2.1 Isolation and antimicrobial activity screening of Streptomyces sp. MS. 10
Streptomyces sp. MS. 10 was recovered from a superficial soil layer sample collected from Ihnasia City in Beni-Suef Governorate, Egypt. It was isolated on International Streptomyces Project (ISP) 4 agar using the soil dilution plate technique, as previously described [13, 14].
Antimicrobial activity screening of Streptomyces sp. MS. 10 was performed using the cup diffusion method  against the following indicator strains: Sarcina lutea (environmental sample), Bacillus subtilis (environmental sample), Enterococcus faecalis (food sample), Salmonella enterica (ATCC 35664), Pseudomonas aeruginosa (ATCC 9027), Proteus sp. (clinical sample), Escherichia coli (clinical sample), and methicillin-resistant Staphylococcus aureus (MRSA; clinical sample). Briefly, tryptone soya agar plates were surface inoculated with the indicator strains, followed by the use of a sterile borer to make 10-mm cups in the agar plates that were filled with 150 μL of the supernatant from a liquid broth culture of Streptomyces sp. MS. 10. The antimicrobial activity against each indicator strain was determined according to the diameter of the inhibition zones around the cups, following incubation for 24 h at the appropriate temperature for growth of each indicator strain. An inhibition zone diameter ≥ 12 mm was recorded as positive for bioactivity.
2.2 Taxonomical identification using 16S rRNA gene sequencing
Genomic DNA of Streptomyces sp. MS. 10 was extracted and purified, as previously described . Then, polymerase chain reaction (PCR) amplification of the 16S rRNA gene was performed using forward primer 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′) . The PCR reaction was performed in a final volume of 50 μL using 10 μL of 5× reaction buffer, 500 ng of genomic DNA, a 10 mM dNTP mixture, 2.5 units of Taq DNA polymerase, and 1 μL of both forward and reverse primers. The PCR cycling conditions were as follows: initial denaturation of the DNA template for 3 min at 94 °C; followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s; and a final extension at 72 °C for 5 min.
Agarose gel electrophoresis (1.5%, w/v) was performed for 60 min at 90 V using 5 μL of each PCR product to determine the success of PCR amplification and the purity of the PCR product. A 1-kb DNA ladder was also included to estimate the size of the PCR products . The PCR product of the 16S rRNA gene was then purified using a PCR purification kit before being sequenced at Macrogen Korea using forward primer 785F (5′-GGATTAGATACCCTGGTA-3′) and reverse primer 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) .
The megaBLAST tool of the National Center for Biotechnology Information (NCBI) was used to compare the good-quality sequences of Streptomyces sp. MS. 10 with the GenBank database to identify the closest related strains that showed a high sequence similarity . Then, we performed multiple sequence alignments of the amplified sequences and those obtained from GenBank, followed by phylogenetic analysis of Streptomyces sp. MS. 10 using the MEGA7 software .
2.3 Fermentation and extraction of the bioactive compounds
Bacterial fermentation of Streptomyces sp. MS. 10 was performed by inoculating tryptone soya broth (TSB) with a single pure colony and incubating for 3 days at 30 °C. Then, ISP4 broth was seeded with 5% bacterial inoculum in TSB and incubated on a rotary shaker at 160 rpm for 7 days at 30 °C. Next, the bacterial broth was filtered through a Whatman No. 1 filter, followed by the addition of ethyl acetate (EtOAc; 1:1 v/v), and the organic-aqueous mixture was shaken frequently in a separating funnel to aid the optimal extraction of the bioactive metabolites from the ISP4 broth. Finally, the organic layer was separated and collected in another flask, and the EtOAc solvent was evaporated using a rotary evaporator .
A small-scale fermentation experiment (1.5 L) was performed using the abovementioned culture conditions and extraction method. Then, a large-scale fermentation experiment (10 L) was performed using the pre-optimized culture conditions, and the bioactive metabolites were extracted using the optimal extraction solvent (1:1 v/v dichloromethane [DCM]), as discussed later.
2.4 High-performance liquid chromatography
The bioactive compounds in the EtOAc crude extract from the small-scale fermentation experiment were chromatographically separated by preparative HPLC on a Dionex Ultimate 3000 HPLC system (Agilent) using a Nucleosil C18 column. The solvent gradient started with 100% H2O (HPLC grade) and 0% acetonitrile (ACN; HPLC grade) at a flow rate of 3 mL/min, and the percentage of ACN was linearly increased to 100% at 25 min. Final washing was performed for 15 min, using 100% ACN Then, a second preparative HPLC run was performed on fraction 25 (retention time [Rt] = 25 min), which showed the highest antimicrobial activity, using the same protocol as the first HPLC run, except that the solvent gradient started with 30% H2O and 70% methanol (HPLC grade) and linearly increased to 100% methanol at 25 min, with final washing for 15 min using 100% methanol.
2.5 Characterization of the major bioactive compounds
We tested the effect of different temperatures and degrading enzymes, including protease, amylase, and α-chymotrypsin, on the bioactivity of the supernatant from a liquid broth culture of Streptomyces sp. MS. 10 to characterize the major bioactive metabolites. Tubes containing 400 μL of the broth supernatant were incubated for 30 min in water baths that were set to different temperatures (60 °C, 80 °C, and 100 °C) to test the effect of temperature. The effect of 121 °C on the major bioactive compounds was determined by autoclaving for 15 min. To test the effect of degrading enzymes, they were separately added to the broth supernatant in different tubes. Their concentrations were adjusted to obtain a final concentration of 1 mg/ml in a final volume of 200 μL, and then the tubes were incubated at 37 °C for 30 min. Finally, the antimicrobial activity of all the treated broth supernatant samples was assessed using the agar diffusion method and compared with the activity of the original broth (positive control). Negative controls for the degrading enzymes were also included.
