcohol 2-methylhexadecan-1-ol. Band 2 displayed extended hydrocarbon chain resonances and a diagnostic amide proton peak at 8.5 parts per million, establishing its identity as (Z)-13-docosenamide. Band 3 produced methylene and methine signals adjacent to multiple hydroxyl groups, identifying the triol 2-ethyl-2-(hydroxymethyl)propane-1,3-diol. Band 4 revealed characteristic terminal methyl peaks at 0.853 parts per million along with carbonyl-adjacent methylene groups and ester-associated methine signals, confirming the structure of the glycerol-derived ester of (Z)-9-octadecenoic acid.

To investigate potential mechanisms of antimicrobial action, in silico molecular docking simulations were carried out against the crystal structures of two microbial virulence regulators: the bacterial transcriptional regulator SrrA from Staphylococcus aureus and the fungal cell-surface adhesin Als3 from Candida albicans. The docking calculations demonstrated that (Z)-13-docosenamide bound to the fungal Als3 protein with a favorable binding affinity of minus 6.3 kilocalories per mole, supported by hydrogen bonding and extensive hydrophobic interactions across twelve target amino acid residues. Compound (Z)-9-octadecenoic acid and 2-methylhexadecan-1-ol also displayed notable affinities toward Als3, yielding binding energies of minus 5.4 and minus 5.2 kilocalories per mole, respectively. When docked against bacterial SrrA, the four compounds exhibited binding energies ranging from minus 4.1 to minus 4.8 kilocalories per mole, indicating favorable physical interactions within regulatory binding pockets.

Computational ADMET profiling using graph-based pharmacokinetic algorithms was conducted to project the physiological safety and drug-likeness parameters of the four identified chemical structures. All four metabolites demonstrated favorable predictive oral bioavailability accompanied by high intestinal absorption rates and low skin permeability indices. Notably, none of the molecules produced mutagenic predictions in the simulated AMES bacterial test, and none were flagged as substrates for renal OCT2 transporters, suggesting a low likelihood of inherent genetic toxicity or direct nephrotoxic liability. Compound 2-methylhexadecan-1-ol was uniquely predicted to traverse the blood-brain barrier, whereas the remaining three compounds showed negligible central nervous system penetration, indicating suitability for systemic applications directed toward peripheral tissue targets.

While these findings represent the first documented identification of bioactive therapeutic secondary metabolites from Streptomyces enissocaesilis, the researchers noted that the antimicrobial mechanisms and predicted virulence disruptions rely largely on in silico modeling. Future research initiatives planned by the investigative team include functional in vitro cellular validation assays to examine the physical disruption of Als3-mediated mucosal adherence and biofilm assembly in Candida albicans. Additionally, the team plans to undertake complete whole-genome sequencing of Streptomyces enissocaesilis SMS712 to map the specific biosynthetic gene clusters governing metabolite synthesis, which may enable optimized production of these candidate chemical scaffolds for drug development pipelines.

Subject of Research: Bioactive secondary metabolites isolated from soil-derived Streptomyces enissocaesilis

Article Title: Isolation, identification, and bioactivity evaluation of novel metabolites from soil-derived actinomycetes with antimicrobial, antioxidant, and anticancer potential: in silico molecular docking insights

Article References: Soliman, M. M., Ibrahim, M., Dawoud, M. A., Mohamed, T. S., & Abo El-Souad, S. M. S. (2026). Isolation, identification, and bioactivity evaluation of novel metabolites from soil-derived actinomycetes with antimicrobial, antioxidant, and anticancer potential: in silico molecular docking insights. Annals of Microbiology, 76(1), Article 17. https://doi.org/10.1186/s13213-025-01836-4

Image Credits: AI Generated

DOI: 10.1186/s13213-025-01836-4

Keywords: Streptomyces enissocaesilis, Actinomycetes, Soil Microbiology, Antibacterial Agents, Antioxidant Activity, Cytotoxicity, Escherichia coli, Staphylococcus aureus, MCF-7 Breast Cancer, Thin Layer Chromatography, Gas Chromatography Mass Spectrometry, Nuclear Magnetic Resonance, Molecular Docking, ADMET Modeling, Secondary Metabolites, Erucamide, Oleic Acid