2.6 PCR amplification of the genes responsible for antibiotic biosynthesis
The genomic DNA of Streptomyces sp. MS. 10 was screened using PCR for the presence of genes responsible for the biosynthesis of natural products, including non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) I, PKS II, and glycopeptide monooxygenase B. The following primer pairs were used: NRPS/A3 F (5′-GCSTACSYSATSTACACSTCSGG-3′) and NRPS/A7 R (5′-SASGTCVCCSGTSGCGTAS-3′) to amplify the NRPS gene, with an expected product size of 700 base pairs (bp) ; PKS/K1 F (5′-TSAAGTCSAACATCCGBCA-3′) and PKS/M6 R (5′-CGCAGGTTSCSGTACCAGTA-3′) to amplify the PKS I gene, with an expected product size of 1200–1400 bp ; ARO-PKS-F (5′-GGCAGCGGITTCGGCGGITTCCAG-3′) and ARO-PKS-R (5′-CGITGTTIACIGCGTAGAACCAGGCG-3′) to amplify the PKS II gene, with an expected product size of 492-630 bp ; and oxyB F (5′-CTGGTCGGCAACCTGATGGAC-3′) and oxyB R (5′-CAGGTACCGGATCAGCTCGTC-3′) to amplify the glycopeptide monooxygenase B gene, with an expected product size of 696 bp . The PCR cycling conditions included initial denaturation for 5 min at 94 °C; followed by 40 successive cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 59 °C, 55 °C, 64 °C, and 60 °C for NRPS, PKS I, PKS II, and glycopeptide monooxygenase primers, respectively; and extension at 72 °C for 2 min. The final extension was at 72 °C for 10 min . The success of PCR amplification of the targeted genes was determined by running the PCR products on a 1.5% (w/v) agarose gel using both 100-bp and 1-kb ladders as size markers .
2.7 Optimization of the culture conditions and the extraction solvents
We optimized the culture conditions for Streptomyces sp. MS. 10 to determine those necessary for the optimal production of the major antimicrobial compounds by comparing different growth media, carbon sources, and nitrogen sources . The best culture media was selected by comparing the antimicrobial activity of Streptomyces sp. MS. 10 inoculated in different culture media, including ISP4, ISP4 supplemented with glucose, and TSB. The fermentation experiments for selecting best culture media were done using a shaker incubator at 160 rpm for 7 days at 30 °C. The best carbon source was determined by replacing the starch in ISP4 broth with other carbon sources, including fructose, glucose, maltose, sucrose, lactose, glycerol, mannitol, and sorbitol, while the best nitrogen source was selected by replacing the ammonium sulfate in ISP4 broth with ammonium citrate, urea, peptone, tryptone, proteose peptone, albumin, casein, casamino acid, and yeast extract. The fermentation experiments for testing optimal carbon and nitrogen sources were done using a shaker incubator at 160 rpm for 11 days at 30 °C with daily sampling starting from day 3.
The extraction conditions were optimized using different organic solvents, including hexane, chloroform, DCM, and EtOAc, at different solvent: broth proportions. The cup diffusion method was used to screen for antimicrobial activity under the different extraction conditions. Extraction success was defined as the absence of activity in the fermentation broth after liquid/liquid extraction, indicating complete extraction of the total metabolites in the bacterial broth.
2.8 Column chromatography
A total of 250 mg of crude extract was obtained from the extraction of the large-scale fermentation broth (10 L) of Streptomyces sp. MS. 10 using DCM (1:1, v/v) for extraction. The bioactive secondary metabolites in the crude extract were chromatographically separated using a normal phase packed silica gel column (column chromatography) and gradient elution with DCM and methanol in 1% increments until 100% methanol. The packed silica column was prepared using 12.5 g of silica gel. Briefly, the dry crude extract of Streptomyces sp. MS. 10 was solubilized in DCM. Then, 0.5 g of silica was added, and the extract was left to dry to make a dry band, which was added to the top of the packed silica column. Fractions of 10 ml each were collected, with a total of 160 fractions. The similarity of the collected fractions was checked using thin-layer chromatography (TLC) plates with DCM-methanol as the solvent system and p-anisaldehyde as the spray reagent, and the similar fractions were pooled. Additionally, the collected fractions were screened for antimicrobial activity using the cup diffusion method. Fractions showing good purity on the TLC plates and high antimicrobial activity were selected for further analysis using proton nuclear magnetic resonance (1H NMR) spectroscopy in an effort to determine their chemical classes.
2.9 Spectroscopic characterization
The most bioactive fraction from the second HPLC run (fraction 14) underwent analysis using liquid chromatography-mass spectrometry (LC-MS) at the Faculty of Postgraduate Studies of Advanced Science, Beni-Suef University, Egypt, to determine the major ion peaks and their mass-to-charge ratios (m/z). LC-MS was performed in both the negative and positive ion modes using electrospray ionization. The bioactive compounds that were isolated from the large-scale fermentation experiment underwent 1H NMR spectroscopy using a 400 MHz NMR system (Bruker) at the Faculty of Pharmacy, Beni-Suef University, Egypt . The NMR solvent was deuterated methanol (MeOD).